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1.
Yakugaku Zasshi ; 140(10): 1199-1206, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999198

RESUMO

Potential risks to the fetus or infant should be considered prior to medication during pregnancy and lactation. It is essential to evaluate the exposure levels of drugs and their related factors in addition to toxicological effects. Epilepsy is one of the most common neurological complications in pregnancy; some women continue to use antiepileptic drugs (AEDs) to control seizures. Benzodiazepines (BZDs) are widely prescribed for several women who experience symptoms such as anxiety and insomnia during the postpartum period. In this review, we describe the 1) transport mechanisms of AEDs across the placenta and the effects of these drugs on placental transporters, and 2) the transfer of BZDs into breast milk. Our findings indicated that carrier systems were involved in the uptake of gabapentin (GBP) and lamotrigine (LTG) in placental trophoblast cell lines. SLC7A5 was the main contributor to GBP transport in placental cells. LTG was transported by a carrier that was sensitive to chloroquine, imipramine, quinidine, and verapamil. Short-term exposure to 16 AEDs had no effect on folic acid uptake in placental cells. However, long-term exposure to valproic acid (VPA) affected the expression of folate carriers (FOLR1, SLC46A1). Furthermore, VPA administration changed the expression levels of various transporters in rat placenta, suggesting that sensitivity to VPA differed across gestational stages. Lastly, we developed a method for quantifying eight BZDs in human breast milk and plasma using LC/MS/MS, and successfully applied it to quantify alprazolam in breast milk and plasma donated by a lactating woman.


Assuntos
Anticonvulsivantes/metabolismo , Benzodiazepinas/metabolismo , Transporte Biológico/genética , Aleitamento Materno , Gabapentina/metabolismo , Lactação/metabolismo , Lamotrigina/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Troca Materno-Fetal , Leite Humano/metabolismo , Placenta/metabolismo , Ácido Valproico/metabolismo , Anticonvulsivantes/efeitos adversos , Benzodiazepinas/efeitos adversos , Benzodiazepinas/uso terapêutico , Linhagem Celular , Epilepsia/tratamento farmacológico , Feminino , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Gabapentina/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Humanos , Lamotrigina/efeitos adversos , Gravidez , Complicações na Gravidez/tratamento farmacológico , Transportador de Folato Acoplado a Próton/genética , Transportador de Folato Acoplado a Próton/metabolismo , Ácido Valproico/efeitos adversos
2.
Nat Commun ; 11(1): 4907, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999289

RESUMO

Global alterations in the metabolic network provide substances and energy to support tumor progression. To fuel these metabolic processes, extracellular matrix (ECM) plays a dominant role in supporting the mass transport and providing essential nutrients. Here, we report a fibrinogen and thrombin based coagulation system to construct an artificial ECM (aECM) for selectively cutting-off the tumor metabolic flux. Once a micro-wound is induced, a cascaded gelation of aECM can be triggered to besiege the tumor. Studies on cell behaviors and metabolomics reveal that aECM cuts off the mass transport and leads to a tumor specific starvation to inhibit tumor growth. In orthotopic and spontaneous murine tumor models, this physical barrier also hinders cancer cells from distant metastasis. The in vivo gelation provides an efficient approach to selectively alter the tumor mass transport. This strategy results in a 77% suppression of tumor growth. Most importantly, the gelation of aECM can be induced by clinical operations such as ultrasonic treatment, surgery or radiotherapy, implying this strategy is potential to be translated into a clinical combination regimen.


Assuntos
Materiais Biomiméticos/administração & dosagem , Matriz Extracelular/química , Neoplasias/terapia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/efeitos da radiação , Materiais Biomiméticos/química , Materiais Biomiméticos/efeitos da radiação , Linhagem Celular Tumoral/transplante , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Quimiorradioterapia/métodos , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos da radiação , Feminino , Fibrinogênio/administração & dosagem , Fibrinogênio/química , Fibrinogênio/efeitos da radiação , Géis , Humanos , Injeções Intravenosas , Metabolômica , Camundongos , Neoplasias/metabolismo , Trombina/administração & dosagem , Trombina/química , Trombina/efeitos da radiação , Terapia por Ultrassom/métodos , Ondas Ultrassônicas
3.
Nat Commun ; 11(1): 4948, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009415

RESUMO

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.


Assuntos
Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Chaperonas Moleculares/metabolismo , Pseudomonas aeruginosa/metabolismo , Regulação Alostérica , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Transporte Biológico , Modelos Moleculares , Domínios Proteicos
4.
Med Hypotheses ; 143: 110201, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33017909

RESUMO

COVID-19 initially an epidemic caused by SARS-CoV-2 has turned out to be a life- threatening global pandemic with increased morbidity and mortality. The presence of cytokine storm has been linked with the pathogenesis of severe lung injury as evinced in COVID-19. Aquaporins (AQPs) are molecular water channels, facilitating water transport across the cell membrane in response to osmotic gradients. Impairment in alveolar fluid clearance due to altered functional expression of respiratory AQPs highlight their pathophysiological significance in pulmonary edema associated respiratory illness. Therefore, we hypothesize that targeted modulation of AQPs in lungs in the intervening period of time, could diminish the dreadful effects of inflammation- induced comorbidity in COVID-19.


Assuntos
Aquaporinas/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Edema Pulmonar/tratamento farmacológico , Animais , Betacoronavirus , Transporte Biológico , Comorbidade , Citocinas/metabolismo , Humanos , Inflamação , Pulmão/imunologia , Pulmão/virologia , Camundongos , Pandemias
5.
Yakugaku Zasshi ; 140(10): 1235-1242, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999202

RESUMO

The central nervous system (CNS) is segregated from the circulating blood and peripheral tissues by endothelial and epithelial barriers. To overcome refractory CNS diseases, it is important to understand the membrane transport systems of drugs and the endogenous compounds that relate to the pathogenesis of CNS diseases at these barriers. The endothelial barrier in the brain is the blood-brain barrier (BBB). Our studies clarified the efflux transport of prostaglandin E2 (PGE2), a modulator of neural excitation and inflammatory responses, across the BBB via plasma membrane transporters such as organic anion transporter 3 (Oat3) and multidrug resistance-associated protein 4 (Mrp4). This efflux transport was attenuated by peripheral inflammation or cerebral treatment with neuroexcitatory l-glutamate, suggesting that BBB-mediated PGE2 elimination was altered under several pathological conditions. We also examined excitatory amino acid transporter (EAAT) 1 and 3 as l-glutamate efflux transporters of the inner blood-retinal barrier (BRB) and blood-cerebrospinal barrier. It was considered that these efflux membrane transporters participated in the homeostasis of neuroexcitatory and neuroinflammatory responses in the brain and retina. Moreover, we identified connexin 43 (Cx43) hemichannels as a new membrane transport system that is activated under pathological conditions and recognizes several monocarboxylate drugs, such as valproate. As it is expected that the action of these membrane transporters across the CNS barriers is of great importance in understanding the pathology of various neuroexcitatory diseases, our studies should contribute to the establishment of therapeutic strategies for refractory CNS diseases.


Assuntos
Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/metabolismo , Encéfalo/metabolismo , Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/metabolismo , Desenvolvimento de Medicamentos , Proteínas de Membrana Transportadoras/metabolismo , Retina/metabolismo , Animais , Doenças do Sistema Nervoso Central/tratamento farmacológico , Conexina 43/metabolismo , Dinoprostona/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Terapia de Alvo Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo
6.
Proc Biol Sci ; 287(1934): 20201506, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32900308

RESUMO

The sea urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires [Formula: see text] concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [Formula: see text] mediated by the [Formula: see text] cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH.


Assuntos
Bicarbonatos/metabolismo , Ouriços-do-Mar/fisiologia , Animais , Transporte Biológico , Calcificação Fisiológica , Carbonato de Cálcio , Concentração de Íons de Hidrogênio , Oceanos e Mares , Prótons , Água do Mar
7.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879484

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribução , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribução , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribução , Vacúolos/metabolismo
8.
Ecotoxicol Environ Saf ; 203: 111054, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888616

RESUMO

Quinclorac (3,7-dichloroquinoline-8-carboxylic acid, QNC) is a highly selective auxin herbicide that is typically applied to paddy rice fields. Its residue is a serious problem in crop rotations. In this study, Oryza sativa L. seedlings was used as a model plant to explore its biochemical response to abiotic stress caused by QNC and nZVI coexposure, as well as the interactions between QNC and nZVI treatments. Exposure to 5 and 10 mg/L QNC reduced the fresh biomass by 26.6% and 33.9%, respectively, compared to the control. The presence of 50 and 250 mg/L nZVI alleviated the QNC toxicity, but the nZVI toxicity was aggravated by the coexist of QNC. Root length was enhanced upon exposure to low or medium doses of both QNC and nZVI, whereas root length was inhibited under high-dose coexposure. Both nZVI and QNC, either alone or in combination, significantly inhibited the biosynthesis of chlorophyll, and the inhibition rate increased with elevated nZVI and QNC concentration. It was indicated that nZVI or QNC can affect the plant photosynthesis, and there was a significant interaction between the two treatments. Effects of QNC on the antioxidant response of Oryza sativa L. differed in the shoots and roots; generally, the introduction of 50 and 250 mg/L nZVI alleviated the oxidative stress (POD in shoots, SOD and MDA in roots) induced by QNC. However, 750 mg/kg nZVI seriously damaged Oryza sativa L. seedlings, which likely resulted from active iron deficiency. QNC could be removed from the culture solution by nZVI; as a result, nZVI suppressed QNC uptake by 20%-30%.


Assuntos
Antioxidantes/metabolismo , Ferro/toxicidade , Nanopartículas/toxicidade , Oryza/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Quinolinas/toxicidade , Poluentes do Solo/toxicidade , Transporte Biológico , Biomassa , Clorofila/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Plântula/metabolismo
9.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: covidwho-744380

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribução , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribução , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribução , Vacúolos/metabolismo
10.
Int J Nanomedicine ; 15: 6385-6399, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922007

RESUMO

Purpose: The mononuclear phagocyte system (MPS) presents a formidable obstacle that hampers the delivery of various nanopreparations to tumors. Therefore, there is an urgent need to improve the off-MPS targeting ability of nanomedicines. In the present study, we present a novel preconditioning strategy to substantially increase the circulation times and tumor targeting of nanoparticles by regulating nanocarrier-MPS interactions. Methods: In vitro, the effect of different vacuolar H+-ATPase inhibitors on macrophage uptake of targeted or nontargeted lipid vesicles was evaluated. Specifically, the clinically approved proton-pump inhibitor esomeprazole (ESO) was selected as a preconditioning agent. Then, we further investigated the blocking effect of ESO on the macrophage endocytosis of nanocarriers. In vivo, ESO was first intravenously administered into A549-tumor-bearing nude mice, and 24 h later, the c(RGDm7)-modified vesicles co-loaded with doxorubicin and gefitinib were intravenously injected. Results: In vitro, ESO was found to reduce the interactions between macrophages and c(RGDm7)-modified vesicles by interfering with the latter's lysosomal trafficking. Studies conducted in vivo confirmed that ESO pretreatment greatly decreased the liver and spleen distribution of the targeted vesicles, enhanced their tumor accumulation, and improved the therapeutic outcome of the drug-loaded nanomedicines. Conclusion: Our findings indicate that ESO can regulate the nanoparticle-MPS interaction, which provides a feasible option for enhancing the off-MPS targeting of nanomedicines.


Assuntos
Portadores de Fármacos/química , Esomeprazol/farmacologia , Sistema Fagocitário Mononuclear/citologia , Nanopartículas/química , Células A549 , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transporte Biológico , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Endocitose , Esomeprazol/farmacocinética , Esomeprazol/uso terapêutico , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Camundongos , Camundongos Nus , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Células RAW 264.7 , Distribuição Tecidual/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo
11.
Int J Nanomedicine ; 15: 6503-6518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922013

RESUMO

Objective: A non-lipolysis nanoemulsion (NNE) was designed to reduce the first-pass metabolism of raloxifene (RAL) by intestinal UDP-glucuronosyltransferases (UGTs) for increasing the oral absorption of RAL, coupled with in vitro and in vivo studies. Methods: In vitro stability of NNE was evaluated by lipolysis and the UGT metabolism system. The oral bioavailability of NNE was studied in rats and pigs. Finally, the absorption mechanisms of NNE were investigated by in situ single-pass intestinal perfusion (SPIP) in rats, Madin-Darby canine kidney (MDCK) cells model, and lymphatic blocking model. Results: The pre-NNE consisted of isopropyl palmitate, linoleic acid, Cremophor RH40, and ethanol in a weight ratio of 3.33:1.67:3:2. Compared to lipolysis nanoemulsion of RAL (RAL-LNE), the RAL-NNE was more stable in in vitro gastrointestinal buffers, lipolysis, and UGT metabolism system (p < 0.05). The oral bioavailability was significantly improved by the NNE (203.30%) and the LNE (205.89%) relative to the suspension group in rats. However, 541.28% relative bioavailability was achieved in pigs after oral NNE intake compared to the suspension and had two-fold greater bioavailability than the LNE (p < 0.05). The RAL-NNE was mainly absorbed in the jejunum and had high permeability at the intestine of rats. The results of both SPIP and MDCK cell models demonstrated that the RAL-NNE was absorbed via endocytosis mediated by caveolin and clathrin. The other absorption route, the lymphatic transport (cycloheximide as blocking agent), was significantly improved by the NNE compared with the LNE (p < 0.05). Conclusion: A NNE was successfully developed to reduce the first-pass metabolism of RAL in the intestine and enhance its lymphatic transport, thereby improving the oral bioavailability. Altogether, NNE is a promising carrier for the oral delivery of drugs with significant first-pass metabolism.


Assuntos
Absorção Fisico-Química , Emulsões/química , Lipólise , Nanopartículas/química , Cloridrato de Raloxifeno/metabolismo , Administração Oral , Animais , Disponibilidade Biológica , Transporte Biológico , Sobrevivência Celular , Cães , Emulsões/administração & dosagem , Feminino , Intestinos/fisiologia , Linfa/metabolismo , Células Madin Darby de Rim Canino , Masculino , Polietilenoglicóis , Ratos Sprague-Dawley , Tensoativos/química , Suínos
12.
PLoS Biol ; 18(8): e3000790, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776918

RESUMO

Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.


Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Uridina/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato , Uridina/metabolismo
13.
Nat Commun ; 11(1): 3825, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732874

RESUMO

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.


Assuntos
Lipídeos , Malária Falciparum/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/parasitologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Transporte Proteico , Vacúolos/metabolismo , Vacúolos/parasitologia
14.
PLoS One ; 15(8): e0234892, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817668

RESUMO

The mosquito Aedes aegypti vectors the arboviral diseases yellow fever, dengue, Zika and chikungunya. Larvae are usually found developing in freshwater; however, more recently they have been increasingly found in brackish water, potential habitats which are traditionally ignored by mosquito control programs. Aedes aegypti larvae are osmo-regulators maintaining their hemolymph osmolarity in a range of ~ 250 to 300 mOsmol l-1. In freshwater, the larvae must excrete excess water while conserving ions while in brackish water, they must alleviate an accumulation of salts. The compensatory physiological mechanisms must involve the transport of ions and water but little is known about the water transport mechanisms in the osmoregulatory organs of these larvae. Water traverses cellular membranes predominantly through transmembrane proteins named aquaporins (AQPs) and Aedes aegypti possesses 6 AQP homologues (AaAQP1 to 6). The objective of this study was to determine if larvae that develop in freshwater or brackish water have differential aquaporin expression in osmoregulatory organs, which could inform us about the relative importance and function of aquaporins to mosquito survival under these different osmotic conditions. We found that AaAQP transcript abundance was similar in organs of freshwater and brackish water mosquito larvae. Furthermore, in the Malpighian tubules and hindgut AaAQP protein abundance was unaffected by the rearing conditions, but in the gastric caeca the protein level of one aquaporin, AaAQP1 was elevated in brackish water. We found that AaAQP1 was expressed apically while AaAQP4 and AaAQP5 were found to be apical and/or basal in the epithelia of osmoregulatory organs. Overall, the results suggest that aquaporin expression in the osmoregulatory organs is mostly consistent between larvae that are developing in freshwater and brackish water. This suggests that aquaporins may not have major roles in adapting to longterm survival in brackish water or that aquaporin function may be regulated by other mechanisms like post-translational modifications.


Assuntos
Aedes/genética , Aquaporinas/genética , Osmorregulação/genética , Aedes/fisiologia , Animais , Aquaporinas/metabolismo , Infecções por Arbovirus , Transporte Biológico , Ecossistema , Água Doce , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Hemolinfa/metabolismo , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Osmorregulação/fisiologia , Osmose , Águas Salinas , Salinidade , Água/metabolismo
15.
PLoS Genet ; 16(8): e1008966, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776922

RESUMO

The vacuole of the yeast Saccharomyces cerevisiae plays an important role in nutrient storage. Arginine, in particular, accumulates in the vacuole of nitrogen-replete cells and is mobilized to the cytosol under nitrogen starvation. The arginine import and export systems involved remain poorly characterized, however. Furthermore, how their activity is coordinated by nitrogen remains unknown. Here we characterize Vsb1 as a novel vacuolar membrane protein of the APC (amino acid-polyamine-organocation) transporter superfamily which, in nitrogen-replete cells, is essential to active uptake and storage of arginine into the vacuole. A shift to nitrogen starvation causes apparent inhibition of Vsb1-dependent activity and mobilization of stored vacuolar arginine to the cytosol. We further show that this arginine export involves Ypq2, a vacuolar protein homologous to the human lysosomal cationic amino acid exporter PQLC2 and whose activity is detected only in nitrogen-starved cells. Our study unravels the main arginine import and export systems of the yeast vacuole and suggests that they are inversely regulated by nitrogen.


Assuntos
Arginina/metabolismo , Nitrogênio/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Aminoácidos/genética , Transporte Biológico/genética , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo
16.
Nat Commun ; 11(1): 4331, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859914

RESUMO

Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet. In addition, ~400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels.


Assuntos
Conexinas/química , Conexinas/metabolismo , Microscopia Crioeletrônica/métodos , Transporte Biológico , Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica
17.
Nat Commun ; 11(1): 4055, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792504

RESUMO

Although metastasis is the most common cause of cancer deaths, metastasis-intrinsic dependencies remain largely uncharacterized. We previously reported that metastatic pancreatic cancers were dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Surprisingly, PGD catalysis was constitutively elevated without activating mutations, suggesting a non-genetic basis for enhanced activity. Here we report a metabolic adaptation that stably activates PGD to reprogram metastatic chromatin. High PGD catalysis prevents transcriptional up-regulation of thioredoxin-interacting protein (TXNIP), a gene that negatively regulates glucose import. This allows glucose consumption rates to rise in support of PGD, while simultaneously facilitating epigenetic reprogramming through a glucose-fueled histone hyperacetylation pathway. Restoring TXNIP normalizes glucose consumption, lowers PGD catalysis, reverses hyperacetylation, represses malignant transcripts, and impairs metastatic tumorigenesis. We propose that PGD-driven suppression of TXNIP allows pancreatic cancers to avidly consume glucose. This renders PGD constitutively activated and enables metaboloepigenetic selection of additional traits that increase fitness along glucose-replete metastatic routes.


Assuntos
Cromatina/metabolismo , Glucose/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Imunoprecipitação da Cromatina , Epigênese Genética/genética , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
18.
Ecotoxicol Environ Saf ; 205: 111145, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32846296

RESUMO

This study characterizes sunflower response to the levels of Cd encountered in moderately Cd-polluted soils. Two sunflower cultivars differing in their ability to sequestrate Cd in roots were exposed to low concentrations of Cd (0.5 nM or 100 nM) in hydroponics and sampled after 18 days (258 degree-days) when ten leaves were fully expanded. Plant growth, Cd uptake and partitioning among organs were monitored along with the ionomic (ICP-MS) and the metabolic (1H-NMR) composition of the xylem sap. Sunflower tolerance to Cd differed between the two cultivars. The cultivar with the highest ability to sequestrate Cd in roots (Kapllan) was more tolerant to Cd than the one with the lowest ability (ES RICA). The 23% penalization of plant growth observed at 100 nM in cultivar ES RICA was associated with reduced xylem loading fluxes of soluble sugars, perhaps pointing to disruption of carbohydrate metabolism. Retention of Cd in the stem was higher at 100 nM than at 0.5 nM in the Cd-sensitive cultivar ES RICA, which can be seen as a sunflower strategy to restrict the amount of Cd delivered to the leaves under Cd stress. No direct connection was found between the speciation of Cd in the xylem sap and the Cd translocation efficiency, although significant changes in the free ionic fraction of Cd were observed between the two cultivars at 0.5 nM. The relevance of these results in promoting the use of sunflower in phytomanagement of Cd-polluted soils is discussed.


Assuntos
Cádmio/metabolismo , Helianthus/metabolismo , Hidroponia , Transporte Biológico , Desenvolvimento Vegetal , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Xilema/metabolismo
19.
Ecotoxicol Environ Saf ; 204: 111148, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32818843

RESUMO

To disclose how phosphorus deficiency influence phytoremediation of Cd contamination using poplars, root architecture, Cd absorption, Cd translocation and antioxidant defense in poplar roots were investigated using a clone of Populus × euramericana. Root growth was unaltered by Cd exposure regardless of P conditions, while the degree of root proliferation upon P deficiency was changed by high level of Cd exposure. The concentration and content of Cd accumulation in roots were increased by P deficiency. This can be partially explained by the increased expression of genes encoding PM H + -ATPase under the combined conditions of P deficiency and high Cd exposure, which enhanced Cd2+-H+ exchanges and led to an increment of Cd uptake under P deficiency. Despite of the increasing Cd accumulation in roots, the translocation of Cd from roots to aerial tissues sharply decreased upon P deficiency. The relative expression of genes responsible for Cd translocation (HMA4) decreased upon P deficiency and thus inhibited Cd translocation via xylem. GR activity was decreased by P deficiency, which can inhibit the form of GSH and GSH-Cd complexes and decrease Cd translocation via GSH-Cd complexes. The transportation of PC-Cd complexes into vacuole decreased under P deficiency as a result of the low expression of PCS and ABCC1, and thus suppressed Cd tolerance and Cd detoxification in roots. Moreover, P deficiency decreased the levels of antioxidase (GR and CAT) and phytohormones including JA, ABA and GA3, which synchronously reduced antioxidant capacity in roots.


Assuntos
Cádmio/metabolismo , Fósforo/metabolismo , Populus/fisiologia , Adaptação Fisiológica , Antioxidantes/metabolismo , Biodegradação Ambiental , Transporte Biológico , Cádmio/toxicidade , Proliferação de Células , Reguladores de Crescimento de Planta/metabolismo , Raízes de Plantas/metabolismo , Populus/metabolismo , Xilema/metabolismo
20.
Chem Biol Interact ; 330: 109228, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32827518

RESUMO

This study aimed at exploring the potential mechanism of decreased in vivo exposure of the antiplatelet agent, ticagrelor and its active metabolite, AR-C124910XX, mediated by tea polyphenols, which was first revealed by our previous study, as well as predicting the in vivo drug-drug interaction (DDI) potential utilizing an in vitro to in vivo extrapolation (IVIVE) approach. The bidirectional transport and uptake kinetics of ticagrelor were determined using Caco-2 cells. Inhibition potency of major components of tea polyphenols, epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were obtained from Caco-2 cells, human intestinal and hepatic microsomes (HIMs and HLMs) in vitro. A mean efflux ratio of 2.28 ± 0.38 and active uptake behavior of ticagrelor were observed in Caco-2 cell studies. Further investigation showed that the IC50 values of EGCG and EGC on the uptake of ticagrelor were 42.0 ± 5.1 µM (95% CI 31.9-54.8 µM) and 161 ± 13 µM (95% CI 136-191 µM), respectively. EGCG and EGC also displayed moderate to weak reversible inhibition on the formation of AR-C124910XX and the inactive metabolite, AR-C133913XX in HIMs and HLMs, while no clinically significant time-dependent inhibition was observed for either compound. IVIVE indicated a significant inhibition effect of EGCG on the uptake process of ticagrelor, while no potential DDI risk was found based on microsomal data. A 45% decrease in ticagrelor in vivo exposure was mechanistically predicted by incorporating intestinal and hepatic metabolism as well as intestinal absorption. This dual inhibition of tea polyphenols on ticagrelor revealed the underlying potential of transporter-enzyme interplay, in which the altered uptake process was more critical.


Assuntos
Modelos Teóricos , Polifenóis/farmacologia , Chá/química , Ticagrelor/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/metabolismo , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular Tumoral , Interações Medicamentosas , Humanos , Absorção Intestinal/efeitos dos fármacos , Cinética , Microssomos Hepáticos/metabolismo , Inibidores da Agregação de Plaquetas/farmacocinética , Antagonistas do Receptor Purinérgico P2Y/farmacocinética , Ticagrelor/metabolismo , Ticagrelor/farmacocinética
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