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1.
Nat Commun ; 11(1): 4368, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868758

RESUMO

Increased extracellular sodium activates Th17 cells, which provide protection from bacterial and fungal infections. Whilst high salt diets have been shown to worsen autoimmune disease, the immunological consequences of clinical salt depletion are unknown. Here, we investigate immunity in patients with inherited salt-losing tubulopathies (SLT). Forty-seven genotyped SLT patients (with Bartter, Gitelman or EAST Syndromes) are recruited. Clinical features of dysregulated immunity are recorded with a standardised questionnaire and immunological investigations of IL-17 responsiveness undertaken. The effects of altering extracellular ionic concentrations on immune responses are then assessed. Patients are hypokalaemic and hypomagnesaemic, with reduced interstitial sodium stores determined by 23Na-magnetic resonance imaging. SLT patients report increased mucosal infections and allergic disease compared to age-matched controls. Aligned with their clinical phenotype, SLT patients have an increased ratio of Th2:Th17 cells. SLT Th17 and Tc17 polarisation is reduced in vitro, yet STAT1 and STAT3 phosphorylation and calcium flux following T cell activation are unaffected. In control cells, the addition of extracellular sodium (+40 mM), potassium (+2 mM), or magnesium (+1 mM) reduces Th2:Th17 ratio and augments Th17 polarisation. Our results thus show that the ionic environment typical in SLT impairs IL-17 immunity, but the intracellular pathways that mediate salt-driven Th17 polarisation are intact and in vitro IL-17 responses can be reinvigorated by increasing extracellular sodium concentration. Whether better correction of extracellular ions can rescue the immunophenotype in vivo in SLT patients remains unknown.


Assuntos
Síndromes de Imunodeficiência/etiologia , Interleucina-17/metabolismo , Túbulos Renais Distais/patologia , Adolescente , Adulto , Idoso de 80 Anos ou mais , Animais , Pré-Escolar , Doença Crônica , Estudos de Coortes , Feminino , Doenças Genéticas Inatas , Humanos , Magnésio/metabolismo , Masculino , Pessoa de Meia-Idade , Potássio/metabolismo , Sais/metabolismo , Sais/uso terapêutico , Sódio/metabolismo , Cloreto de Sódio/metabolismo , Cloreto de Sódio na Dieta/uso terapêutico , Células Th17/metabolismo , Células Th2/metabolismo , Adulto Jovem
2.
Cell ; 182(6): 1560-1573.e13, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32783916

RESUMO

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.


Assuntos
Metiltransferases/química , RNA Helicases/química , RNA Replicase/química , Proteínas não Estruturais Virais/química , Replicação Viral , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Holoenzimas/química , Holoenzimas/metabolismo , Magnésio/metabolismo , Metiltransferases/metabolismo , Ligação Proteica , RNA Helicases/metabolismo , RNA Replicase/metabolismo , RNA Viral/química , Proteínas não Estruturais Virais/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(33): 20235-20243, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32753384

RESUMO

All cells require Mg2+ to replicate and proliferate. The macrophage protein Slc11a1 is proposed to protect mice from invading microbes by causing Mg2+ starvation in host tissues. However, the Mg2+ transporter MgtB enables the facultative intracellular pathogen Salmonella enterica serovar Typhimurium to cause disease in mice harboring a functional Slc11a1 protein. Here, we report that, unexpectedly, the Salmonella small protein MgtR promotes MgtB degradation by the protease FtsH, which raises the question: How does Salmonella preserve MgtB to promote survival inside macrophages? We establish that the Salmonella small protein MgtU prevents MgtB proteolysis, even when MgtR is absent. Like MgtB, MgtU is necessary for survival in Slc11a1 +/+ macrophages, resistance to oxidative stress, and growth under Mg2+ limitation conditions. The Salmonella Mg2+ transporter MgtA is not protected by MgtU despite sharing 50% amino acid identity with MgtB and being degraded in an MgtR- and FtsH-dependent manner. Surprisingly, the mgtB, mgtR, and mgtU genes are part of the same transcript, providing a singular example of transcript-specifying proteins that promote and hinder degradation of the same target. Our findings demonstrate that small proteins can confer pathogen survival inside macrophages by altering the abundance of related transporters, thereby furthering homeostasis.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Macrófagos/microbiologia , Magnésio/metabolismo , Salmonella typhimurium/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Macrófagos/fisiologia , Camundongos , Plasmídeos/genética , Salmonella typhimurium/genética , Virulência
4.
Food Chem ; 332: 127381, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32603917

RESUMO

In this work, three different polyether-modified siloxanes (PMS1, PMS2, and PMS3) were applied to stabilize water-in-oil emulsions, and sodium caseinate (SC) was used to establish water-in-oil-in-water (W/O/W) emulsions. Here, PMS polymers were modified by Isolan GPS and SC by Tween 80. The impact of modifications on the physical stability and controlled release of W/O/W emulsions were investigated. It was found that the storage stability and control release of double emulsions were dependent on the types of PMS used, percent of Isolan GPS, and Tween 80. When PMS1 and PMS2 were combined with low percent of Isolan GPS and Tween 80, the dispersed droplet sizes were reduced, lower percent in the gravitational sedimentation were achieved than using PMS3 emulsions. The controlled releases of Mg2+ from W/O/W emulsions by using PMS3 were slower than using other PMS. PMS3 had a strong influence in controlling the release of Mg2+ from the double emulsions.


Assuntos
Emulsões/química , Siloxanas/química , Caseínas/química , Condutividade Elétrica , Magnésio/metabolismo , Óleos/química , Tamanho da Partícula , Polissorbatos/química , Tensão Superficial , Água/química
5.
Nucleic Acids Res ; 48(15): 8663-8674, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32663277

RESUMO

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.


Assuntos
Cátions Bivalentes/metabolismo , Ferro/metabolismo , Clivagem do RNA/genética , Ribossomos/genética , Sítios de Ligação , Cátions Bivalentes/química , Ferro/química , Magnésio/química , Magnésio/metabolismo , Metais/química , Metais/metabolismo , Oxirredução/efeitos dos fármacos , Ribossomos/química
6.
Nat Commun ; 11(1): 3228, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591529

RESUMO

Plasmodium falciparum (Pf) relies solely on the salvage pathway for its purine nucleotide requirements, making this pathway indispensable to the parasite. Purine nucleotide levels are regulated by anabolic processes and by nucleotidases that hydrolyse these metabolites into nucleosides. Certain apicomplexan parasites, including Pf, have an IMP-specific-nucleotidase 1 (ISN1). Here we show, by comprehensive substrate screening, that PfISN1 catalyzes the dephosphorylation of inosine monophosphate (IMP) and is allosterically activated by ATP. Crystal structures of tetrameric PfISN1 reveal complex rearrangements of domain organization tightly associated with catalysis. Immunofluorescence microscopy and expression of GFP-fused protein indicate cytosolic localization of PfISN1 and expression in asexual and gametocyte stages of the parasite. With earlier evidence on isn1 upregulation in female gametocytes, the structures reported in this study may contribute to initiate the design for possible transmission-blocking agents.


Assuntos
5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Biocatálise , Plasmodium falciparum/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/metabolismo , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Proteínas Mutantes/química , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Especificidade por Substrato
7.
Proc Natl Acad Sci U S A ; 117(23): 12624-12635, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32434915

RESUMO

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.


Assuntos
Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Hidrogênio/metabolismo , Magnésio/metabolismo , Oxirredução , Oxigênio/metabolismo , Fótons , Complexo de Proteína do Fotossistema II/química , Quinonas/metabolismo , Água/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(23): 12799-12805, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457155

RESUMO

Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.


Assuntos
Alquil e Aril Transferases/química , Diatomáceas/enzimologia , Dimetilaliltranstransferase/química , Ácido Caínico/análogos & derivados , Neurotoxinas/biossíntese , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Diatomáceas/metabolismo , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Ácido Glutâmico/metabolismo , Ácido Caínico/metabolismo , Magnésio/metabolismo , Prenilação , Ligação Proteica
9.
PLoS One ; 15(4): e0232015, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330166

RESUMO

Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and modulate protein stability and activity. Quinary intreactions are readily detected by in-cell NMR spectroscopy as severe broadening of the NMR signals. Intact ribosome particles were shown to be necessary for the interactions that give rise to the NMR protein signal broadening observed in cell lysates and sufficient to mimic quinary interactions present in the crowded cytosol. Recovery of target protein NMR spectra that were broadened in lysates, in vitro and in the presence of purified ribosomes was achieved by RNase A digestion only after the structure of the ribosome was destabilized by removing magnesium ions from the system. Identifying intact ribosomal particles as the major protein-binding component of quinary interactions and consequent spectral peak broadening will facilitate quantitative characterization of macromolecular crowding effects in live cells and streamline models of metabolic activity.


Assuntos
Conformação Proteica , Proteínas/metabolismo , Ribossomos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Magnésio/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica/fisiologia , Estabilidade Proteica , Ribonuclease Pancreático/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-32272354

RESUMO

Calcium is an essential element for plants' survival and ability to deal with environmental stresses. However, it can cause cell death due to cellular disequilibrium. Serpentine plants are sensitive to high concentrations of Ca2+, which induces lethal symptoms, especially under environmental stress. In this study, the direct effects of Ca2+ on cell death were investigated in cell cultures of Alyssum inflatum, a serpentine plant native to Western Iran, and results were compared to a non-serpentinitic congeneric species A. saxatile. The results were also compared to the effects of Mg2+ treatments in both species, as another determinative factor in serpentinite soil is high Mg2+ content. Plasma membrane permeability, reactive oxygen species (ROS), and malondialdehyde (MDA) production were measured as physiological cell injury indices. In A. inflatum higher levels of ROS and MDA were observed in Ca2+-treated cells (5 mM or more), while in A. saxatile they were measured in Mg2+-treated cells (5 mM or more). In serpentine species, results indicated that cell death by Ca2+ was more intensive than the cell death by Mg2+, which were observed with less intensity in non-serpentine plants. Microscopic studies showed that cell death occurred via apoptosis-like programmed cell death (AL-PCD). Therefore, Ca2+ sensitivity and AL-PCD as mechanistic reasons for their non-serpentine intolerance would be a crucial consideration in cellular researches concerning serpentine plants, which could be employed in green technologies such as phytoremediation.


Assuntos
Brassicaceae , Cálcio , Morte Celular , Técnicas de Cultura , Magnésio , Brassicaceae/metabolismo , Cálcio/metabolismo , Morte Celular/fisiologia , Irã (Geográfico) , Magnésio/metabolismo , Solo/química
11.
Proc Natl Acad Sci U S A ; 117(15): 8486-8493, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234785

RESUMO

Nucleic acid aptamers hold great promise for therapeutic applications due to their favorable intrinsic properties, as well as high-throughput experimental selection techniques. Despite the utility of the systematic evolution of ligands by the exponential enrichment (SELEX) method for aptamer determination, complementary in silico aptamer design is highly sought after to facilitate virtual screening and increased understanding of important nucleic acid-protein interactions. Here, with a combined experimental and theoretical approach, we have developed two optimal epithelial cellular adhesion molecule (EpCAM) aptamers. Our structure-based in silico method first predicts their binding modes and then optimizes them for EpCAM with molecular dynamics simulations, docking, and free energy calculations. Our isothermal titration calorimetry experiments further confirm that the EpCAM aptamers indeed exhibit enhanced affinity over a previously patented nanomolar aptamer, EP23. Moreover, our study suggests that EP23 and the de novo designed aptamers primarily bind to EpCAM dimers (and not monomers, as hypothesized in previous published works), suggesting a paradigm for developing EpCAM-targeted therapies.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Molécula de Adesão da Célula Epitelial/química , Molécula de Adesão da Célula Epitelial/metabolismo , Magnésio/metabolismo , Calorimetria , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Multimerização Proteica , Técnica de Seleção de Aptâmeros
12.
Artigo em Inglês | MEDLINE | ID: mdl-32203778

RESUMO

Panax notoginseng is a traditional medicinal herb in China. However, the high capacity of its roots to accumulate cadmium (Cd) poses a potential risk to human health. Our previous study showed that nitrate reductase (NR)-dependent nitric oxide (NO) production promoted Cd accumulation in P. notoginseng root cell walls. In this study, the role of Mg in the regulation of NO production and Cd accumulation in P. notoginseng roots was characterized. Exposure of P. notoginseng roots to increasing concentrations of Cd resulted in a linear increase in NO production. The application of 2 mM Mg for 24 h significantly alleviated Cd-induced NO production and Cd accumulation in roots, which coincided with a significant decrease in the NR activity. Western analysis suggested that Mg increased the interaction between the 14-3-3 protein and NR, which might have been a reason for the Mg-mediated decrease in NR activity and NO production under Cd stress. These results suggested that Mg-mediated alleviation of Cd-induced NO production and Cd accumulation is achieved by enhancement of the interaction between the 14-3-3 protein and NR in P. notoginseng roots.


Assuntos
Cádmio/metabolismo , Magnésio/metabolismo , Óxido Nítrico/metabolismo , Panax notoginseng/metabolismo , Poluentes do Solo/metabolismo , Bioacumulação , Magnésio/administração & dosagem , Raízes de Plantas , Plantas Medicinais/metabolismo
13.
Ecotoxicol Environ Saf ; 193: 110355, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32120164

RESUMO

In the Montado system, in Portuguese Alentejo region, some Eutric Cambisols are known to promote manganese (Mn) toxicity in wheat. Variation on bioavailable Mn concentration depends on soil acidity, which can be increased by natural events (e.g. waterlogging) or human activity (e.g. excess use of chemical fertilizers). The effect of increasing soil Mn on crop element uptake, element distribution and oxidative stress was evaluated on winter wheat (Triticum aestivum). Plants were grown for 3 weeks in an acidic Cambisol spiked with increasing Mn concentrations (0, 45.2 and 90.4 mg MnCl2/Kg soil). Calcium (Ca), phosphorus (P), magnesium (Mg) and Mn were quantified in the soil solution, root and shoot tissues and respective subcellular fractions. The activity of the antioxidant enzymes ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX) and superoxide dismutase (SOD) were determined in extracts of wheat shoots and roots. Overall, increase in soil bioavailable Mn inhibited the uptake of other elements, increased the Ca proportion in the root apoplast, promoted the translocation of Mn and P to shoot tissues and increased their proportion in the shoot vacuoles. Wheat roots showed greater antioxidant enzymes activities than shoots. These activities decreased at the highest soil Mn concentration in both plant parts. Wheat roots appear to be more sensitive to oxidative stress derived from excess soil Mn and promote Mn translocation and storage in shoot vacuoles, probably in Mn and P complexes, as a detoxification strategy. Improvement in wheat production, in acidic soils, may rely on the enhancement of its Mn detoxification strategies.


Assuntos
Manganês/toxicidade , Poluentes do Solo/toxicidade , Triticum/efeitos dos fármacos , Antioxidantes/metabolismo , Ascorbato Peroxidases/metabolismo , Transporte Biológico , Cálcio/metabolismo , Catalase/metabolismo , Glutationa Redutase/metabolismo , Magnésio/metabolismo , Manganês/farmacocinética , Estresse Oxidativo , Peroxidase/metabolismo , Fósforo/metabolismo , Solo/química , Poluentes do Solo/farmacocinética , Superóxido Dismutase/metabolismo , Triticum/enzimologia , Triticum/metabolismo
14.
Ecotoxicol Environ Saf ; 193: 110342, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32109585

RESUMO

Agricultural production of Ligusticum chuanxiong Hort. is often affected by heavy metal pollution in soil, especially mixtures of cadmium (Cd) and lead (Pb). We assessed metal-induced phytotoxicity in L. chuanxiong by exposing the plants to soil treated with Cd, Pb, or Cd/Pb mixtures. A combined Cd/Pb treatment alleviated the inhibition in plant growth, photosynthesis, and secondary metabolite generation seen in single-metal exposures in three of the four combinations. Most combined Cd/Pb treatments resulted in preferential uptake of magnesium, copper, and nitrogen in underground plant parts and accumulation of phosphorus and calcium in aboveground plant parts, thereby leading to improvements in photosynthetic potential. Compared with single-metal exposures, combined Cd/Pb treatment significantly decreased the contents of Cd by 16.67%-40.12% and Pb by 10.68%-21.70% in the plant, respectively. At the subcellular level, the Pb presence increased the Cd percentage associated with cell wall from 64.79% to 67.93% in rhizomes and from 32.76% to 45.32% in leaves, while Cd reduced Pb contents by 9.36%-46.39% in the subcellular fractions. A combined Cd/Pb treatment decreased the contents of water- and ethanol-extractable metal forms and increased the contents of acetic acid- and hydrochloric acid-extractable forms. The lower toxic effects of the Cd/Pb mixture in L. chuanxiong were associated with photosynthetic potential, subcellular distribution, the chemical forms of Cd and Pb, and synthesis of secondary metabolites. These findings are useful for plant production strategies in soils contaminated by heavy metals.


Assuntos
Cádmio/toxicidade , Chumbo/toxicidade , Ligusticum/efeitos dos fármacos , Poluentes do Solo/toxicidade , Cádmio/farmacocinética , Cálcio/metabolismo , Cobre/metabolismo , Interações Medicamentosas , Chumbo/farmacocinética , Ligusticum/metabolismo , Magnésio/metabolismo , Nitrogênio/metabolismo , Fósforo/metabolismo , Fotossíntese/efeitos dos fármacos , Folhas de Planta/metabolismo , Plantas Medicinais/efeitos dos fármacos , Plantas Medicinais/metabolismo , Metabolismo Secundário/efeitos dos fármacos , Poluentes do Solo/farmacocinética
15.
PLoS One ; 15(2): e0228938, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074140

RESUMO

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.


Assuntos
Calcinose/patologia , Microvasos/fisiologia , alfa-2-Glicoproteína-HS/metabolismo , Animais , Calcinose/genética , Difosfatos/metabolismo , Modelos Animais de Doenças , Feminino , Rim/patologia , Fígado/patologia , Magnésio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microvasos/metabolismo , Minerais , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Insuficiência Renal Crônica/complicações , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , alfa-2-Glicoproteína-HS/fisiologia , alfa-Fetoproteínas
16.
Int J Mol Sci ; 21(1)2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31947724

RESUMO

Long-term use of proton pump inhibitors (PPIs) is common in patients with muscle wasting-related chronic diseases. We explored the hypothesis that the use of PPIs may contribute to a reduction in muscle mass and function in these patients. Literature indicates that a PPI-induced reduction in acidity of the gastrointestinal tract can decrease the absorption of, amongst others, magnesium. Low levels of magnesium are associated with impaired muscle function. This unwanted side-effect of PPIs on muscle function has been described in different disease backgrounds. Furthermore, magnesium is necessary for activation of vitamin D. Low vitamin D and magnesium levels together can lead to increased inflammation involved in muscle wasting. In addition, PPI use has been described to alter the microbiota's composition in the gut, which might lead to increased inflammation. However, PPIs are often provided together with nonsteroidal anti-inflammatory drugs (NSAIDs), which are anti-inflammatory. In the presence of obesity, additional mechanisms could further contribute to muscle alterations. In conclusion, use of PPIs has been reported to contribute to muscle function loss. Whether this will add to the risk factor for development of muscle function loss in patients with chronic disease needs further investigation.


Assuntos
Caquexia/etiologia , Músculos/efeitos dos fármacos , Inibidores da Bomba de Prótons/efeitos adversos , Sarcopenia/etiologia , Animais , Caquexia/fisiopatologia , Doença Crônica , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Magnésio/metabolismo , Músculos/fisiopatologia , Obesidade/complicações , Obesidade/fisiopatologia , Sarcopenia/fisiopatologia
17.
J Bacteriol ; 202(7)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-31964700

RESUMO

Transition metals are essential for life but are toxic when in excess. Metal ion intoxication may result from the mismetallation of essential metal-dependent enzymes with a noncognate metal. To begin to identify enzymes and processes that are susceptible to mismetallation, we have selected for strains with increased resistance to Mn(II) and Co(II). In Bacillus subtilis, cells lacking the MntR metalloregulator are exquisitely sensitive to Mn(II) but can easily become resistant by acquiring mutations affecting the MntH Mn(II) importer. Using transposon mutagenesis, and starting with an mntR mntH strain, we recovered mariner insertions that inactivated the mpfA gene encoding a putative Mg(II) efflux system. Loss of MpfA leads to elevated intracellular Mg(II), increased sensitivity to high Mg(II), and reduced Mn(II) sensitivity. Consistently, we also recovered an insertion disrupting the mgtE riboswitch, which normally restricts expression of the major Mg(II) importer. These results suggest that Mn(II) intoxication results from disruption of a Mg(II)-dependent enzyme or process. Mutations that inactivate MpfA were also recovered in a selection for Co(II) resistance beginning with sensitized strains lacking the major Co(II) efflux pump, CzcD. Since both Mn(II) and Co(II) may mismetallate iron-dependent enzymes, we repeated the selections under conditions of iron depletion imposed by expression of the Listeria monocytogenes FrvA iron exporter. Under conditions of iron depletion, a wider variety of suppressor mutations were recovered, but they still point to a central role for Mg(II) in maintaining metal ion homeostasis.IMPORTANCE Cellular metal ion homeostasis is tightly regulated. When metal ion levels are imbalanced, or when one metal is at toxic levels, enzymes may bind to the wrong metal cofactor. Enzyme mismetallation can impair metabolism, lead to new and deleterious reactions, and cause cell death. Beginning with Bacillus subtilis strains genetically sensitized to metal intoxication through loss of efflux or by lowering intracellular iron, we identified mutations that suppress the deleterious effects of excess Mn(II) or Co(II). For both metals, mutations in mpfA, encoding a Mg(II) efflux pump, suppressed toxicity. These mutant strains have elevated intracellular Mg(II), suggesting that Mg(II)-dependent processes are very sensitive to disruption by transition metals.


Assuntos
Bacillus subtilis/metabolismo , Cobalto/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Adaptação Biológica , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Modelos Biológicos , Óperon
18.
Medicine (Baltimore) ; 99(2): e18709, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31914079

RESUMO

Kidney handling of electrolytes varies in different stages of chronic kidney disease (CKD). Diabetes mellitus (DM) plays an important role in CKD. Fractional excretion (FE) is an important means in clinical practice. The relationship between FE of electrolytes in patients at different stages of CKD is worth further investigating.We designed a cross-sectional study in 1 teaching hospital, consecutive CKD patients were enrolled between February 2016 and January 2017. Including clinical demographic features, laboratory examination including spot urine electrolytes, blood biochemistries, and relevant medications were determined.A total of 762 CKD patients completed the study. Of these, 218 (28.6%) had DM. Participants were grouped according to estimated glomerular filtration rate into 7 categories: hyperfiltration (HF), CKD1, CKD2, CKD3a, CKD3b, CKD4, and CKD5. Groups HF, CKD1, 2, 3a, 3b, 4 and 5 contained 83, 143, 192, 94, 82, 82, and 86 patients, respectively. FE of electrolytes tended to increase along with the decline of renal function (CKD1-CKD5) (P < .001). The relationship was similar between the DM and non-DM groups. Diabetic patients demonstrated higher FE of magnesium compared with non-DM subjects at CKD2 and CKD5 (P < .05).CKD patients showed a progressive increase in the FE of electrolytes; FE of magnesium seemed to increase more among diabetic patients with CKD, and could be a potential predictor of CKD progression.


Assuntos
Eletrólitos/metabolismo , Taxa de Filtração Glomerular/fisiologia , Insuficiência Renal Crônica/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Eletrólitos/urina , Feminino , Humanos , Magnésio/metabolismo , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença
19.
Biophys Chem ; 258: 106330, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31981743

RESUMO

The glycolytic pathway is present in most organisms and represents a central part of the energy production mechanism in a cell. For a general understanding of glycolysis, the investigation from a thermodynamic point of view is essential and allows realising thermodynamic feasibility analyses under in vivo conditions. However, available literature standard Gibbs energies of reaction, ΔRg'0, are calculated using equilibrium-molality ratios Km', which might lead to a misinterpretation of the glycolytic pathway. It was the aim of this work to thermodynamically investigate the triosephosphate isomerase (TPI) reaction to provide new activity-based reaction data. In vitro equilibrium experiments were performed, and activity coefficients were predicted with the equation of state electrolyte PC-SAFT (ePC-SAFT). The combination of experimental concentrations and predicted activity coefficients yielded the thermodynamic equilibrium constant Ka and a new value for ΔRg'0(298.15 K, pH 7) = 7.1 ± 0.3 kJ mol­1. The availability of the new ΔRg'0 value allowed predicting influences of the reaction medium on the reaction equilibrium of the TPI reaction. In this work, influences of the initial substrate concentration, pH and Mg2+ concentration on the reaction equilibrium were investigated and a method is presented to predict these influences. The higher the substrate concentration and the higher the temperature, the stronger the reaction equilibrium is shifted on the product side. While the pH did not have a significant influence on the reaction equilibrium, Mg2+ yielded a shift of the reaction equilibrium to the substrate side. All these effects were predicted correctly with ePC-SAFT. Based on the ePC-SAFT predictions we concluded that a charge-reduction of the product by complexation of the product with Mg2+ was responsible for the strong influence of Mg2+ on the reaction equilibrium. Finally, the standard enthalpy of reaction of ΔRh'0(pH 7) = 18 ± 7 kJ mol­1 was determined with the equilibrium constants Ka at 298.15 K, 304.15 K and 310.15 K using the van 't Hoff equation.


Assuntos
Termodinâmica , Triose-Fosfato Isomerase/metabolismo , Fosfato de Di-Hidroxiacetona/química , Fosfato de Di-Hidroxiacetona/metabolismo , Magnésio/análise , Magnésio/metabolismo , Modelos Estatísticos
20.
Arch Biochem Biophys ; 681: 108258, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31917961

RESUMO

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.


Assuntos
Glioxilatos/metabolismo , Ácidos Mandélicos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Animais , Cálcio/metabolismo , Magnésio/metabolismo , Suínos
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