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1.
Proc Natl Acad Sci U S A ; 117(34): 20943-20949, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32817465

RESUMO

The reiterative process of lateral root (LR) formation is widespread and underlies root system formation. However, early LR primordium (LRP) morphogenesis is not fully understood. In this study, we conducted both a clonal analysis and time-lapse experiments to decipher the pattern and sequence of pericycle founder cell (FC) participation in LR formation. Most commonly, LRP initiation starts with the specification of just one FC longitudinally. Clonal and anatomical analyses suggested that a single FC gradually recruits neighboring pericycle cells to become FCs. This conclusion was validated by long-term time-lapse live-imaging experiments. Once the first FC starts to divide, its immediate neighbors, both lengthwise and laterally, are recruited within the hour, after which they recruit their neighboring cells within a few hours. Therefore, LRP initiation is a gradual, multistep process. FC recruitment is auxin-dependent and is abolished by treatment with a polar auxin transport inhibitor. Furthermore, FC recruitment establishes a morphogenetic field where laterally peripheral cells have a lower auxin response, which is associated with a lower proliferation potential, compared to centrally located FCs. The lateral boundaries of the morphogenetic field are determined by phloem-adjacent pericycle cells, which are the last cells to be recruited as FCs. The proliferation potential of these cells is limited, but their recruitment is essential for root system formation, resulting in the formation of a new vascular connection between the nascent and parent root, which is crucial for establishing a continuous and efficient vascular system.


Assuntos
Arabidopsis/genética , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/metabolismo , Transporte Biológico/fisiologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Meristema/metabolismo , Morfogênese/genética , Organogênese Vegetal/fisiologia , Floema/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
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
3.
PLoS Comput Biol ; 16(7): e1007884, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32614821

RESUMO

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a "competitive" environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest.


Assuntos
Transporte Biológico/fisiologia , Simulação por Computador , Computadores Moleculares , Biologia de Sistemas/métodos , Algoritmos , Cadeias de Markov , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Método de Monte Carlo , Termodinâmica
4.
PLoS Comput Biol ; 16(7): e1007789, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32614861

RESUMO

Membrane transport is generally thought to occur via an alternating access mechanism in which the transporter adopts at least two states, accessible from two different sides of the membrane to exchange substrates from the extracellular environment and the cytoplasm or from the cytoplasm and the intracellular matrix of the organelles (only in eukaryotes). In recent years, a number of high resolution structures have supported this general framework for a wide class of transport molecules, although additional states along the transport pathway are emerging as critically important. Given that substrate binding is often weak in order to enhance overall transport rates, there exists the distinct possibility that transporters may transport the incorrect substrate. This is certainly the case for many pharmaceutical compounds that are absorbed in the gut or cross the blood brain barrier through endogenous transporters. Docking studies on the bacterial sugar transporter vSGLT reveal that many highly toxic compounds are compatible with binding to the orthosteric site, further motivating the selective pressure for additional modes of selectivity. Motivated by recent work in which we observed failed substrate delivery in a molecular dynamics simulation where the energized ion still goes down its concentration gradient, we hypothesize that some transporters evolved to harness this 'slip' mechanism to increase substrate selectivity and reduce the uptake of toxic molecules. Here, we test this idea by constructing and exploring a kinetic transport model that includes a slip pathway. While slip reduces the overall productive flux, when coupled with a second toxic molecule that is more prone to slippage, the overall substrate selectivity dramatically increases, suppressing the accumulation of the incorrect compound. We show that the mathematical framework for increased substrate selectivity in our model is analogous to the classic proofreading mechanism originally proposed for tRNA synthase; however, because the transport cycle is reversible we identified conditions in which the selectivity is essentially infinite and incorrect substrates are exported from the cell in a 'detoxification' mode. The cellular consequences of proofreading and membrane slippage are discussed as well as the impact on future drug development.


Assuntos
Sítios de Ligação , Transporte Biológico/fisiologia , Proteínas de Membrana Transportadoras , Modelos Biológicos , Ligação Proteica/fisiologia , Biologia Computacional , Humanos , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Simulação de Dinâmica Molecular , Transportador 1 de Glucose-Sódio , Especificidade por Substrato
5.
Am J Physiol Regul Integr Comp Physiol ; 319(3): R315-R322, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32697652

RESUMO

During exhaustive ramp-incremental cycling tests, the incidence of O2 uptake (V̇o2) plateaus is low. To verify the attainment of maximum V̇o2 (V̇o2max), it is recommended that a trial at a power output (PO) corresponding to 110% of the ramp-derived peak (POpeak) is performed. It remains unclear whether verification trials set at this PO can be tolerated for long enough to allow attainment of V̇o2max. Eleven recreationally trained individuals performed five ramp tests of varying slope (5, 10, 15, 25, and 30 W/min), each followed, in series, by two verification trials: the first at 110% POpeak of the 25 W/min ramp and the second at 110% POpeak attained in the preceding ramp test. Exercise duration of the first verification trial was on average 81 ± 15 s (CV = 9 ± 3%) versus 162 ± 32, 121 ± 24, 103 ± 15, and 73 ± 10 s for the second verification trials at 110% of POpeak of the 5, 10, 15, and 30 W/min ramp tests, respectively (P < 0.05). Compared with the highest V̇o2 recorded during ramp tests, V̇o2 from the subsequent verification trials was not different for the 5, 10, and 15 W/min ramp tests (P > 0.05) but was lower for the 25 and 30 W/min ramp tests (P < 0.05). Verification trials at 110% POpeak of rapidly incrementing ramp tests (i.e., 25 W/min) were not sustained for long enough to allow the attainment of V̇o2max. With commonly used rapidly incrementing ramp tests engendering exhaustion within 8-12 min, verification trials less than POpeak should be preferred as they can be sustained sufficiently long to allow the attainment of V̇o2max.


Assuntos
Transporte Biológico/fisiologia , Exercício Físico/fisiologia , Consumo de Oxigênio/fisiologia , Esforço Físico/fisiologia , Adulto , Teste de Esforço/métodos , Frequência Cardíaca/fisiologia , Humanos , Masculino
6.
Am J Physiol Endocrinol Metab ; 319(2): E345-E353, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32543943

RESUMO

Type 1 and type 2 diabetes are both tightly associated with impaired glucose control. Although both pathologies stem from different mechanisms, a reduction in insulin action coincides with drastic metabolic dysfunction in skeletal muscle and metabolic inflexibility. However, the underlying explanation for this response remains poorly understood, particularly since it is difficult to distinguish the role of attenuated insulin action from the detrimental effects of reactive lipid accumulation, which impairs mitochondrial function and promotes reactive oxygen species (ROS) emission. We therefore utilized streptozotocin to examine the effects of acute insulin deprivation, in the absence of a high-lipid/nutrient excess environment, on the regulation of mitochondrial substrate sensitivity and ROS emission. The ablation of insulin resulted in reductions in absolute mitochondrial oxidative capacity and ADP-supported respiration and reduced the ability for malonyl-CoA to inhibit carnitine palmitoyltransferase I (CPT-I) and suppress fatty acid-supported respiration. These bioenergetic responses coincided with increased mitochondrial-derived H2O2 emission and lipid transporter content, independent of major mitochondrial substrate transporter proteins and enzymes involved in fatty acid oxidation. Together, these data suggest that attenuated/ablated insulin signaling does not affect mitochondrial ADP sensitivity, whereas the increased reliance on fatty acid oxidation in situations where insulin action is reduced may occur as a result of altered regulation of mitochondrial fatty acid transport through CPT-I.


Assuntos
Ácidos Graxos/fisiologia , Insulina/deficiência , Mitocôndrias Musculares/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Transporte Biológico/fisiologia , Carnitina O-Palmitoiltransferase/metabolismo , Peróxido de Hidrogênio/metabolismo , Insulina/fisiologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Masculino , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/ultraestrutura , Oxirredução , Consumo de Oxigênio , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Estreptozocina/farmacologia
7.
Nat Rev Mol Cell Biol ; 21(10): 585-606, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32457507

RESUMO

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications.


Assuntos
Vesículas Extracelulares/metabolismo , Mamíferos/metabolismo , RNA/metabolismo , Animais , Transporte Biológico/fisiologia , Comunicação Celular/fisiologia , Humanos
8.
Am J Physiol Cell Physiol ; 319(1): C136-C147, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32401606

RESUMO

The cortical collecting duct (CCD) of the mammalian kidney plays a major role in the maintenance of total body electrolyte, acid/base, and fluid homeostasis by tubular reabsorption and excretion. The mammalian CCD is heterogeneous, composed of Na+-absorbing principal cells (PCs) and acid-base-transporting intercalated cells (ICs). Perturbations in luminal flow rate alter hydrodynamic forces to which these cells in the cylindrical tubules are exposed. However, most studies of tubular ion transport have been performed in cell monolayers grown on or epithelial sheets affixed to a flat support, since analysis of transepithelial transport in native tubules by in vitro microperfusion requires considerable expertise. Here, we report on the generation and characterization of an in vitro, perfusable three-dimensional kidney CCD model (3D CCD), in which immortalized mouse PC-like mpkCCD cells are seeded within a cylindrical channel embedded within an engineered extracellular matrix and subjected to luminal fluid flow. We find that a tight epithelial barrier composed of differentiated and polarized PCs forms within 1 wk. Immunofluorescence microscopy reveals the apical epithelial Na+ channel ENaC and basolateral Na+/K+-ATPase. On cessation of luminal flow, benzamil-inhibitable cell doming is observed within these 3D CCDs consistent with the presence of ENaC-mediated Na+ absorption. Our 3D CCD provides a geometrically and microphysiologically relevant platform for studying the development and physiology of renal tubule segments.


Assuntos
Túbulos Renais Coletores/anatomia & histologia , Túbulos Renais Coletores/fisiologia , Modelos Biológicos , Perfusão/métodos , Impressão Tridimensional , Animais , Transporte Biológico/fisiologia , Linhagem Celular Transformada , Camundongos , Microscopia de Fluorescência/métodos
9.
PLoS Comput Biol ; 16(4): e1007853, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32352964

RESUMO

The structure of tubular transport networks is thought to underlie much of biological regularity, from individuals to ecosystems. A core assumption of transport network models is either area-preserving or area-increasing branching, such that the summed cross-sectional area of all child branches is equal to or greater than the cross-sectional area of their respective parent branch. For insects, the most diverse group of animals, the assumption of area-preserving branching of tracheae is, however, based on measurements of a single individual and an assumption of gas exchange by diffusion. Here we show that ants exhibit neither area-preserving nor area-increasing branching in their abdominal tracheal systems. We find for 20 species of ants that the sum of child tracheal cross-sectional areas is typically less than that of the parent branch (area-decreasing). The radius, rather than the area, of the parent branch is conserved across the sum of child branches. Interpretation of the tracheal system as one optimized for the release of carbon dioxide, while readily catering to oxygen demand, explains the branching pattern. Our results, together with widespread demonstration that gas exchange in insects includes, and is often dominated by, convection, indicate that for generality, network transport models must include consideration of systems with different architectures.


Assuntos
Formigas/fisiologia , Transporte Biológico/fisiologia , Biologia Computacional/métodos , Modelos Biológicos , Traqueia/fisiologia , Animais , Dióxido de Carbono/metabolismo , Oxigênio/metabolismo
10.
Xenobiotica ; 50(10): 1208-1219, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32352885

RESUMO

Liver enzymes and transporters play an essential role in xenobiotic metabolism, distribution and elimination. Pre-clinical safety assessment relies on studies on animal models, including the (mini)pig. The pig shares many anatomical and physiological characteristics with humans, and there is currently a gap in information about porcine metabolism and disposition pathways and their similarities and differences from human ones.Three different sample preparation methods (filter-aided sample preparation (FASP), enhanced FASP (eFASP) and in-solution sample preparation) were used to prepare porcine liver tissue (two samples) for proteomic analysis. The analysis relied on rapid-separation liquid chromatography coupled to Orbitrap mass spectrometry in data-dependent acquisition mode. MASCOT was used for identification and relative label-free quantification was based on spectral counting.The three sample preparation methods provided complementary results, allowing characterisation of approximately 70 pharmacologically relevant proteins. The main quantified proteins included 16 cytochrome P450 (CYP) enzymes, 5 UGT enzymes, and 11 transporters. In addition, 20 Phase I and 14 Phase II enzymes were also characterised. Inter-operator differences were negligible and the pig liver pies for CYP, UGT and efflux transporter proteins were established. Human homologues of the quantified CYP, UGT and transporter proteins were identified.


Assuntos
Transporte Biológico/fisiologia , Inativação Metabólica/fisiologia , Taxa de Depuração Metabólica/fisiologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado , Proteômica , Suínos
11.
Am J Physiol Gastrointest Liver Physiol ; 318(6): G1070-G1087, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390462

RESUMO

Lipopolysaccharides (LPS) are potent pro-inflammatory molecules that enter the systemic circulation from the intestinal lumen by uncertain mechanisms. We investigated these mechanisms and the effect of exogenous glucagon-like peptide-2 (GLP-2) on LPS transport in the rodent small intestine. Transmucosal LPS transport was measured in Ussing-chambered rat jejunal mucosa. In anesthetized rats, the appearance of fluorescein isothiocyanate (FITC)-LPS into the portal vein (PV) and the mesenteric lymph was simultaneously monitored after intraduodenal perfusion of FITC-LPS with oleic acid and taurocholate (OA/TCA). In vitro, luminally applied LPS rapidly appeared in the serosal solution only with luminal OA/TCA present, inhibited by the lipid raft inhibitor methyl-ß-cyclodextrin (MßCD) and the CD36 inhibitor sulfosuccinimidyl oleate (SSO), or by serosal GLP-2. In vivo, perfusion of FITC-LPS with OA/TCA rapidly increased FITC-LPS appearance into the PV, followed by a gradual increase of FITC-LPS into the lymph. Rapid PV transport was inhibited by the addition of MßCD or by SSO, whereas transport into the lymph was inhibited by chylomicron synthesis inhibition. Intraveous injection of the stable GLP-2 analog teduglutide acutely inhibited FITC-LPS transport into the PV, yet accelerated FITC-LPS transport into the lymph via Nω-nitro-l-arginine methyl ester (l-NAME)- and PG97-269-sensitive mechanisms. In vivo confocal microscopy in mouse jejunum confirmed intracellular FITC-LPS uptake with no evidence of paracellular localization. This is the first direct demonstration in vivo that luminal LPS may cross the small intestinal barrier physiologically during fat absorption via lipid raft- and CD36-mediated mechanisms, followed by predominant transport into the PV, and that teduglutide inhibits LPS uptake into the PV in vivo.NEW & NOTEWORTHY We report direct in vivo confirmation of transcellular lipopolysaccharides (LPS) uptake from the intestine into the portal vein (PV) involving CD36 and lipid rafts, with minor uptake via the canonical chylomicron pathway. The gut hormone glucagon-like peptide-2 (GLP-2) inhibited uptake into the PV. These data suggest that the bulk of LPS absorption is via the PV to the liver, helping clarify the mechanism of LPS transport into the PV as part of the "gut-liver" axis. These data do not support the paracellular transport of LPS, which has been implicated in the pathogenesis of the "leaky gut" syndrome.


Assuntos
Gorduras/metabolismo , Intestino Delgado/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Fármacos Gastrointestinais/farmacologia , Células HEK293 , Humanos , Intestino Delgado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
12.
PLoS One ; 15(4): e0232141, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320440

RESUMO

INTRODUCTION: 3,4-dihydroxy-6-[18F]fluoro-L-phenylalanine (FDOPA) uptake quantification in glioma assessment can be distorted using a non-optimal time frame binning of time-activity curves (TAC). Under-sampling or over-sampling dynamic PET images induces significant variations on kinetic parameters quantification. We aimed to optimize temporal time frame binning for dynamic FDOPA PET imaging. METHODS: Fourteen patients with 33 tumoral TAC with biopsy-proven gliomas were analysed. The mean SUVmax tumor-to-brain ratio (TBRmax) were compared at 20 min and 35 min post-injection (p.i). Five different time frame samplings within 20 min were compared: 11x10sec-6x15sec-5x20sec-3x300sec; 8x15sec- 2x30sec- 2x60sec- 3x300sec; 6x20sec- 8x60sec- 2x300sec; 10x30sec- 3x300sec and 4x45sec- 3x90sec- 5x150sec. The reversible single-tissue compartment model with blood volume parameter (VB) was selected using the Akaike information criterion. K1 values extracted from 1024 noisy simulated TAC using Monte Carlo method from the 5 different time samplings were compared to a target K1 value as the objective, which is the average of the K1 values extracted from the 33 lesions using an imaging-derived input function for each patient. RESULTS: The mean TBRmax was significantly higher at 20 min p.i. than at 35 min p.i (respectively 1.4 +/- 0.8 and 1.2 +/- 0.6; p <0.001). The target K1 value was 0.161 mL/ccm/min. The 8x15sec- 2x30sec- 2x60sec- 3x300sec time sampling was the optimal time frame binning. K1 values extracted using this optimal time frame binning were significantly different with K1 values extracted from the other time frame samplings, except with K1 values obtained using the 11x10sec- 6x15sec -5x20sec-3x300sec time frame binning. CONCLUSIONS: This optimal sampling schedule design (8x15sec- 2x30sec- 2x60sec- 3x300sec) could be used to minimize bias in quantification of FDOPA uptake in glioma using kinetic analysis.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Fenilalanina/metabolismo , Adulto , Idoso , Transporte Biológico/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Método de Monte Carlo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/metabolismo , Adulto Jovem
13.
Life Sci ; 253: 117696, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32334013

RESUMO

AIMS: We have previously demonstrated that p-tyramine (TYR), an endogenous trace amine-associated receptor 1 agonist, passage across neuronal membranes involves a transporter exhibiting the pharmacological profile of Organic Cation Transporter 2 (OCT2). Since TYR is also a constituent of foodstuffs and produced by the intestinal microbiota, here we have investigated whether similar processes are involved in the passage of 100 nM TYR across apical and basolateral membranes of the Caco-2 human intestinal epithelial cell line. MATERIALS AND METHODS: [3H]TYR transport across apical and basolateral membranes of Caco-2 cell monolayers was measured in the presence of inhibitors of TYR metabolizing enzymes. Cellular, apical, and basolateral compartments were collected at various timepoints, TYR concentrations calculated, and transport properties pharmacologically characterized. KEY FINDINGS: Apical transport resulted in equimolar accumulation of TYR within cells. Pentamidine (OCT1/OCT2 inhibitor) decreased apical transport (P = 0.001) while atropine (OCT1 inhibitor) had no effect, suggesting apical transport involved OCT2. In contrast, basolateral transport resulted in 500-1000 nM cellular concentrations (P < 0.0001) indicating the presence of an active transporter. Replacement of Na+ on an equimolar basis with choline resulted in loss of TYR transport (P = 0.017). Unexpectedly, this active transport was also atropine-sensitive (P = 0.020). Kinetic analysis of the active transporter revealed Vmax = 43.0 nM/s with a Kt = 33.1 nM. SIGNIFICANCE: We have demonstrated for the first time that TYR is transported across Caco-2 apical membranes via facilitated diffusion by OCT2, whereas transport across basolateral membranes is by a Na+-dependent, atropine-sensitive, active transporter.


Assuntos
Mucosa Intestinal/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Sódio/metabolismo , Tiramina/metabolismo , Atropina/metabolismo , Transporte Biológico/fisiologia , Transporte Biológico Ativo/fisiologia , Células CACO-2 , Humanos
14.
Development ; 147(6)2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32229613

RESUMO

Auxin is a key signal regulating plant growth and development. It is well established that auxin dynamics depend on the spatial distribution of efflux and influx carriers on the cell membranes. In this study, we employ a systems approach to characterise an alternative symplastic pathway for auxin mobilisation via plasmodesmata, which function as intercellular pores linking the cytoplasm of adjacent cells. To investigate the role of plasmodesmata in auxin patterning, we developed a multicellular model of the Arabidopsis root tip. We tested the model predictions using the DII-VENUS auxin response reporter, comparing the predicted and observed DII-VENUS distributions using genetic and chemical perturbations designed to affect both carrier-mediated and plasmodesmatal auxin fluxes. The model revealed that carrier-mediated transport alone cannot explain the experimentally determined auxin distribution in the root tip. In contrast, a composite model that incorporates both carrier-mediated and plasmodesmatal auxin fluxes re-capitulates the root-tip auxin distribution. We found that auxin fluxes through plasmodesmata enable auxin reflux and increase total root-tip auxin. We conclude that auxin fluxes through plasmodesmata modify the auxin distribution created by efflux and influx carriers.


Assuntos
Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Plasmodesmos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal/fisiologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Distribuição Tecidual
15.
Phys Chem Chem Phys ; 22(14): 7537-7545, 2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32219231

RESUMO

Understanding how electrons and protons move in a coupled manner and affect one another is important to the design of proton-electron conductors and achieving biological transport in synthetic materials. In this study, a new methodology is proposed that allows for the quantification of the degree of coupling between electrons and protons in tyrosine-rich peptides and metal oxide hybrid films at room temperature under a voltage bias. This approach is developed according to the Onsager principle, which has been thoroughly established for the investigation of mixed ion-electron conductors with electron and oxide ion vacancies as carriers at high temperatures. Herein, a new device platform using electron-blocking electrodes provides a new strategy to investigate the coupling of protons and electrons in bulk materials beyond the molecular level investigation of coupled proton and electron transfer. Two Onsager transport parameters, αi* and σe', are obtained from the device, and the results of these transport parameters demonstrate that the coupled transport of electrons and protons inside the hybrid film plays an important role in the macroscopic-scale conduction. The results suggest that an average of one electron is dragged by one proton in the absence of a direct driving force for electron movement ∇ηe.


Assuntos
Técnicas de Química Analítica/instrumentação , Transporte de Elétrons/fisiologia , Elétrons , Compostos de Manganês/química , Óxidos/química , Peptídeos/química , Prótons , Transporte Biológico/fisiologia
16.
Nat Chem Biol ; 16(4): 469-478, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152546

RESUMO

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.


Assuntos
Resistência a Medicamentos/genética , Proteínas Carreadoras de Solutos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Antineoplásicos , Fenômenos Bioquímicos , Transporte Biológico/genética , Transporte Biológico/fisiologia , Sistemas CRISPR-Cas , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Resistência a Medicamentos/fisiologia , Testes Genéticos , Humanos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Transporte Proteico/fisiologia , Proteínas Carreadoras de Solutos/fisiologia , Simportadores/genética , Simportadores/metabolismo
17.
PLoS Genet ; 16(3): e1008561, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32134924

RESUMO

Intraflagellar transport (IFT) is required for ciliary assembly and maintenance. While disruption of IFT may trigger ciliary disassembly, we show here that IFT mediated transport of a CDK-like kinase ensures proper ciliary disassembly. Mutations in flagellar shortening 2 (FLS2), encoding a CDK-like kinase, lead to retardation of cilia resorption and delay of cell cycle progression. Stimulation for ciliary disassembly induces gradual dephosphorylation of FLS2 accompanied with gradual inactivation. Loss of FLS2 or its kinase activity induces early onset of kinesin13 phosphorylation in cilia. FLS2 is predominantly localized in the cell body, however, it is transported to cilia upon induction of ciliary disassembly. FLS2 directly interacts with IFT70 and loss of this interaction inhibits its ciliary transport, leading to dysregulation of kinesin13 phosphorylation and retardation of ciliary disassembly. Thus, this work demonstrates that IFT plays active roles in controlling proper ciliary disassembly by transporting a protein kinase to cilia to regulate a microtubule depolymerizer.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteína Quinase CDC2/metabolismo , Chlamydomonas/metabolismo , Cílios/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Transporte Biológico/fisiologia , Ciclo Celular/fisiologia , Flagelos/metabolismo , Fosforilação/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais/fisiologia
18.
J Exp Med ; 217(4)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32211826

RESUMO

The blood vessels vascularizing the central nervous system exhibit a series of distinct properties that tightly control the movement of ions, molecules, and cells between the blood and the parenchyma. This "blood-brain barrier" is initiated during angiogenesis via signals from the surrounding neural environment, and its integrity remains vital for homeostasis and neural protection throughout life. Blood-brain barrier dysfunction contributes to pathology in a range of neurological conditions including multiple sclerosis, stroke, and epilepsy, and has also been implicated in neurodegenerative diseases such as Alzheimer's disease. This review will discuss current knowledge and key unanswered questions regarding the blood-brain barrier in health and disease.


Assuntos
Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/fisiologia , Animais , Transporte Biológico/fisiologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiologia , Humanos
19.
Dev Cell ; 53(1): 60-72.e4, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32109385

RESUMO

Many eukaryotic cells distribute their intracellular components asymmetrically through regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors and unveil MAP9 as a positive modulator of kinesin-3 motility. From these data, we propose a general "MAP code" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.


Assuntos
Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Transporte Proteico/fisiologia , Animais , Transporte Biológico/fisiologia , Movimento Celular/fisiologia , Citoplasma/metabolismo , Cinesina/metabolismo
20.
Fluids Barriers CNS ; 17(1): 17, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046769

RESUMO

BACKGROUND: Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2-20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. METHODS: Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined. RESULTS: The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2-20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). CONCLUSION: These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/líquido cefalorraquidiano , Células Epiteliais/metabolismo , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Homeostase , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Transporte Biológico/genética , Transporte Biológico/fisiologia , Células Cultivadas , Plexo Corióideo/metabolismo , Feminino , Cadeias Leves da Proteína-1 Reguladora de Fusão/genética , Ácido Glutâmico/metabolismo , Homeostase/fisiologia , Masculino , Camundongos Knockout , Pré-Albumina/metabolismo
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