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1.
Diagn. tratamento ; 27(3): 94-101, jul-set. 2022. ilus, tab, tab
Artigo em Português | LILACS | ID: biblio-1380679

RESUMO

Contexto: A obesidade infantil ocasiona diversas doenças e uma das formas para combatê-la é a atividade física, que exerce um papel fundamental. Objetivo: Comparar as diferentes intensidades da atividade física mensurada objetivamente de acordo com o transporte ativo, a prática de esportes e as atividades físicas estruturadas e seu impacto na gordura corporal e índice de massa corporal (IMC) em escolares. Desenho e local: Estudo transversal de amostra por critério de conveniência, realizado em São Caetano do Sul pelo Centro de Estudos do Laboratório de Aptidão Física de São Caetano do Sul (CELAFISCS). Métodos: Foram avaliadas um total de 584 crianças (277 meninos) que atenderam aos critérios de inclusão. A amostra foi dividida em grupos segundo o transporte (ativo e passivo) e a prática esportiva (sim e não). Para análise estatística foi utilizado o teste t Student e o teste U de Mann-Whitney. Para o ajuste das variáveis foi utilizada a análise de covariância (ANCOVA). Resultados: Os meninos demonstraram que, independentemente do tempo de transporte, há efeito do tipo do transporte sobre a atividade física (AF) durante a semana, de intensidade moderada, moderada-vigorosa, AF durante o final de semana de intensidade moderada, moderada-vigorosa e vigorosa. As meninas demonstraram efeito do tipo de transporte sobre a AF durante a semana na AF de intensidade moderada e de intensidade moderada-vigorosa. A gordura corporal e o IMC não apresentaram diferenças entre os grupos. As práticas esportivas não tiveram diferenças significativas em nenhuma das variáveis. Conclusões: O transporte ativo atingiu os níveis de intensidade moderada, moderada-vigorosa durante a semana, tanto no masculino como no feminino. No final de semana, além dessas, a intensidade vigorosa foi encontrada nos meninos.


Assuntos
Transporte Biológico Ativo , Exercício Físico , Índice de Massa Corporal , Demografia , Volta ao Esporte
2.
J Phys Chem Lett ; 13(24): 5405-5412, 2022 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-35679158

RESUMO

Biological cells frequently exhibit a so-called secondary active transport by moving various species across their membranes. In this mode of transport, an energetically favorable transmembrane gradient of one type of molecule is used to drive another type of molecule in the energetically unfavorable direction against their gradient. Although it is well established that conformational transitions play a critical role in functioning of transporters, the molecular details of underlying mechanisms remain not well understood. Here, we utilize a recently developed theoretical method to understand better the microscopic picture of secondary active transport. Specifically, we evaluate how mutations in different parts of transporters affect their dynamic properties. In addition, we present a possible explanation on existence of different stoichiometries in the secondary active transport. Our theoretical analysis clarifies several important aspects of complex biological transport phenomena.


Assuntos
Proteínas de Membrana Transportadoras , Transporte Biológico Ativo , Proteínas de Membrana Transportadoras/metabolismo , Mutação
3.
PLoS One ; 17(5): e0267599, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35503771

RESUMO

Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, ß-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.


Assuntos
Transporte Biológico Ativo , Túbulos Renais Proximais , Metalotioneína 3 , Células Epiteliais/metabolismo , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Simulação de Acoplamento Molecular , RNA Mensageiro/genética
4.
J Chem Phys ; 156(8): 085102, 2022 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-35232188

RESUMO

Successful functioning of biological cells relies on efficient translocation of different materials across cellular membranes. An important part of this transportation system is membrane channels that are known as antiporters and symporters. They exploit the energy stored as a trans-membrane gradient of one type of molecules to transport the other types of molecules against their gradients. For symporters, the directions of both fluxes for driving and driven species coincide, while for antiporters, the fluxes move in opposite directions. There are surprising experimental observations that despite differing only by the direction of transport fluxes, the molecular mechanisms of translocation adopted by antiporters and symporters seem to be drastically different. We present chemical-kinetic models to quantitatively investigate this phenomenon. Our theoretical approach allows us to explain why antiporters mostly utilize a single-site transportation when only one molecule of any type might be associated with the channel. At the same time, the transport in symporters requires two molecules of different types to be simultaneously associated with the channel. In addition, we investigate the kinetic constraints and efficiency of symporters and compare them with the same properties of antiporters. Our theoretical analysis clarifies some important physical-chemical features of cellular trans-membrane transport.


Assuntos
Antiporters , Simportadores , Antiporters/química , Antiporters/metabolismo , Transporte Biológico , Transporte Biológico Ativo , Modelos Teóricos , Simportadores/metabolismo
5.
Biosens Bioelectron ; 203: 114011, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35124343

RESUMO

Motor proteins, such as myosin and kinesin, are biological molecular motors involved in force generation and intracellular transport in living cells. They were proposed to drive molecular shuttles for the active transport of analytes, thus significantly accelerating the sensing process of biosensors. Integrating motor proteins into biosensors requires their immobilisation on the operating surfaces. However, this process makes some motor proteins defective, slowing analyte detection. Here, we investigated the movements of molecular shuttles on surfaces in the presence of active and defective motors using a Brownian dynamics simulation, and elucidated the effects of defective motor proteins on the transport efficiency of the shuttles. We found that the motility of shuttles depends on the fraction of active motors relative to defective ones and that over 90% of the surface-bound motor proteins must remain active for efficient transport. The high fraction of active motors required for efficient transport can be attributed to the difference in the binding lifetimes of active and defective motors to shuttles. These results provide insights into how motors accumulate on sensor surfaces and set a guideline for the choice of polymer materials for biosensors powered by motor proteins.


Assuntos
Técnicas Biossensoriais , Transporte Biológico Ativo , Microtúbulos/química , Microtúbulos/metabolismo , Miosinas
6.
Int J Mol Sci ; 23(3)2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35163125

RESUMO

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug-drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP+. Uptake of MPP+ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP+ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP+ transporting solute carrier proteins (SLCs) in general.


Assuntos
Impedância Elétrica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , 1-Metil-4-fenilpiridínio/efeitos adversos , Transporte Biológico , Transporte Biológico Ativo , Células HEK293 , Herbicidas/efeitos adversos , Humanos , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/antagonistas & inibidores , Transportador 1 de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico/antagonistas & inibidores , Transportador 2 de Cátion Orgânico/genética
7.
Int J Mol Sci ; 23(3)2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-35162943

RESUMO

Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson's disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. Here, we show that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of ß-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.


Assuntos
Ácido Aspártico/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Desacoplamento Mitocondrial/genética , Proteínas de Desacoplamento Mitocondrial/metabolismo , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/biossíntese , Transporte Biológico Ativo , Clonagem Molecular , Citosol/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , beta-Alanina/biossíntese
8.
J Pharm Pharm Sci ; 25: 77-83, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35041802

RESUMO

PURPOSE: This narrative review explores the currently published studies that have evaluated tenapanor for the treatment of hyperphosphatemia in end-stage kidney disease (ESKD) patients on hemodialysis. This medication's new phosphate lowering mechanism of action reduces intestinal phosphate absorption predominantly through reduction of passive paracellular phosphate flux by inhibition of the sodium/hydrogen exporter isoform 3 (NHE3). Tenapanor additionally prevents active transcellular phosphate absorption compensation by decreasing the expression of sodium phosphorus 2b transport protein (NaPi2b). METHODS: A comprehensive search of the literature was conducted using PubMed and ClinicalTrials.gov search engines. The search term "tenapanor hyperphosphatemia" was used for study retrieval. Results were limited to studies published in the English language and excluded review articles. Human, animal, and in vitro studies were included. No date range was specified. RESULTS: A total of 11 primary studies were identified and included in this review, the largest human study of which enrolled 236 patients. Each study is presented in table format along with measured end points. CONCLUSIONS: Tenapanor is the first drug in its class that lowers hyperphosphatemia in ESKD patients through a novel mechanism of action involving paracellular inactive transport. Although more studies are needed, early results indicate that tenapanor may have a place in managing hyperphosphatemia in ESKD patients both as monotherapy and as an adjunct to existing phosphate binder therapy.


Assuntos
Hiperfosfatemia/tratamento farmacológico , Hiperfosfatemia/etiologia , Isoquinolinas/farmacocinética , Isoquinolinas/uso terapêutico , Falência Renal Crônica/complicações , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêutico , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450 , Interações Medicamentosas , Humanos , Absorção Intestinal/efeitos dos fármacos , Fosfatos/metabolismo , Ratos , Trocador 3 de Sódio-Hidrogênio/efeitos dos fármacos
9.
Bioorg Med Chem ; 53: 116520, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34847494

RESUMO

The increase of concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in the serum of postmenopausal women is the important risk factor of the high morbidity of cardiovascular diseases of old women worldwide. To test the anti-hypercholesterolemia function of dihydroartemisinin (DHA) in postmenopausal women, ovariectomized (OVX) mice were generated, and DHA were administrated to OVX mice for 4 weeks. The blood and liver tissues were collected for biochemical and histological tests respectively. The mRNA and protein expression levels of genes related to metabolism and transport of cholesterol, bile acid and fatty acid in the liver or ileum were checked through qPCR and western blot. DHA could significantly reduce the high concentrations of TC and LDL-C in the serum and the lipid accumulation in the liver of ovariectomized mice. The expression of ABCG5/8 was reduced in liver of OVX mice, and DHA could up-regulate the expression of them. Genes of transport proteins for bile salt transport from blood to bile, including Slc10a1, Slco1b2 and Abcb11, were also significantly up-regulated by DHA. DHA also down-regulated the expression of Slc10a2 in the ileum of OVX mice to reduce the absorption of bile salts. Genes required for fatty acid synthesis and uptake, such as Fasn and CD36, were reduced in the liver of OVX mice, and DHA administration could significantly up-regulate the expression of them. These results demonstrated that DHA could improve hypercholesterolemia in OVX mice through enhancing the vectorial transport of cholesterol and bile acid from blood to bile.


Assuntos
Anticolesterolemiantes/farmacologia , Artemisininas/farmacologia , Ácidos e Sais Biliares/metabolismo , Bile/metabolismo , Colesterol/metabolismo , Hipercolesterolemia/tratamento farmacológico , Animais , Anticolesterolemiantes/química , Artemisininas/química , Bile/química , Ácidos e Sais Biliares/sangue , Transporte Biológico Ativo/efeitos dos fármacos , Colesterol/sangue , Relação Dose-Resposta a Droga , Feminino , Hipercolesterolemia/patologia , Hipercolesterolemia/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Ovariectomia , Relação Estrutura-Atividade
10.
Amino Acids ; 54(8): 1115-1122, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34694500

RESUMO

L-Carnosine (ß-alanyl-L-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of data demonstrating its efficiency as a neuroprotector, there is a distinct lack of data regarding the mechanism of its take up by neurons. According to literature, cultures of rat astrocytes, SKPT cells and rat choroid plexus epithelial cells take up carnosine via the H+-coupled PEPT2 membrane transporter. We've assessed the effectiveness and mechanism of carnosine transport, and its stability in primary rat cortical culture neurons. We demonstrated that neurons take up carnosine via active transport with Km = 119 µM and a maximum velocity of 0.289 nmol/mg (prot)/min. Passive transport speed constituted 0.21∙10-4 nmol/mg (prot)/min (with 119 µM concentration in the medium)-significantly less than active transport speed. However, carnosine concentrations over 12.5 mM led to passive transport speed becoming greater than active transport speed. Using PEPT2 inhibitor zofenopril, we demonstrated that PEPT2-dependent transport is one of the main modes of carnosine take up by neurons. Our experiments demonstrated that incubation with carnosine does not affect PEPT2 amount present in culture. At the same time, after removing carnosine from the medium, its elimination speed by culture cells reached 0.035 nmol/mg (prot)/min, which led to a decrease in carnosine quantity to control levels in culture within 1 h. Thus, carnosine is taken up by neurons with an effectiveness comparable to that of other PEPT2 substrates, but its elimination rate suggests that for effective use as a neuroprotector it's necessary to either maintain a high concentration in brain tissue, or increase the effectiveness of glial cell synthesis of endogenous carnosine and its shuttling into neurons, or use more stable chemical modifications of carnosine.


Assuntos
Carnosina , Simportadores , Animais , Transporte Biológico Ativo , Carnosina/metabolismo , Carnosina/farmacologia , Plexo Corióideo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ratos , Simportadores/metabolismo
11.
PLoS Pathog ; 17(12): e1010132, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34910768

RESUMO

Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1.


Assuntos
Transporte Biológico Ativo/fisiologia , Capsídeo/metabolismo , Cromatina/metabolismo , Membrana Nuclear/metabolismo , Simplexvirus/metabolismo , Animais , Chlorocebus aethiops , Herpes Simples , Células Vero , Replicação Viral/fisiologia
12.
Sci Rep ; 11(1): 24150, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34921178

RESUMO

Capillary endothelial cells of the human blood-brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico Ativo , Células HEK293 , Humanos , Especificidade de Órgãos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
14.
J Biol Chem ; 297(5): 101312, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34673028

RESUMO

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs.


Assuntos
Acrossomo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Espermatócitos/metabolismo , Substituição de Aminoácidos , Animais , Transporte Biológico Ativo/genética , Proteínas de Ciclo Celular/genética , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/genética , Fosfoproteínas/genética
15.
J Biol Chem ; 297(5): 101334, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34688652

RESUMO

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.


Assuntos
Complexo de Golgi/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Ativo , Complexo de Golgi/genética , Complexos Multiproteicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
16.
Molecules ; 26(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34684822

RESUMO

Cyclopeptidic photosensitizer prodrugs (cPPPs) are compounds designed to specifically target overexpressed hydrolases such as serine proteases, resulting in their specific activation in close proximity to tumor cells. In this study, we explored a series of conjugates that can be selectively activated by the urokinase plasminogen activator (uPA). They differ from each other by their pheophorbide a (Pha) loading, their number of PEG chains and the eventual presence of black hole quenchers (BHQ3). The involvement of a peptidic linker between the drugs and the cyclopeptidic carrier allows specific cleavage by uPA. Restoration of the photophysical activity was observed in vitro on A549 lung and MCF7 breast cancer cells that exhibited an increase in red fluorescence emission up to 5.1-fold and 7.8-fold, respectively for uPA-cPPQ2+2/5. While these cPPP conjugates do not show dark toxicity, they revealed their phototoxic potential in both cell lines at 5 µM of Phaeq and a blue light fluence of 12.7 J/cm2 that resulted in complete cell death with almost all conjugates. This suggests, in addition to the promising use for cancer diagnosis, a use as a PDT agent. Intravenous injection of tetrasubstituted conjugates in fertilized hen eggs bearing a lung cancer nodule (A549) showed that a double PEGylation was favorable for the selective accumulation of the unquenched Pha moieties in the tumor nodules. Indeed, the diPEGylated uPA-cPPP4/52 induced a 5.2-fold increase in fluorescence, while the monoPEGylated uPA-cPPP4/5 or uPA-cPPQ2+2/5 led to a 0.4-fold increase only.


Assuntos
Membrana Corioalantoide/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Pró-Fármacos/metabolismo , Células A549 , Animais , Transporte Biológico Ativo , Embrião de Galinha , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Técnicas In Vitro , Células MCF-7 , Modelos Biológicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacocinética , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacocinética , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
ACS Biomater Sci Eng ; 7(10): 4679-4693, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34490771

RESUMO

Chronic kidney disease affects one in six people worldwide. Due to the scarcity of donor kidneys and the complications associated with hemodialysis (HD), a cell-based bioartificial kidney (BAK) device is desired. One of the shortcomings of HD is the lack of active transport of solutes that would normally be performed by membrane transporters in kidney epithelial cells. Specifically, proximal tubule (PT) epithelial cells play a major role in the active transport of metabolic waste products. Therefore, a BAK containing an artificial PT to actively transport solutes between the blood and the filtrate could provide major therapeutic advances. Creating such an artificial PT requires a biocompatible tubular structure which supports the adhesion and function of PT-specific epithelial cells. Ideally, this scaffold should structurally replicate the natural PT basement membrane which consists mainly of collagen fibers. Fiber-based technologies such as electrospinning are therefore especially promising for PT scaffold manufacturing. This review discusses the use of electrospinning technologies to generate an artificial PT scaffold for ex vivo/in vivo cellularization. We offer a comparison of currently available electrospinning technologies and outline the desired scaffold properties required to serve as a PT scaffold. Discussed also are the potential technologies that may converge in the future, enabling the effective and biomimetic incorporation of synthetic PTs in to BAK devices and beyond.


Assuntos
Células Epiteliais , Túbulos Renais Proximais , Transporte Biológico Ativo , Biologia , Biomimética , Humanos , Túbulos Renais Proximais/metabolismo
18.
Sci Rep ; 11(1): 18053, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508127

RESUMO

Fluoro-Gold is a fluorescent neuronal tracer suitable for targeted deep imaging of the nervous system. Widefield fluorescence microscopy enables visualization of Fluoro-Gold, but lacks depth discrimination. Though scanning laser confocal microscopy yields volumetric data, imaging depth is limited, and optimal single-photon excitation of Fluoro-Gold requires an unconventional ultraviolet excitation line. Two-photon excitation microscopy employs ultrafast pulsed infrared lasers to image fluorophores at high-resolution at unparalleled depths in opaque tissue. Deep imaging of Fluoro-Gold-labeled neurons carries potential to advance understanding of the central and peripheral nervous systems, yet its two-photon spectral and temporal properties remain uncharacterized. Herein, we report the two-photon excitation spectrum of Fluoro-Gold between 720 and 990 nm, and its fluorescence decay rate in aqueous solution and murine brainstem tissue. We demonstrate unprecedented imaging depth of whole-mounted murine brainstem via two-photon excitation microscopy of Fluoro-Gold labeled facial motor nuclei. Optimal two-photon excitation of Fluoro-Gold within microscope tuning range occurred at 720 nm, while maximum lifetime contrast was observed at 760 nm with mean fluorescence lifetime of 1.4 ns. Whole-mount brainstem explants were readily imaged to depths in excess of 450 µm via immersion in refractive-index matching solution.


Assuntos
Transporte Biológico Ativo , Corantes Fluorescentes , Microscopia de Fluorescência por Excitação Multifotônica , Neurônios/metabolismo , Estilbamidinas , Animais , Biomarcadores , Feminino , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Molecular
19.
Sci Rep ; 11(1): 18636, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545146

RESUMO

Age, apolipoprotein E (apoE) isoform, sex, and diet can independently affect the risk for the development of Alzheimer's disease (AD). Additionally, synergy between some of these risk factors have been observed. However, the relation between the latter three risk factors has not been investigated. Central nervous system (CNS) insulin resistance is commonly involved in each of these risk factors. CNS insulin is primarily derived from the periphery in which insulin must be transported across the blood-brain barrier (BBB). Additionally, insulin can bind the brain endothelial cell to affect intracellular signaling. Therefore, we hypothesized CNS access to insulin could be affected by the combination of apoE isoform, sex, and diet. We analyzed insulin BBB pharmacokinetics in aged apoE targeted replacement (E3 and E4) male and female mice on a low-fat and high-fat diet. There were differences within males and females due to apoE genotype and diet in insulin interactions at the BBB. These sex-, diet-, and apoE isoform-dependent differences could contribute to the cognitive changes observed due to altered CNS insulin signaling.


Assuntos
Apolipoproteínas E/sangue , Barreira Hematoencefálica/metabolismo , Insulina/metabolismo , Envelhecimento/sangue , Envelhecimento/genética , Envelhecimento/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Apolipoproteína E3/sangue , Apolipoproteína E3/genética , Apolipoproteína E4/sangue , Apolipoproteína E4/genética , Apolipoproteínas E/genética , Transporte Biológico Ativo , Sistema Nervoso Central/metabolismo , Dieta com Restrição de Gorduras , Dieta Hiperlipídica/efeitos adversos , Feminino , Genótipo , Humanos , Insulina/sangue , Insulina/farmacocinética , Resistência à Insulina , Radioisótopos do Iodo , Masculino , Camundongos , Fatores de Risco , Fatores Sexuais , Transdução de Sinais , Distribuição Tecidual
20.
Drug Metab Dispos ; 49(12): 1038-1046, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34548392

RESUMO

Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.


Assuntos
Inativação Metabólica/fisiologia , Intestino Delgado , Proteínas de Membrana Transportadoras/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Fatores Etários , Transporte Biológico Ativo , Criança , Cromatografia Líquida/métodos , Citocromo P-450 CYP3A/metabolismo , Ensaios Enzimáticos/métodos , Ontologia Genética , Glucuronosiltransferase/metabolismo , Humanos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/enzimologia , Intestino Delgado/metabolismo , Taxa de Depuração Metabólica , Proteínas de Neoplasias/metabolismo , Transportador 1 de Peptídeos/metabolismo , Espectrometria de Massas em Tandem/métodos
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