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1.
Adv Exp Med Biol ; 1131: 183-213, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646511

RESUMO

Ca2+ binding proteins (CBP) are of key importance for calcium to play its role as a pivotal second messenger. CBP bind Ca2+ in specific domains, contributing to the regulation of its concentration at the cytosol and intracellular stores. They also participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e. decoding Ca2+ signals. Since CBP are integral to normal physiological processes, possible roles for them in a variety of diseases has attracted growing interest in recent years. In addition, research on CBP has been reinforced with advances in the structural characterization of new CBP family members. In this chapter we have updated a previous review on CBP, covering in more depth potential participation in physiopathological processes and candidacy for pharmacological targets in many diseases. We review intracellular CBP that contain the structural EF-hand domain: parvalbumin, calmodulin, S100 proteins, calcineurin and neuronal Ca2+ sensor proteins (NCS). We also address intracellular CBP lacking the EF-hand domain: annexins, CBP within intracellular Ca2+ stores (paying special attention to calreticulin and calsequestrin), proteins that contain a C2 domain (such as protein kinase C (PKC) or synaptotagmin) and other proteins of interest, such as regucalcin or proprotein convertase subtisilin kexins (PCSK). Finally, we summarise the latest findings on extracellular CBP, classified according to their Ca2+ binding structures: (i) EF-hand domains; (ii) EGF-like domains; (iii) ɣ-carboxyl glutamic acid (GLA)-rich domains; (iv) cadherin domains; (v) Ca2+-dependent (C)-type lectin-like domains; (vi) Ca2+-binding pockets of family C G-protein-coupled receptors.


Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Espaço Intracelular/metabolismo
2.
Adv Exp Med Biol ; 1131: 371-394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646518

RESUMO

Ca2+ signals are probably the most common intracellular signaling cellular events, controlling an extensive range of responses in virtually all cells. Many cellular stimuli, often acting at cell surface receptors, evoke Ca2+ signals by mobilizing Ca2+ from intracellular stores. Inositol trisphosphate (IP3) was the first messenger shown to link events at the plasma membrane to release Ca2+ from the endoplasmic reticulum (ER), through the activation of IP3-gated Ca2+ release channels (IP3 receptors). Subsequently, two additional Ca2+ mobilizing messengers were discovered, cADPR and NAADP. Both are metabolites of pyridine nucleotides, and may be produced by the same class of enzymes, ADP-ribosyl cyclases, such as CD38. Whilst cADPR mobilizes Ca2+ from the ER by activation of ryanodine receptors (RyRs), NAADP releases Ca2+ from acidic stores by a mechanism involving the activation of two pore channels (TPCs). In addition, other pyridine nucleotides have emerged as intracellular messengers. ADP-ribose and 2'-deoxy-ADPR both activate TRPM2 channels which are expressed at the plasma membrane and in lysosomes.


Assuntos
Cálcio , ADP-Ribose Cíclica , Piridinas , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Espaço Intracelular/metabolismo , NADP/metabolismo , Piridinas/química , Piridinas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
3.
Bioresour Technol ; 289: 121744, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31323718

RESUMO

Haematococcus pluvialis is a prominent feedstock of astaxanthin. The ratio of carbon to nitrogen (C/N) strongly influences the metabolic pathways of mixotrophic-grown microalgae, however, its role involved in astaxanthin biosynthesis is still not fully understood. In this study, integrative metabolic and physiologic profiles were analyzed in elucidating how C/N affected carbon and nitrogen assimilation and thereby exerted influence on astaxanthin biosynthesis. It was demonstrated that high C/N up-regulated the activities of acetate kinase by increase of 5.76 folds in early logarithmic phase, leading a significant increase of acetyl-CoA. The increased carbon skeletons were then funneled into astaxanthin biosynthesis. Additionally, high C/N increased the proportion of carotenoid-intermediates in cytoplasm from chloroplast. Finally, a fed-batch cultivation strategy based on C/N gradient was developed. Biomass attained 9.18 g L-1 in 100% type of immotile cyst cells, which presented astaxanthin productivity at 15.45 mg L-1 d-1 afterward, exhibiting a promising paradigm in commercial production.


Assuntos
Carbono/metabolismo , Clorofíceas/metabolismo , Nitrogênio/metabolismo , Biomassa , Espaço Intracelular/metabolismo , Microalgas/metabolismo , Xantofilas/metabolismo
4.
Artif Cells Nanomed Biotechnol ; 47(1): 1476-1487, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31070063

RESUMO

Polymeric micelles (PMs) play a vital role in multidrug co-delivery and cancer treatment. However, the development of intelligent PMs further allows PMs to accurately -target tumour, selectively release cargo multidrug and increase uptake. Therefore, targets, controlled release and uptake of intelligent PMs should be paid more attention to improvement synergistic therapeutic outcomes and minimize side effects. In this review, tumour targeting of co-delivery intelligent PMs and its intracellular trafficking mechanisms were overviewed. And this review provides a comprehensive summarization of several intelligent co-delivery PMs. Such a system could control the multidrug to be released simultaneously or sequentially by special properties of tumour microenvironment (TME) (including acidic PH, redox, overexpressed enzyme, excessive temperature) and external environment trigger. Additionally, limitations, clinical translation and future perspectives of intelligent co-delivery PMs were also being discussed in this article.


Assuntos
Portadores de Fármacos/química , Micelas , Neoplasias/tratamento farmacológico , Polímeros/química , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Neoplasias/patologia , Microambiente Tumoral/efeitos dos fármacos
5.
Adv Neurobiol ; 22: 125-153, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31073934

RESUMO

The enormous advances made over the last 50 years in materials science, microelectronics, and nanoelectronics, together with the acknowledgment that substrate-integrated planar multielectrode arrays (MEA) are limited to recording of extracellular field potentials (FPs) rather than the entire electrophysiological signaling repertoire of the brain, have prompted a number of laboratories to merge the advantages of planar MEA technologies (non-damaging and durable) with those of the classical sharp and patch electrodes for intracellular recordings. Unlike extracellular planar electrode-based MEAs, the new generation of three-dimensional (3D) vertical nanoelectrodes are designed to functionally penetrate the plasma membrane of cultured cells and operate in a similar manner to classical intracellular microelectrodes. Although only approximately 10 years has elapsed since the development of the first vertical 3D nanostructure-based MEAs, this technology has progressed to enable recordings of attenuated intracellular action potentials (APs) and synaptic potentials from individual neurons, cardiomyocytes, and striated myotubes. Furthermore, recently the scaling advantages of nanochip/microchip fabrication technologies enabled simultaneously intracellular recordings of APs from hundreds of cultured cardiomyocytes, thus heralding a new milestone in MEA technology.In this chapter we present the earliest and today's cutting-edge achievements of this "young vertical nano-sensors MEA technology" at the single-cell and network levels, explain the biophysical principles and the various configurations used to form functional nanoelectrode/cell hybrids, and describe the quality and characteristic features of the recorded intracellular APs and subthreshold synaptic potentials by the vertical nanoelectrode-based MEA. Basic cell-biological mechanisms that curtail the length of time intracellular access by the nanoelectrodes are discussed, and approaches to overcome this problem are offered.Recent development of biotechnologies that use induced human pluripotent stem cells taken from healthy subjects and patients, and in vitro drug screening for the development of personalized medicine as well as basic brain research will benefit tremendously from the use of MEAs that record the entire brain electrophysiological signaling repertoire from individual cells within an operational network rather than only extracellular FPs.


Assuntos
Potenciais de Ação , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Espaço Intracelular/metabolismo , Microeletrodos , Neurônios/citologia , Encéfalo/citologia , Humanos
6.
J Opt Soc Am A Opt Image Sci Vis ; 36(4): 665-677, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31044988

RESUMO

Intracellular dynamics in living tissue are dominated by active transport driven by bioenergetic processes far from thermal equilibrium. Intracellular constituents typically execute persistent walks. In the limit of long mean free paths, the persistent walks are ballistic, exhibiting a "Doppler edge" in light scattering fluctuation spectra. At shorter transport lengths, the fluctuations are described by lifetime-broadened Doppler spectra. Dynamic light scattering from transport in the ballistic, diffusive, or the crossover regimes is derived analytically, including the derivation of autocorrelation functions through a driven damped harmonic oscillator analog for light scattering from persistent walks. The theory is validated through Monte Carlo simulations. Experimental evidence for the Doppler edge in three-dimensional (3D) living tissue is obtained using biodynamic imaging based on low-coherence interferometry and digital holography.


Assuntos
Espaço Intracelular/metabolismo , Espaço Intracelular/efeitos da radiação , Luz , Sobrevivência de Tecidos , Animais , Humanos , Imagem Tridimensional , Método de Monte Carlo , Espalhamento de Radiação
7.
Bioresour Technol ; 287: 121414, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31078813

RESUMO

An increase in the total lipid content in algal cells under stress conditions is often accompanied by an increase in reactive oxygen species (ROS). However, the link between these two events is unclear. In this study, the regulatory mechanism of ROS formation on lipid accumulation in C. pyrenoidosa was investigated using a Fenton-like reaction. A high Spearman correlation coefficient of 0.901 was obtained between the Hydroxyl radical (OH) level and lipid content. Importantly, the increase in the total lipid content was clearly coupled with a significant increase in the intracellular OH concentration rather than increases in the H2O2 and O2- concentrations. Transcriptome data confirms that most of the differential expression genes (DEGs) involved in fatty acid and glycerolipid biosynthesis were up-regulated by the increased OH under stress conditions. These results reveal that lipid accumulation in algal cells was promoted by OH.


Assuntos
Chlorella/metabolismo , Lipídeos/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio/metabolismo , Radical Hidroxila/metabolismo , Espaço Intracelular/metabolismo
8.
Electromagn Biol Med ; 38(2): 143-148, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31032646

RESUMO

Except for relatively few polarity reversals the magnitude of the magnetic dipole moment of the earth has remained constant since life first began, allowing evolutionary processes to integrate the geomagnetic field (GMF) into several biological functions. One of these, bearing the classical signature of an ion cyclotron resonance (ICR)-like interaction, results in biological change associated with enhanced proton transport. The wide range of cation masses over which this effect is found suggest a fundamental biological dependence on the GMF, one that functions equally well for electric as well as magnetic fields. Such generalization of ICR requires two things: transparency of tissues to the GMF and suitably tuned ELF resonant magnetic or electric fields. To complement the widely reported ICR responses to applied AC magnetic fields, we hypothesize the existence of weak endogenous ICR electric field oscillations within the cell. This equivalence implies that even in the absence of applied AC magnetic fields, biological systems will exhibit intrinsic GMF-dependent ion cyclotron resonance intracellular interactions. Many ICR effects that have been reported appear as antagonist pairs suggesting that the characteristics of the GMF have not only been incorporated into the genome but also appear to function in an endocrine-like manner.


Assuntos
Ciclotrons , Campos Magnéticos , Espaço Intracelular/metabolismo
9.
Eur J Pharmacol ; 852: 231-243, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959046

RESUMO

Multidrug resistance (MDR) remains an obstacle to chemotherapy related with the overexpression of several efflux membrane proteins, and p-glycoprotein (P-gp) is the most studied among them. Thus, continuous investigational efforts are necessary to find valuable MDR reversal agents, and the flavonoid compound glabridin (GBD) seems to be a promising candidate. This study aimed to investigate the potential of GBD against MDR and explore the possible mechanisms. First, we found that GBD could decrease the half maximal inhibitory concentration of paclitaxel and doxorubicin (DOX) in breast cancer cells like MDA-MB-231/MDR1 cells and MCF-7/ADR cells. It was further explained that GBD enhanced the apoptosis of MDA-MB-231/MDR1 cells induced by DOX, due to the increased accumulation of DOX. Then, tests were performed to explore the possible MDR reversal mechanisms. On one hand, GBD can suppress the expression of P-gp. On the other hand, GBD can downregulate the activity of P-gp ATPase when cotreated with DOX or verapamil, revealing that GBD was a substrate of P-gp. Moreover, the obtained kinetic inhibition parameters proved that GBD was a competitive inhibitor of P-gp, and in molecular docking simulation modeling, GBD exhibited stronger binding affinity with P-gp than DOX. In conclusion, GBD can increase the accumulation of DOX in MDA-MB-231/MDR1 cells by suppressing the expression of P-gp and competitively inhibiting the P-gp efflux pump and enhance the apoptosis of MDA-MB-231/MDR1 cells induced by DOX, and thus realize reversal effects on MDR. Therefore, the combination therapy of anticancer drugs and flavonoid-like GBD is a promising strategy to overcome P-gp-mediated MDR.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoflavonas/farmacologia , Fenóis/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Isoflavonas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Fenóis/metabolismo , Conformação Proteica
10.
Phys Rev E ; 99(3-1): 032409, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30999457

RESUMO

Morphogen gradients play a vital role in developmental biology by enabling embryonic cells to infer their spatial location and determine their developmental fate accordingly. The standard mechanism for generating a morphogen gradient involves a morphogen being produced from a localized source and subsequently degrading. While this mechanism is effective over the length and time scales of tissue development, it fails over typical subcellular length scales due to the rapid dissipation of spatial asymmetries. In a recent theoretical work, we found an alternative mechanism for generating concentration gradients of diffusing molecules, in which the molecules switch between spatially constant diffusivities at switching rates that depend on the spatial location of a molecule. Independently, an experimental and computational study later found that Caenorhabditis elegans zygotes rely on this mechanism for cell polarization. In this paper, we extend our analysis of switching diffusivities to determine its role in protein concentration gradient formation. In particular, we determine how switching diffusivities modifies the standard theory and show how space-dependent switching diffusivities can yield a gradient in the absence of a localized source. Our mathematical analysis yields explicit formulas for the intracellular concentration gradient which closely match the results of previous experiments and numerical simulations.


Assuntos
Quimiotaxia/fisiologia , Modelos Biológicos , Proteínas/metabolismo , Animais , Caenorhabditis elegans , Simulação por Computador , Difusão , Drosophila , Espaço Intracelular/metabolismo , Morfogênese/fisiologia , Conformação Proteica
11.
J Photochem Photobiol B ; 194: 96-106, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30953915

RESUMO

Photodynamic therapy (PDT) is a promising approach for the treatment of different types of cancer and has been brought into focus for its synergy, compatibility, repeatability, relatively inexpensive cost and it's typically more efficacious nature. Photosensitizers (PSs) play a major role in PDT and are the core of this specific therapy. Al (III) Phthalocyanine Chloride Tetra sulfonic Acid (AlPcS4Cl) PS is an aromatic macrocyclic metal-based compound that is synthetically derived. It aids in deep tissue penetration due to its far red light activation wavelength, low photo bleaching, increased quantum yields and stability. Lung cancer is a leading cause of cancer related deaths worldwide accounting for approximately 1 in 5 of all cancer-related deaths. In this study, we explored the photochemical properties of AlPcS4Cl, its uptake into lung cancer, the intracellular localization and photodynamic action on lung cancer (A549 cells). Results indicated that AlPcS4Cl is a stable PS that localizes in intracellular organelles including the mitochondrion and lysosomes. PDT using AlPcS4Cl indicated an increase in cell death and decrease in cell proliferation and viability. AlPcS4Cl showed to be effective in treating lung cancer in vitro, however the resulting PDT efficacy will finally depend on the biological features such as tumour vasculature and tumour specific accumulation when used as a clinical application. It is noted that PDT can be considered as an adjunct therapy until standard protocols for various tumour types along with a relevant PS has been validated.


Assuntos
Alumínio/química , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Indóis/farmacologia , Neoplasias Pulmonares/patologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Células A549 , Transporte Biológico , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Relação Dose-Resposta a Droga , Humanos , Indóis/metabolismo , Espaço Intracelular/metabolismo , Cinética , Fotoquimioterapia , Fármacos Fotossensibilizantes/metabolismo
12.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934807

RESUMO

Lipid accumulation in renal cells has been implicated in the pathogenesis of obesity-related kidney disease, and lipotoxicity in the kidney can be a surrogate marker for renal failure or renal fibrosis. Fatty acid oxidation provides energy to renal tubular cells. Ca2+ is required for mitochondrial ATP production and to decrease reactive oxygen species (ROS). However, how nifedipine (a calcium channel blocker) affects lipogenesis is unknown. We utilized rat NRK52E cells pre-treated with varying concentrations of nifedipine to examine the activity of lipogenesis enzymes and lipotoxicity. A positive control exposed to oleic acid was used for comparison. Nifedipine was found to activate acetyl Coenzyme A (CoA) synthetase, acetyl CoA carboxylase, long chain fatty acyl CoA elongase, ATP-citrate lyase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductase, suggesting elevated production of cholesterol and phospholipids. Nifedipine exposure induced a vast accumulation of cytosolic free fatty acids (FFA) and stimulated the production of reactive oxygen species, upregulated CD36 and KIM-1 (kidney injury molecule-1) expression, inhibited p-AMPK activity, and triggered the expression of SREBP-1/2 and lipin-1, underscoring the potential of nifedipine to induce lipotoxicity with renal damage. To our knowledge, this is the first report demonstrating nifedipine-induced lipid accumulation in the kidney.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Rim/metabolismo , Lipogênese/efeitos dos fármacos , Nifedipino/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transcrição Genética/efeitos dos fármacos , Animais , Vias Biossintéticas/efeitos dos fármacos , Antígenos CD36/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Regulação para Baixo/efeitos dos fármacos , Espaço Intracelular/metabolismo , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/lesões , Modelos Biológicos , PPAR alfa/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos
13.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934953

RESUMO

Manganese, as an essential trace element, participates in many physiological reactions by regulating Mn associated enzymes. Magnaporthe oryzae is a serious pathogen and causes destructive losses for rice production. We identified a cytochrome P450 gene, MoMCP1, involving the alleviation of manganese toxin and pathogenicity. To identify the underlying mechanisms, transcriptomics were performed. The results indicated that many pathogenicity related genes were regulated, especially hydrophobin related genes in ∆Momcp1. Furthermore, the Mn2+ toxicity decreased the expressions of genes involved in the oxidative phosphorylation and energy production, and increased the reactive oxygen species (ROS) levels, which might impair the functions of mitochondrion and vacuole, compromising the pathogenicity and development in ∆Momcp1. Additionally, our results provided further information about Mn associated the gene network for Mn metabolism in cells.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Genes Fúngicos , Magnaporthe/genética , Manganês/toxicidade , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Hifas/metabolismo , Espaço Intracelular/metabolismo , Magnaporthe/patogenicidade , Mutação/genética , Oryza/microbiologia , Superóxido Dismutase/metabolismo , Transcriptoma/genética
14.
Artif Cells Nanomed Biotechnol ; 47(1): 1357-1366, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30977425

RESUMO

Senile osteoporosis is closely related to the loss of function of stem cells. In this study, we tried to investigate the potential of secretome from human umbilical cord-derived mesenchymal stem cells (hUCMSCs) in recovering stem cell ability from senescence and then delaying bone loss. We first harvested bone marrow-derived mesenchymal stem cells (BMSCs) from young and old rats and then compared their cellular characteristics such as cell growth, anti-senescence and differentiation. The results showed that these abilities were negatively affected by animal aging. Subsequently, aged BMSCs were exposed to secretome from hUCMSCs, and we found that this loss of cell potential can be modified by secretome treatment. Thereafter, the secretome was loaded into silk fibroin-based hydrogels and used for an in vivo animal study. The results showed that compared to the old untreated group, the bone formation capacity of aged rats was improved by local treatment of secretome-loaded silk fibroin hydrogels. In conclusion, these findings demonstrated that secretome from hUCMSCs has the capacity to recover stem cell potential and delay local bone loss in age-related osteoporosis, which could potentially be applied in osteoporosis therapy in the future.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Osteoporose/terapia , Cordão Umbilical/citologia , Animais , Fenômenos Biomecânicos , Diferenciação Celular , Sobrevivência Celular , Humanos , Espaço Intracelular/metabolismo , Masculino , Osteoporose/patologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
15.
PLoS Pathog ; 15(4): e1007704, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30951565

RESUMO

Typhoid toxin is a virulence factor for Salmonella Typhi and Paratyphi, the cause of typhoid fever in humans. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. Typhoid toxin is uniquely adapted to humans, recognizing surface glycoprotein sialoglycans terminated in acetyl neuraminic acid, which are preferentially expressed by human cells. The transport pathway to its cellular targets followed by typhoid toxin after receptor binding is currently unknown. Through a genome-wide CRISPR/Cas9-mediated screen we have characterized the mechanisms by which typhoid toxin is transported within human cells. We found that typhoid toxin hijacks specific elements of the retrograde transport and endoplasmic reticulum-associated degradation machineries to reach its subcellular destination within target cells. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Degradação Associada com o Retículo Endoplasmático , Salmonella typhi/patogenicidade , Febre Tifoide/microbiologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Transporte Biológico , Sistemas CRISPR-Cas , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Ligação Proteica , Salmonella typhi/genética , Febre Tifoide/genética , Febre Tifoide/metabolismo
16.
J Phys Chem A ; 123(17): 3928-3934, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30957999

RESUMO

Detailed knowledge of the water status in living organisms is crucial for understanding their physiology and pathophysiology. Here, we developed a technique to spectroscopically image water at high resolution using ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy equipped with a supercontinuum light source. This system allows for the visualization of a wide spectrum of CARS signals from the fingerprint to the end of O-H stretching at a spectral resolution of ∼10 cm-1. Application of the system to living mammalian cells revealed a spectral red shift of the O-H stretching vibrational band inside compared to outside the cells, suggesting the existence of stronger hydrogen bonds inside the cells. Furthermore, potential changes in spectra were examined by adding mannitol to the extracellular solution, which increases the osmolality outside the cells and thereby induces dehydration of the cells. Under this treatment, the red shift of the O-H stretching band was further enhanced, revealing the effects of mannitol on water states inside the cells. The methodology developed here should serve as a powerful tool for the chemical imaging of water in living cells in various biological and medical contexts.


Assuntos
Espaço Extracelular/metabolismo , Espaço Intracelular/metabolismo , Análise Espectral Raman , Água/metabolismo , Animais , Células CHO , Cricetulus , Osmose
17.
Soft Matter ; 15(18): 3655-3658, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31012474

RESUMO

We developed biocompatible ATP responsive DNA nanogels, by grafting DNA strands on carboxymethyl chitosan polymer chains, and then hybridizing with ATP aptamers to form core-shell nanogels. The DNA duplex structure could embed DOX in the G-C sequences, and realize simultaneous sol-gel transition and DOX release when suffering enrich ATP in tumor cells.


Assuntos
Trifosfato de Adenosina/metabolismo , Antineoplásicos/química , Materiais Biocompatíveis/química , DNA/química , Portadores de Fármacos/química , Nanoestruturas/química , Células A549 , Antineoplásicos/metabolismo , Transporte Biológico , Doxorrubicina/química , Doxorrubicina/metabolismo , Liberação Controlada de Fármacos , Géis , Humanos , Espaço Intracelular/metabolismo
18.
Methods Mol Biol ; 1944: 95-114, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840237

RESUMO

We present the development, optimization, and application of constructs, cell lines, covalent cross-linking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome cross-linking and perform immunoprecipitations are then presented. We conclude with a discussion of methods to elute collagen interactomes and prepare samples for mass spectrometry-mediated identification of interactors. Throughout, caveats and potential problems researchers may encounter when working with collagen are discussed. We note that the protocols presented herein may be readily adapted to define interactomes of other collagen types, as well as to determine comparative interactomes of normal and disease-causing collagen variants using quantitative isotopic labeling (SILAC)- or isobaric mass tags (iTRAQ or TMT)-based mass spectrometry analysis.


Assuntos
Colágeno/metabolismo , Espaço Intracelular/metabolismo , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Colágeno/análise , Humanos , Imunoprecipitação
19.
Biomater Sci ; 7(5): 1825-1832, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30892297

RESUMO

As drug-delivery carriers for cancer chemotherapy, gatekeeper-capped mesoporous silica nanoparticles (MSNs) have been widely studied due to their high drug-loading capability, controlled drug release property and good biocompatibility. However, the currently reported gatekeeper-capped MSNs suffer from complex synthetic procedures, potential toxicity of gatekeepers, unsatisfactory control on drug stimuli-release, etc. In this work, we develop a simple but efficient approach to fabricate PEGylated organosilica-capped mesoporous silica nanoparticles (POMSNs) by employing a disulfide-doped organosilica coating as the gatekeeper formed by the hydrolysis and condensation of a silane coupling agent 3-(mercaptopropyl)trimethoxysilane (MPTMS) to block the mesopores of MSNs. Owing to the glutathione (GSH)-responsive biodegradation behavior of the disulfide-doped organosilica gatekeeper, the DOX-loaded POMSNs exhibit only 20% cell viability towards SMMC-7721 tumor cells, and almost no toxicity towards L-02 cells at a DOX concentration of 50 µg mL-1 was measured, demonstrating their selective cytotoxicity in vitro. More importantly, it is demonstrated that the DOX-loaded POMSNs exhibit a tumor inhibition rate of 71.3% and negligible systematic toxicity. Consequently, the resultant POMSNs show great potential as drug nanocarriers for redox-responsive drug release and passive-targeting tumor chemotherapy.


Assuntos
Portadores de Fármacos/química , Portadores de Fármacos/síntese química , Liberação Controlada de Fármacos , Nanoestruturas/química , Dióxido de Silício/química , Dióxido de Silício/síntese química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Técnicas de Química Sintética , Doxorrubicina/química , Doxorrubicina/metabolismo , Portadores de Fármacos/metabolismo , Feminino , Glutationa/metabolismo , Humanos , Espaço Intracelular/metabolismo , Camundongos , Oxirredução , Porosidade , Dióxido de Silício/metabolismo
20.
Cancer Immunol Immunother ; 68(6): 883-895, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30847498

RESUMO

We recently reported that pretreatment of IL-2 activated human natural killer (NK) cells with the drugs dimethyl fumarate (DMF) and monomethyl fumarate (MMF) upregulated the expression of surface chemokine receptor CCR10. Ligands for CCR10, namely CCL27 and CCL28, induced the chemotaxis of these cells. Here, we performed a bioinformatics analysis to see which chemokines might be expressed by the human HCT-116 colorectal cancer cells. We observed that, in addition to CCL27 and CCL28, HCT-116 colorectal cancer cells profoundly express CXCL16 which binds CXCR6. Consequently, NK92 cells were treated with DMF and MMF for 24 h to investigate in vitro chemotaxis towards CXCL16, CCL27, and CCL28. Furthermore, supernatants collected from HCT-116 cells after 24 or 48 h incubation induced the chemotaxis of NK92 cells. Similar to their effects on human IL-2-activated NK cells, MMF and DMF enhanced the expression of CCR10 and CXCR6 in NK92 cells. Neutralizing anti-CXCL16 or anti-CCL28 inhibited the chemotactic effects of 24 and 48 supernatants, whereas anti-CCL27 only inhibited the 48 h supernatant activity, suggesting that 24 h supernatant contains CXCL16 and CCL28, whereas HCT-116 secretes all three chemokines after 48 h in vitro cultures. CXCL16, CCL27, and CCL28, as well as the supernatants collected from HCT-116, induced the mobilization of (Ca)2+ in NK92 cells. Cross-desensitization experiments confirmed the results of the chemotaxis experiments. Finally, incubation of NK92 cells with HCT-116 induced the lysis of the tumor cells. In summary, these results might have important implications in directing the anti-tumor effectors NK cells towards tumor growth sites.


Assuntos
Cálcio/metabolismo , Quimiocinas/biossíntese , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Receptores de Quimiocinas/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Linhagem Celular Tumoral , Quimiocinas/imunologia , Quimiocinas/farmacologia , Quimiotaxia/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Meios de Cultivo Condicionados/farmacologia , Fumarato de Dimetilo/farmacologia , Fumaratos/química , Fumaratos/farmacologia , Células HCT116 , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia
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