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1.
Proc Natl Acad Sci U S A ; 117(23): 12674-12685, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32430322

RESUMO

Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-ß, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-ß). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-ß plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.


Assuntos
Imunoterapia/métodos , Interferon beta/genética , Neoplasias Experimentais/terapia , Linfócitos T/imunologia , Transfecção/métodos , Ondas Ultrassônicas , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos da radiação , Movimento Celular , Humanos , Interferon beta/metabolismo , Camundongos , Microbolhas/uso terapêutico , Linfócitos T/fisiologia
2.
Biochim Biophys Acta Bioenerg ; 1861(5-6): 148115, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32204904

RESUMO

Green plants protect against photodamage by dissipating excess energy in a process called non-photochemical quenching (NPQ). In vivo, NPQ is activated by a drop in the luminal pH of the thylakoid membrane that triggers conformational changes of the antenna complexes, which activate quenching channels. The drop in pH also triggers de-epoxidation of violaxanthin, one of the carotenoids bound within the antenna complexes, into zeaxanthin, and so the amplitude of NPQ in vivo has been shown to increase in the presence of zeaxanthin. In vitro studies on light-harvesting complex II (LHCII), the major antenna complex in plants, compared different solubilization environments, which give rise to different levels of quenching and so partially mimic NPQ in vivo. However, in these studies both completely zeaxanthin-independent and zeaxanthin-dependent quenching have been reported, potentially due to the multiplicity of solubilization environments. Here, we characterize the zeaxanthin dependence of the photophysics in LHCII in a near-physiological membrane environment, which produces slightly enhanced quenching relative to detergent solubilization, the typical in vitro environment. The photophysical pathways of dark-adapted and in vitro de-epoxidized LHCIIs are compared, representative of the low-light and high-light conditions in vivo, respectively. The amplitude of quenching as well as the dissipative photophysics are unaffected by zeaxanthin at the level of individual LHCIIs, suggesting that zeaxanthin-dependent quenching is independent of the channels induced by the membrane. Furthermore, our results demonstrate that additional factors beyond zeaxanthin incorporation in LHCII are required for full development of NPQ.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Complexos de Proteínas Captadores de Luz/metabolismo , Luz , Zeaxantinas/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Transferência de Energia , Fluorescência , Concentração de Íons de Hidrogênio , Modelos Moleculares , Spinacia oleracea/metabolismo , Zeaxantinas/química
3.
Science ; 367(6482): 1091-1097, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139536

RESUMO

Many disease pathologies can be understood through the elucidation of localized biomolecular networks, or microenvironments. To this end, enzymatic proximity labeling platforms are broadly applied for mapping the wider spatial relationships in subcellular architectures. However, technologies that can map microenvironments with higher precision have long been sought. Here, we describe a microenvironment-mapping platform that exploits photocatalytic carbene generation to selectively identify protein-protein interactions on cell membranes, an approach we term MicroMap (µMap). By using a photocatalyst-antibody conjugate to spatially localize carbene generation, we demonstrate selective labeling of antibody binding targets and their microenvironment protein neighbors. This technique identified the constituent proteins of the programmed-death ligand 1 (PD-L1) microenvironment in live lymphocytes and selectively labeled within an immunosynaptic junction.


Assuntos
Antígeno B7-H1/metabolismo , Membrana Celular/metabolismo , Microambiente Celular , Linfócitos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Catálise , Membrana Celular/efeitos da radiação , Transferência de Energia , Humanos , Células Jurkat , Linfócitos/efeitos da radiação , Metano/análogos & derivados , Metano/química , Metano/efeitos da radiação , Processos Fotoquímicos , Raios Ultravioleta
4.
Proc Natl Acad Sci U S A ; 117(14): 7729-7738, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213584

RESUMO

Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol-protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.


Assuntos
Diglicerídeos/química , Lipídeos/química , Proteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Sobrevivência Celular , Isoenzimas/metabolismo , Cinética , Luz , Modelos Biológicos , Proteína Quinase C/metabolismo , Transdução de Sinais
5.
Proc Natl Acad Sci U S A ; 116(38): 18822-18826, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31481620

RESUMO

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.


Assuntos
Viabilidade Microbiana , Triptofano/química , Aminoácidos/química , Aminoácidos/efeitos da radiação , Bacillus subtilis/fisiologia , Bacillus subtilis/efeitos da radiação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Escherichia coli/fisiologia , Escherichia coli/efeitos da radiação , Fluorescência , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação , Fotólise , Desdobramento de Proteína , Espectrometria de Fluorescência , Triptofano/efeitos da radiação , Raios Ultravioleta
6.
Nano Lett ; 19(9): 6182-6191, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31369284

RESUMO

Determining the surface concentration and diffusivity of cell-membrane-bound molecules is central to the understanding of numerous important biochemical processes taking place at cell membranes. Here we use the high aspect ratio and lightguiding properties of semiconductor nanowires (NWs) to detect the presence of single freely diffusing proteins bound to a lipid bilayer covering the NW surface. Simultaneous observation of light-emission dynamics of hundreds of individual NWs occurring on the time scale of only a few seconds is interpreted using analytical models and employed to determine both surface concentration and diffusivity of cholera toxin subunit B (CTxB) bound to GM1 gangliosides in supported lipid bilayer (SLB) at surface concentrations down to below one CTxB per µm2. In particular, a decrease in diffusivity was observed with increasing GM1 content in the SLB, suggesting increasing multivalent binding of CTxB to GM1. The lightguiding capability of the NWs makes the method compatible with conventional epifluorescence microscopy, and it is shown to work well for both photostable and photosensitive dyes. These features make the concept an interesting complement to existing techniques for studying the diffusivity of low-abundance cell-membrane-bound molecules, expanding the rapidly growing use of semiconductor NWs in various bioanalytical sensor applications and live cell studies.


Assuntos
Toxina da Cólera/isolamento & purificação , Nanotecnologia , Nanofios/química , Imagem Individual de Molécula , Membrana Celular/química , Membrana Celular/efeitos da radiação , Toxina da Cólera/química , Gangliosídeo G(M1)/química , Luz , Bicamadas Lipídicas/química , Microscopia de Fluorescência , Ligação Proteica , Semicondutores
7.
Plant Cell Environ ; 42(9): 2554-2566, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31069808

RESUMO

Due to the preeminence of reductionist approaches, understanding of plant responses to combined stresses is limited. We speculated that light-quality signals of neighbouring vegetation might increase susceptibility to heat shocks because shade reduces tissue temperature and hence the likeness of heat shocks. In contrast, plants of Arabidopsis thaliana grown under low-red/far-red ratios typical of shade were less damaged by heat stress than plants grown under simulated sunlight. Neighbour signals reduce the activity of phytochrome B (phyB), increasing the abundance of PHYTOCHROME-INTERACTING FACTORS (PIFs). The phyB mutant showed high tolerance to heat stress even under simulated sunlight, and a pif multiple mutant showed low tolerance under simulated shade. phyB and red/far-red ratio had no effects on seedlings acclimated with nonstressful warm temperatures before the heat shock. The phyB mutant showed reduced expression of several fatty acid desaturase (FAD) genes and less proportion of fully unsaturated fatty acids and electrolyte leakage of membranes exposed to heat shocks. Red-light-activated phyB also reduced thermotolerance of dark-grown seedlings but not via changes in FADs expression and membrane stability. We propose that the reduced photosynthetic capacity linked to thermotolerant membranes would be less costly under shade, where the light input limits photosynthesis.


Assuntos
Arabidopsis/efeitos da radiação , Membrana Celular/efeitos da radiação , Fitocromo B/metabolismo , Termotolerância/efeitos da radiação , Aclimatação , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Aquecimento Global , Resposta ao Choque Térmico , Fitocromo B/genética , Plântula/efeitos da radiação , Termotolerância/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
8.
J BUON ; 24(1): 158-162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30941965

RESUMO

PURPOSE: The present study explored the potential of microwaves on membrane fluidity changes in diethylnitrosamine (DEN) induced hepatocellular carcinoma (HCC), in vivo. METHODS: Rats were segregated into four groups: normal control, DEN-treated, microwave-treated, DEN+microwave-treated. Brush border membranes (BBM) were isolated from the rats and, using the membrane extrinsic fluorophore pyrene, we assessed the viscosities as well as fluidity parameters. RESULTS: DEN treatment resulted in a significant rise in lipid peroxidation (LPO). Reduced glutathione levels (GSH) and the activities of glutathione reductase (GR), glutathione transferase (GST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were found to be significantly decreased following DEN treatment. On the other hand, microwave treatment in DEN-treated rats resulted in a significant decrease in the levels of lipid peroxidation but caused a significant rise in the levels of GSH as well in the activities of GR, GST, SOD, CAT and GPx. The results further demonstrated a marked decrease in membrane microviscosity following DEN treatment. On the other hand, a significant increase was observed in the excimer/monomer ratio and fluidity parameter of DEN-treated rats when compared to normal control rats. However, the alterations in membrane microviscosity and the fluidity parameters were significantly restored after microwave treatment. CONCLUSION: The study, therefore, concludes that microwave proved quite useful in the modulation of membrane stability parameters following DEN-induced hepatic cancer.


Assuntos
Membrana Celular/fisiologia , Dietilnitrosamina/toxicidade , Neoplasias Hepáticas/prevenção & controle , Fluidez de Membrana/fisiologia , Micro-Ondas/uso terapêutico , Alquilantes/toxicidade , Animais , Catalase/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos da radiação , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Masculino , Fluidez de Membrana/efeitos dos fármacos , Fluidez de Membrana/efeitos da radiação , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo
9.
J Labelled Comp Radiopharm ; 62(7): 310-320, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31033025

RESUMO

The purpose of this study was to develop preclinical evaluation of a novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor targeting peptide for prostate cancer therapy. The new antiproliferative agent of GnRH-I analogue was developed on the basis of the D-Trp6 -GnRH-I scaffold, and in vivo pharmacokinetics and receptor binding affinity were enhanced by the substitution of Gly-NHNH2 for Gly-NH2 at position 10 in D-Trp6 -GnRH-I. To evaluate 177 Lu-DOTA-triptorelin-hydrazide as radionuclide therapy of tumor, the quality control tests and preclinical stage assessment were carried out. Solid-phase method was used to synthesize new peptide. Characterization and purity of peptide were done by mass spectroscopy and high-performance liquid chromatography (HPLC). In order to be utilized in targeted therapy, the new GnRH-I agonist was coupled with pSCN-Bn-DOTA. The precipitate crude of DOTA-triptorelin-hydrazide was then purified via preparative HPLC. At optimal conditions of time, temperature, ligand amount, and lutetium content, DOTA-triptorelin-hydrazide was labeled with 177 Lu (specific activity not less than 925 GBq/mg). Investigation of the in vivo biodistribution and in vitro studies for 177 Lu-DOTA-TRPHYD was performed in three different ways, and the binding of radiopeptide to GnRH receptors was expressed on the human cell lines using 125 I-labeled D-TRP6 GnRH-I as a tracer, respectively. Synthesized novel GnRH-I was obtained with purity greater than 98%. Paper chromatography was found to be the most suitable with Rf of the complex and observed radiochemical purity of RTLC and HPLC greater than 97%. For in vivo studies, 177 Lu-DOTA-triptorelin-hydrazide showed promising results with fast clearance from the blood and resulted in good T/NT ratios at 1, 4, and 24 hours postinjection and satisfactory biodistribution with no significant activity seen in normal tissue. The values of internalization efficiency and receptor affinity of new radiopeptide binding were IC50 = 0.47 ± 0.06 vs 0.13 ± 0.01 nM for triptorelin and cellular uptake: 3.4 ± 0.7% at 1 hour and 6.8 ± 1.17% at 4 hours of the internal reference. The results showed a good stability and radiochemical purity of the obtained radioconjugate. For in vivo and in vitro studies, new radiopeptide showed a high uptake of 177 Lu conjugate in tumor and rapid clearance from the blood stream almost entirely via the renal/urinary pathway and binding to the GnRH receptors with high specificity and affinity, respectively.


Assuntos
Lutécio/uso terapêutico , Oligopeptídeos/química , Oligopeptídeos/uso terapêutico , Radioisótopos/uso terapêutico , Receptores LHRH/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos da radiação , Feminino , Humanos , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Traçadores Radioativos , Ratos , Distribuição Tecidual
10.
Cells ; 8(3)2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875802

RESUMO

The intracellular transport of receptor tyrosine kinases results in the differential activation of various signaling pathways. In this study, optogenetic stimulation of fibroblast growth factor receptor type 1 (FGFR1) was performed to study the effects of subcellular targeting of receptor kinases on signaling and neurite outgrowth. The catalytic domain of FGFR1 fused to the algal light-oxygen-voltage-sensing (LOV) domain was directed to different cellular compartments (plasma membrane, cytoplasm and nucleus) in human embryonic kidney (HEK293) and pheochromocytoma (PC12) cells. Blue light stimulation elevated the pERK and pPLCγ1 levels in membrane-opto-FGFR1-transfected cells similarly to ligand-induced receptor activation; however, no changes in pAKT levels were observed. PC12 cells transfected with membrane-opto-FGFR1 exhibited significantly longer neurites after light stimulation than after growth factor treatment, and significantly more neurites extended from their cell bodies. The activation of cytoplasmic FGFR1 kinase enhanced ERK signaling in HEK293 cells but not in PC12 cells and did not induce neuronal differentiation. The stimulation of FGFR1 kinase in the nucleus also did not result in signaling changes or neurite outgrowth. We conclude that FGFR1 kinase needs to be associated with membranes to induce the differentiation of PC12 cells mainly via ERK activation.


Assuntos
Diferenciação Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Diferenciação Celular/efeitos da radiação , Membrana Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Ligantes , Luz , Neuritos/metabolismo , Neuritos/efeitos da radiação , Neurônios/efeitos da radiação , Optogenética , Células PC12 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais
11.
Lasers Med Sci ; 34(8): 1555-1566, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30887233

RESUMO

In this study, we combine heat diffusion equation and modified Hodgkin-Huxley axonal model to investigate how an action potential is generated during infrared neural stimulation. The effects of temporal and spatial distribution of heat induced by infrared pulsed lasers on variation of electrical membrane capacitance are investigated. These variations can lead to depolarize the membrane and generate an action potential. We estimate the threshold values of laser light parameters such as energy density, pulse duration, and repetition rate are needed to trigger an action potential. In order to do it, we present an analytic solution to heat diffusion equation. Then, the analytic results are verified by experimental results. Furthermore, the modified Hodgkin-Huxley axonal model is applied to simulate the generation of action potential during infrared neural stimulation by taking into account the temperature dependence of electrical membrane capacitance. Results show that the threshold temperature increase induced by a train infrared pulse laser can be smaller if repetition rate is higher. These results also indicate that temperature rise time and axon diameter influence on threshold temperature increase. To verify threshold values estimated by the presented method, we use a train infrared pulsed laser (λ = 1450 nm with repetition rate of 3.8 Hz, pulse duration of 18 ms and energy density of 5 J/cm2) to optically pace an adult rat heart, and we are able to successfully pace the rat heart during an open-heart surgery. The presented method can be used to estimate threshold values of laser parameters required for generating an action potential, and it can provide an insight to how the temperature changes lead to neural stimulation during INS.


Assuntos
Raios Infravermelhos , Lasers , Sistema Nervoso/efeitos da radiação , Potenciais de Ação/efeitos da radiação , Animais , Axônios/efeitos da radiação , Membrana Celular/efeitos da radiação , Masculino , Imagens de Fantasmas , Ratos , Temperatura , Fatores de Tempo
12.
Bioelectromagnetics ; 40(2): 104-117, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30786058

RESUMO

A polysaccharide of Irpex lacteus, a white-rot fungus with lignocellulose-degrading activities, has been used as a commercial medicine for nephritis treatment. Previously, a low-intensity electromagnetic field (LI-EMF) was found to increase the biomass and polysaccharide content of Irpex lacteus and induce twists on the cell surface. In this study, RNA-sequencing (RNA-seq) technology was used to analyze the underlying mechanism of LI-EMF's influence on Irpex lacteus. We identified 3268, 1377, and 941 differentially expressed genes (DEGs) in the LI-EMF-treated samples at recovery times of 0 h, 3 h, and 6 h, respectively, indicating a significant decline in the influence of the LI-EMF treatment on Irpex lacteus with the passage of recovery time. Moreover, 30 upregulated and 14 downregulated DEGs overlapped in the LI-EMF-treated samples at the recovery times of 0 h, 3 h, and 6 h, implying the important lasting effects of LI-EMF. The reliability of the RNA-seq data were validated by quantitative real-time PCR (qRT-PCR). The DEGs related to transcription factors, cell proliferation, cell wall, membrane components, amino acid biosynthesis and metabolism, and polysaccharide biosynthesis and metabolism were significantly enriched in the LI-EMF-treated samples. The experiments confirmed that the LI-EMF treatment significantly increased the content of amino acids with a considerable increase in the content of essential amino acids. Therefore, the global gene expression changes explained the pleiotropic effects of Irpex lacteus induced by the LI-EMF treatment. These findings provide the requisite data for the appropriate design and application of LI-EMF in the fermentation of microorganisms to increase production. Bioelectromagnetics. 40:104-117, 2019. © 2019 Bioelectromagnetics Society.


Assuntos
Basidiomycota/metabolismo , Campos Eletromagnéticos/efeitos adversos , Regulação da Expressão Gênica/efeitos da radiação , Aminoácidos/análise , Aminoácidos/efeitos da radiação , Sequência de Bases , Biomassa , Membrana Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Biblioteca Gênica , Polissacarídeos/efeitos da radiação , Polissacarídeos/toxicidade , Fatores de Tempo
13.
PLoS One ; 14(1): e0206713, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30699112

RESUMO

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations.


Assuntos
Neoplasias da Mama/patologia , Comunicação Celular/efeitos da radiação , Membrana Celular/metabolismo , Produtos do Gene env/metabolismo , Proteínas da Gravidez/metabolismo , Tolerância a Radiação , Apoptose/efeitos da radiação , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Membrana Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Feminino , Produtos do Gene env/genética , Humanos , Proteínas da Gravidez/genética , Radiação Ionizante , Alinhamento de Sequência , Ensaio Tumoral de Célula-Tronco/métodos
14.
Plant Signal Behav ; 14(2): 1561107, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30601076

RESUMO

Stomatal opening is induced by red light as well as blue light. Recently, we established an immunohistochemical technique using whole leaves to study plasma membrane (PM) H+-ATPase in guard cells, which is an important enzyme driving stomatal opening. Our technique revealed that red light illuminated to whole leaves induces photosynthesis-dependent phosphorylation of C-terminal penultimate residue of PM H+-ATPase, threonine, in guard cells, which has been considered to be important for activation of PM H+-ATPase, and we proposed that red light promotes stomatal opening via activation of PM H+-ATPase in guard cells in whole leaves. Here, using our new immunohistochemical technique, we investigated fluence rate dependence of red light-induced phosphorylation of PM H+-ATPase. We found that illumination of red light at 50 µmol m-2 s-1, which was suggested to initiate photosynthesis, saturates phosphorylation of PM H+-ATPase. Furthermore, we immunohistochemically confirmed decrease in the amount of PM H+-ATPase protein in a knock-out mutant of AHA1, an isogene encoding the major isoform of PM H+-ATPase in guard cells, implying the importance of AHA1 as the major PM H+-ATPase protein in guard cells for light-induced stomatal opening.


Assuntos
Membrana Celular/metabolismo , Luz , Estômatos de Plantas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Membrana Celular/efeitos da radiação , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Fosforilação/efeitos da radiação , Estômatos de Plantas/efeitos da radiação , ATPases Translocadoras de Prótons/genética
15.
J Pharm Biomed Anal ; 164: 557-573, 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30466024

RESUMO

Raman micro-spectroscopy was performed in vitro on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). The acquired spectra were analyzed by principal component analysis (PCA) and interval-PCA (i-PCA) methods. Biochemical changes occurring in the different regions of single cells as a consequence of the radiation exposure were observed in cells fixed immediately after the irradiation. The most relevant effects arose from the analysis of the spectra from the cell nucleus region. The observed changes were discussed in terms of the modifications in the cell cycle, resulting in an increase in the DNA-related signal, a protein rearrangement and changes in lipid and carbohydrates profiles within the nucleus. Potential markers of an apoptotic process in cell population irradiated with 6 and 8-Gy X-ray doses could have been singled out. No significant effects were found in spectra from cells fixed 24 h after the irradiation, thus suggesting the occurrence of repairing processes of the X-ray induced damage.


Assuntos
Membrana Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Neuroblastoma/radioterapia , Análise de Célula Única/métodos , Análise Espectral Raman/métodos , Apoptose/efeitos da radiação , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Humanos , Doses de Radiação , Terapia por Raios X
16.
Lasers Med Sci ; 34(1): 15-21, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29980944

RESUMO

This study aimed to analyze the effects of laser irradiation on the membrane integrity and viability of stem cells from human exfoliated deciduous teeth (SHED) that were kept in serum starvation. Nutritional deficit was used to mimic the cellular stress conditions of SHED isolation for regenerative dental approaches, where laser therapy could be beneficial. SHED were cultured under serum starvation (MEMα + 1%FBS) for 1 or 24 h pre-irradiation (protocols A and B, respectively). Then, cells received low-level laser therapy (LLLT; 660 nm) at 2.5 J/cm2 (0.10 W; groups I and V), 5.0 J/cm2 (0.20 W; groups II and VI), 7.5 J/cm2 (0.30 W; groups III and VII), or remained non-irradiated (groups IV and VIII). During irradiation, cells were maintained in 1% FBS (groups I-IV) or 10% FBS (normal culture conditions; groups V-VIII). Membrane integrity was evaluated by quantifying lactate dehydrogenase (LDH) release (immediately after irradiation), and cell viability was assessed by the MTT assay (24, 48, and 72 h post-irradiation). Serum starvation did not alter LDH release by non-irradiated SHED, while LDH release decreased significantly in groups irradiated in 1% FBS (I and III), but not in groups irradiated in 10% FBS (V-VII), regardless the pre-irradiation conditions (protocols A/B). Cell viability was significantly higher 24 h after irradiation, in most protocol A groups. In contrast, cell viability remained mostly unaltered in protocol B groups. LLLT contributed to maintain membrane integrity in SHED subjected to nutritional deficit before and during irradiation with 0.10 or 0.30 W. Short serum starvation before irradiation improved SHED viability at 24 h post-irradiation.


Assuntos
Membrana Celular/metabolismo , Lasers , Fenômenos Fisiológicos da Nutrição , Células-Tronco/patologia , Células-Tronco/efeitos da radiação , Esfoliação de Dente/patologia , Dente Decíduo/efeitos da radiação , Membrana Celular/patologia , Membrana Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Humanos , L-Lactato Desidrogenase/metabolismo , Soro
17.
Plant Physiol Biochem ; 135: 51-60, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30500518

RESUMO

Chloroplast movement mediated by the plant-specific phototropin blue light photoreceptors is crucial for plants to cope with fluctuating light conditions. While chloroplasts accumulate at weak light-illuminated areas, chloroplast avoidance response mediated primarily by the phototropin2 (phot2) receptor is induced by strong light illumination. Although extensive studies have been performed on phot2-mediated chloroplast avoidance in the model plant Arabidopsis, little is known on the role of the corresponding PHOT2 orthologs in chloroplast movement in cotton. In this study, we found that chloroplast avoidance movement also occurs in the tetraploid G. hirsutum and two diploid species, G. arboreum and G. raimondii, albeit with distinct features. Further bioinformatics and genetic analysis identified the cotton PHOT2 ortholog, GhPHOT2-1, which retained a conserved role in plant chloroplast avoidance movement under strong blue light. Ghphot2-1was localized in the plasma membrane and formed aggregates after high blue light irradiation. Constitutive expression of GhPHOT2-1 restored chloroplast avoidance and accumulation response, as well as phototropism, and leaf flattening characteristics of the Arabidopsis phot2 or phot1 phot2 mutants. On the contrary, silencing of GhPHOT2-1 by virus-induced gene silencing (VIGS) disrupted high blue light-induced chloroplast avoidance movement and caused photo damage in cotton leaves. Taken together, these findings demonstrated that GhPHOT2-1 is a conserved PHOT2 ortholog in regulating chloroplast avoidance and the other aforementioned phot2-mediated responses, implicating its potential role for improving high light tolerance in cotton cultivars.


Assuntos
Cloroplastos/efeitos da radiação , Genes de Plantas/fisiologia , Gossypium/efeitos da radiação , Fototropinas/fisiologia , Arabidopsis/genética , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Membrana Celular/fisiologia , Membrana Celular/efeitos da radiação , Cloroplastos/fisiologia , Genes de Plantas/genética , Gossypium/genética , Gossypium/fisiologia , Luz , Fototropinas/genética , Filogenia , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência
18.
J Neurophysiol ; 121(2): 480-489, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30565960

RESUMO

We report a novel phenomenon produced by focused ultrasound (US) that may be important for understanding its effects on cell membranes. When a US burst (2.1 MHz, 1-mm focal diameter, 0.1-1 MPa) was focused on a motor axon of the crayfish neuromuscular junction, it consistently produced a fast hyperpolarization, which was followed or superseded by subthreshold depolarizations or action potentials in a stochastic manner. The depolarization persisted in the presence of voltage-gated channel blockers [1 µM TTX ( INa), 50 µM ZD7288 ( Ih), and 200 µM 4-aminopyridine ( IK)] and typically started shortly after the onset of a 5-ms US burst, with a mean latency of 3.35 ± 0.53 ms (SE). The duration and amplitude of depolarizations averaged 2.13 ± 0.87 s and 10.1 ± 2.09 mV, with a maximum of 200 s and 60 mV, respectively. The US-induced depolarization was always associated with a decrease in membrane resistance. By measuring membrane potential and resistance during the US-induced depolarization, the reversal potential of US-induced conductance ( gus) was estimated to be -8.4 ± 2.3 mV, suggesting a nonselective conductance. The increase in gus was 10-100 times larger than the leak conductance; thus it could significantly influence neuronal activity. This change in conductance may be due to stimulation of mechanoreceptors. Alternatively, US may perturb the lateral motion of phospholipids and produce nanopores, which then increase gus. These results may be important for understanding mechanisms underlying US-mediated modulation of neuronal activity and brain function. NEW & NOTEWORTHY We report a specific increase in membrane conductance produced by ultrasound (US) on neuronal membrane. When a 5-ms US tone burst was focused on a crayfish motor axon, it stochastically triggered either depolarization or a spike train. The depolarization was up to 60 mV in amplitude and 200 s in duration and therefore could significantly influence neuronal activity. Depolarization was still evoked by US burst in the presence of Na+ and Ca2+ channel blockers and had a reversal potential of -8.4 ± 2.3 mV, suggesting a nonselective permeability. US can be applied noninvasively in the form of a focused beam to deep brain areas through the skull and has been shown to modulate brain activity. Understanding the depolarization reported here should be helpful for improving the use of US for noninvasive modulation and stimulation in brain-related disease.


Assuntos
Axônios/efeitos da radiação , Potenciais da Membrana , Ondas Ultrassônicas , Animais , Astacoidea , Axônios/efeitos dos fármacos , Axônios/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/efeitos da radiação , Bloqueadores dos Canais de Potássio/farmacologia , Pirimidinas/farmacologia , Tetrodotoxina/farmacologia
19.
IEEE Trans Biomed Eng ; 66(5): 1353-1360, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30281431

RESUMO

OBJECTIVE: Unipolar pulses have been used in cell electrofusion over the last decades. However, the problem of high mortality with unipolar pulses has not been solved effectively. The cell fusion rate is restricted by cell mortality. By using the advantages of bipolar pulses which cause less cell damage, this paper attempts to use bipolar pulses to increase the cell fusion rate. METHODS: the transmembrane voltage and pore density of cells subjected to unipolar/bipolar pulses were simulated in COMSOL software. In an experiment, two 40 µs unipolar and two 20-20 µs bipolar pulses with electric fields of 2, 2.5, and 3 kV/cm were applied to SP2/0 murine myeloma cells. To determine the cell fusion rate and cell mortality, cells were stained with Hoechst 33342 and propidium iodide. RESULTS: the simulation in this paper showed that a high transmembrane voltage and a high pores density were concentrated only at the contact area of cells when bipolar pulses were used. The results of the cell staining experiment verified the simulation analysis. When bipolar pulses were applied, the cell mortality was significantly reduced. In addition, the cell fusion rate with bipolar pulses was almost two times higher than that with unipolar pulses. CONCLUSION: for cell electrofusion, compared with unipolar pulses, bipolar pulses can not only reduce the cell mortality remarkably but also improve the cell fusion rate obviously. SIGNIFICANCE: this paper introduces a novel way to increase the fusion rate of cells.


Assuntos
Fusão Celular/métodos , Membrana Celular/efeitos da radiação , Eletroporação/métodos , Animais , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Simulação por Computador , Camundongos , Porosidade
20.
Photosynth Res ; 140(1): 39-49, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30315435

RESUMO

The trimeric nature of the Fenna-Matthews-Olson (FMO) protein antenna complex from green sulfur phototrophic bacteria was investigated. Mutations were introduced into the protein at positions 142 and 198, which were chosen to destabilize the intra-trimer salt bridges between adjacent monomers. Strains bearing the mutations R142L, R198L, or their combination, exhibited altered optical absorption spectra of purified membranes and fluoresced more intensely than the wild type. In particular, the introduction of the R142L mutation resulted in slower culture growth rates, as well as an FMO complex that was not able to be isolated in appreciable quantities, while the R198L mutation yielded an FMO complex with increased sensitivity to sodium thiocyanate and Triton X-100 treatments. Native and denaturing PAGE experiments suggest that much of the FMO complexes in the mutant strains pool with the insoluble material upon membrane solubilization with n-dodecyl ß-D-maltoside, a mild nonionic detergent. Taken together, our results suggest that the quaternary structure of the FMO complex, the homotrimer, is an important factor in the maintenance of the complex's tertiary structure.


Assuntos
Proteínas de Bactérias/química , Bacterioclorofilas/química , Chlorobi/química , Complexos de Proteínas Captadores de Luz/química , Estrutura Quaternária de Proteína , Substituição de Aminoácidos , Membrana Celular/efeitos da radiação , Chlorobi/efeitos da radiação , Modelos Moleculares , Complexos Multiproteicos , Mutação , Fotossíntese , Estabilidade Proteica
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