RESUMO
A fundamental variable in culture medium is its pH, which must be controlled by an appropriately formulated buffering regime, since biological processes are exquisitely sensitive to acid-base chemistry. Although awareness of the importance of pH is fostered early in the training of researchers, there are no consensus guidelines for best practice in managing pH in cell cultures, and reporting standards relating to pH are typically inadequate. Furthermore, many laboratories adopt bespoke approaches to controlling pH, some of which inadvertently produce artefacts that increase noise, compromise reproducibility or lead to the misinterpretation of data. Here, we use real-time measurements of medium pH and intracellular pH under live-cell culture conditions to describe the effects of various buffering regimes, including physiological CO2/HCO3- and non-volatile buffers (e.g. HEPES). We highlight those cases that result in poor control, non-intuitive outcomes and erroneous inferences. To improve data reproducibility, we propose guidelines for controlling pH in culture systems.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Animais , Bicarbonatos/química , Soluções Tampão , Células CACO-2 , Proliferação de Células , HEPES/química , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Laboratórios , Mamíferos , Pesquisadores/educação , Cloreto de Sódio/químicaRESUMO
The intracellular pH (pHi) in the cytosol of mammalian central neurons is tightly regulated and small pHi-fluctuations are deemed to modulate inter-/intracellular signaling, excitability, and synaptic plasticity. The resting pHi of young rodent hippocampal pyramidal neurons is known to decrease alongside aging for about 0.1 pH-units. There is no information about the relationship between age and pHi of human central neurons. We addressed this knowledge gap using 26 neocortical slices from 12 patients (1-56-years-old) who had undergone epilepsy surgery. For fluorometric recordings, the slice-neurons were loaded with the pHi-sensitive dye BCECF-AM. We found that the pyramidal cells' resting pHi (n = 26) descended linearly alongside aging (r = - 0.71, p < 0.001). This negative relationship persisted, when the sample was confined to specific brain regions (i.e., middle temporal gyrus, 23 neurons, r = - 0.68, p < 0.001) or pathologies (i.e., hippocampus sclerosis, 8 neurons, r = - 0.78, p = 0.02). Specifically, neurons (n = 9, pHi 7.25 ± 0.12) from young children (1.5 ± 0.46-years-old) were significantly more alkaline than neurons from adults (n = 17, 38.53 ± 12.38 years old, pHi 7.08 ± 0.07, p < 0.001). Although the samples were from patients with different pathologies the results were in line with those from the rodent hippocampal pyramidal neurons. Like a hormetin, the age-related mild pHi-decrease might contribute to neuroprotection, e.g., via limiting excitotoxicity. On the other hand, aging cortical neurons could become more vulnerable to metabolic overstress by a successive pHi-decrease. Certainly, its impact for the dynamics in short and long-term synaptic plasticity and, ultimately, learning and memory provides a challenge for further research.
Assuntos
Envelhecimento/metabolismo , Neocórtex/citologia , Neurônios/metabolismo , Adulto , Células Cultivadas , Pré-Escolar , Epilepsia Resistente a Medicamentos/cirurgia , Feminino , Fluoresceínas/análise , Fluorometria , Humanos , Concentração de Íons de Hidrogênio , Lactente , Líquido Intracelular/química , Masculino , Pessoa de Meia-Idade , Neocórtex/metabolismo , Adulto JovemRESUMO
OBJECTIVE: To compare the genomic profiles of blastocoel fluid (BF), inner cell mass (ICM), and trophectoderm (TE) cells derived from the same blastocyst. DESIGN: Prospective study. SETTING: Academic and in vitro fertilization units. PATIENT(S): Sixteen donated cryopreserved embryos at blastocyst stage. INTERVENTION(S): BF, TE, and ICM cells were retrieved from each blastocyst for chromosome analysis by means of next-generation sequencing (NGS). MAIN OUTCOME MEASURE(S): Aneuploidy screening and assessment of mosaicism in BF, TE and ICM samples with subsequent comparison of genomic profiles between the three blastocyst compartments. RESULT(S): Out of 16 blastocysts, 10 BF samples and 14 TE and ICM samples provided reliable NGS data for comprehensive chromosome analysis. Only 40.0% of BF-DNA karyotypes were fully concordant with TE or ICM, compared with 85.7% concordance between TE and ICM. In addition, BF-DNA was burdened with mosaic aneuploidies and the total number of affected chromosomes in BF was significantly higher compared with the TE and ICM. CONCLUSION(S): BF-DNA can be successfully amplified and subjected to NGS, but owing to increased discordance with ICM and TE, BF does not adequately represent the status of the rest of the embryo. To overcome biologic and technical challenges associated with BF sampling and processing, blastocentesis would require improvement in both laboratory protocols and aneuploidy calling algorithms. Therefore, TE biopsy remains the most effective way to predict embryonic karyotype, and the use of BF as a single source of DNA for preimplantation genetic screening is not yet advised.
Assuntos
Massa Celular Interna do Blastocisto/patologia , Blastocisto/patologia , Ectoderma/patologia , Líquido Intracelular/química , Cariotipagem , Diagnóstico Pré-Implantação , Aneuploidia , Massa Celular Interna do Blastocisto/metabolismo , Células Cultivadas , Ectoderma/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Líquido Intracelular/metabolismo , Cariótipo , Cariotipagem/métodos , Cariotipagem/normas , Mosaicismo , Diagnóstico Pré-Implantação/métodos , Diagnóstico Pré-Implantação/normas , Reprodutibilidade dos TestesRESUMO
AIMS: Our previous studies demonstrated that serum 1,5-anhydroglucitol (1,5-AG) levels increased slightly rather than declined after an acute glucose load. Therefore, the current study aims at exploring the transport and metabolic characteristics of 1,5-AG, as well as the effect of glucose on 1,5-AG transport. METHODS: Km and Vmax were determined to measure the affinity of glucose oxidase (GOD) and hexokinase (HK) for 1,5-AG and glucose. HepG2, C2C12, and primary mouse hepatocytes were incubated for 2 h with 1,5-AG at concentrations of 0, 80, and 160 µg/mL. Then, intracellular and extracellular concentrations of 1,5-AG were measured before and after washing with PBS to evaluate the transport and metabolic rates of 1,5-AG. In addition, the influence of an acute glucose load on the transport of 1,5-AG was studied. RESULTS: The affinity of GOD and HK for 1,5-AG is 5 and 42.5% of that for glucose, respectively. Moreover, there is no de novo synthesis of 1,5-AG, and its metabolic rate is < 3%. After a 2 h incubation with additional 1,5-AG, the intracellular levels of 1,5-AG were 50-80% of extracellular levels. Moreover, intracellular 1,5-AG concentrations decreased rapidly and reached zero following the removal of 1,5-AG from the external medium. In addition, an acute glucose load can affect the dynamic balance of 1,5-AG, causing the intracellular 1,5-AG levels to decline significantly and the extracellular levels to increase slightly in HepG2 cells. CONCLUSIONS: Unlike glucose, 1,5-AG is hard to be metabolized in vivo, and its transport is influenced by an acute glucose load in hepatocytes.
Assuntos
Desoxiglucose/metabolismo , Hepatócitos/metabolismo , Líquido Intracelular/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Desoxiglucose/farmacocinética , Glucose/metabolismo , Glucose Oxidase/metabolismo , Células Hep G2 , Hexoquinase/metabolismo , Humanos , Líquido Intracelular/química , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.
Assuntos
Membrana Celular/química , Plexo Corióideo/metabolismo , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Líquido Intracelular/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/administração & dosagem , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Encéfalo/fisiologia , Membrana Celular/metabolismo , Líquido Cefalorraquidiano/química , Líquido Cefalorraquidiano/enzimologia , Líquido Cefalorraquidiano/fisiologia , Plexo Corióideo/química , Plexo Corióideo/citologia , Plexo Corióideo/ultraestrutura , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/efeitos adversos , Citometria de Fluxo , Líquido Intracelular/enzimologia , Líquido Intracelular/metabolismo , Macrolídeos/administração & dosagem , Macrolídeos/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Sódio/metabolismo , Tionucleotídeos/metabolismoRESUMO
PURPOSE: To develop an assay that can enable the quantification of intra- and extracellular nitric oxide (NO) levels in liver biopsies without application of potentially harmful exogenous NO traps. THEORY: Electron paramagnetic resonance (EPR) spectroscopy is currently the most appropriate method of measuring NO in biological samples due to the outstanding specificity resulting from the interaction of NO with exogenous NO traps. Because such traps are not allowed in clinical settings, we tested the reliability of endogenous NO traps for the determination of NO levels in blood and liver compartments. METHODS: Rats were injected with 0-8 mg/kg lipopolysaccharide (LPS) to gradually induce a systemic inflammatory response. Specific features of NO-hemoglobin and NO-Fe EPR signals were quantified using a specifically developed calibration procedure. RESULTS: Whereas both NO-hemoglobin (NO-HbLIVER BLOOD ) and NO-Fe (NO-FeLIVER ) complexes were detected in nonperfused liver tissue, only NO-Fe complexes were detected in perfused tissue and only NO-Hb complexes were detected in blood (NO-HbBLOOD ). The NO concentrations increased in the sequence NO-HbBLOOD < NO-FeLIVER < NO-HbLIVER BLOOD (9.4, 18.5, 27.9 nmol/cm3 , respectively at 2.5 mg/kg LPS). The detection limit of the method was 0.61 nmol/cm3 for NO-Hb and 0.52 nmol/cm3 for NO-Fe. CONCLUSION: The assay reported here does not influence natural NO pathways and enables the quantification of NO distribution in two liver compartments using a single liver biopsy. Magn Reson Med 77:2372-2380, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Líquido Extracelular/química , Hepatite/metabolismo , Líquido Intracelular/química , Fígado/química , Fígado/patologia , Óxido Nítrico/análise , Animais , Biomarcadores/análise , Biópsia , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Hyper-activation of the Na+/H+ exchanger NHE1 occurs at the onset of oncogenic transformation and plays a critical role in breast cancer carcinogenesis. Dysregulation of NHE1 activity results in intracellular alkalinization and the acidification of the extracellular tumor microenvironment that promotes metastasis. Hence, the use of chemical inhibitors of NHE1 as chemotherapeutic agents is an alluring prospect. We previously demonstrated that two structurally different NHE1 inhibitors, EMD87580 [(2-methyl-4,5-di-(methylsulfonyl)-benzoyl)-guanidine], and HMA [5-(N,N-hexamethylene)-amiloride], were effective as co-adjuvants to potentiate paclitaxel-mediated cytotoxic chemotherapy in triple-negative breast cancer (TNBC) cells. Both these drugs, however, had reduced or minimal anti-cancer effects when used alone. Here, we tested KR-33028 (4-cyano (benzo[b]thiophene-2-carbonyl)guanidine), a potent and selective inhibitor of NHE1, to determine its efficacy in inhibition of metastatic potential of TNBC cells. In highly invasive MDA-MB-231, moderately invasive MDA-MB-468, and lowly invasive Hs578T TNBC cells, KR-33028 considerably reduced rates of cell migration and anchorage-independent colony growth. Invasion of MDA-MB-231 and MDA-MB-468 cells through extracellular matrix was also dramatically decreased in response to KR-33028. We further tested the effect of KR-33028 on MDA-MB-231 cells lacking NHE1 expression (231koNHE1); no differences were observed between untreated control and KR-33028-treated 231koNHE1 cells. Taken together, our results highlight the in vitro efficacy of KR-33028-mediated NHE1 inhibition on limiting cellular functions that are predictive of metastasis in vivo. We suggest that targeting NHE1 in the development of novel chemotherapeutics could be highly effective in combatting triple-negative breast cancer and that KR-33028 is potentially useful in prevention of metastasis.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Guanidinas/farmacologia , Moduladores de Transporte de Membrana/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Tiofenos/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Líquido Intracelular/química , Líquido Intracelular/efeitos dos fármacos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/efeitos dos fármacos , Ensaio Tumoral de Célula-TroncoAssuntos
Cálcio/metabolismo , Eritrócitos/química , Proteínas de Escherichia coli/farmacologia , Proteínas Hemolisinas/farmacologia , Acilação , Trifosfato de Adenosina/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas Hemolisinas/química , Líquido Intracelular/química , Transporte de Íons , Processamento de Proteína Pós-Traducional , Coelhos , Receptores Purinérgicos/fisiologia , Imagem com Lapso de TempoRESUMO
Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an intracellular milieu is discussed.
Assuntos
Argônio/química , Radicais Livres/análise , Líquido Intracelular/química , Gases em Plasma/química , Soluções/química , Linhagem Celular , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Citometria de Fluxo/métodos , Humanos , Líquido Intracelular/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Soluções/efeitos da radiação , Detecção de Spin/métodos , Raios XRESUMO
Triblock copolymers, Monomethoxy (Polyethylene glycol)-b-P(D,L-lactic-co-glycolic acid)-b-P(L-glutamic acid) (mPEG-PLGA-PGlu) with different molecular weights, were synthesized and mPEG(5k)-PLGA(20.5k)-PGlu(7.9k) were self-assembled into negatively charged nanoparticles with a hybrid core of PLGA and PGlu, and a stealth PEG shell. Because of electrostatic interaction with the negative hybrid-core, the model drug, doxorubicin (DOX), could be easily loaded into the hybrid-core nanoparticles with a high drug loading of ca. 25%. The hydrophobic interaction provided by PLGA could increase the stability of drug-loaded nanoparticles with no change in particle size for at least 3 days and only minor drug leakage (< 0.5%) in pH7.4 physiological media. Due to protonation of PGlu block in pH5.0 medium, the hybrid-core of these nanoparticles was destroyed, as shown by transmission electron microscopy, and this resulted in an increase in the pH-triggered release of DOX from 38.9% in pH7.4 release medium to 71% in pH5.0 release medium at 24 h. In vitro cytotoxicity testing involving MCF-7 and NCI-H460 cells showed that DOX-loaded nanoparticles were more cytotoxic to both types of cells than free DOX. Time-dependent cellular uptake of the drug-loaded nanoparticles was observed and at least 4 hours was required for rapid internalization through caveolinmediated endocytosis and macropinocytosis by MCF-7 cells into the endosomes where pH-trigged release of DOX from the nanoparticles occurred. The hybrid-core nanoparticles represent a potentially useful therapeutic delivery system for cationic drugs due to their high drug loading, high stability in physiological media and intracellular pH-triggered release.
Assuntos
Preparações de Ação Retardada/síntese química , Doxorrubicina/administração & dosagem , Nanocápsulas/química , Neoplasias Experimentais/tratamento farmacológico , Poliésteres/química , Polietilenoglicóis/química , Ácido Poliglutâmico/química , Adsorção , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalização/métodos , Preparações de Ação Retardada/administração & dosagem , Difusão , Doxorrubicina/química , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Nanocápsulas/administração & dosagem , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Resultado do TratamentoRESUMO
The alkalization of intracellular pH (pHin) advances together with enhancement of aerobic glycolysis within tumor cells (the Warburg effect), and that is responsible for the progression of tumor malignancy together with hypoxia and angiogenesis. But how they correlate each other during tumor growth is poorly understood, partly due to the lack of suitable imaging methods. In present study, we propose a novel method to visually determine the pHin of tumor xenograft model from fluorescent image ratios. We utilized tandemly-linked two fluorescent proteins as a pH indicator; yellow fluorescent protein (YFP, pH sensitive) as an indicator, and red fluorescent protein (RFP, pH insensitive) as a reference. This method can eliminate the influence of optical factors from tissue as well as of the diverse expression level of pH indicator in the grafted cells. In addition, that can be operated by filter-based fluorescent imagers that are generally used in small animal study. The efficacy of the pH indicator, RFP-YFP, was confirmed by studies using recombinant protein in vitro and HeLa cells expressing RFP-YFP in vivo. Furthermore, we prepared nude mice subcutaneously xenografted HeLa cells expressing RFP-YFP cells as tumor model. The image ratios (YFP/RFP) of the tumor at the day 5 after surgery clearly showed the heterogeneous distribution of diverse pHin cells in the tumor tissue. Concomitantly acquired angiography using near-infrared fluorescence (680 nm for emission) also indicated that the relative alkaline pHin cells located in the region far from tumor vessels in which tumor aerobic glycolysis would be facilitated by progression of hypoxia and nutrient starvation. Applying the present method for a multi-wavelength imaging concerning pO2 and/or nutrient starvation states in addition to pHin and angiogenesis would provide valuable information about complicated alteration of tumoral cell states during tumorigenesis.
Assuntos
Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Proteínas Luminescentes/química , Microscopia de Fluorescência/métodos , Neoplasias Experimentais/química , Neoplasias Experimentais/patologia , Animais , Fluorescência , Corantes Fluorescentes , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos NusAssuntos
Técnicas Biossensoriais , Líquido Intracelular/química , MicroRNAs/genética , Biologia Sintética/métodos , Animais , Compartimento Celular , Linhagem Celular , Desenho de Equipamento , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes , Humanos , Líquido Intracelular/metabolismo , Modelos Genéticos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Repressoras/fisiologia , Biologia Sintética/tendências , Fatores de Transcrição/fisiologia , Transcrição Gênica/genéticaRESUMO
Directional cell migration requires cell polarization with respect to the distribution of the guidance cue. Cell polarization often includes asymmetric distribution of response components as well as elements of the motility machinery. Importantly, the function and regulation of most of these molecules are known to be pH dependent. Intracellular pH gradients were shown to occur in certain cells migrating in vitro, but the functional relevance of such gradients for cell migration and for the response to directional cues, particularly in the intact organism, is currently unknown. In this study, we find that primordial germ cells migrating in the context of the developing embryo respond to the graded distribution of the chemokine Cxcl12 by establishing elevated intracellular pH at the cell front. We provide insight into the mechanisms by which a polar pH distribution contributes to efficient cell migration. Specifically, we show that Carbonic Anhydrase 15b, an enzyme controlling the pH in many cell types, including metastatic cancer cells, is expressed in migrating germ cells and is crucial for establishing and maintaining an asymmetric pH distribution within them. Reducing the level of the protein and thereby erasing the pH elevation at the cell front resulted in abnormal cell migration and impaired arrival at the target. The basis for the disrupted migration is found in the stringent requirement for pH conditions in the cell for regulating contractility, for the polarization of Rac1 activity, and hence for the formation of actin-rich structures at the leading edge of the migrating cells.
Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Quimiocinas/metabolismo , Células Germinativas/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Actinas/metabolismo , Animais , Quimiocina CXCL12/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Células Germinativas/metabolismo , Líquido Intracelular/metabolismo , Peixe-ZebraRESUMO
OBJECTIVE: To evaluate if pRCCs demonstrate intracellular lipid (i-lipid) at chemical-shift (CS) MRI, and assess T2W-MRI and pathologic characteristics. METHODOLOGY: Sixty-two patients with a pRCC diagnosis underwent MRI over 11 years (IRB-approved). Two radiologists independently assessed for presence of i-lipid on CS-MRI and homogeneity on T2W-MRI. Inter-observer agreement was assessed via an intraclass correlation and results were compared using the Chi-square test. Discordant cases were reviewed to establish consensus. T2W SI-ratios (SI.tumor/SI.kidney) and CS-SI index were compared using independent t-tests and Spearman correlation. Two pathologists re-evaluated the histopathology. RESULTS: Nine of the 62 pRCCs (14.5%) demonstrated i-lipid; agreement was moderate (ICC = 0.63). Pathology review depicted clear cells in four tumours and foamy histiocytes in five tumours. 25.8-35.4% (ICC = 0.65) of tumours were homogeneous on T2W-MRI. No pRCC with i-lipid was considered homogeneous (p = 0.01-0.04). Overall, T2W SI-ratio and CS-SI index were 0.89 (±0.29) and -3.63 % (-7.27 to 11.42). pRCC with i-lipid had significantly higher T2W SI-ratio (p = 0.003). There was a correlation between the CS-SI index and T2W SI-ratio, (r = 0.44, p < 0.001). CONCLUSIONS: Intracellular lipid is uncommonly detected in pRCCs due to clear cell changes and foamy histiocytes. These tumours are associated with heterogeneously-increased SI in T2W-MRI. KEY POINTS: ⢠A minority of pRCCs demonstrate intracellular lipid in CS-MRI. ⢠Quantitatively, intracellular lipid in pRCCs is minimal (<25%). ⢠Intracellular lipid in pRCCs are from clear cell heterogeneity or foamy histiocytes. ⢠pRCCs with intracellular lipid are heterogeneously hyperintense at T2W-MRI. ⢠pRCCs that are homogeneously hypointense at T2W-MRI do not contain intracellular lipid.
Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Lipídeos/análise , Carcinoma de Células Renais/metabolismo , Feminino , Humanos , Líquido Intracelular/química , Neoplasias Renais/metabolismo , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Estudos RetrospectivosRESUMO
A current limitation of MR spectroscopic imaging of multiple skeletal muscles is prolonged scan duration. A significant reduction in the total scan duration using the echo-planar correlated spectroscopic imaging (EP-COSI) sequence was accomplished using two bipolar readout trains with different phase-encoded echoes for one of two spatial dimensions within a single repetition time (TR). The second bipolar readout was used for spatially encoding the outer k-space, whereas the first readout was used for the central k-space only. The performance of this novel sequence, called multi-echo based echo-planar correlated spectroscopic imaging (ME-EPCOSI), was demonstrated by localizing specific key features in calf muscles and bone marrow of 11 healthy volunteers and five subjects with type 2 diabetes (T2D). A 3 T MRI-MRS scanner equipped with a transmit-receive extremity coil was used. Localization of the ME-EPCOSI sequence was in good agreement with the earlier single-readout based EP-COSI sequence and the required scan time was reduced by a factor of two. In agreement with an earlier report using single-voxel based 2D MRS, significantly increased unsaturated pools of intramyocellular lipid (IMCL) and extramyocellular lipid (EMCL) and decreased IMCL and EMCL unsaturation indices (UIs) were observed in the soleus and tibialis anterior muscle regions of subjects with T2D compared with healthy controls. In addition, significantly decreased choline content was observed in the soleus of T2D subjects compared with healthy controls. Multi-voxel characterization of IMCL and EMCL ratios and UI in the calf muscle may be useful for the non-invasive assessment of altered lipid metabolism in the pathophysiology of T2D.
Assuntos
Imagem Ecoplanar/métodos , Músculo Esquelético/química , Adulto , Medula Óssea/química , Colina/análise , Creatina/análise , Diabetes Mellitus Tipo 2/metabolismo , Líquido Extracelular/química , Humanos , Líquido Intracelular/química , Lipídeos/análise , Masculino , Pessoa de Meia-Idade , Células Musculares/química , Projetos PilotoRESUMO
Localized ion fluxes at the plasma membrane provide electrochemical gradients at the cell surface that contribute to cell polarization, migration, and division. Ion transporters, local pH gradients, membrane potential, and organization are emerging as important factors in cell polarization mechanisms. The power of electrochemical effects is illustrated by the ability of exogenous electric fields to redirect polarization in cells ranging from bacteria, fungi, and amoebas to keratocytes and neurons. Electric fields normally surround cells and tissues and thus have been proposed to guide cell polarity in development, cancer, and wound healing. Recent studies on electric field responses in model systems and development of new biosensors provide new avenues to dissect molecular mechanisms. Here, we review recent advances that bring molecular understanding of how electrochemistry contributes to cell polarity in various contexts.
Assuntos
Polaridade Celular/fisiologia , Animais , Ânions/metabolismo , Cátions/metabolismo , Divisão Celular , Movimento Celular , Forma Celular , Dictyostelium/citologia , Eletroquímica , Campos Eletromagnéticos , Peixes , Fungos/citologia , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Transporte de Íons/fisiologia , Potenciais da Membrana/fisiologia , Regeneração , Eletricidade Estática , CicatrizaçãoRESUMO
PURPOSE: We investigated succinate metabolism in cells undergoing clinically relevant cyclic stretch and in spontaneously hypertensive rat (SHR) retina. METHODS: We seeded ARPE-19 cells on 6-well BioFlex collagen I-coated, silicone elastomer-bottomed culture plates. Cells then were subjected to pulsatile stretch using a computer-controlled vacuum stretch apparatus. A physiologic stretch frequency of 60 cycles per minute and 5% to 15% prolongation of the elastomer-bottomed plates were used. Succinate concentration was assessed by enzymatic analysis and high-performance liquid chromatography-mass spectrometry. The VEGF was measured using enzyme-linked immunosorbent assays. The 12-week-old male SHRs and weight-matched Wistar-Kyoto (WKY) control rats were treated with or without 100 mg·kg(-1)·day(-1) captopril for 1 week. The vitreous body and retina of each rat were extracted after 1 week of therapy, and the vitreoretinal succinate concentration was measured. RESULTS: Cells exposed to cyclic stretch accumulated intracellular succinate in a time- and magnitude-dependent manner, and also accumulated VEGF protein levels. Moreover, BAPTA/AM, an intracellular calcium chelate reagent, significantly inhibited the stretch-induced succinate increase. After cyclic stretch, levels of intracellular fumarate, a citric acid cycle intermediate, also were significantly increased compared to controls. The BAPTA/AM inhibited this increase. For the in vivo experiments, hypertension increased vitreoretinal succinate and fumarate in SHRs compared to the normotensive WKY controls. When hypertension was reduced using captopril, vitreoretinal succinate returned to baseline levels. CONCLUSIONS: These findings suggest that cyclic stretch and hypertension increased intracellular succinate in cultured retinal pigment epithelial cells and the vitreoretinal succinate of SHRs through a calcium-dependent pathway.
Assuntos
Hipertensão/complicações , Retina/química , Neovascularização Retiniana/etiologia , Estresse Mecânico , Ácido Succínico/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Hipertensão/metabolismo , Líquido Intracelular/química , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Acidic pH inside cells indicates cellular dysfunctions such as cancer. Therefore, the development of optical pH sensors for measuring and imaging intracellular pH is a demanding challenge. The available pH-sensitive probes are vulnerable to e.g. photobleaching or autofluorescence background in biological materials. Our approach circumvents these problems due to near infrared excitation and upconversion photoluminescence. We introduce a nanosensor based on upconversion resonance energy transfer (UC-RET) between an upconverting nanoparticle (UCNP) and a fluorogenic pH-dependent dye pHrodo™ Red that was covalently bound to the aminosilane surface of the nanoparticles. The sensitized fluorescence of the pHrodo™ Red dye increases strongly with decreasing pH. By referencing the pH-dependent emission of pHrodo™ Red with the pH-insensitive upconversion photoluminescence of the UCNP, we developed a pH-sensor which exhibits a dynamic range from pH 7.2 to 2.5. The applicability of the introduced pH nanosensor for pH imaging was demonstrated by imaging the two emission wavelengths of the nanoprobe in living HeLa cells with a confocal fluorescence microscope upon 980 nm excitation. This demonstrates that the presented pH-nanoprobe can be used as an intracellular pH-sensor due to the unique features of UCNPs: excitation with deeply penetrating near-infrared light, high photostability, lack of autofluorescence and biocompatibility due to an aminosilane coating.
Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Medições Luminescentes/métodos , Nanopartículas/química , Células HeLa , Humanos , Nanopartículas/análise , Fotometria/métodosRESUMO
Amyloid-ß protein precursor intracellular domain (AICD), which exerts intracellular effects by interacting with proteins involved in a plethora of biological processes, is a key player behind the pathophysiology of Alzheimer's disease (AD). Keeping in mind that overwhelming presence of AICD would mimic AD-like conditions in neuroblastoma cell lines, we hypothesized alteration in the proteomic expression pattern in these cells in the presence of AICD compared to their normal proteome. The rationale behind the study was to distinguish between symptomatic pathophysiological effects as opposed to any artifactual consequence due to protein overload in the cell lines. Using 2D-DIGE analysis and MALDI-MS identifications in neuro2A (mouse) and SHSY5Y (human) cell lines, we have identified several proteins belonging to different functional classes and involved in several biological pathways including protein folding, cytoskeletal dynamics, metabolism, and stress. Many of these were being upregulated or downregulated due to AICD effects and could be correlated directly with AD phenotypes.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Líquido Intracelular/fisiologia , Neuroblastoma/genética , Proteoma/genética , Precursor de Proteína beta-Amiloide/biossíntese , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Humanos , Líquido Intracelular/química , Líquido Intracelular/enzimologia , Camundongos , Neuroblastoma/química , Neuroblastoma/enzimologia , Estrutura Terciária de Proteína/genética , Proteoma/biossíntese , Regulação para Cima/genéticaRESUMO
A lanthanide complex-based ratiometric luminescence probe, [4'-(2,4-dinitrobenzenesulfonyloxy)-2,2':6',2''-terpyridine-6,6''-diyl] bis(methylenenitrilo) tetrakis(acetate)-Eu(3+)/Tb(3+) (NSTTA-Eu(3+)/Tb(3+)), has been designed and synthesized for the specific recognition and time-gated luminescence detection of biothiols in physiological pH aqueous media. The probe itself is almost nonluminescent due to the presence of photoinduced electron transfer (PET) from the terpyridine-Ln(3+) moiety to the 2,4-dinitrobenzenesulfonyl (DNBS) moiety. In the presence of biothiols, the reaction of NSTTA-Eu(3+)/Tb(3+) with biothiols results in the cleavage of DNBS to afford the deprotonated (4'-hydroxy-2,2':6',2''-terpyridine-6,6''-diyl) bis(methylenenitrilo) tetrakis(acetate)-Eu(3+)/Tb(3+) (HTTA-Eu(3+)/Tb(3+)), which terminates the PET process. After the reaction, the intensity of Eu(3+) emission at 610 nm is unchanged, while that of Tb(3+) emission at 540 nm is remarkably increased, which provides a ~36-fold enhanced intensity ratio of Tb(3+) emission to Eu(3+) emission (I540/I610). This unique luminescence response allows NSTTA-Eu(3+)/Tb(3+) to be used as a ratiometric probe for the time-gated luminescence detection of biothiols, using the intensity ratio of I540/I610 as a signal. Thus, based on the probe NSTTA-Eu(3+)/Tb(3+), a ratiometric time-gated luminescence detection method for biothiols was established and successfully used for the quantitative detection of the total biothiols in several living cell samples.