RESUMO
Sheep are important domestic animals for the production of wool and meat. Although numerous cultured cell lines from humans and mice have been established, the number of cell lines derived from sheep is limited. To overcome this issue, the efficient establishment of a sheep-derived cell line and its biological characterization is reported. Mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase were introduced into sheep muscle-derived cells in an attempt to immortalize primary cells using the K4DT method. Furthermore, the SV40 large T oncogene was introduced into the cells. The successful immortalization of sheep muscle-derived fibroblasts was shown using the K4DT method or SV40 large T antigen. Furthermore, the expression profile of established cells showed close biological characteristics of ear-derived fibroblasts. This study provides a useful cellular resource for veterinary medicine and cell biology.
Assuntos
Telomerase , Transcriptoma , Humanos , Animais , Camundongos , Ovinos , Linhagem Celular , Ciclo Celular , Telomerase/genética , Telomerase/metabolismo , Fibroblastos/metabolismoRESUMO
Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. GCN2-mediated phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) restores homeostasis in response to amino acid starvation (AAS) through the inhibition of the general translation and upregulation of amino acid biosynthetic enzymes and transporters by activating the translation of Gcn4 and ATF4 in yeast and mammals, respectively. GCN1 is a GCN2-binding protein that possesses an RWD binding domain (RWDBD) in its C-terminus. In yeast, Gcn1 is essential for Gcn2 activation by AAS; however, the roles of GCN1 in mammals need to be established. Here, we revealed a novel role of GCN1 that does not depend on AAS by generating two Gcn1 mutant mouse lines: Gcn1-knockout mice (Gcn1 KO mice (Gcn1-/-)) and RWDBD-deleted mutant mice (Gcn1ΔRWDBD mice). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice, such that the Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation, while the Gcn1ΔRWDBD embryos showed mild growth retardation and died soon after birth, most likely due to respiratory failure. Extension of pregnancy by 24 h through the administration of progesterone to the pregnant mothers rescued the expression of differentiation markers in the lungs and prevented lethality of the Gcn1ΔRWDBD pups, indicating that perinatal lethality of the Gcn1ΔRWDBD embryos was due to simple growth retardation. Similar to the yeast Gcn2/Gcn1 system, AAS- or UV irradiation-induced elF2α phosphorylation was diminished in the Gcn1ΔRWDBD mouse embryonic fibroblasts (MEFs), suggesting that GCN1 RWDBD is responsible for GCN2 activity. In addition, we found reduced cell proliferation and G2/M arrest accompanying a decrease in Cdk1 and Cyclin B1 in the Gcn1ΔRWDBD MEFs. Our results demonstrated, for the first time, that GCN1 is essential for both GCN2-dependent stress response and GCN2-independent cell cycle regulation.
Assuntos
Ciclo Celular , Proliferação de Células , Desenvolvimento Fetal , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico , Transativadores/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Ciclina B1/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/genética , Transativadores/genéticaRESUMO
Channelrhodopsins have been utilized in gene therapy to restore vision in patients with retinitis pigmentosa and their channel kinetics are an important factor to consider in such applications. We investigated the channel kinetics of ComV1 variants with different amino acid residues at the 172nd position. Patch clamp methods were used to record the photocurrents induced by stimuli from diodes in HEK293 cells transfected with plasmid vectors. The channel kinetics (τon and τoff) were considerably altered by the replacement of the 172nd amino acid and was dependent on the amino acid characteristics. The size of amino acids at this position correlated with τon and decay, whereas the solubility correlated with τon and τoff. Molecular dynamic simulation indicated that the ion tunnel constructed by H172, E121, and R306 widened due to H172A variant, whereas the interaction between A172 and the surrounding amino acids weakened compared with H172. The bottleneck radius of the ion gate constructed with the 172nd amino acid affected the photocurrent and channel kinetics. The 172nd amino acid in ComV1 is a key residue for determining channel kinetics as its properties alter the radius of the ion gate. Our findings can be used to improve the channel kinetics of channelrhodopsins.
Assuntos
Aminoácidos , Luz , Humanos , Channelrhodopsins/genética , Células HEK293 , CinéticaRESUMO
Electroretinograms (ERGs) are often used to evaluate retinal function. However, assessing local retinal function can be challenging; therefore, photopic and scotopic ERGs are used to record whole-retinal function. This study evaluated focal retinal function in rats exposed to continuous light using a multifocal ERG (mfERG) system. The rats were exposed to 1000 lux of fluorescent light for 24 h to induce photoreceptor degeneration. After light exposure, the rats were reared under cyclic light conditions (12 h: 5 lux, 12 h: dark). Photopic and multifocal ERGs and single-flash and multifocal visual evoked potentials (mfVEPs) were recorded 7 days after light exposure. Fourteen days following light exposure, paraffin-embedded sections were prepared from the eyes for histological evaluation. The ERG and VEP responses dramatically decreased after 24 h of light exposure, and retinal area-dependent decreases were observed in mfERGs and mfVEPs. Histological assessment revealed severe damage to the superior retina and less damage to the inferior retina. Considering the recorded visual angles of mfERGs and mfVEPs, the degenerated area shown on the histological examinations correlates well with the responses from multifocal recordings.
Assuntos
Potenciais Evocados Visuais , Degeneração Retiniana , Ratos , Animais , Retina/fisiologia , Eletrorretinografia , Degeneração Retiniana/etiologiaRESUMO
Calpains are Ca2+-dependent cysteine proteases involved in various intercellular physiological functions. Although most calpains exist in the cytosol, four isoforms of calpain (calpains-1, -2, -5, -10) are also localized in the mitochondria. In the present study, we examined the mitochondrial localization of calpain-13, as a novel mitochondrial calpain, in C57BL/6J mice. The tissue distribution and mitochondrial subfractionation of calpain-13 were investigated using western blotting. Calpain-13 was present in both the mitochondrial membrane (outer membrane and inner membrane) and soluble (intermembrane space and matrix) fractions. Through immunohistochemistry, calpain-13 was found to be expressed in the cerebral cortex and hippocampus of the mouse brain. We further confirmed the localization of calpain-13 in the mitochondria of the mouse brain using immunoelectron microscopy. Our present study thus revealed that calpain-13 is localized in the mitochondria, in addition to the cytosol, in the mouse brain. Future studies investigating the enzymatic properties and physiological functions of both cytosolic and mitochondrial calpain-13 will shed light on the potential involvement of calpain-13 in neurodegenerative diseases including Parkinson's disease and Alzheimer's disease.
Assuntos
Calpaína , Mitocôndrias , Animais , Calpaína/metabolismo , Córtex Cerebral/metabolismo , Citosol/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismoRESUMO
Calpains are cysteine proteases activated in response to intracellular calcium signaling. Activated calpains regulate various cellular functions by degrading substrate molecules in a site-specific manner. Although most calpains are localized in the cytosol, we previously reported that calpain-5 exists in the mitochondria. The mitochondrial calpain-5 is activated during endoplasmic reticulum (ER) stress. However, the substrate of calpain-5, as well as the physiological significance of calpain-5 activation, has not yet been elucidated. In the present study, we treated HeLa cells with A23187, tunicamycin, or hydrogen peroxide to induce intracellular calcium increase, resulting in cell death. The cells treated with A23187 or tunicamycin exhibited the activation of calpain-5 and truncation of caspase-4. The truncation of caspase-4 was inhibited by the repression of calpain-5 expression with the appropriate siRNA. Additionally, both calpain-5 and caspase-4 were observed in the mitochondria. Our study is the first to demonstrate that the activation of mitochondrial calpain-5 triggers the truncation of caspase-4, suggesting that mitochondrial calpain-5 regulates the downstream pathway of caspase-4, including cell death and the inflammatory cascade. The results of the present study provide new insights into ER-stress-related diseases such as Alzheimer's disease and cancer. These perspectives allow us to propose new therapeutic strategies such as the development of inhibitors or activators of calpain-5, which may be useful in the development of treatment for ER-stress-related diseases.
Assuntos
Calpaína , Caspases Iniciadoras , Estresse do Retículo Endoplasmático , Mitocôndrias , Apoptose , Calcimicina , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Calpaína/metabolismo , Caspases Iniciadoras/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Tunicamicina/farmacologiaRESUMO
Dysregulation of autophagy, one of the major processes through which abnormal proteins are degraded, is a cardinal feature of synucleinopathies, including Lewy body diseases [Parkinson's disease (PD) and dementia with Lewy bodies (DLB)] and multiple system atrophy (MSA), which are characterized by the presence of abnormal α-synuclein in neurons and glial cells. Although several research groups have reported that Rubicon family proteins can regulate autophagosome-lysosome fusion or positioning, little is known about their involvement in synucleinopathies. In the present study, by studying patients with PD (N = 8), DLB (N = 13), and MSA (N = 5) and controls (N = 16), we explored the involvement of Rubicon family proteins [Rubicon, Pacer and differentially expressed in FDCP8 (DEF8)] in synucleinopathies. Immunohistochemical analysis showed that not only brainstem-type Lewy bodies but also cortical Lewy bodies were immunoreactive for DEF8 in Lewy body diseases, whereas Rubicon and Pacer were detectable in only a few brainstem-type Lewy bodies in PD. Glial cytoplasmic inclusions in patients with MSA were not immunoreactive for Rubicon, Pacer or DEF8. Immunoblotting showed significantly increased protein levels of DEF8 in the substantia nigra and putamen of patients with PD and the temporal cortex of patients with DLB. In addition, the smear band of DEF8 appeared in the insoluble fraction where that of phosphorylated α-synuclein was detected. These findings indicate the involvement of DEF8 in the formation of Lewy bodies. Quantitative and qualitative alterations in DEF8 may reflect the dysregulation of autophagy in Lewy body diseases.
Assuntos
Doença por Corpos de Lewy , Atrofia de Múltiplos Sistemas , Doença de Parkinson , Sinucleinopatias , Autofagia , Encéfalo/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Age-related macular degeneration is a progressive retinal disease that is associated with factors such as oxidative stress and inflammation. In this study, we evaluated the protective effects of SIG-1451, a non-steroidal anti-inflammatory compound developed for treating atopic dermatitis and known to inhibit Toll-like receptor 4, in light-induced photoreceptor degeneration. SIG-1451 was intraperitoneally injected into rats once per day before exposure to 1000 lx light for 24 h; one day later, optical coherence tomography showed a decrease in retinal thickness, and electroretinogram (ERG) amplitude was also found to have decreased 3 d after light exposure. Moreover, SIG-1451 partially protected against this decrease in retinal thickness and increase in ERG amplitude. One day after light exposure, upregulation of inflammatory response-related genes was observed, and SIG-1451 was found to inhibit this upregulation. Iba-1, a microglial marker, was suppressed in SIG-1451-injected rats. To investigate the molecular mechanism underlying these effects, we used lipopolysaccharide (LPS)-stimulated rat immortalised Müller cells. The upregulation of C-C motif chemokine 2 by LPS stimulation was significantly inhibited by SIG-1451 treatment, and Western blot analysis revealed a decrease in phosphorylated I-κB levels. These results indicate that SIG-1451 indirectly protects photoreceptor cells by attenuating light damage progression, by affecting the inflammatory responses.
Assuntos
Lipopolissacarídeos , Degeneração Retiniana , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Eletrorretinografia , Luz , Lipopolissacarídeos/farmacologia , Células Fotorreceptoras de Vertebrados , Ratos , Retina , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/etiologiaRESUMO
GCN1 is an evolutionarily-conserved ribosome-binding protein that mediates the amino acid starvation response as well as the ribotoxic stress response. We previously demonstrated that Gcn1 mutant mice lacking the GCN2-binding domain suffer from growth retardation and postnatal lethality via GCN2-independent mechanisms, while Gcn1-null mice die early in embryonic development. In this study, we explored the role of GCN1 in adult mice by generating tamoxifen-inducible conditional knockout (CKO) mice. Unexpectedly, the Gcn1 CKO mice showed body weight loss during tamoxifen treatment, which gradually recovered following its cessation. They also showed decreases in liver weight, hepatic glycogen and lipid contents, blood glucose and non-esterified fatty acids, and visceral white adipose tissue weight with no changes in food intake and viability. A decrease of serum VLDL suggested that hepatic lipid supply to the peripheral tissues was primarily impaired. Liver proteomic analysis revealed the downregulation of mitochondrial ß-oxidation that accompanied increases of peroxisomal ß-oxidation and aerobic glucose catabolism that maintain ATP levels. These findings show the involvement of GCN1 in hepatic lipid metabolism during tamoxifen treatment in adult mice.
Assuntos
Proteínas de Saccharomyces cerevisiae , Animais , Lipídeos , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Camundongos , Camundongos Knockout , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases , Proteômica , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tamoxifeno/efeitos adversos , Tamoxifeno/metabolismo , Transativadores/metabolismo , Redução de PesoRESUMO
The death of photoreceptor cells is induced by continuous light exposure. However, it is unclear whether light damage was induced in retinal ganglion cells with photosensitivity by transduction of optogenetic genes. In this study, we evaluated the phototoxicities of continuous light exposure on retinal ganglion cells after transduction of the optogenetic gene mVChR1 using an adeno-associated virus vector. Rats were exposed to continuous light for a week, and visually evoked potentials (VEPs) were recorded. The intensities of continuous light (500, 1000, 3000, and 5000 lx) increased substantially after VEP recordings. After the final recording of VEPs, retinal ganglion cells (RGCs) were retrogradely labeled with a fluorescein tracer, FluoroGold, and the number of retinal ganglion cells was counted under a fluorescent microscope. There was no significant reduction in the amplitudes of VEPs and the number of RGCs after exposure to any light intensity. These results indicated that RGCs were photosensitive after the transduction of optogenetic genes and did not induce any phototoxicity by continuous light exposure.
Assuntos
Optogenética/métodos , Células Ganglionares da Retina/fisiologia , Rodopsina/genética , Animais , Dependovirus/genética , Potenciais Evocados Visuais , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Luz/efeitos adversos , Técnicas de Patch-Clamp , Estimulação Luminosa , Ratos , Células Ganglionares da Retina/patologia , Rodopsina/metabolismo , Estilbamidinas/química , Estilbamidinas/metabolismo , Transdução Genética , Volvox/genéticaRESUMO
ES1 homologs are conserved among prokaryotes and eukaryotes, and the gene expression of ES1 homologs has been confirmed in diverse mammalian tissues. However, the localization and function of mammalian ES1 proteins remain poorly understood. ES1 protein was found specifically expressed in the cone cells of zebrafish and was proposed to contribute to the formation of mega mitochondria. We also observed mega mitochondria in the cone cells of porcine retinas, which raised the question regarding the localization of the porcine ES1. Therefore, in the present study, we aimed to determine the localization of ES1 in porcine retinas. We prepared a rabbit polyclonal antibody against the ES1 C-terminal and performed western blotting analysis and immunoelectron microscopy. The ES1 was found to be localized mainly in the mitochondrial intermembrane space of the porcine retinal cells. Immunopositive signals for ES1 were observed in the mitochondria of almost all retinal cells, and not specifically in cone cells. Our results and the ES1 sequences indicated that the glyoxalase III activity of ES1 might contribute to the stable functionality of the active mitochondria in a protective manner.
Assuntos
Proteínas do Olho/metabolismo , Membranas Mitocondriais/metabolismo , Retina/citologia , Homologia de Sequência de Aminoácidos , Suínos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Olho/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestrutura , Retina/ultraestrutura , SolubilidadeRESUMO
Calpains are Ca2+-dependent cysteine proteases that are widely distributed in animal tissues and modulate a variety of cellular processes. There are 15 members of the calpain family in mammals. In animal cells, there are three types of calpains, viz., calpain-1, calpain-2, and calpain-10 in the mitochondria. The three types of calpains have been shown to play significant roles in pathophysiological conditions, including in apoptosis- and necrosis-like cell death. One of the severe retinal diseases, autosomal dominant neovascular inflammatory vitreoretinopathy, is known to be induced by mutations of the calpain-5 gene. However, the distribution of calpain-5 in the retina has not been elucidated. Therefore, in the present study, we determined the localization of calpain-5 in the porcine retina. We detected calpain-5 in the inner segment of photoreceptor cells using immunohistochemistry. With immunoelectron microscopy, calpain-5 was localized in the mitochondria of photoreceptor cells. Western blot analyses showed that calpain-5 was present in each mitochondrial subfraction. Furthermore, we showed that the molecular weight of mitochondrial calpain-5 was slightly smaller than cytosolic one. Our results demonstrated that a novel mitochondrial calpian, calpain-5, was localized in the mitochondria of retinal photoreceptor cells.
Assuntos
Calpaína/metabolismo , Mitocôndrias/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Animais , Citosol/metabolismo , Elétrons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Inflamação , Microscopia Imunoeletrônica , Células Fotorreceptoras de Vertebrados/metabolismo , Frações Subcelulares , SuínosRESUMO
Channelrhodopsin-2 (ChR2), a light-activated cation-selective ion channel, has been widely used as a tool in optogenetic research. ChR2 is specifically sensitive to wavelengths less than 550â¯nm. One of the methods to expand the sensitivity of a channelrhodopsin to a wider range of wavelengths is to express another channelrhodopsin in the cells by the transduction of an additional gene. Here, we report the characteristic features of cells expressing two types of channelrhodopsins, each having different wavelength sensitivities. In HEK293â¯cells stably expressing ChR2, photocurrents were elicited at stimuli of 400-550â¯nm, and the wavelength sensitivity range was expanded by the additional transduction of the modified Volvox channelrhodopsin-1 (mVChR1) gene, which has broad wavelength sensitivities, ranging from 400 to 600â¯nm. However, the photocurrent at 550â¯nm was lower than that of the mVChR1-expressing cell; moreover, the turning-on and turning-off constants were delayed, and the deactivation rates were decreased. Meanwhile, the response to lower light intensity was improved by the additional gene. Thus, the transduction of an additional gene is a useful method to improve the light and wavelength sensitivities, as well as photocurrent kinetic profiles, of channelrhodopsins.
Assuntos
Channelrhodopsins/fisiologia , Channelrhodopsins/efeitos da radiação , Ativação do Canal Iônico/fisiologia , Ativação do Canal Iônico/efeitos da radiação , Transdução de Sinal Luminoso/fisiologia , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Relação Dose-Resposta à Radiação , Células HEK293 , Humanos , Cinética , Luz , Doses de RadiaçãoRESUMO
P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-d-glucamine(+) (NMDG(+)). The effects of genetic modification of the C-terminus on NMDG(+) permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATP-gated current measured under bi-ionic NMDG(+)/Na(+) condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus.
Assuntos
Citoplasma/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Benzoquinonas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Meglumina/metabolismo , Proteínas Mutantes/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Ratos , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Various serotypes of adeno-associated virus (AAV) vectors have been used for gene therapy and as research tools. Among these serotypes, the AAV type 2 vector has been used successfully in human gene therapies. However, the transduction efficiency of AAV2 depends on the cell type, and this poses a problem in the efficacy of gene therapy. To improve the transduction efficiency of AAV2, we designed a small peptide consisting of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor peptide and the HIV-Tat sequence Tat-Y1068. Pre- or co-treatment of CYNOM-K1 cells from cynomolgus monkey embryo skin with Tat-Y1068 increased the transduction efficiencies in a dose-dependent manner and caused p38 phosphorylation. The transduction efficiency of AAV2 into the rat fibroblast cell line RAT-1 highly expressing EGFR was less than the transduction efficiency of AAV2 into CYNOM-K1 cells. Tat-Y1068 increased the transduction efficiency in RAT-1 cells in the same manner as in CYNOM-K1 cells. In conclusion, cell-permeable peptides possessing the EGFR tyrosine kinase inhibitor function might serve as a useful ingredient of AAV2 vector solution for increasing the transduction efficiency of gene therapies.
Assuntos
Peptídeos Penetradores de Células/farmacologia , Dependovirus/genética , Fibroblastos/efeitos dos fármacos , Pele/efeitos dos fármacos , Transdução Genética/métodos , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Peptídeos Penetradores de Células/síntese química , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/genética , Macaca fascicularis , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Ratos , Reprodutibilidade dos Testes , Pele/citologia , Pele/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Intracellular Ca(2+)-dependent cysteine proteases such as calpains have been suggested as critical factors in retinal ganglion cell (RGC) death. However, it is unknown whether mitochondrial calpains are involved in RGC death. The purpose of the present study was to determine whether the inhibition of mitochondrial µ-calpain activity protects against RGC death during ischemia/reperfusion (I/R) injury. This study used a well-established rat model of experimental acute glaucoma involving I/R injury. A specific peptide inhibitor of mitochondrial µ-calpain, Tat-µCL, was topically applied to rats via eye drops three times a day for 5 days after I/R. RGC death was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The truncation of apoptosis-inducing factor (AIF) was determined by western blot analyses. Retinal morphology was determined after staining with hematoxyline and eosin. In addition, the number of Fluoro Gold-labeled RGCs in flat-mounted retinas was used to determine the percentage of surviving RGCs after I/R injury. After 1 day of I/R, RGC death was observed in the ganglion cell layer. Treatment with Tat-µCL eye drops significantly prevented the death of RGCs and the truncation of AIF. After 5 days of I/R, RGC death decreased by approximately 40%. However, Tat-µCL significantly inhibited the decrease in the retinal sections and flat-mounted retinas. The results suggested that mitochondrial µ-calpain is associated with RGC death during I/R injury via truncation of AIF. In addition, the inhibition of mitochondrial µ-calpain activity by Tat-µCL had a neuroprotective effect against I/R-induced RGC death.
Assuntos
Calpaína/antagonistas & inibidores , Proteínas Mitocondriais/antagonistas & inibidores , Peptídeos/farmacologia , Traumatismo por Reperfusão/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/antagonistas & inibidores , Fator de Indução de Apoptose/metabolismo , Western Blotting , Calpaína/metabolismo , Glaucoma/metabolismo , Glaucoma/fisiopatologia , Microscopia Confocal , Proteínas Mitocondriais/metabolismo , Soluções Oftálmicas , Peptídeos/administração & dosagem , Peptídeos/síntese química , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/síntese química , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Traumatismo por Reperfusão/fisiopatologia , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/fisiopatologia , Células Ganglionares da Retina/metabolismoRESUMO
We previously showed that blind rats whose vision was restored by gene transfer of Chlamydomonas channelrhodopsin-2 (ChR2) could only detect wavelengths less than 540 nm because of the action spectrum of the transgene product. Volvox-derived channelrhodopsin-1, VChR1, has a broader spectrum than ChR2. However, the VChR1 protein was mainly localized in the cytoplasm and showed weak ion channel properties when the VChR1 gene was transfected into HEK293 cells. We generated modified Volvox channelrhodopsin-1 (mVChR1), which is a chimera of Volvox channelrhodopsin-1 and Chlamydomonas channelrhodopsin-1 and demonstrated increased plasma membrane integration and dramatic improvement in its channel properties. Under whole-cell patch clamp, mVChR1-expressing cells showed a photo-induced current upon stimulation at 468-640 nm. The evoked currents in mVChR1-expressing cells were ~30 times larger than those in VChR1-expressing cells. Genetically, blind rats expressing mVChR1 via an adeno-associated virus vector regained their visual responses to light with wavelengths between 468 and 640 nm and their recovered visual responses were maintained for a year. Thus, mVChR1 is a candidate gene for gene therapy for restoring vision, and gene delivery of mVChR1 may provide blind patients access to the majority of the visible light spectrum.
Assuntos
Cegueira/terapia , Terapia Genética/métodos , Retina/fisiopatologia , Rodopsina/metabolismo , Volvox/genética , Animais , Cegueira/metabolismo , Chlamydomonas/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/uso terapêutico , Células HEK293 , Humanos , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/genéticaRESUMO
Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) was originally identified as a positive regulator of drug detoxifying enzyme gene expression during exposure to environmental electrophiles. Currently, Nrf2 is known to regulate the expression of hundreds of cytoprotective genes to counteract endogenously or exogenously generated oxidative stress. Furthermore, when activated in human tumors by somatic mutations, Nrf2 confers growth advantages and chemoresistance by regulating genes involved in various processes such as the pentose phosphate pathway and nucleotide synthesis in addition to antioxidant proteins. Interestingly, increasing evidence shows that Nrf2 is associated with mitochondrial biogenesis during environmental stresses in certain tissues such as the heart. Furthermore, SKN-1, a functional homolog of Nrf2 in C. elegans, is activated by mitochondrial reactive oxygen species and extends life span by promoting mitochondrial homeostasis (i.e., mitohormesis). Similarly, Nrf2 activation was recently observed in the heart of surfeit locus protein 1 (Surf1) -/- mice in which cellular respiration was decreased due to cytochrome c oxidase defects. In this review, we critically examine the relationship between Nrf2 and mitochondria and argue that the Nrf2 stress pathway intimately communicates with mitochondria to maintain cellular homeostasis during oxidative stress.
RESUMO
This study investigated the levels of public trust in organizations associated with the Great East Japan Earthquake. In Study 1 (N = 639), the levels of trust in eight organizations as well as the determinants of trust--perceived salient value similarity (SVS), ability, and motivation--were measured twice, first immediately after the earthquake and then a year later. The results indicated that the trust levels for six of the eight organizations had been preserved, supporting the double asymmetric effect of trust. The results of structural equation modeling (SEM) revealed that SVS explained trust more when the organization had been less trusted. Trust in the organization explains well the perceived reduction of the target risk. The results of SEM in Study 2 (N = 1,030) replicated those of Study 1, suggesting the stability of the explanatory power of the determinants of trust. Implications of the study for risk management practices are discussed.
Assuntos
Terremotos , Organizações , Risco , Confiança , Adulto , Feminino , Humanos , Japão , MasculinoRESUMO
Calpains are calcium-dependent cysteine proteases activated by intracellular Ca2+. Although calpains mainly exist in the cytosol, calpain-13 is present in the mitochondria in mouse brains; however, the enzymatic properties and physiological functions of calpain-13 remain unknown. Hence, in this study, we predicted and evaluated the enzymatic properties of calpain-13. Based on our bioinformatic approaches, calpain-13 possessed a catalytic triad and EF-hand domain, similar to calpain-1, a well-studied calpain. Therefore, we hypothesized that calpain-13 had calpain-1-like enzymatic properties; however, calpain-13 was not proteolyzed in C57BL/6J mouse brains. Subsequently, cerebral ischemia/reperfusion (I/R) injury caused proteolysis of mitochondrial calpain-13. Thus, our study showed that mitochondrial calpain-13 was proteolyzed in the mitochondria of the I/R injured mouse brain. This finding could be valuable in further research elucidating the involvement of calpain-13 in cell survival or death in brain diseases, such as cerebral infarction.