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A homozygous mutation of the DNAJC6 gene causes autosomal recessive familial type 19 of Parkinson's disease (PARK19). To test the hypothesis that PARK19 DNAJC6 mutations induce the neurodegeneration of dopaminergic cells by reducing the protein expression of functional DNAJC6 and causing DNAJC6 paucity, an in vitro PARK19 model was constructed by using shRNA-mediated gene silencing of endogenous DANJC6 in differentiated human SH-SY5Y dopaminergic neurons. shRNA targeting DNAJC6 induced the neurodegeneration of dopaminergic cells. DNAJC6 paucity reduced the level of cytosolic clathrin heavy chain and the number of lysosomes in dopaminergic neurons. A DNAJC6 paucity-induced reduction in the lysosomal number downregulated the protein level of lysosomal protease cathepsin D and impaired macroautophagy, resulting in the upregulation of pathologic α-synuclein or phospho-α-synucleinSer129 in the endoplasmic reticulum (ER) and mitochondria. The expression of α-synuclein shRNA or cathepsin D blocked the DNAJC6 deficiency-evoked degeneration of dopaminergic cells. An increase in ER α-synuclein or phospho-α-synucleinSer129 caused by DNAJC6 paucity activated ER stress, the unfolded protein response and ER stress-triggered apoptotic signaling. The lack of DNAJC6-induced upregulation of mitochondrial α-synuclein depolarized the mitochondrial membrane potential and elevated the mitochondrial level of superoxide. The DNAJC6 paucity-evoked ER stress-related apoptotic cascade, mitochondrial malfunction and oxidative stress induced the degeneration of dopaminergic neurons via activating mitochondrial pro-apoptotic signaling. In contrast with the neuroprotective function of WT DNAJC6, the PARK19 DNAJC6 mutants (Q789X or R927G) failed to attenuate the tunicamycin- or rotenone-induced upregulation of pathologic α-synuclein and stimulation of apoptotic signaling. Our data suggest that PARK19 mutation-induced DNAJC6 paucity causes the degeneration of dopaminergic neurons via downregulating protease cathepsin D and upregulating neurotoxic α-synuclein. Our results also indicate that PARK19 mutation (Q789X or R927G) impairs the DNAJC6-mediated neuroprotective function.
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Catepsina D , Neuronas Dopaminérgicas , Estrés del Retículo Endoplásmico , Proteínas del Choque Térmico HSP40 , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Apoptosis/genética , Catepsina D/metabolismo , Catepsina D/genética , Línea Celular Tumoral , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Regulación hacia Abajo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP40/genética , Lisosomas/metabolismo , Mitocondrias/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Regulación hacia ArribaRESUMEN
Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA-APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN , Humanos , Sitios de Unión , ADN/metabolismo , ADN/química , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Microscopía de Fuerza Atómica , Unión ProteicaRESUMEN
Introduction: Chemotherapy and radiation therapy for breast cancer cause side effects, such as cardiovascular changes, which can be monitored with echocardiography. However, more convenient methods are always encouraged. Radial arterial waves that are used to detect cardiovascular changes can be used to assist in confirming cardiovascular changes. Aim: This retrospective study aimed to analyze the frequency and time domains of the radial artery pulse wave in patients with breast cancer to understand its effectiveness in identifying cardiovascular changes. Methods: Patients with breast cancer were screened from the pulse examination records in Changhua Christian Hospital and divided into the treatment and remission groups. After unlinking the data, the pulse data were analyzed for the breast cancer treatment and remission group, including the average value of the parameters of four consecutive pulse diagnosis records in four consecutive months to test the difference in pulse waves due to breast cancer treatment between the two groups. Additionally, the pulse wave stability of the two groups was compared using the coefficient of variation. Results and conclusion: The comparison of the pulse wave data between 19 patients in the treatment group and 40 patients in the remission group revealed 45 parameters in time and 50 in frequency domains. D3, ND3, NA1, and NT1 are the four parameters with significant differences (p < 0.05), which are all related to heart function, and mainly related to cardiac output and peripheral resistance, indicating that patients in the treatment period have poor heart function. No difference was found in the degree of data dispersion between the two groups. Cardiovascular side effects caused by breast cancer treatment can mainly be shown in the pulse wave time domain.
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BACKGROUND: Type 2 hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant disease and is associated with ALK1 gene mutations. Type 2 HHT patients primarily suffer from recurrent bleeding. There is currently no promising treatment. CASE SUMMARY: A 5-year-old Chinese patient (III23) was admitted to Zhongshan Hospital for recurrent melena occurring over 2 mo. She had been experiencing epistaxis for years and had been diagnosed with idiopathic pulmonary hypertension 4 mo before presentation. Abdominal computed tomography examination showed hepatic arteriovenous malformation. Gene testing revealed a c.1121G>A mutation on the ALK1 gene. According to the international diagnostic criteria, this patient was diagnosed with HHT. In addition, 8 more family members exhibited HHT symptoms to varying degrees. Gene testing in 5 family members (2 with HHT symptoms and 3 without HHT symptoms) revealed the ALK1 c.1121G>A mutation in the 2 family members with HHT symptoms. This missense mutation results in the substitution of arginine for glutamine at amino acid position 374 (R374Q) in the conserved functional kinase domain of ALK1. Biological studies revealed that this mutation decreased the kinase activity of ALK1 and impeded the phosphorylation of its substrate Smad1. Moreover, the R374Q mutant downregulated the protein level of collagen-1, a fibrogenic factor, indicating abnormal fiber generation during vascular formation. CONCLUSION: The R374Q mutant of ALK1 and its subsequent influence on fiber generation highly indicated its pathogenic role in this family with type 2 HHT. Detection of this gene mutation will facilitate early diagnosis of suspected type 2 HHT patients, and mechanistic studies will provide insights for future therapy.
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The base excision repair (BER) is the primary damage repair pathway for repairing most of the endogenous DNA damage including oxidative base lesions, apurinic/apyrimidinic (AP) sites, and single-strand breaks (SSBs) in the genome. Repair of these damages in cells relies on sequential recruitment and coordinated actions of multiple DNA repair enzymes, which include DNA glycosylases (such as OGG1), AP-endonucleases (APE1), DNA polymerases, and DNA ligases. APE1 plays a key role in the BER pathway by repairing the AP sites and SSBs in the genome. Several methods have been developed to generate a map of endogenous AP sites or SSBs in the genome and the binding of DNA repair proteins. In this chapter, we describe detailed approaches to map genome-wide occupancy or enrichment of APE1 in human cells using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). Further, we discuss standard bioinformatics approaches for analyzing ChIP-seq data to identify APE1 enrichment or binding peaks in the genome.
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MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.
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Objective: Ultrasound (US) detection acupuncture (UDA) is an innovative acupuncture technique that uses ultrasonography (USG) to detect the depth of the lung before performing acupuncture on the points around the chest to avoid puncturing the lungs. For acupuncturists to use UDA appropriately, it is crucial to have a good operating method to identify the pleura with USG. This study compared 2 US operating methods through active learning in a "flipped classroom" setting for acupuncture students. Materials and Methods: Students and interns were recruited to complete the UDA flipped classroom course and evaluate the operations of 2 US methods on either of 2 simulation models: (1) a single B-mode or (2) a combined M-mode + B-mode. Participants were interviewed and satisfaction surveys were administered to obtain feedback. Results: A total of 37 participants completed the course and evaluations. The combined mode had better measurement accuracy, acupuncture safety, and operating time (P < 0.05), and no pneumothoraxes occurred. Among both participant groups, the combined mode allowed the student group to learn quickly and the intern group to become more proficient. Both interviews and satisfaction surveys yielded positive feedback. Conclusions: Using a combined mode for UDA can improve its performance greatly. The combined mode is definitely helpful for learning and promotion of UDA.
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The reflex auriculo-cardiac (RAC), dynamic pulse reaction (Nogier reflex), or vascular autonomic signal was proposed by Nogier. It refers to the pulse changes that can occur in the radial artery immediately after auricular acupuncture is performed. RAC is helpful for the clinical practice of auricular acupuncture, but there is a lack of objective verification methods. Photoplethysmography (PPG) has been used to objectively calculate radial artery blood flow. This study used PPG via a smartphone to measure RAC induced by auricular acupuncture. Thirty subjects without major diseases were recruited to receive traditional needle and laser acupuncture. The Shen Men ear point and control points were stimulated for 20 s. PPG was continuously measured during the acupuncture. The PPG data were tested for differences with a paired t-test. The results showed that there were no statistical differences in the frequency and amplitude of PPG obtained before and after acupuncture, either with a traditional needle or laser acupuncture. However, interestingly, it was found that one patient with insomnia, one patient with viral respiratory symptoms, and two menstruating females exhibited changes in PPG within five seconds of needle placement. We hypothesized that RAC might be induced by auricular acupuncture and could be quantified by PPG, even among subjects suffering from mild diseases; however, auricular acupuncture might not induce a measurable RAC in totally healthy subjects.
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Deletion and missense or nonsense mutation of RAB39B gene cause familial Parkinson's disease (PD). We hypothesized that deletion and mutation of RAB39B gene induce degeneration of dopaminergic neurons by decreasing protein level of functional RAB39B and causing RAB39B deficiency. Cellular model of deletion or mutation of RAB39B gene-induced PD was prepared by knocking down endogenous RAB39B in human SH-SY5Y dopaminergic cells. Transfection of shRNA-induced 90% reduction in RAB39B level significantly decreased viability of SH-SY5Y dopaminergic neurons. Deficiency of RAB39B caused impairment of macroautophagy/autophagy, which led to increased protein levels of α-synuclein and phospho-α-synucleinSer129 within endoplasmic reticulum (ER) and mitochondria. RAB39B deficiency-induced increase of ER α-synuclein and phospho-α-synucleinSer129 caused activation of ER stress, unfolded protein response, and ER stress-induced pro-apoptotic cascade. Deficiency of RAB39B-induced increase of mitochondrial α-synuclein decreased mitochondrial membrane potential and increased mitochondrial superoxide. RAB39B deficiency-induced activation of ER stress pro-apoptotic pathway, mitochondrial dysfunction, and oxidative stress caused apoptotic death of SH-SY5Y dopaminergic cells by activating mitochondrial apoptotic cascade. In contrast to neuroprotective effect of wild-type RAB39B, PD mutant (T168K), (W186X), or (G192R) RAB39B did not prevent tunicamycin- or rotenone-induced increase of neurotoxic α-synuclein and activation of pro-apoptotic pathway. Our results suggest that RAB39B is required for survival and macroautophagy function of dopaminergic neurons and that deletion or PD mutation of RAB39B gene-induced RAB39B deficiency induces apoptotic death of dopaminergic neurons via impairing autophagy function and upregulating α-synuclein.
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Estrés del Retículo Endoplásmico , Neuroblastoma , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Autofagia , Neuronas Dopaminérgicas/metabolismo , Mitocondrias/metabolismo , Neuroblastoma/metabolismo , Estrés Oxidativo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The dynamic pulse reaction (Nogier reflex), Reflex-Auriculo-Cardiac (RAC), or vascular autonomic signal is a physiologic phenomenon that is not fully accepted and widely understood in contemporary medical practice. In order to provide appropriate scientific evidence for better understanding, qualitative and-above all-quantitative research in this area is necessary. In this short report, 2 recordings of dynamic pulse reactions based on an analysis performed on a modified smartphone are demonstrated during laser acupuncture experiments using stimulation at ear acupuncture points.
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Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients' tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.
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Carcinoma Ductal Pancreático , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , G-Cuádruplex , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Carcinoma Ductal Pancreático/genética , ADN/química , Endonucleasas/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMEN
BACKGROUND: Cyclin C (CCNC) was reported to take part in regulating mitochondria-derived oxidative stress under cisplatin stimulation. However, its effect in gastric cancer is unknown. This study aimed to investigate the role of cyclin C and its ubiquitylation in regulating cisplatin resistance in gastric cancer. METHODS: The interaction between HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1 (HACE1) and cyclin C was investigated by GST pull-down assay, co-immunoprecipitation and ubiquitylation assay. Mitochondria-derived oxidative stress was studied by MitoSOX Red assay, seahorse assay and mitochondrial membrane potential measurement. Cyclin C-associated cisplatin resistance was studied in vivo via xenograft. RESULTS: HACE1 catalysed the ubiquitylation of cyclin C by adding Lys11-linked ubiquitin chains when cyclin C translocates to cytoplasm induced by cisplatin treatment. The ubiquitin-modified cyclin C then anchor at mitochondira, which induced mitochondrial fission and ROS synthesis. Depleting CCNC or mutation on the ubiquitylation sites decreased mitochondrial ROS production and reduced cell apoptosis under cisplatin treatment. Xenograft study showed that disrupting cyclin C ubiquitylation by HACE1 conferred impairing cell apoptosis response upon cisplatin administration. CONCLUSIONS: Cyclin C is a newly identified substrate of HACE1 E3 ligase. HACE1-mediated ubiquitylation of cyclin C sheds light on a better understanding of cisplatin-associated resistance in gastric cancer patients. Ubiquitylation of cyclin C by HACE1 regulates cisplatin-associated sensitivity in gastric cancer. With cisplatin-induced nuclear-mitochondrial translocation of cyclin C, its ubiquitylation by HACE1 increased mitochondrial fission and mitochondrial-derived oxidative stress, leading to cell apoptosis.
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Cisplatino , Neoplasias Gástricas , Cisplatino/farmacología , Ciclina C/genética , Humanos , Neoplasias Gástricas/tratamiento farmacológico , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Medulloblastoma (MB) is a malignant pediatric brain tumor with a poor prognosis. Post-surgical radiation and cisplatin-based chemotherapy have been a mainstay of treatment, which often leads to substantial neurocognitive impairments and morbidity, highlighting the need for a novel therapeutic target to enhance the sensitivity of MB tumors to cytotoxic therapies. We performed a comprehensive study using a cohort of 71 MB patients' samples and pediatric MB cell lines and found that MB tumors have elevated levels of nucleosome remodeling FACT (FAcilitates Chromatin Transcription) complex and DNA repair enzyme AP-endonuclease1 (APE1). FACT interacts with APE1 and facilitates recruitment and acetylation of APE1 to promote repair of radiation and cisplatin-induced DNA damage. Further, levels of FACT and acetylated APE1 both are correlate strongly with MB patients' survival. Targeting FACT complex with CBL0137 inhibits DNA repair and alters expression of a subset of genes, and significantly improves the potency of cisplatin and radiation in vitro and in MB xenograft. Notably, combination of CBL0137 and cisplatin significantly suppressed MB tumor growth in an intracranial orthotopic xenograft model. We conclude that FACT complex promotes chemo-radiation resistance in MB, and FACT inhibitor CBL0137 can be used as a chemo-radiation sensitizer to augment treatment efficacy and reduce therapy-related toxicity in high-risk pediatric patients.
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Cisplatino/administración & dosificación , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Meduloblastoma/tratamiento farmacológico , Factores de Elongación Transcripcional/genética , Adolescente , Adulto , Animales , Carbazoles/administración & dosificación , Carbazoles/efectos adversos , Niño , Preescolar , Cisplatino/efectos adversos , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Xenoinjertos , Proteínas del Grupo de Alta Movilidad/antagonistas & inhibidores , Chaperonas de Histonas/genética , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/patología , Meduloblastoma/radioterapia , Ratones , Factores de Elongación Transcripcional/antagonistas & inhibidores , Adulto JovenRESUMEN
Our previous study suggests that upregulated RAB35 is implicated in etiology of Parkinson's disease (PD). We hypothesized that upregulated RAB35 results from single nucleotide polymorphisms (SNPs) in RAB35 gene promoter. We identified SNPs within RAB35 gene promoter by analyzing DNA samples of discovery cohort and validation cohort. SNP rs17525453 within RAB35 gene promoter (T>C at position of -66) was significantly associated with idiopathic PD patients. Compared to normal controls, sporadic PD patients had higher C allele frequency. CC and CT genotype significantly increased risk of PD compared with TT genotype. SNP rs17525453 within RAB35 gene promoter leads to formation of transcription factor TFII-I binding site. Results of EMSA and supershift assay indicated that TFII-I binds to rs17525453 sequence of RAB35 gene promoter. Luciferase reporter assays showed that rs17525453 variant of RAB35 gene promoter possesses an augmented transcriptional activity. Our results suggest that functional variant rs17525453 within RAB35 gene promoter is likely to enhance transcriptional activity and upregulate RAB35 protein, which could lead to increased risk of PD in Taiwanese population.
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Estudios de Asociación Genética , Enfermedad de Parkinson/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Proteínas de Unión al GTP rab/genética , Pueblo Asiatico/genética , Estudios de Cohortes , Frecuencia de los Genes , Genética de Población , Genotipo , Humanos , Enfermedad de Parkinson/epidemiología , Riesgo , Taiwán/epidemiología , Transcripción Genética/genética , Regulación hacia Arriba/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Achilles tendinopathy has a high re-injury rate and poor prognosis. Development of effective therapy for Achilles tendinopathy is important. Excessive accumulation of ROS and resulting oxidative stress are believed to cause tendinopathy. Overproduction of hydrogen peroxide (H2O2), the most common ROS, could lead to the tendinopathy by causing oxidative damage, activation of endoplasmic reticulum (ER) stress and apoptotic death of tenocytes. Activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) is expected to alleviate oxidative stress and ER stress. Alda-1 is a selective and potent activator of ALDH2. In this study, we examined the cytoprotective benefit of Alda-1, an activator of ALDH2, on H2O2-induced Achilles tendinopathy in cellular and mouse models. We prepared cellular and mouse models of Achilles tendinopathy by treating cultured Achilles tenocytes and Achilles tendons with oxidative stressor H2O2. Subsequently, we studied the protective benefit of Alda-1 on H2O2-induced Achilles tendinopathy. Alda-1 pretreatment attenuated H2O2-induced cell death of cultured Achilles tenocytes. Treatment of Alda-1 prevented H2O2-induced oxidative stress and depolarization of mitochondrial membrane potential in tenocytes. Application of Alda-1 attenuated H2O2-triggered mitochondria- and ER stress-mediated apoptotic cascades in cultured tenocytes. Alda-1 treatment ameliorated the severity of H2O2-induced Achilles tendinopathy in vivo by preventing H2O2-induced pathological histological features of Achilles tendons, apoptotic death of Achilles tenocytes and upregulated expression of inflammatory cytokines IL-1ß and TNF-α. Our results provide the evidence that ALDH2 activator Alda-1 ameliorates H2O2-induced Achilles tendinopathy. Alda-1 could be used for preventing and treating Achilles tendinopathy.
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Tendón Calcáneo/metabolismo , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Benzamidas/uso terapéutico , Benzodioxoles/uso terapéutico , Modelos Animales de Enfermedad , Tendinopatía/tratamiento farmacológico , Tendinopatía/metabolismo , Tendón Calcáneo/efectos de los fármacos , Tendón Calcáneo/patología , Animales , Benzamidas/farmacología , Benzodioxoles/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/toxicidad , Ratones , Ratones Endogámicos C57BL , Tendinopatía/patología , Tenocitos/efectos de los fármacos , Tenocitos/metabolismo , Tenocitos/patologíaRESUMEN
MicroRNAs (miRs) downregulate or upregulate the mRNA level by binding to the 3'-untranslated region (3'UTR) of target gene. Dysregulated miR levels can be used as biomarkers of Parkinson's disease (PD) and could participate in the etiology of PD. In the present study, 45 brain-enriched miRs were evaluated in serum samples from 50 normal subjects and 50 sporadic PD patients. The level of miR-204-5p was upregulated in serum samples from PD patients. An upregulated level of miR-204-5p was also observed in the serum and substantia nigra (SN) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Expression of miR-204-5p increased the level of α-synuclein (α-Syn), phosphorylated (phospho)-α-Syn, tau, or phospho-tau protein and resulted in the activation of endoplasmic reticulum (ER) stress in SH-SY5Y dopaminergic cells. Expression of miR-204-5p caused autophagy impairment and activation of c-Jun N-terminal kinase (JNK)-mediated apoptotic cascade in SH-SY5Y dopaminergic cells. Our study using the bioinformatic method and dual-luciferase reporter analysis suggests that miR-204-5p positively regulates mRNA expression of dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) by directly interacting with 3'UTR of DYRK1A. The mRNA and protein levels of DYRK1A were increased in SH-SY5Y dopaminergic cells expressing miR-204-5p and SN of MPTP-induced PD mouse model. Knockdown of DYRK1A expression or treatment of the DYRK1A inhibitor harmine attenuated miR-204-5p-induced increase in protein expression of phospho-α-Syn or phospho-tau, ER stress, autophagy impairment, and activation of JNK-mediated apoptotic pathway in SH-SY5Y dopaminergic cells or primary cultured dopaminergic neurons. Our results suggest that upregulated expression of miR-204-5p leads to the death of dopaminergic cells by targeting DYRK1A-mediated ER stress and apoptotic signaling cascade.
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Purpose: To investigate the mitochondria-related mechanism of Gynura segetum (GS)-induced apoptosis and the protective effect of phosphocreatine (PCr), a mitochondrial respiration regulator. Methods: First, the mechanism was explored in human hepatocyte cell line. The mitochondrial oxidative stress was determined by fluorescence assay. The level of sirtuin 3 (SIRT3), acetylated superoxide dismutase 2 (Ac-SOD2), SOD2, and apoptosis were detected by Western blotting. Mito-TEMPO and cell lines of viral vector-mediated overexpression of SIRT3 and SIRT3H248Y were used to further verify the mechanism of GS-induced apoptosis. GS-induced liver injury mice models were built by GS through intragastric administration and interfered by PCr through intraperitoneal injection. A total of 30 C57BL/6J mice were assigned to 5 groups and treated with either saline, PCr (100 mg/kg), GS (30 g/kg), or PCr (50 or 100 mg/kg)+GS (30 g/kg). Liver hematoxylin and eosin (HE) staining, immunohistochemical analysis, and blood biochemical evaluation were performed. Results: GS induced hepatocyte apoptosis and elevated levels of mitochondrial ROS in L-02 cells. The expression of SIRT3 was decreased. Downregulation of SIRT3 was associated with increased levels of Ac-SOD2, which is the inactivated enzymatic form of SOD2. Conversely, when overexpressing SIRT3 in GS-treated cells, SOD2 activity was restored, and mitochondrial ROS levels and hepatocyte apoptosis declined. Upon administration of PCr to GS-treated cells, they exhibited a significant upregulation of SIRT3 and were protected against apoptosis. In animal experiments, serum ALT level and mitochondrial ROS of the mice treated with GS and 50 mg/kg PCr were significantly attenuated compared with only GS treated. The changes in SIRT3 expression were also consistent with the in vitro results. In addition, immunohistochemical analysis of the mouse liver showed that Ac-SOD2 was decreased in the PCr and GS co-treated group compared with GS treated group. Conclusion: GS caused liver injury by dysregulating mitochondrial ROS generation via a SIRT3-SOD2 pathway. PCr is a potential agent to treat GS-induced liver injury by mitochondrial protection.
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Apoptosis/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Hepatocitos/efectos de los fármacos , Fosfocreatina/farmacología , Sustancias Protectoras/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/análisis , Sirtuina 3/antagonistas & inhibidores , Sirtuina 3/metabolismo , Relación Estructura-Actividad , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/metabolismoRESUMEN
PARK14 patients with homozygous (D331Y) PLA2G6 mutation display motor deficits of pure early-onset Parkinson's disease (PD). The aim of this study is to investigate the pathogenic mechanism of mutant (D331Y) PLA2G6-induced PD. We generated knockin (KI) mouse model of PARK14 harboring homozygous (D331Y) PLA2G6 mutation. Then, we investigated neuropathological and neurological phenotypes of PLA2G6D331Y/D331Y KI mice and molecular pathogenic mechanisms of (D331Y) PLA2G6-induced degeneration of substantia nigra (SN) dopaminergic neurons. Six-or nine-month-old PLA2G6D331Y/D331Y KI mice displayed early-onset cell death of SNpc dopaminergic neurons. Lewy body pathology was found in the SN of PLA2G6D331Y/D331Y mice. Six-or nine-month-old PLA2G6D331Y/D331Y KI mice exhibited early-onset parkinsonism phenotypes. Disrupted cristae of mitochondria were found in SNpc dopaminergic neurons of PLA2G6D331Y/D331Y mice. PLA2G6D331Y/D331Y mice displayed mitochondrial dysfunction and upregulated ROS production, which may lead to activation of apoptotic cascade. Upregulated protein levels of Grp78, IRE1, PERK, and CHOP, which are involved in activation of ER stress, were found in the SN of PLA2G6D331Y/D331Y mice. Protein expression of mitophagic proteins, including parkin and BNIP3, was downregulated in the SN of PLA2G6D331Y/D331Y mice, suggesting that (D331Y) PLA2G6 mutation causes mitophagy dysfunction. In the SN of PLA2G6D331Y/D331Y mice, mRNA levels of eight genes that are involved in neuroprotection/neurogenesis were decreased, while mRNA levels of two genes that promote apoptotic death were increased. Our results suggest that PARK14 (D331Y) PLA2G6 mutation causes degeneration of SNpc dopaminergic neurons by causing mitochondrial dysfunction, elevated ER stress, mitophagy impairment, and transcriptional abnormality.
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Neuronas Dopaminérgicas/patología , Estrés del Retículo Endoplásmico , Técnicas de Sustitución del Gen , Fosfolipasas A2 Grupo VI/genética , Mitofagia , Degeneración Nerviosa/patología , Enfermedad de Parkinson/genética , Sustancia Negra/patología , Animales , Apoptosis , Secuencia de Bases , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Regulación de la Expresión Génica , Homocigoto , Humanos , Cuerpos de Lewy/patología , Ratones Endogámicos C57BL , Mitocondrias/patología , Mitocondrias/ultraestructura , Mutación/genética , Neuroprotección , Enfermedad de Parkinson/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
HACE1 E3 ligase was discovered to be down-regulated in several cancers while its role in regulating tumors was merely understood. This study aimed to explore the specific effect of HACE1 played in gastric tumorigenesis and its potential mechanism. HACE1's expression was found significantly lower in gastric cancer tissues compared with the adjacent normal tissues (P < 0.001). Its protein level in gastric cancer negatively correlated to tumor pathological differentiation (P = 0.019). And in gastric cancer patients with TNM I-IIIa, those with lower HACE1 protein level had poorer overall survival (P = 0.025). Studies, in vivo and in vitro, showed that overexpressing HACE1 inhibited tumor proliferation and migration, and enhanced cell apoptosis. Besides, ectopic expression of HACE1 down-regulated the protein level of ß-catenin and inhibited the activity of the Wnt/ß-catenin signaling pathway. All the cellular functions were abolished when we overexpressed inactive HACE1-deltaHECT. Above all, we demonstrated that HACE1 E3 ligase played a suppressive role in gastric tumorigenesis and inhibited the activity of the Wnt/ß-catenin signaling pathway. Circumventing the decline of HACE1 in early stage of carcinoma may impede the tumorigenesis and malignant process of gastric cancer.
Asunto(s)
Expresión Génica , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Ubiquitina-Proteína Ligasas/genética , Adulto , Anciano , Animales , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Técnicas de Inactivación de Genes , Humanos , Masculino , Ratones , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mutations in the gene encoding Ca2+-independent phospholipase A2 group 6 (PLA2G6) cause the recessive familial type 14 of Parkinson's disease (PARK14). Mitochondrial dysfunction is involved in the pathogenesis of Parkinson's disease (PD). PLA2G6 is believed to be required for maintaining mitochondrial function. In the present study, rotenone-induced cellular model of PD was used to investigate possible molecular pathogenic mechanism of PARK14 mutant PLA2G6-induced PD. Overexpression of wild-type (WT) PLA2G6 ameliorated rotenone-induced apoptotic death of SH-SY5Y dopaminergic cells. PARK14 mutant (D331Y), (G517C), (T572I), (R632W), (N659S) or (R741Q) PLA2G6 failed to prevent rotenone-induced activation of mitochondrial apoptotic pathway and exert a neuroprotective effect. WT PLA2G6, but not PARK14 mutant PLA2G6, prevented rotenone-induced mitophagy impairment. In contrast to WT PLA2G6, PARK14 mutant PLA2G6 was ineffective in attenuating rotenone-induced decrease in mitochondrial membrane potential and increase in the level of mitochondrial superoxide. WT PLA2G6, but not PARK14 PLA2G6 mutants, restored enzyme activity of mitochondrial complex I and cellular ATP content in rotenone-treated SH-SY5Y dopaminergic cells. In contrast to WT PLA2G6, PARK14 mutant PLA2G6 failed to prevent rotenone-induced mitochondrial lipid peroxidation and cytochrome c release. These results suggest that PARK14 PLA2G6 mutants lose their ability to maintain mitochondrial function and are defective inpreventing mitochondrial dysfunction, ROS production and activation of mitochondrial apoptotic pathway in rotenone-induced cellular model of PD.