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1.
Emerg Microbes Infect ; 9(1): 680-686, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32207377

RESUMEN

Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Sueros Inmunes/inmunología , Pruebas de Neutralización , Neumonía Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Línea Celular , Humanos , Límite de Detección , Glicoproteínas de Membrana/inmunología , Ratones , Plásmidos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/genética , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas del Envoltorio Viral/inmunología , Internalización del Virus
2.
Chem Biol Interact ; 321: 109025, 2020 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-32135139

RESUMEN

Epigenetic regulation is one of the driving forces in the process of carcinogenesis. Corosolic acid (CA); triterpenoid abundantly found in Lagerstroemia speciosa L. is known to modulate various cellular process including cellular oxidative stress and signaling kinases in various diseases, including skin cancer. Genetic mutations in early stages of skin cancer are well-documented, the epigenetic alterations remain elusive. In the present study, we identified the transcriptomic gene expression changes with RNAseq and genome-wide DNA CpG methylation changes with DNA methylseq to profile the early stage transcriptomic and epigenomic changes using tumor promoter TPA-mediated mouse epidermal epithelial JB6 P+ cells. JB6 P+ cells were treated with TPA and Corosolic acid by 7.5uM optimized by MTS assay. Differentiated expressed genes (DEGs) and Differentially methylated genes (DMRs) were analyzed by R software. Ingenuity Pathway Analysis (IPA) was employed to understand the differential regulation of specific pathways. Novel TPA induced differentially overexpressed genes like tumor promoter Prl2c2, small prolin rich protein (Sprr2h) was reported which was downregulated by corosolic acid treatment. Several cancer related pathways were identified by Ingenuity Pathways Analysis (IPA) including p53, Erk, TGF beta signaling pathways. Moreover, differentially methylated regions (DMRs) in genes like Dusp22 (Dual specificity protein phosphatase 22), Rassf (tumor suppressor gene family, Ras association domain family) in JB6 P+ cells were uncovered which are altered by TPA and are reversed by CA treatment. Interestingly, genes like CDK1 (Cyclin-dependent kinases 1) and RASSF2 (Ras association domain family member 2) observed to be differentially methylated and expressed which was further modulated by corosolic acid treatment, validated by qPCR. Given study indicated gene expression changes to DNA CpG methylation epigenomic changes modulated various molecular pathways in TPA-induced JB6 cells and revealed that CA can potentially reverse these changes which deciphering novel molecular targets for future prevention of early stages of skin cancer studies in human.


Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Metilación de ADN/efectos de los fármacos , Células Epidérmicas/efectos de los fármacos , Células Epidérmicas/metabolismo , Triterpenos/farmacología , Animales , Carcinógenos/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Islas de CpG/efectos de los fármacos , Células Epidérmicas/patología , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones , Fitoquímicos/farmacología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Acetato de Tetradecanoilforbol/toxicidad , Transcriptoma/efectos de los fármacos
3.
Chem Biol Interact ; 321: 109031, 2020 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-32142722

RESUMEN

Reactive oxygen species (ROS) is mainly produced as a by-product from electron transport chain (ETC) of mitochondria and effectively eliminated by cellular antioxidants. However, 2-chloroethyl ethyl sulfide (CEES) exposure to keratinocytes declined antioxidant capacity and increased accumulation of ROS triggered alteration of mitochondrial activity and apoptosis is lacking. Our findings demonstrated that the electron leakage from the impaired ETC, leading to the accumulation of ROS was gradually elevating with increasing concentration of CEES exposure, which decline the activity of superoxide dismutase (SOD), manganese SOD (MnSOD) and copper-zinc SOD (Cu-ZnSOD) in keratinocytes. Further, excess accumulation of ROS, decreased the mitochondrial membrane potential (ΔΨm) and increased the mitochondrial mass with increasing dose of CEES. CEES exposure provoked the decrease in expression of transcription factor A mitochondrial (TFAM), augmented mitochondrial DNA (mtDNA) damage and altered the mtDNA-encoded oxidative phosphorylation (OXPHOS) subunits. Moreover, fragmented mtDNA translocated into cytosol, where it activated cGAS-STING and interferon regulatory factor3 (IRF3), coinciding with the increased expression of inflammatory mediators and alteration of cell-to-cell communication markers. Pre-treatment of N-acetyl-l-cysteine (NAC) or L-Nω-nitroarginine methyl ester (NAME), hydralazine hydrochloride (Hyd·HCl) or ERK1/2 or phosphoinositide3-kinase (PI3-K)/Akt inhibitors in keratinocyte cells significantly restored the CEES effect. Our findings suggest that CEES-induced mitochondrial ROS production and accumulation leads to mitochondrial dysfunction and inflammatory response in keratinocytes. However, treatment of antioxidants or ERK1/2 or PI3-K/Akt inhibitors is a novel therapeutic option for the keratinocytes complication.


Asunto(s)
Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Gas Mostaza/análogos & derivados , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Sustancias para la Guerra Química/toxicidad , Daño del ADN , ADN Mitocondrial/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Irritantes/toxicidad , Queratinocitos/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Pelados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Gas Mostaza/toxicidad , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos
4.
Chem Biol Interact ; 321: 109044, 2020 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-32151596

RESUMEN

Overconsumption of alcohol could lead to severe liver injury that connects with oxidative stress, apoptosis, and inflammatory response. Previously, we proved that p-coumaric acid prevents ethanol induced reproductive toxicity; however, p-coumaric acid (PCA) on ethanol mediated hepatotoxicity has not been examined yet. In our work, we sought to study the potential of PCA in contradiction of ethanol induced hepatoxicity which linking with MAPKs, apoptosis, oxidative stress, and Nrf2 signaling. Foremost, we found that PCA could protect ethanol induced both L-02 and HepG2 hepatic cells by inhibiting cytotoxicity, ROS production, mitochondrial depolarization, and nuclear fragmentation. Also, in vivo experiments showed that the ethanol increasing the lipid markers (TBARS, CD) and depletes the antioxidants thereby increased phosphorylation of JNK, ERK, and p38 in rat liver tissues. Interestingly, PCA treatments inhibit ethanol exposed lipid markers and depletion of antioxidants, which directs the inhibition of MAPKs activation in rat liver tissues. We also noticed that the PCA protected ethanol induced apoptosis and liver markers by inhibiting the expression of Bax, caspases; AST, ALT, ALS, and LDH in liver tissue. Overall, the ameliorative consequence of PCA on ethanol induced oxidative stress and apoptosis was achieved by suppressing the expression of CYP2E1 and overexpressing Nrf2 and its target protein HO-1 in rat liver tissue. As a result, PCA was marked to be an effective antioxidant with notable hepatoprotection by inhibiting MAPKs and apoptosis signaling via enhancing Nrf2 signaling.


Asunto(s)
Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/prevención & control , Propionatos/farmacología , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Etanol/toxicidad , Células Hep G2 , Humanos , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/lesiones , Hígado/metabolismo , Hepatopatías Alcohólicas/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Sustancias Protectoras/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos
5.
Emerg Microbes Infect ; 9(1): 664-675, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32193996

RESUMEN

The H7N9 viruses have been circulating for six years. The insertion of a polybasic cleavage site in the haemagglutinin (HA) protein of H7N9 has resulted in the emergence of a highly pathogenic (HP) avian influenza virus. Currently, there are limited studies on neutralizing monoclonal antibodies(mAbs) against HP H7N9 AIVs. In this study, mice were immunized with inactivated H7N9 vaccine of A/ZJU01/PR8/2013 to produce murine mAbs. Finally, two murine mAbs against the HA of low pathogenic (LP) virus were produced and characterized. Characterization included determining mAbs binding breadth and affinity, in vitro neutralization capacity, and potential in vivo protection. Two of these mAbs, 1H10 and 2D1, have been identified to have therapeutic and prophylactic efficacy against the HP strain in mouse passive transfer-viral challenge experiments. The mAb 1H10 was most efficacious, even if the treatment-time was as late as 72 h post-infection, or the therapeutic dose was as low as 1 mg/kg; and it was confirmed to have haemagglutination inhibition and neutralizing activity on both LP-and HP-H7N9 strains. Further study indicated that the protection provided by 2D1 was mediated by antibody-dependent cellular cytotoxicity. The mAbs described here provide promising results and merit further development into potential antiviral therapeutics for H7N9 infection.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Subtipo H7N9 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/terapia , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos , Línea Celular , Mapeo Epitopo , Femenino , Pruebas de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza , Inmunización Pasiva , Subtipo H7N9 del Virus de la Influenza A/metabolismo , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Ratones , Ratones Endogámicos BALB C , Mutación , Pruebas de Neutralización , Filogenia
6.
Cell Physiol Biochem ; 54(2): 230-251, 2020 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-32153152

RESUMEN

BACKGROUND/AIMS: Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2). METHODS: Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot. RESULTS: In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process. CONCLUSION: Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.


Asunto(s)
Aerosoles/química , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Material Particulado/toxicidad , Calcio/metabolismo , Línea Celular , Colorantes Fluorescentes/química , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Confocal , Mitocondrias/efectos de los fármacos , Material Particulado/química , Humo/efectos adversos , Factores de Tiempo , Productos de Tabaco/análisis
7.
Emerg Microbes Infect ; 9(1): 571-581, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32172658

RESUMEN

Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the Flaviviridae family. To date, the host factors required for CSFV entry remain poorly characterized. To identify the functional membrane protein(s) involved in CSFV infection, we analyzed the transcriptomic data from previous studies describing gene expression profiles for CSFV, and found twelve novel candidate proteins. One of these proteins, MERTK, significantly reduced CSFV protein expression by RNA interference screening using a recombinant CSFV that contains a luciferase reporter to measure CSFV protein expression. Furthermore, our results demonstrated that either anti-MERTK antibodies or soluble MERTK ectodomain could reduce CSFV infection in PK-15 cells in a dose-dependent manner. Mechanistically, MERTK interacted with the E2 protein of CSFV and facilitated virus entry. After virus entry, MERTK downregulates of mRNA expression of IFN-ß and promotes CSFV infection. Interestingly, the soluble MERTK ectodomain could also reduce the infection of bovine viral diarrhea virus (BVDV), another pestivirus. Taken together, our results suggested that MERTK is a CSFV entry factor that synergistically dampens innate immune responses in PK-15 cells and is also involved in BVDV infection.


Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/inmunología , Inmunidad Innata , Internalización del Virus , Tirosina Quinasa c-Mer/metabolismo , Animales , Bovinos , Línea Celular , Humanos , Recombinación Genética , Porcinos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Tirosina Quinasa c-Mer/genética
8.
Emerg Microbes Infect ; 9(1): 586-596, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32174269

RESUMEN

Since 2015, the prevalence of severe hepatitis-hydropericardium syndrome, which is caused by the novel genotype fowl adenovirus serotype 4 (FAdV-4), has increased in China and led to considerable economic losses. The replication cycle of FAdV-4, especially the emerging highly pathogenic novel genotype FAdV-4, remains largely unknown. The adenovirus fibre interacts with the cellular receptor as the initial step in adenovirus (AdV) infection. In our previous studies, the complete genome sequence showed that the fibre patterns of FAdV-4 were distinct from all other AdVs. Here, protein-blockage and antibody-neutralization assays were performed to confirm that the novel FAdV-4 short fibre was critical for binding to susceptible leghorn male hepatocellular (LMH) cells. Subsequently, fibre 1 was used as bait to investigate the receptor on LMH cells via mass spectrometry. The chicken coxsackie and adenovirus receptor (CAR) protein was confirmed as the novel FAdV-4 receptor in competition assays. We further identified the D2 domain of CAR (D2-CAR) as the active domain responsible for binding to the short fibre of the novel FAdV-4. Taken together, these findings demonstrate for the first time that the chicken CAR homolog is a cellular receptor for the novel FAdV-4, which facilitates viral entry by interacting with the viral short fibre through the D2 domain. Collectively, these findings provide an in-depth understanding of the mechanisms of the emerging novel genotype FAdV-4 invasion and pathogenesis.


Asunto(s)
Adenoviridae/inmunología , Receptores Virales/inmunología , Adenoviridae/genética , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Pollos , Expresión Génica , Humanos , Receptores Virales/genética , Solubilidad , Proteínas Virales/genética , Proteínas Virales/inmunología
9.
Science ; 367(6484): 1366-1371, 2020 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-32193326

RESUMEN

Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria-ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIß [PI(4)KIIIß], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P-containing vesicles at mitochondria-ER contact sites may trigger final events leading to mitochondrial scission.


Asunto(s)
Mitocondrias/metabolismo , Dinámicas Mitocondriales , Fosfatos de Fosfatidilinositol/metabolismo , Red trans-Golgi/metabolismo , 1-Fosfatidilinositol 4-Quinasa/genética , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Animales , Células COS , Línea Celular , Dinaminas/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Células HeLa , Humanos , Microdominios de Membrana , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Interferencia de ARN
10.
Clin Sci (Lond) ; 134(5): 543-545, 2020 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-32167153

RESUMEN

A new coronavirus, referred to as SARS-CoV-2, is responsible for the recent outbreak of severe respiratory disease. This outbreak first detected in Wuhan, China in December 2019, has spread to other regions of China and to 25 other countries as of January, 2020. It has been known since the 2003 SARS epidemic that the receptor critical for SARS-CoV entry into host cells is the angiotensin-converting enzyme 2 (ACE2). The S1 domain of the spike protein of SARS-CoV attaches the virus to its cellular receptor ACE2 on the host cells. We thought that it is timely to explain the connection between the SARS-CoV, SARS-CoV-2, ACE2 and the rationale for soluble ACE2 as a potential therapy.


Asunto(s)
Betacoronavirus/patogenicidad , Infecciones por Coronavirus/tratamiento farmacológico , Peptidil-Dipeptidasa A , Neumonía Viral/tratamiento farmacológico , Virus del SRAS/patogenicidad , Acoplamiento Viral , Animales , Línea Celular , Haplorrinos , Humanos , Peptidil-Dipeptidasa A/fisiología , Proteínas Recombinantes/uso terapéutico , Solubilidad , Replicación Viral
11.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 283-289, 2020 Feb.
Artículo en Chino | MEDLINE | ID: mdl-32027290

RESUMEN

OBJECTIVE: To investigate the effect of bone marrow stromal cell glycosyltransferase B4GALT1 expression on hematopoietic cell proliferation and its upstream regulation mechanism. METHODS: B4GALT1 was overexpressed in human bone marrow stromal cell line HS5, which was then co-cultured with acute myeloid leukemia cell line KG1a. And its effect on hematopoietic cell proliferation was detected by flow cytometry. Dual luciferase reporter assay, real-time PCR and Western blot were used to predict and validate upstream transcription factors that regulate stromal cell B4GALT1 expression. RESULTS: Overexpression of B4GALT1 in HS5 significantly promoted the proliferation of KG1a in the co-culture system. B4GALT1 expression in stromal cells positively correlated with upstream c-Jun expression, which was verified by JNK/c-Jun inhibitors. CONCLUSION: The differential expression of glycosyltransferases and their corresponding glycosylation in the hematopoietic microenvironment play an important role.


Asunto(s)
Galactosiltransferasas/metabolismo , Leucemia Mieloide Aguda , Células Madre Mesenquimatosas , Células de la Médula Ósea , Línea Celular , Proliferación Celular , Técnicas de Cocultivo , Glicosiltransferasas , Humanos , Células del Estroma , Microambiente Tumoral
12.
Gen Physiol Biophys ; 39(1): 27-36, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32039822

RESUMEN

The G protein-coupled estrogen receptor (GPER) was proved to be a new type of estrogen receptor (ER). It is unknown that whether estrogen can regulate the secretion of gonadotrophin releasing hormone (GnRH) in GT1-7 cells through the mechanism with the involvement of GPER. The GnRH, estradiol (17ß-estradiol, E2) and GPER in peripheral blood of precocious puberty children were detected by ELISA and RT-qPCR assays. After E2 treatment, the levels of GPER and GnRH in GT1-7 cells were detected. Following G1 treatment, cell proliferation was examined using a CCK-8 assay. The levels of GnRH, KISS1, GPR54, nNOS, c-FOS in GT1-7 cells were assessed following GT1-7 cells were induced by E2 combined with G1 or G15. GnRH, E2 and GPER were significantly increased in precocious puberty children. After E2 treatment, GT1-7 cells expressed more GnRH and GPER was markedly elevated and reached a peak at 8 h. The KISS1, GPR54 and nNOS in GT1-7 cells were significantly increased with G1 induction, but were significantly decreased with G15 induction compared with E2 induction alone. Collectively, GPER cannot promote the release of GnRH via affecting the proliferation of GT1-7 cells, but it may regulate GnRH through KISS1/GPR54 pathway, which provides novel ideas for precocious puberty children treatment.


Asunto(s)
Pubertad Precoz , Animales , Línea Celular , Estradiol , Estrógenos , Hormona Liberadora de Gonadotropina , Ratones , Receptores Estrogénicos , Receptores Acoplados a Proteínas G
13.
Gen Physiol Biophys ; 39(1): 79-87, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32039827

RESUMEN

Glucose triggers glucagon-like peptide (GLP)-1 secretion from L cells involving several glucose sensors including sodium-glucose transporter (SGLT)1, glucose transporter (GLUT)2, and sweet taste receptors (STRs). This study investigated the effects of different glucose concentrations on GLP-1 secretion, intracellular concentrations of Ca2+ and cAMP, glucose uptake, and protein levels of SGLT1, GLUT2, and STRs in STC-1 cells. Low glucose (5.6 mM) increased GLP-1 secretion, intracellular Ca2+ concentration, and SGLT1 protein level compared with glucose-free group. GLP-1 secretion and intracellular Ca2+ concentration triggered by low glucose were inhibited by the SGLT1 inhibitor. GLP-1 secretion or intracellular Ca2+ concentration in high-glucose (25, 100, 200 mM) groups was significantly higher than that of low-glucose group. Elevation of cAMP level was observed in concentration-dependent manner, and decreased glucose uptake was observed in 100 or 200 mM glucose group. High glucose increased protein levels of STRs and GLUT2 in comparison to low-glucose group. GLP-1 secretion and intracellular levels of Ca2+ and cAMP triggered by high glucose were inhibited in the presence of the GLUT2 or STR inhibitor. These results suggest that SGLT1 is dominantly responsible for GLP-1 secretion triggered by low glucose, and that STRs and GLUT2 are involved in GLP-1 secretion induced by high glucose.


Asunto(s)
Células Enteroendocrinas , Línea Celular , Péptido 1 Similar al Glucagón , Glucosa
14.
Chem Pharm Bull (Tokyo) ; 68(2): 133-139, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32009080

RESUMEN

C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) is a secretory protein that participates in the regulation of glucose and lipid metabolism in obesity and diabetes. Its role in cardiovascular disease, particularly sepsis-induced cardiac injury, is unclear. Here, we stimulated cardiomyocytes with lipopolysaccharide (LPS) to establish an in vitro cardiomyocyte injury model and CTRP12 was overexpressed with an adenovirus delivery system. Overexpression of CTRP12 reduced the transcription and release of pro-inflammatory cytokines from LPS-stimulated cardiomyocytes, including TNFα, interleukin-1 (IL-1), and IL-6. Reactive oxygen species (ROS) level increased and the oxidation/redox system was disturbed in LPS-stimulated cardiomyocytes, as evident from the decrease in superoxide dismutase activity and an increase in reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and malondialdehyde level. CTRP12 overexpression decreased the increasing level of ROS and ameliorated the unbalance in the oxidation/redox system in LPS-stimulated cardiomyocytes. The viability of cardiomyocytes decreased after LPS stimulation, and the cells underwent apoptosis. CTRP12-overexpressing cardiomyocytes showed a decrease in the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells, and the ratio of B cell lymphoma (Bcl)-1/Bax in these cells was recovered. In comparison with the control group, LPS-stimulated cardiomyocytes showed reduced expression of nuclear factor E2-related factor 2 (NRF2), while CTRP12-overexpressing cardiomyocytes showed elevated NRF2 expression. Small-interfering RNA-mediated silencing of NRF2 expression in cardiomyocytes resulted in the inhibition of the protective effects of CTRP12. Thus, CTRP12 ameliorated injury in LPS-stimulated cardiomyocytes in an NRF2-dependent manner.


Asunto(s)
Inflamación/genética , Lipopolisacáridos/inmunología , Miocitos Cardíacos/inmunología , Regulación hacia Arriba , Adenoviridae/genética , Animales , Línea Celular , Células Cultivadas , Regulación hacia Abajo , Técnicas de Transferencia de Gen , Inflamación/inmunología , Inflamación/patología , Interleucina-1/genética , Interleucina-1/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología
15.
Gene ; 735: 144404, 2020 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-32018013

RESUMEN

Glucose uptake in adipocytes is crucial for regulating systemic metabolism. Long noncoding RNAs (lncRNAs), defined as being transcripts with lengths exceeding 200 nucleotides that are not translated, are recently identified regulators of cellular functions. Previously, we have shown that an lncRNA, "down-regulated expression by hepatitis B virus X" (dreh), is involved in glucose transport in skeletal muscle cells. Here, we aimed to examine the involvement of dreh in glucose transport in 3T3-L1 adipocytes. Expression analysis showed that dreh was expressed in 3T3-L1 fibroblasts and adipocytes. Knockdown of dreh expression using its specific siRNAs lowered the glucose concentration of the medium and facilitated [3H]-2-deoxyglucose transport in adipocytes. Additionally, dreh silencing enhanced the protein expression of glucose transporter (GLUT4) in the plasma membrane of adipocytes. Treatment with siRNA against vimentin attenuated the glucose-lowering effect of dreh depletion. These results suggest that the repression of dreh facilitates glucose transport via increased GLUT4 expression in the plasma membrane through the involvement of vimentin in 3T3-L1 adipocytes. In conclusion, dreh is the first observed lncRNA that regulates glucose transport in adipocytes and could serve as a novel therapeutic target for diabetes by modulating adipocyte function. Considering the new function of dreh, we propose that dreh be renamed "down-regulated expression-related hexose/glucose transport enhancer."


Asunto(s)
Adipocitos/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , ARN Largo no Codificante/genética , Vimentina/metabolismo , Animales , Línea Celular , Fibroblastos/metabolismo , Ratones , ARN Largo no Codificante/metabolismo
16.
J Photochem Photobiol B ; 204: 111799, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32018156

RESUMEN

CdSe/CdS core shelled quantum dots (QDs) were prepared by colloidal synthesis using a binary ligand system and a non-coordinating, reusable solvent n-octadecane (nOD). Both the synthesis of CdSe and CdSe/CdS core shelled quantum dots were achieved by hot injection technique at much lower temperatures than reported earlier. The use of binary ligand facilitated enough nucleation and growth. Red shift in absorption spectra, an enhanced crystallite and particle size is evidenced by XRD and TEM respectively, confirming the formation of core shell structure of CdSe/CdS. The synthesized core shells exhibited high fluorescence intensity, long term stability and good mono dispersion, making it a potential material for bio-imaging and sensing. Core shell QDs were modified with mercapto propionic acid (MPA) to impart aqueous solubility. Studies on cytotoxicity of shelled QDs reveal good bio compatibility with a very minimum toxicity of IC50 = 20 µg/L. These QDs were used for sensing E. coli. Ordinary glass slide, modified using plasma etching is surface modified through APTES aiding conjugation of antibodies. Anti- E. coli polyclonal antibody on glass matrix (slide) and antibody conjugated QDs were used for detection of E. coli in a typical sandwich model. The excellent optical transparency of glass and high emission of QDs lead to detection of E.coli with a limit of detection of 50 CFU/mL.


Asunto(s)
Escherichia coli/efectos de los fármacos , Vidrio/química , Puntos Cuánticos/química , Animales , Compuestos de Cadmio/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ratones , Microscopía Fluorescente , Puntos Cuánticos/toxicidad , Compuestos de Selenio/química , Sulfuros/química , Propiedades de Superficie
17.
J Photochem Photobiol B ; 204: 111810, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32028189

RESUMEN

Vitis vinifera L. water extract from red grapevine leaves contains high levels of polyphenols in quantities similar to those found in red grape and grape seeds. Phenolic compounds are the largest group of natural antioxidants with also an anti-inflammatory activity, widely demonstrated both in vitro and in vivo. Interestingly, their antioxidant effect relies not only on the direct radical scavenging activity but also on their ability in modulating cellular signalling transduction pathways. UV radiation exerts multiple effects on skin cells inducing apoptosis, senescence and carcinogenesis. The aim of this study was to investigate the antioxidant and the DNA protective potentials of Vitis vinifera L. water extract against UV-A and UV-B radiation in HaCaT cells, a human keratinocytes cell line. Comet and É£H2AX assays were used to assess DNA damage in UV irradiated cells pre-treated or not with the extract (100 µg/mL). For UV-B, DNA damage resulted significantly increased at 40 mJ/cm2 dose determining cell cycle arrest and apoptosis. For UV-A, DNA damage was significant at 10 J/cm2 while cell cycle arrest and apoptosis were evident only at 25 J/cm2. The extract (1h of pre-treatment) highlights the antioxidant and scavenger activity on the UV-A, while the maintenance of the apoptosis with both UV-A and UV-B must be interpreted as an anti-mutagenic effect.


Asunto(s)
Apoptosis/efectos de los fármacos , Daño del ADN/efectos de la radiación , Extractos Vegetales/farmacología , Rayos Ultravioleta , Vitis/química , Antioxidantes/química , Antioxidantes/farmacología , Apoptosis/efectos de la radiación , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular , Daño del ADN/efectos de los fármacos , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Extractos Vegetales/química , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Vitis/metabolismo
18.
J Photochem Photobiol B ; 204: 111806, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32044619

RESUMEN

The cotton fabrics are a cosmopolitan in usage due to their extraordinary features. The clothes are a very good medium for the growth of pathogenic microorganisms. The nanoparticles have diverse benefits in the biomedical field like drug carrier and as antimicrobials. The current investigation was aimed to synthesize the metallic silver nanoparticles (AgNPs) from the aqueous extract of Curcuma longa leaf and evaluating their antimicrobial and wound healing potential of AgNPs coated cotton fabric. The synthesized AgNPs were characterized by HR-TEM and FT-IR examinations. The formulated AgNPs were coated with cotton fabrics to test their efficiency against the pathogenic microorganisms. The existence of AgNPs in the cotton fabrics was confirmed via the SEM along with EDX analysis. The antimicrobial potential of fabricated AgNPs and its coated cotton fabrics was inspected against the human pathogenic strains. The wound healing efficacy was examined in the L929 cells. The HR-TEM analysis proved the existence of spherical shaped AgNPs. In the antimicrobial activity, the CL-AgNPs loaded cotton fabric was exhibited an appreciable decrease in the growth of pathogenic microorganisms. The crude extract, as well as formulated AgNPs, also exhibited the noticeable antimicrobial potency against the S.aureus, P.aeruginosa, S.pyogenes, and C.albicans. The AgNPs loaded cotton fabrics was displayed the potent wound healing activity in the fibroblast (L929) cells. Consequently, it was concluded that the formulated AgNPs from C.longa coated cotton fabrics may be utilized for the variety of applications in hospital patients and even medical workers to prevent the microbial infection.


Asunto(s)
Antiinfecciosos/química , Fibra de Algodón/análisis , Curcuma/química , Nanopartículas del Metal/química , Plata/química , Animales , Antiinfecciosos/farmacología , Candida albicans/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Curcuma/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Tecnología Química Verde , Nanopartículas del Metal/toxicidad , Ratones , Pruebas de Sensibilidad Microbiana , Extractos Vegetales/química , Hojas de la Planta/química , Hojas de la Planta/metabolismo
19.
Cell Physiol Biochem ; 54(2): 195-210, 2020 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-32083406

RESUMEN

BACKGROUND/AIMS: Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive and chronic interstitial lung disease of unknown cause. IPF is characterized by excessive deposition of extracellular matrix (ECM) and destructive pathological remodeling due to epithelial-to-mesenchymal transition (EMT). Eventually, lung interstitium thickens and stiffens and breathing becomes difficult. It has been well established that the transforming growth factor-ß1 (TGF-ß1)/Smad signaling pathway plays a critical role in the pathogenesis of pulmonary fibrosis. TGF-ß1-mediated activation of mitogen activated protein kinase (MAPK) family affects Smad signaling. p90RSK is a serine/threonine kinase and is activated by the extracellular signal-regulated kinase (ERK) signaling pathway. However, the roles played by p90RSK in TGF-ß1 signaling and the pathogenesis of pulmonary fibrosis remain unknown. METHODS: We investigated whether p90RSK regulates the pathogenesis of pulmonary fibrosis using in vitro and in vivo systems and Western blotting, real-time quantitative PCR, transcriptional activity assays and immunofluorescence studies. RESULTS: Pharmacological inhibition of p90RSK by FMK or inhibition of p90RSK with adenoviral vector encoding a dominant negative form of p90RSK suppressed TGF-ß1-induced ECM accumulation and EMT in lung epithelial cells and fibroblasts. Interestingly, FMK significantly inhibited TGF-ß1-induced Smad3 nuclear translocation and smad binding element-dependent transcriptional activity, but not Smad3 phosphorylation. Furthermore, in a mouse model of bleomycin-induced lung fibrosis, FMK ameliorated pulmonary fibrosis. CONCLUSION: These findings indicate that p90RSK plays critical roles in pulmonary fibrosis, which suggests it be viewed as a novel therapeutic target for the treatment of lung fibrosis.


Asunto(s)
Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteína smad3/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Transición Epitelial-Mesenquimal/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Isoquinolinas/farmacología , Cetonas/farmacología , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Piridinas/farmacología , Pirroles/farmacología , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteína smad3/antagonistas & inhibidores , Proteína smad3/genética , Activación Transcripcional/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
20.
Emerg Microbes Infect ; 9(1): 278-290, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32013758

RESUMEN

Cell death mechanisms are central to combat infections and to drive inflammation. The inflammasome controls infection through activation of caspase-1 leading to either IL-1ß dependent inflammation, or pyroptotic cell death in infected cells. Hemolysins, which are pore-forming toxins (PFTs), alter the permeability of the host target membrane, often leading to cell death. We previously discovered a leukocidin domain-containing PFT produced by the Gram-negative bacterium Vibrio proteolyticus, named VPRH. VPRH constitutes a distinct, understudied class within the leukocidin superfamily, which is distributed among several photogenic Vibrios. Since PFTs of other pathogens were shown to activate the inflammasome pathway, we hypothesized that VPRH-induced cell death is mediated by direct activation of the inflammasome in mammalian immune host cells. Indeed, we found that VPRH induced a two-step cell death in macrophages. The first, a rapid step, was mediated by activating the NLRP3 inflammasome, leading to caspase-1 activation that resulted in IL-1ß secretion and pyroptosis. The second step was independent of the inflammasome; however, its mechanism remains unknown. This study sets the foundation for better understanding the immunological consequences of inflammasome activation by a new leukocidin class of toxins, which may be shared between marine bacteria and give rise to new pathogenic isolates.


Asunto(s)
Inflamasomas/metabolismo , Leucocidinas/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Ratones Endogámicos C57BL , Vibrio/química
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