RESUMEN
The microecological stability of the gut microbiota plays a pivotal role in both preventing and treating colorectal cancer (CRC). This study investigated whether Lactobacillus plantarum CBT (LP-CBT) prevents CRC by inducing alterations in the gut microbiota composition and associated metabolites. The results showed that LP-CBT inhibited colorectal tumorigenesis in azoxymethane/dextran sulfate sodium (AOM/DSS)-treated mice by repairing the intestinal barrier function. Furthermore, LP-CBT decreased pro-inflammatory cytokines and anti-inflammatory cytokines. Importantly, LP-CBT remodeled intestinal homeostasis by increasing probiotics (Coprococcus, Mucispirillum, and Lactobacillus) and reducing harmful bacteria (Dorea, Shigella, Alistipes, Paraprevotella, Bacteroides, Sutterella, Turicibacter, Bifidobacterium, Clostridium, Allobaculum), significantly influencing arginine biosynthesis. Therefore, LP-CBT treatment regulated invertases and metabolites associated with the arginine pathway (carbamoyl phosphate, carboxymethyl proline, L-lysine, 10,11-epoxy-3-geranylgeranylindole, n-(6)-[(indol-3-yl)acetyl]-L-lysine, citrulline, N2-succinyl-L-ornithine, and (5-L-glutamyl)-L-glutamate). Furthermore, the inhibitory effect of LP-CBT on colorectal cancer was further confirmed using the MC38 subcutaneous tumor model. Collectively, these findings offer compelling evidence supporting the potential of LP-CBT as a viable preventive strategy against CRC.
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Colitis , Neoplasias Colorrectales , Microbioma Gastrointestinal , Lactobacillus plantarum , Animales , Ratones , Lactobacillus plantarum/metabolismo , Lisina/farmacología , Citocinas/metabolismo , Metaboloma , Neoplasias Colorrectales/metabolismo , Arginina/metabolismo , Sulfato de Dextran/farmacología , Modelos Animales de Enfermedad , Colitis/microbiología , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: The conversion from estrogen-dependent to estrogen-independent state of ER+ breast cancer cells is the key step to promote resistance to endocrine therapies. Although the crucial role of MAPK/ERK signaling pathway in estrogen-independent breast cancer cell growth is well established, the underlying mechanism is not fully understood. METHODS: In this study, we profiled lncRNA expression against a focused group of lncRNAs selected from lncRNA database. CRISPR/Cas9 was employed to knockout (KO) linc-RoR in MCF-7 cells, while rescue experiments were carried out to re-express linc-RoR in KO cells. Colony formation and MTT assays were used to examine the role of linc-RoR in estrogen-independent growth and tamoxifen resistance. Western blot and qRT-PCR were used to determine the change of protein and lncRNA levels, respectively. The expression of DUSP7 in clinical specimens was downloaded from Oncomine ( www.oncomine.org ) and the dataset from Kaplan-Meier Plotter ( http://kmplot.com ) was used to analyze the clinical outcomes in relation to DUSP7. RESULTS: We identified that linc-RoR functions as an onco-lncRNA to promote estrogen-independent growth of ER+ breast cancer. Under estrogen deprivation, linc-RoR causes the upregulation of phosphorylated MAPK/ERK pathway which in turn activates ER signaling. Knockout of linc-RoR abrogates estrogen deprivation-induced ERK activation as well as ER phosphorylation, whereas re-expression of linc-RoR restores all above phenotypes. Moreover, we show that the ERK-specific phosphatase Dual Specificity Phosphatase 7 (DUSP7), also known as MKP-X, is involved in linc-RoR KO-induced repression of MAPK/ERK signaling. Interestingly, linc-RoR KO increases the protein stability of DUSP7, resulting in repression of ERK phosphorylation. Clinical data analysis reveal that DUSP7 expression is lower in ER+ breast cancer samples than that in ER- breast cancer. Moreover, downregulation of DUSP7 expression is associated with poor patient survival. CONCLUSION: Taken together, these results suggest that linc-RoR promotes estrogen-independent growth and activation of MAPK/ERK pathway of breast cancer cells by regulating the ERK-specific phosphatase DUSP7. Thus, this study might help not only in establishing a role for linc-RoR in estrogen-independent and tamoxifen resistance of ER+ breast cancer, but also suggesting a link between linc-RoR and MAPK/ERK pathway.
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Neoplasias de la Mama/metabolismo , ARN Largo no Codificante/metabolismo , Antineoplásicos Hormonales/farmacología , Western Blotting , Neoplasias de la Mama/genética , Sistemas CRISPR-Cas/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Resistencia a Antineoplásicos/genética , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/metabolismo , Humanos , Células MCF-7 , ARN Largo no Codificante/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tamoxifeno/farmacologíaRESUMEN
Although methylguanine-DNA-methyltransferase (MGMT) plays an important role in resistance to temozolomide (TMZ) in glioma, 40% of gliomas with MGMT inactivation are still resistant to TMZ. The underlying mechanism is not clear. Here, we report that forkhead box M1 (FoxM1) transcriptionally activates the expression of DNA repair gene replication factor C5 (RFC5) to promote TMZ resistance in glioma cells independent of MGMT activation. We showed that RFC5 expression is positively correlated with FoxM1 expression in human glioma cells and FoxM1 is able to transcriptionally activate RFC expression by interaction with the RFC5 promoter. Furthermore, knockdown of FoxM1 or RFC5 partially re-sensitizes glioma cells to TMZ. Consistently, thiostrepton, a FoxM1 inhibitor, in combination with TMZ significantly inhibits proliferation and promotes apoptosis in glioma cells. Taken together, these findings suggest that the FoxM1-RFC5 axis may mediate TMZ resistance and thiostrepton may serve as a potential therapeutic agent against TMZ resistance in glioma cells.
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Dacarbazina/análogos & derivados , Proteína Forkhead Box M1/genética , Glioma/tratamiento farmacológico , Proteína de Replicación C/genética , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Reparación del ADN , Dacarbazina/farmacología , Resistencia a Antineoplásicos , Proteína Forkhead Box M1/metabolismo , Glioma/genética , Glioma/metabolismo , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Regiones Promotoras Genéticas , Proteína de Replicación C/biosíntesis , Proteína de Replicación C/metabolismo , Temozolomida , Tioestreptona/farmacologíaRESUMEN
Neopanaxadiol (NPD), the main panaxadiol constituent of Panax ginseng C. A. Meyer (Araliaceae), has been regarded as the active component for the treatment of Alzheimer's disease. However, few references are available about pharmacokinetic evaluation for NPD. Accordingly, a rapid and sensitive method for quantitative analysis of NPD in beagle dog plasma based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry was developed and validated. Analytes were extracted from plasma by liquid-liquid extraction and chromatographic separation was achieved on an Agilent Zorbax Stable Bond C18 column. Detection was performed in the positive ion mode using multiple reaction monitoring of the transitions both at m/z 461.4 â 425.4 for NPD and internal standard of panaxadiol. All validation parameters, such as lower limit of quantitation, linearity, specificity, precision, accuracy, extraction recovery, matrix effect and stability, were within acceptable ranges and the method was appropriate for multitude sample determination. After oral intake, NPD was slowly absorbed and eliminated from circulatory blood system and corresponding plasma exposure was low. Application of this quantitative method will yield the first pharmacokinetic profile after oral administration of NPD to beagle dog. The information obtained here will be useful to understand the pharmacological effects of NPD.
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Cromatografía Líquida de Alta Presión/métodos , Ginsenósidos/sangre , Ginsenósidos/farmacocinética , Espectrometría de Masas/métodos , Administración Oral , Animales , Perros , Femenino , Ginsenósidos/administración & dosificación , MasculinoRESUMEN
AIM: To investigate the role of interferon regulatory factor 5 (IRF5) in reversing polarization of lung macrophages during severe acute pancreatitis (SAP) in vitro. METHODS: A mouse SAP model was established by intraperitoneal (ip) injections of 20 µg/kg body weight caerulein. Pathological changes in the lung were observed by hematoxylin and eosin staining. Lung macrophages were isolated from bronchoalveolar lavage fluid. The quantity and purity of lung macrophages were detected by fluorescence-activated cell sorting and evaluated by real-time polymerase chain reaction (RT-PCR). They were treated with IL-4/IRF5 specific siRNA (IRF5 siRNA) to reverse their polarization and were evaluated by detecting markers expression of M1/M2 using RT-PCR. RESULTS: SAP associated acute lung injury (ALI) was induced successfully by ip injections of caerulein, which was confirmed by histopathology. Lung macrophages expressed high levels of IRF5 as M1 phenotype during the early acute pancreatitis stages. Reduction of IRF5 expression by IRF5 siRNA reversed the action of macrophages from M1 to M2 phenotype in vitro. The expressions of M1 markers, including IRF5 (S + IRF5 siRNA vs S + PBS, 0.013 ± 0.01 vs 0.054 ± 0.047, P < 0.01), TNF-α (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.019 ± 0.018, P < 0.001), iNOS (S + IRF5 siRNA vs S + PBS, 0.0003 ± 0.0002 vs 0.026 ± 0.018, P < 0.001) and IL-12 (S + IRF5 siRNA vs S + PBS, 0.000005 ± 0.00004 vs 0.024 ± 0.016, P < 0.001), were decreased. In contrast, the expressions of M2 markers, including IL-10 (S + IRF5 siRNA vs S + PBS, 0.060 ± 0.055 vs 0.0230 ± 0.018, P < 0.01) and Arg-1 (S + IRF5 siRNA vs S + PBS, 0.910 ± 0.788 vs 0.0036 ± 0.0025, P < 0.001), were increased. IRF5 siRNA could reverse the lung macrophage polarization more effectively than IL-4. CONCLUSION: Treatment with IRF5 siRNA can reverse the pancreatitis-induced activation of lung macrophages from M1 phenotype to M2 phenotype in SAP associated with ALI.
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Lesión Pulmonar Aguda/metabolismo , Factores Reguladores del Interferón/metabolismo , Activación de Macrófagos , Macrófagos Alveolares/metabolismo , Pancreatitis/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Células Cultivadas , Ceruletida , Modelos Animales de Enfermedad , Femenino , Factores Reguladores del Interferón/genética , Macrófagos Alveolares/patología , Masculino , Ratones Endogámicos C57BL , Pancreatitis/inducido químicamente , Pancreatitis/genética , Pancreatitis/patología , Fenotipo , Interferencia de ARN , Índice de Severidad de la Enfermedad , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Previous studies suggest that early growth response gene-1 (Egr-1) plays an important role in hypoxia-induced drug-resistance. However, the mechanism still remains to be clarified. Herein, we investigated the role of Egr-1 in hypoxia-induced autophagy and its resulted hypoxia-driven chemo-resistance in Hepatocellular Carcinoma (HCC) cells. Our data demonstrated that Egr-1 was overexpressed in HCC tissues and cells and conferred them drug resistance under hypoxia. Mechanistically, Egr-1 transcriptionally regulated hypoxia-induced autophagy by binding to LC3 promoter in HCC cells, which resulted in resistance of HCC cells to chemotherapeutic agents; while dominant negative Egr-1 could inhibit autophagy level, and thus enhanced the sensitivity of HCC cells to chemotherapeutic agents, indicating that hypoxia-induced Egr-1 expression enhanced drug resistance of HCC cells likely through autophagy. Accordingly, it is suggested that a mechanism of hypoxia/Egr-1/autophagy axis might be involved in drug resistance in HCC.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Autofagia , Carcinoma Hepatocelular/tratamiento farmacológico , Resistencia a Antineoplásicos , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Autofagia/efectos de los fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Ciclo Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The growth factor PDGF controls the development of glioblastoma (GBM), but its contribution to the function of GBM stem-like cells (GSC) has been little studied. Here, we report that the transcription factor FoxM1 promotes PDGFA-STAT3 signaling to drive GSC self-renewal and tumorigenicity. In GBM, we found a positive correlation between expression of FoxM1 and PDGF-A. In GSC and mouse neural stem cells, FoxM1 bound to the PDGF-A promoter to upregulate PDGF-A expression, acting to maintain the stem-like qualities of GSC in part through this mechanism. Analysis of the human cancer genomic database The Cancer Genome Atlas revealed that GBM expresses higher levels of STAT3, a PDGF-A effector signaling molecule, as compared with normal brain. FoxM1 regulated STAT3 transcription through interactions with the ß-catenin/TCF4 complex. FoxM1 deficiency inhibited PDGF-A and STAT3 expression in neural stem cells and GSC, abolishing their stem-like and tumorigenic properties. Further mechanistic investigations defined a FoxM1-PDGFA-STAT3 feed-forward pathway that was sufficient to confer stem-like properties to glioma cells. Collectively, our findings showed how FoxM1 activates expression of PDGF-A and STAT3 in a pathway required to maintain the self-renewal and tumorigenicity of glioma stem-like cells.
Asunto(s)
Factores de Transcripción Forkhead/biosíntesis , Glioblastoma/genética , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Factor de Transcripción STAT3/biosíntesis , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/patología , Humanos , Ratones , Células Madre Neoplásicas/patología , Factor de Crecimiento Derivado de Plaquetas/genética , Regiones Promotoras Genéticas , Factor de Transcripción STAT3/genética , Transducción de SeñalRESUMEN
Decreasing oocyte competence with maternal aging is a major factor in mammalian infertility. One of the factors contributing to this infertility is changes to chromatin modifications, such as histone acetylation in old MII stage oocytes. Recent studies indicate that changes in histone acetylation at MII arise at the germinal vesicle (GV) stage. We hypothesised that histone methylation could also change in old GV oocytes. To test this hypothesis, we examined mono-, di- and trimethylation of histone H3 lysine 4 (H3K4 me1, me2 and me3, respectively) in young and older oocytes from 6-8- and 42-44-week-old mice, respectively. We found that H3K4 me2 and me3 decreased in older compared with young GV oocytes (100% vs. 81% and 100% vs. 87%, respectively; P<0.05). H3K4 me2 later increased in older MII oocytes (21% vs. 56%; P<0.05). We also examined the expression of genes encoding the H3K4 demethylases lysine (K)-specific demethylase 1A (Kdm1a) and retinol binding protein 2 (Rbp2). Expression of Kdm1a increased at both the mRNA and protein levels in older GV oocytes, but decreased in older MII oocytes (P<0.05), and was negatively correlated with H3K4 me2 levels. Conversely, expression of Rbp2 mRNA and protein decreased in older GV oocytes (P<0.05), and this was not correlated with H3K4 me3 levels. Finally, we showed that inhibition of Kdm1a of older oocytes at the GV stage restored levels of H3K4 me2 at the MII stage to those seen in 'young' oocytes (41% vs. 38%; P>0.05). These results suggest that changes in expression of H3K4 me2 and Kdm1a in older GV oocytes may represent a molecular mechanism underlying human infertility caused by aging.
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Envejecimiento/fisiología , Núcleo Celular/metabolismo , Metilación de ADN/fisiología , Histonas/metabolismo , Infertilidad Femenina/etiología , Oocitos/metabolismo , Animales , Cartilla de ADN/genética , Femenino , Histona Demetilasas/metabolismo , Inmunohistoquímica , Técnicas de Maduración In Vitro de los Oocitos/métodos , Ratones , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Celulares de Unión al Retinol/metabolismo , TranilciprominaRESUMEN
Extensive and dynamic chromatin remodeling occurs after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate early embryonic development. Histone H3 lysine 4 methylation (H3K4me) is an important epigenetic mechanism that associates with gene-specific activation and functions in development. However, dynamic regulation of H3K4me during early embryonic development remains unclear. Herein, the authors examined the dynamic changes of H3K4me and its key regulators (Ash1l, Ash2l, Kmt2a, Kmt2b, Kmt2c, Setd1a, Setd7, Kdm1a, Kdm1b, Kdm5a, Kdm5b, Kdm5c, and Kdm5d) in mouse oocytes and preimplantation embryos. An increase in levels of H3K4me2 and me3 was observed at the one- to two-cell stages (P < 0.05), corresponding to the period of embryonic genome activation (EGA). Subsequently, the H3K4me2 level dramatically decreased at the four-cell stage and remained at low level until the blastocyst stage (P < 0.05), whereas the H3K4me3 level transiently decreased in the four-cell embryos but steadily increased to the peak in the blastocysts (P < 0.05). The high level of H3K4me2 during the EGA was coinciding with a peak expression of its methyltransferase, ASH2L, which may stabilize this methylation level during this period. Correspondingly, a concomitant decrease in levels of its demethylases, KDM5B and KDM1A, was observed. H3K4me3 was correlated to the expression of its methyltransferase (KMT2B) and demethylase (KDM5A). Thus, these enzymes may function for the EGA and the first lineage segregation in preimplantation mouse embryos.
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Ensamble y Desensamble de Cromatina/fisiología , Metilación de ADN/fisiología , Desarrollo Embrionario/fisiología , Epigénesis Genética/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , N-Metiltransferasa de Histona-Lisina/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Cartilla de ADN/genética , Femenino , Técnica del Anticuerpo Fluorescente , Histona Metiltransferasas , Histonas/metabolismo , Masculino , Ratones , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Recent studies have discovered that nuclear translocation of nerve growth factor (NGF) and its receptor fragments function differently from the traditional model. This study aimed to uncover the nuclear expression of NGF in astrocytoma and its biological significance. METHODS: Ninety-four paraffin-embedded astrocytoma specimens were subjected to immunohistochemical (IHC) and hemotoxylin & eosin (HE) staining. Preoperative cerebrospinal fluid (CSF) specimens and intraoperative snap-frozen astrocytoma tissues were assayed for NGF expression by ELISA and Western blotting. The outcome of patients who contributed samples was tracked. Each ten tissue samples from patients with traumatic brain injury who had received decompression surgery and CSF samples from patients undergoing spinal anesthesia but with no history of nervous system disease were taken as control. RESULTS: NGF-positive immunoreactive products were distributed in both the cytoplasm and nucleus of astrocytoma, but were only located in the cytoplasm of traumatic brain injury (TBI) tissue. NGF nuclear-positive rate (NPR) of grades III - IV astrocytomas (70.0%) was higher than that of grades I - II astrocytoma (28.6%, P < 0.05). NGF-NP expression positively correlated with the NGF concentration in cerebrospinal fluid (CSF) (r = 0.755, P < 0.01). Kaplan-Meier survival analysis indicated that the median survival time was 25 months for NGF-NP astrocytoma grade I - II patients and 42 months in NGF nuclear negative (NGF-NN) astrocytoma grade I - II patients (P < 0.05). In astrocytoma III - IV patients, the median survival was 7 months for NGF-NP patients and 24 months for NGF-NN patients (P < 0.01). Two types of NGF with molecular weights of 13 and 36 kDa were present in astrocytoma, but only the 36 kDa NGF was found in the CSF. NGF expression elevated as the malignancy increased. CONCLUSIONS: NGF-NP expression and NGF level in CSF were significant prognostic factors in astrocytoma patients. Because of the easy access of CSF, it may be developed as an index for early diagnosis and surveillance of astrocytoma.
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Astrocitoma/metabolismo , Biomarcadores/metabolismo , Líquido Cefalorraquídeo/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
We have used semi-quantitative RT-PCR, Western blot, and immunofluorescence imaging approaches to detect the expression levels of Slit2 and its receptor Robo1 in the rat spinal cord after traumatic lesions. Our results revealed that both the mRNA and protein levels of Slit2 were up-regulated in the injured spinal cord. The Slit2 expression level was increased at day 7 until day 14, and then returned to normal level at day 21 after injury. A double-immunolabelling study showed that Slit2 and neurofilament (NF) proteins were both localized in neurons of spinal corda cinerea. Slit2 immunopositivity was detected in neuronal plasma membranes but not in the axonal fibers. In contrast, the immunolabelling of Robo1 in the normal spinal cord was at a low level, mostly in the neurons of spinal corda cinerea, and remained unchanged at all time points following spinal cord injury (SCI). The regulation levels of Slit2 and Robo1 after traumatic lesions in the rat spinal cord are different. Our results indicate that Slit2-Robo1 might not be involved in the inhibitory environment after SCI.
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Axones/metabolismo , Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Proteínas del Tejido Nervioso , Neuronas/metabolismo , Receptores Inmunológicos , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Animales , Axones/patología , Western Blotting , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Proteínas RoundaboutRESUMEN
Discovery of histone lysine specific demethylase 1 (LSD1) indicates that even histone methylation is reversible. Structural analysis shows that LSD1 is a flavin-dependent amine oxidase, which is able to catalyze the specific removal of methyl groups from mono- and dimethylated Lys4 and Lys9 of histone H3. Functional studies demonstrate that LSD1 regulates activation and inhibition of gene transcription in the nucleus, which is known as the innermost gene switch of cells. LSD1 plays important roles in embryonic development and tumorigenesis. Here, we review recent insights into the structure and chemical mechanism of LSD1, and its regulatory roles in development and cancer.
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Histona Demetilasas/metabolismo , Animales , Desarrollo Embrionario , Histona Demetilasas/química , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
BACKGROUND: Implantation of tissue-engineered scaffolds is one of the most promising therapeutic strategies for inducing nerve regenerations following spinal cord injuries. In this paper, we report a novel bioengineered hybrid scaffold comprised of three major extracellular matrix (ECM) proteins. METHODS: ECM-scaffolds (ECM-S) were prepared by gelling fibrinogen, fibronectin and laminin using fresh rat plasma. Olfactory ensheathing cells (OECs) were isolated from fresh rat olfactory mucosa, purified under differential adhesion, and assessed by immunofluorescent staining. OECs were seeded onto ECM-S and cultured. The effects of the scaffolds on the seeded cells were detected using the immunofluorescent staining, Western blotting, scanning electron microscopy and transmission electron microscopy. RESULTS: Tissue-engineered ECM-S could be easily molded into mat-like or cylindrical shapes and gelled by addition of fresh plasma. Observations by electron microscopy show that the ECM-S forms a stable three-dimensional porous network. Studies on the effects of the ECM-S on the biological behaviors of OECs in vitro indicate that the scaffold can promote OEC adhesion, proliferation and process extensions. Additionally, OECs seeded on the scaffold maintained the expression of nerve growth factor, matrix metalloproteinase-3 and matrix metalloproteinase-9. CONCLUSION: We developed a biosynthetic hybrid gel which could be used as a scaffold for OEC transplantation; this gel can promote nerve regeneration following spinal cord injuries.
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Bulbo Olfatorio/citología , Mucosa Olfatoria/citología , Traumatismos de la Médula Espinal/terapia , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Células Cultivadas , Immunoblotting , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Bulbo Olfatorio/fisiología , Bulbo Olfatorio/trasplante , Mucosa Olfatoria/fisiología , Mucosa Olfatoria/trasplante , RatasRESUMEN
A method was developed for the determination of As, Sb, Bi, Cd and Sn in degrees C, iodides of As, Sb, Bi, Cd and Sn were formed and sublimated from the sample matrix and then extracted by diluted HCl. The prepared solution was determined by ICP-AES. The sample treatment procedure, such as the ratio of NH4 I to sample, and sublimation and extraction conditions, was studied. The instrument determination parameters, including ICP power, Ar gas flow rate, spectral wavelengths, background subtraction, spectral interferences correction and internal standard, were selected and optimized. The established procedure is rapid and simple, the detection limits for the five elements are in the range of 0.01-0.35 microg x g(-1), and the analytical results for soil and sediment reference materials were in good agreement with the reference values.
RESUMEN
Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos.
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Epigénesis Genética , Impresión Genómica , Histonas/genética , Histonas/metabolismo , Técnicas de Transferencia Nuclear , Animales , Blastocisto/fisiología , Clonación de Organismos , Femenino , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Masculino , Metilación , Ratones , Ratones Endogámicos ICR , Mórula/fisiología , Oocitos/fisiología , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
An alkaline sample digestion procedure using fusion with lithium metaborate (LiBO2) after dry ashing was developed. Silicon, aluminium and other, a total of 10 elements in different kinds of biological samples, were measured by ICP-AES. The detection limit of this method was 0.15 microg x mL(-1) for Si and 0.03 microg x mL(-1) for Al. The accuracy and precision of the method were demonstrated by analyzing certified reference materials poplar leaves (GBW07604). The precision was less than 3% and the accuracy was -0.03% for Si and -3% for Al. This method was applied to the certification of a series of ten biological reference materials. The results were satisfied.
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Aluminio/análisis , Boratos/química , Litio/química , Silicio/análisis , Espectrofotometría Atómica , Aluminio/química , Análisis de los Alimentos , Límite de Detección , Compuestos de Organosilicio/química , Plantas/química , Estándares de Referencia , Silicio/química , Solventes/química , Espectrofotometría Atómica/normas , TemperaturaRESUMEN
Numerous previous studies demonstrated that gene expression was influenced by histone modifications. However, little information is available about the relation of histone methylation with embryonic gene expression. Here, we examine the significance of histone H3 dimethyl-lysine 4 (H3K4me2) during mouse zygotic genome activation (ZGA) by inhibiting demethylation with the specific histone H3 lysine 4 demethylase inhibitor bisguanidine 1c (1c). A 1c treatment of one-cell embryos did not significantly affect the level of eIF-4C transcripts but did affect Oct4 levels by the two-cell stage. Furthermore, 1c treatment significantly inhibited cleavage of the embryos to the four-cell stage (from 82.7% to 18.2%), and the inhibitory effect was identified to be irreversible. These results suggest that histone methylation may be closely correlated with the formation of a transcriptionally repressive state during ZGA and that the repressive state actually dictates the appropriate pattern of gene expression required for further development.
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Blastocisto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Animales , Factor 1 Eucariótico de Iniciación/metabolismo , Femenino , Metilación , Ratones , Embarazo , Cigoto/metabolismoRESUMEN
OBJECTIVE: To culture olfactory ensheathing cells (OECs) of rats in vitro and to investigate its morphology, mitosis and immunocytochemistry, and to explore if the OECs could be a new donation for transplantation. METHODS: OECs were harvested from olfactory mucosa of Sprague Dawley rats based on the differing rates of attachment of the various cell types, followed by glial fibrillary acidic protein (GFAP), nerve growth factor (NGF), anti-low affinity receptor for NGF (NGFRp75), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and S-100 immunocytochemistry. The morphological changes and mitosis were observed under a phase contrast microscope at different culture time. RESULTS: Three morphologically distinct types of cells, bipolar, multipolar and flat morphology were present in the primary culture of adult rat olfactory mucosa. Mitosis was characterized by a retraction of all processes, forming a sphere that divided into spherical daughter cells, the daughter cells sent out their processes. The OECs were immunoreactive for GFAP, NGFRp75, S-100, NGF, BDNF and NT-3. CONCLUSIONS: The OECs from nasal olfactory mucosa cultivated in the medium with fetal bovine serum could survive, divide, differentiate, and express the neurotrophin. It may become an accessible source for autologous grafting in spinal cord injury.