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BACKGROUND: TRIzolTM is widely used for RNA and DNA extraction. However, this method is laborious and time-consuming. The objective of this study was to validate a time-effective and labor-saving protocol. METHODS: The TRIzol method was used to separate the aqueous phase, protein, and phenol layer of bone marrow samples from 12 patients with hematological diseases. Subsequently, RNA and DNA were extracted from the aqueous layer containing RNA and phenol layer containing DNA, respectively, using magnetic bead extraction kits. The quantity and purity of extracted RNA and DNA were examined using a NanoDrop spectrophotometer. Quantitative fluorescence PCR amplification of the ABL1 gene was performed to verify the effectiveness of the extracted RNA and DNA for downstream experiments. RNA and DNA from another 16 bone marrow samples were extracted to compare the performance of the two methods. RESULTS: Co-extraction of RNA and DNA was completed within 1 h. The data showed that RNA and DNA yield ranged from 13.1 to 204.5 ng/µL and 33.1 to 228.8 ng/µL, respectively. The A260/A280 ratios of RNA and DNA samples ranged from 1.82 to 2.01 and 1.73 to 1.91, respectively. RNA and DNA extracted using this scheme exhibited ideal performance in quantitative fluorescence PCR. The present protocol showed better quality and effectiveness in extracting RNA and DNA compared to the TRIzol method. CONCLUSIONS: This protocol for RNA and DNA co-extraction is fast, labor-saving, and high throughput. It can be adopted for routine molecular biology analyses, particularly for non-reproducible specimens.
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BACKGROUND: T315I mutations of the BCR::ABL1 gene lead to resistance to tyrosine kinase inhibitors (TKIs). This study evaluated the performance of digital droplet polymerase chain reaction (ddPCR) in quantifying T315I mutations and their frequency in Philadelphia chromosome (Ph) positive hematological patients. METHODS: The course of disease and BCR::ABL1 fusion transcripts (e13a2, e14a2 and e1a2) were retrospectively reviewed in 21 patients with acute lymphoblastic leukemia (ALL) and 85 patients with chronic myeloid leukemia (CML). T315I mutation analysis was carried out using ddPCR and the limit of detection was assessed using mutant T315I DNA at varying variant allele fractions. RESULTS: T315I mutations were found in two ALL patients and one CML patient without remission in molecular biology and with mutation burdens of 29.20%, 40.85%, and 3.00%, respectively. The mutation burden of ALL patients was higher than that of CML patients, but there was no significant difference between the two (p-value = 0.0536). The test's limit of detection was 0.02% with a correlation coefficient greater than 0.99 between the expected and actual detection abundances. CONCLUSION: T315I mutations have a high incidence in Ph-positive ALL patients even if the course of disease is short. In molecular biology, T315I mutation detection is indicated for CML patients not in remission.
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INTRODUCTION: HCMV infection is widespread in humans. This retrospective study aimed to explore the relationship between human cytomegalovirus (HCMV) glycoprotein B (gB) genotype distribution, viral load, and the demographic and clinical features of symptomatic infants. The detection rate of HCMV in blood and urine samples was also compared. METHODOLOGY: Retrospective data from 265 infants who underwent urine HCMV DNA testing were analyzed. The viral load and gB genotype were detected in 91 HCMV positive infants by quantitative fluorescence polymerase chain reaction (PCR) and DNA sequencing, respectively. RESULTS: The positive rate of HCMV infection was 46.04% (122/265) in all infants, and increased rapidly with age. Among the 91 infants investigated, liver function abnormality was the most common diagnosis (34/91, 37.36%), followed by pneumonia (21/91, 23.07%). Sequence analysis of gB yielded two genetic subtypes: the most prevalent gB3 (47/91, 51.65%), followed by gB1 (44/91, 48.35%). The gB3 HCMV infection was more prevalent in infants aged 0-2 months than in infants aged 3-12 months (χ2 = 4.38, p = 0.0364). The data showed that ALT and AST levels were significantly higher in the anti-HCMV IgM+IgG- group than in the anti-HCMV IgM+IgG+ and IgM-IgG+ groups. In addition, this study showed that the detection rate of HCMV DNA in the blood was significantly lower than that in the urine (χ2 = 6.7131, p = 0.0096). CONCLUSIONS: This study presents the HCMV infection status of infants and its relationship with their demographic characteristics and clinical manifestations. In addition, this study suggests that urinary PCR is the most appropriate assay for detecting HCMV infections.
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Anticuerpos Antivirales , Citomegalovirus , Proteínas del Envoltorio Viral , Lactante , Humanos , Estudios Retrospectivos , Carga Viral , Citomegalovirus/genética , Genotipo , Inmunoglobulina G , ADN , Inmunoglobulina MRESUMEN
BACKGROUND: High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation. OBJECTIVES: This study evaluated HRM analysis as an option for detecting the single nucleotide polymorphism (SNP) of cytokine, and profiled the distribution of cytokine gene polymorphism in the lung transplant recipients (LTRs). MATERIAL AND METHODS: High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (-308 A/G), tumor growth factor beta 1 (TGF-ß1) (+869 T/C), interleukin 10 (IL-10) (-592 C/A, -819 T/C, -1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) SNPs were developed on the LightCycler® 480. The SNPs of the aforementioned cytokine genes in 322 LTRs and 266 normal controls were detected using HRM-PCR approach. To confirm the accuracy of the HRM-PCR assay, we randomly selected 100 samples from the LTRs and detected the aforementioned SNPs with sequence-specific primer-polymerase chain reaction (SSP-PCR) method, using a commercial kit. RESULTS: The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0). Our data also show that the allele and genotype frequencies of the abovementioned cytokine are not significantly different between the LTRs and the control groups (p > 0.05). In addition, we found the genotypes of TGF-ß1 +869 associated with high expression phenotype were prevalent in the LTRs. On the contrary, for TNF-α -308, IL-10 and IFN-γ, the genotypes associated with low expression phenotype were most common in the LTRs. CONCLUSIONS: In this study, we described a rapid, low-cost and high-throughput HRM-PCR technology for genotyping cytokine SNPs. Our data may be utilized for future studies examining the associations of cytokine gene polymorphisms with the prognosis of the LTRs.
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Interleucina-10 , Factor de Necrosis Tumoral alfa , Citocinas , Genotipo , Humanos , Interferón gamma/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/genética , Pulmón/metabolismo , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta1/genética , Receptores de Trasplantes , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PURPOSE: This study investigated the effects of dexmedetomidine on cardiovascular response during the decannulation period of general anesthesia in patients with different genotypes of angiotensin-converting enzyme (ACE) and essential hypertension. METHODS: The present study enrolled patients with essential hypertension and American Society of Anesthesiologists class II or III who were scheduled to undergo abdominal surgery under general anesthesia. Patients were assigned to 1 of 6 groups according to ACE genotype, as detected by polymerase chain reaction-restriction fragment length polymorphism, as follows: DD; ID; II; and DD, ID, and II each with dexmedetomidine (Dex). Dexmedetomidine was intravenously infused at 0.5 µg/kg/h for 30 min before the end of surgery in groups DD (Dex), ID (Dex), and II(Dex). Anesthesia was induced and maintained by the same anesthetics in all patients. Systolic and diastolic blood pressure, heart rate (HR), ECG, and rate-pressure product were recorded before anesthesia induction; at 30 min before the end of surgery; at the end of surgery; and at 0, 1.5, 5, and 10 min after extubation. FINDINGS: A total of 210 patients were enrolled (n = 35 per genotype). After extubation, systolic and diastolic blood pressure, HR, and RPP were increased markedly from baseline in groups DD, ID, and II; the increases were greater in groups DD and ID than in group II. No significant changes in blood pressure, HR, or RPP were found, and proper sedative was achieved in groups DD (Dex), ID (Dex), and II(Dex). The prevalences of cardiac arrhythmia were higher in groups DD and ID than in groups II, DD (Dex), ID (Dex), and II(Dex). IMPLICATIONS: Patients essential hypertension and the ACE D allele had a strong hemodynamic response to tracheal extubation, on which dexmedetomidine was found to have both a prevention and treatment effect.
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Anestesia General/métodos , Dexmedetomidina/administración & dosificación , Hipertensión Esencial/fisiopatología , Peptidil-Dipeptidasa A/genética , Alelos , Presión Sanguínea/fisiología , Dexmedetomidina/farmacología , Femenino , Genotipo , Frecuencia Cardíaca/fisiología , Hemodinámica/fisiología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Clear cell renal cell carcinoma (ccRCC) is one of the most common and lethal types of cancer within the urinary system. Great efforts have been made to elucidate the pathogeny. However, the molecular mechanism of ccRCC is still not well understood. The aim of this study is to identify key genes in the carcinogenesis and progression of ccRCC. The mRNA microarray dataset GSE53757 was downloaded from the Gene Expression Omnibus database. The GSE53757 dataset contains tumor and matched paracancerous specimens from 72 ccRCC patients with clinical stage I to IV. The linear model of microarray data (limma) package in R language was used to identify differentially expressed genes (DEGs). The protein-protein interaction (PPI) network of the DEGs was constructed using the search tool for the retrieval of interacting genes (STRING). Subsequently, we visualized molecular interaction networks by Cytoscape software and analyzed modules with MCODE. A total of 1,284, 1,416, 1,610 and 1,185 up-regulated genes, and 932, 1,236, 1,006 and 929 down-regulated genes were identified from clinical stage I to IV ccRCC patients, respectively. The overlapping DEGs among the four clinical stages contain 870 up-regulated and 645 down-regulated genes. The enrichment analysis of DEGs in the top module was carried out with DAVID. The results showed the DEGs of the top module were mainly enriched in microtubule-based movement, mitotic cytokinesis and mitotic chromosome condensation. Eleven up-regulated genes and one down-regulated gene were identified as hub genes. Survival analysis showed the high expression of CENPE, KIF20A, KIF4A, MELK, NCAPG, NDC80, NUF2, TOP2A, TPX2 and UBE2C, and low expression of ACADM gene could be involved in the carcinogenesis, invasion or recurrence of ccRCC. Literature retrieval results showed the hub gene NDC80, CENPE and ACADM might be novel targets for the diagnosis, clinical treatment and prognosis of ccRCC. In conclusion, the findings of present study may help us understand the molecular mechanisms underlying the carcinogenesis and progression of ccRCC, and provide potential diagnostic, therapeutic and prognostic biomarkers.
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Traumatic brain injury (TBI) is a common cause of death and disability. Enhancing the midline-crossing of the contralateral corticospinal tract (CST) to the denervated side of spinal cord facilitates functional recovery after TBI. Activation of the gamma isoform of PKC (PKCγ) in contralateral CST implicates its roles in promoting CST remodeling after TBI. In this study, we deployed loss and gain of function strategies in N2a cells and primary cortical neurons in vitro, and demonstrated that PKCγ is not only important but necessary for neuronal differentiation, neurite outgrowth and axonal branching but not for axonal extension. Mechanically, through the phosphorylation of GSK3ß, PKCγ stabilizes the expression of cytosolic ß-catenin and increase GAP43 expression, thus promoting axonal outgrowth. Further, rAAV2/9-mediated delivery of constitutive PKCγ in the corticospinal tract after unilateral TBI in vivo additionally showed that specifically delivery of active PKCγ mutant to cortical neuron promotes midline crossing of corticospinal fibers from the uninjured side to the denervated cervical spinal cord. This PKCγ-mediated injury response promoted sensorimotor functional recovery. In conclusion, PKCγ mediates stability of ß-catenin through the phosphorylation of GSK3ß to facilitate neuronal differentiation, neurite outgrowth and axonal branching, and PKCγ maybe a novel therapeutic target for physiological and functional recovery after TBI.
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Axones/efectos de los fármacos , Lesiones Traumáticas del Encéfalo/fisiopatología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neuronas/citología , Proteína Quinasa C/farmacología , Tractos Piramidales/citología , beta Catenina/metabolismo , Animales , Axones/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Regeneración Nerviosa , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Tractos Piramidales/efectos de los fármacos , Tractos Piramidales/metabolismo , Recuperación de la Función , Transducción de Señal , beta Catenina/genéticaRESUMEN
To date, the molecular mechanism underlying constitutive signal transducer and activator of transcription 3 (STAT3) activation in gliomas is largely unclear. In this study, we report that Smad6 is overexpressed in nuclei of glioma cells, which correlates with poor patient survival and regulates STAT3 activity via negatively regulating the Protein Inhibitors of Activated STAT3 (PIAS3). Mechanically, Smad6 interacts directly with PIAS3, and this interaction is mediated through the Mad homology 2 (MH2) domain of Smad6 and the Ring domain of PIAS3. Smad6 recruits Smurf1 to facilitate PIAS3 ubiquitination and degradation, which also depends on the MH2 domain and the PY motif of Smad6. Consequently, Smad6 reduces PIAS3-mediated STAT3 inhibition and promotes glioma cell growth and stem-like cell initiation. Moreover, the Smad6 MH2 transducible protein restores PIAS3 expression and subsequently reduces gliomagenesis. Collectively, we conclude that nuclear-Smad6 enhances glioma development by inducing PIAS3 degradation and subsequent STAT3 activity upregulation.
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Neoplasias Encefálicas/patología , Glioma/patología , Chaperonas Moleculares/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteína smad6/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Carcinogénesis/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/genética , Estudios de Cohortes , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/mortalidad , Células HEK293 , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Dominios Proteicos , Proteolisis , Transducción de Señal/genética , Tasa de Supervivencia , Ubiquitina-Proteína Ligasas , Ubiquitinación/genética , Regulación hacia Arriba , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
Although an association between obesity and the occurrence of renal cell carcinoma (RCC) has been identified, the mechanism by which obesity functions to increase this risk of cancer remains unclear. Leptin, visfatin, apelin, resistin and adiponectin are peptide hormones secreted by adipocytes; it is considered that these may affect RCC development by exerting effects on proliferation, cell growth and inflammation. The aim of the present study was to investigate the association between the aforementioned adipokine genes and clear cell RCC (CC-RCC). The GSE6344 dataset was downloaded from the Gene Expression Omnibus database, and the relative expression levels of the adipokine genes were analyzed. To verify the results of the mRNA microarray, 77 paired samples of CC-RCC and corresponding adjacent normal tissue were allocated into two groups. The extraction of total RNA was conducted, and the mRNA expression of adipokine genes was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The data from the GSE6344 dataset indicated that the expression of visfatin and apelin was upregulated (P<0.0001 and P<0.01, respectively), and adiponectin was downregulated (P<0.001) in the CC-RCC tissues compared with the adjacent normal tissues. The data from RT-qPCR demonstrated that visfatin and resistin gene expression was increased (P<0.01 and P<0.05, respectively) in the CC-RCC tissues. Furthermore, the mRNA expression level of leptin and adiponectin in the adjacent normal tissue was higher than those in the cancer tissue (P<0.01). The current study verifies that visfatin and adiponectin are associated with an increased risk of CC-RCC, which presents further insights into the molecular mechanisms of CC-RCC tumorigenesis.
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Although several studies have documented the role of leptin receptor gene polymorphisms in cancers, the association between leptin receptor gene polymorphisms and renal cell carcinoma (RCC) remains unknown. The aim of this study was to develop a high-resolution melting (HRM) approach for genotyping single nucleotide polymorphisms of leptin receptor gene on the LightCycler 480, and to explore the relation between polymorphisms of the leptin receptor gene and RCC. The study population consisted of 83 patients with renal cell carcinoma and 161 healthy control subjects. The Lys109Arg (A/G) and Gln223Arg (A/G) polymorphisms of leptin receptor gene were examined with HRM assay. Direct DNA sequencing and PCR-restriction fragment length polymorphisms were used as a reference method for genotyping Lys109Arg and Gln223Arg, respectively. Three genotypes of Lys109Arg or Gln223Arg were clearly distinguishable from the melting curve shapes with HRM assay. The data also showed the results of the direct DNA sequencing or PCR-restriction fragment length polymorphisms analysis were in complete concordance to genotyping results obtained by HRM (kappa=1.0). In addition, the data showed the G-G haplotype frequency was higher (p<0.05), and that the A-G (p<0.001) and G-A (p<0.01) haplotypes frequencies were lower in the RCC than controls. We developed a rapid, low cost, high-throughput and reliable single-tube technology for genotyping Lys109Arg and Gln223Arg polymorphisms. In addition, our data suggest that Lys109Arg and Gln223Arg gene polymorphisms are associated with RCC in Chinese Han studied population.
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Carcinoma de Células Renales/genética , Técnicas de Genotipaje/métodos , Neoplasias Renales/genética , Desnaturalización de Ácido Nucleico/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple/genética , Receptores de Leptina/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Secuencia de Bases , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes/genética , Haplotipos/genética , Humanos , Desequilibrio de Ligamiento/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
Endotracheal intubation elicits a hemodynamic response associated with increased heart rate and blood pressure that is influenced by the angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) genetic polymorphism which may be of importance also for the pressure response to anesthesia. A total of 337 patients underwent abdominal surgery in general anesthesia and 16% were D/D-homozygotes, 45% were I/D heterozygotes and 39% of the patients were I/I homozygotes. Before surgery most patients were in treatment for arterial hypertension. Systolic and diastolic pressure, heart rate and concentrations of catecholamines in blood were determined before and after induction of anesthesia and for up to 10 minutes following endotracheal intubation. Anesthesia decreased blood pressure and for patients presenting ID and DD, blood pressure and heart rate reached similar levels but compared to II-homozygotes, D-carriers demonstrated significantly higher levels for systolic pressure and heart rate before and after intubation (p < 0.05). The blood levels of catercholamines were similar in the three genotype groups. The incidence of ECG-determined myocardial ischemia was higher in D-allele carriers compared to I-allele homozygotes (DD 22%, ID 25% vs. II 5%). In response to anesthesia and intubation and independent of sympathetic nervous activity, D-allele carriers for ACE polymorphism increased blood pressure response and higher risk for development of cardiovascular complications compared to patients homozygous for the I-allele.
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Hemodinámica , Hipertensión , Intubación Intratraqueal/efectos adversos , Peptidil-Dipeptidasa A/genética , Anciano , Anestesia , Presión Sanguínea , Catecolaminas/sangre , Femenino , Humanos , Hipertensión/sangre , Hipertensión/enzimología , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/etiología , Isquemia Miocárdica/genética , Peptidil-Dipeptidasa A/sangre , Polimorfismo GenéticoRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a rare, progressive, and lethal interstitial lung disease with unknown etiology. Divergent observations have suggested that genetic factors contribute to IPF susceptibility. This study investigated the relationship between human leukocyte antigen (HLA), cytokine gene polymorphisms, and IPF in a Chinese Han population. The gene polymorphisms of HLA-A, -B, -DRB1, tumor necrosis factor alpha [TNF-α (-308 A/G)], transforming growth factor beta [TGF-ß1 (+869 T/C)], interleukin 10 [IL-10 (-592 C/A, -819 T/C, and -1082 G/A)], and interferon gamma [IFN-γ (+874 T/A)] were detected in 102 individuals with IPF and 266 unrelated normal controls using PCR with sequence-specific primers and a high-resolution melt (HRM) approach. The data showed that there was no difference in HLA allele frequencies between the IPF and control groups. However, the data showed the frequency of HLA-A*02-DRB1*04 haplotype in the IPF group was significantly higher than that in the control group [odds ratio (OR) = 4.69, 95% confidence interval (CI) = 1.82-12.08, p < 0.001]. In addition, no differences in the allele and genotype distributions of the cytokines were found between the IPF and control groups (p > 0.01). Our findings suggest that there is an association between specific HLA haplotype and IPF genetic susceptibility and that the genetic variability of some cytokines may not be involved in the pathogenesis of IPF.
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Citocinas/genética , Predisposición Genética a la Enfermedad , Antígenos HLA/genética , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/inmunología , Polimorfismo de Nucleótido Simple/genética , Alelos , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Haplotipos/genética , Humanos , Desequilibrio de Ligamiento/genética , Masculino , Persona de Mediana EdadRESUMEN
We previously reported that glucocorticoid receptor ß (GRß) regulates injury-mediated astrocyte activation and contributes to glioma pathogenesis via modulation of ß-catenin/T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activity. The aim of this study was to characterize the mechanism behind cross-talk between GRß and ß-catenin/TCF in the progression of glioma. Here, we reported that GRß knockdown reduced U118 and Shg44 glioma cell proliferation in vitro and in vivo. Mechanistically, we found that GRß knockdown decreased TCF/LEF transcriptional activity without affecting ß-catenin/TCF complex. Both GRα and GRß directly interact with TCF-4, while only GRß is required for sustaining TCF/LEF activity under hormone-free condition. GRß bound to the N-terminus domain of TCF-4 its influence on Wnt signaling required both ligand- and DNA-binding domains (LBD and DBD, respectively). GRß and TCF-4 interaction is enough to maintain the TCF/LEF activity at a high level in the absence of ß-catenin stabilization. Taken together, these results suggest a novel cross-talk between GRß and TCF-4 which regulates Wnt signaling and the proliferation in gliomas.
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Glioma/patología , Proteínas de Neoplasias/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Neoplasias de la Mama/patología , Células COS , División Celular , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Xenoinjertos , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Transfección , Ensayo de Tumor de Célula MadreRESUMEN
Multidrug resistance (MDR) to chemotherapeutic agents is a major obstacle to curative treatment of cancer. In various types of cancers, overexpression of glucosylceramide synthase (GCS) has been observed to be associated with MDR, thus making GCS a target for reversal of resistance. Our previous work demonstrated that GCS and Bcl-2 are co-overexpressed in the K562/A02 leukemia multidrug-resistant cell line compared with its sensitive counterpart, K562. In the present study, we investigated the effects of GCS on apoptosis in K562/A02 and the associated molecular mechanisms. Our results indicate that the inhibition of GCS caused downregulation of Bcl-2 as well as apoptosis enhancement in response to ADM via the ERK pathway, while JNK or p38 MAPK signaling appeared to play less significant roles in the regulation of apoptosis and MDR in K562/A02 cells. Targeting GCS by siRNA also enhanced ceramide accumulation, which is involved in GCS knockdown-induced inhibition of ERK activation and Bcl-2 expression levels.
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Glucosiltransferasas/metabolismo , Leucemia/genética , Leucemia/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Butadienos/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glucosiltransferasas/genética , Humanos , Células K562 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Nitrilos/farmacología , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Infection and rejection are common complications faced by lung transplant recipients (LTRs) and have become major impediments to long-term survival. Cytokines may play an important role in the development of these complications. In this study, we explored the correlation between TNF-α (-308 A/G), TGF-ß1 (+869 T/C, +915 G/C), IL-10 (-592 C/A, -819 T/C, -1082 G/A), IL-6 (-174 G/C), and IFN-γ (+874 T/A) gene polymorphisms and the incidence of acute rejection and infection. Transplant outcomes were reviewed in a retrospective cohort of 113 LTRs from a single center between December 2004 and November 2012. Cytokine polymorphisms were measured using sequence-specific primer-based PCR. HLA typing was performed for the donors and recipients. We found that the LTRs with the IL-10 -819 CC and -592 CC genotypes had a significantly decreased risk of infection (p = 0.017, OR = 0.177, 95% CI = 0.04-0.85). However, we found no significant association between cytokine polymorphisms and acute rejection. Furthermore, the data revealed that the occurrence of acute rejection was strongly associated with infection episodes (χ(2) = 8.5256, p < 0.01). These results suggest that LTRs possessing the IL-10 -819 CC and -592 CC genotype may be protected from the occurrence of infection. Our results demonstrated that infection is an important cause of acute rejection for LTRs.
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Citocinas/genética , Rechazo de Injerto/genética , Infecciones/genética , Enfermedades Pulmonares/genética , Trasplante de Pulmón , Polimorfismo Genético/genética , Adolescente , Adulto , Anciano , Biomarcadores/análisis , Femenino , Estudios de Seguimiento , Tasa de Filtración Glomerular , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Prueba de Histocompatibilidad , Humanos , Interferón gamma/genética , Interleucina-10/genética , Interleucina-6/genética , Enfermedades Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Pronóstico , Factores de Riesgo , Tasa de Supervivencia , Factor de Crecimiento Transformador beta1/genética , Receptores de Trasplantes , Factor de Necrosis Tumoral alfa/genética , Adulto JovenRESUMEN
Amyloid-ß (Aß) protein and its precursor, amyloid-ß precursor protein (ß-APP), have traditionally been used in the diagnosis of Alzheimer disease. Their use in diagnosis of traumatic brain injury by forensic analysis is becoming more widespread. However, to date, no reliable small animal model exists to evaluate these brain injury indicators. To address this, we have studied primary brain-stem injury in rats to assess the appearance of diffuse axonal injury in brain sections and correlate these findings with appearance of Aß and relative ß-APP mRNA levels. Using an EnVision 2-step immunohistochemical staining method to measure axon diameter, we found that there was significant difference in axon diameters within the medulla oblongata and several time points after brain injury, ranging from 3 to 24 hours. In addition, mRNA expression levels of ß-APP increased following brain injury, peaking 3 hours following injury and decreasing back to baseline levels by 24 hours after injury. These results suggest that using immunohistochemistry and reverse transcription-polymerase chain reaction to detect changes in Aß-associated axonal changes and ß-APP mRNA levels, respectively, can be useful for the diagnosis of diffuse axonal injury during autopsy at early time points following fatal brain injury.
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Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Axones/metabolismo , Tronco Encefálico/lesiones , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Axones/patología , Tronco Encefálico/patología , Lesión Axonal Difusa/metabolismo , Lesión Axonal Difusa/patología , Patologia Forense , Inmunohistoquímica , Modelos Animales , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Although roles of genetic polymorphisms of leptin receptor (LEPR) gene in several cancers have been documented, the association between polymorphisms of LEPR and clear cell renal cell carcinoma (CC-RCC) remains unknown. The aim of this study was to explore any relation. MATERIALS AND METHODS: The study population consisted of 77 patients with CC-RCC and 161 healthy control subjects. Polymorphism analyses of Lys109Arg and Gln223Arg were performed by direct DNA sequencing and PCR-restriction fragment length polymorphism approaches respectively. RESULTS: Comparisons of allelic and genotypic frequencies in Lys109Arg and Gln223Arg showed no significant difference between the cases and controls. However, when evaluating the combined genotype of Lys109Arg and Gln223Arg, risk with GG/GG was increased (OR=1.85, 95%CI=1.04-3.30) and with GA/GG or GG/GA was decreased (OR=0.07, 95%CI=0.01-0.54; OR and 95%CI of the latter could not be calculated for a value of zero) . Furthermore, the G-G haplotype frequency of Lys109Arg and Gln223Arg in the cases was higher (OR=1.68; 95%CI=1.02-2.76). In contrast, the A-G and G-A haplotype frequencies in the cases were lower than those in the controls (OR=0.06; 95%CI=0.01 to 0.47; OR and 95%CI of the latter could not be calculated for a value of zero). In addition, the Lys109Arg A allele was in LD with the Gln223Arg A allele (d'=0.9399) in the CC-RCC subjects, but not in the controls. CONCLUSIONS: Our data suggest that the GG/GG combined genotype and G-G haplotype of Lys109Arg and Gln223Arg can act as evaluating factors for CC-RCC risk.
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Carcinoma de Células Renales/genética , Receptores de Leptina/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Riesgo , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
OBJECTIVE: To investigate whether transcription factor-kappaB (NF-κ B) is involved in the modulation of P-glycoprotein (P-gp) by glucosylceramide synthase (GCS) in a multidrug resistance leukemia cell line K562/A02 and to explore the relationship between NF-κ B and extracelluar signal-regulated kinase (ERK). METHODS: K562/A02 cells were treated with GCSsiRNA, pyrrolidine dithiocarbamate (PDTC, a NF-κ B specific inhibitor) and U0126 (a MEK1/2 inhibitor), respectively. The expression of GCS and multidrug resistance protein 1 (MDR1) mRNA were analyzed with qRT-PCR. Various proteins of different groups were measured by Western blotting. RESULTS: After transfected with GCSsiRNA for 48 h, GCS mRNA were reduced by 62% (51%-73%) and MDR1 mRNA was reduced by 52% (43%-61%) in the K562/A02 cells. Compared with the negative control, relative expression of NF-κ B p65 in nuclear and P-ERK1/2 were both down-regulated, and P-gp was also inhibited significantly at 72 h after transfected with GCSsiRNA (P< 0.05). In addition, the expression of P-gp was decreased at 24 h with 80 µ mol/L PDTC and 48 h with 20 µ mol/L PDTC. P-ERK1/2 was inhibited significantly when the cells were treated with 20 µ mol/L U0126 for 48 h. The expression of NF-κ B p65 in nuclear and P-gp were also down-regulated. CONCLUSION: NF-κ B can modulate the effect of GCS on P-gp in K562/A02 cells. P-ERK1/2 can activate NF-κ B in above signal transduction pathway.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Leucemia/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Células K562RESUMEN
Astrocytes react to central nervous system (CNS) injury and participate in gliotic responses, imparting negative, as well as positive effects on axonal regeneration. Despite the considerable biochemical and morphological changes astrocytes undergo following insult, and the known influence of steroids on glial activation, details surrounding glucocorticoid receptor expression and activity are lacking. Such mechanistic information is essential for advancing and enhancing therapies in the treatment of CNS injuries. Using an in vitro wound-healing assay, we found glucocorticoid receptor ß (GRß), not GRα, is upregulated and acts as a regulator of gliosis after injury. In addition, our results suggest that GRß interacts with ß-catenin and is a necessary component for proliferation and migration in both injured astrocytes and glioma cells. Further analysis indicated GRß/ß-catenin interaction as a key modulator of astrocyte reactivity through sustained Wnt/ß-catenin/TCF signaling in its dominant-negative effect on GRα mediated trans-repression by a GSK-3ß-independent manner. These findings expand our knowledge of the mechanism of GRß action in promoting astrocyte proliferation and migration following injury and in glioma. This information furthers our understanding the function of glucocorticoid receptor in CNS injury and disease, as well as in the basic biochemical responses astrocytes undergo in response to injury and glioma pathogenesis.
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Astrocitos/metabolismo , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Glioma/patología , Receptores de Glucocorticoides/metabolismo , Factores de Transcripción TCF/metabolismo , beta Catenina/metabolismo , Astrocitos/efectos de los fármacos , Ciclo Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Invasividad Neoplásica , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de Glucocorticoides/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de Transcripción TCF/genética , Activación Transcripcional , beta Catenina/genéticaRESUMEN
OBJECTIVE: Suppressors of cytokine signaling (SOCS) have been shown to play an important role in regulating cytokines, such as intracellular interferon (IFN) and interleukin (IL), in the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. At present, the association between SOCS and asthma is still under study. The aim of this study is to explore the relationship of SOCS-1 and SOCS-3 mRNA expression in peripheral blood mononuclear cells (PBMCs) with the intracellular IFN-'/IL-4 ratio in CD4+ T cells and specific IgE (sIgE) level in children with asthma. METHODS: BMCs were collected from 44 children with allergic asthma (4-14 years) and 30 healthy children. The intracellular IFN-'/IL-4 ratio in CD4+ T cells was measured by flow cytometry. Total RNAs were extracted from the PBMCs and SOCS-1 and SOCS-3 mRNA expression was measured by SYBR Green I quantitative RT-PCR. RESULTS: Compared with the healthy children, children with allergic asthma showed a lower level of intracellular IFN-' in peripheral blood [(15.7±2.0)% vs (19.1±2.7)%] and IFN-'/IL-4 ratio (3.4±1.5 vs 4.8±2.9) and higher SOCS-1 mRNA expression (-Ct, 11.1±1.9 vs 12.6±2.8). There was a negative relationship between SOCS-1 mRNA expression and the percentage of IFN-'-producing CD4+ T cells in peripheral blood in both asthmatic and healthy children (P<0.05). No correlation was found between SOCS-1 and SOCS-3 expression and sIgE level. CONCLUSIONS: Children with allergic asthma have elevated levels of SOCS-1 mRNA in PBMCs, which is associated with Th2-skewed immune response.