RESUMO
Gemcitabine plus cisplatin (GP) chemotherapy is the standard of care for nasopharyngeal carcinoma (NPC). However, the mechanisms underpinning its clinical activity are unclear. Here, using single-cell RNA sequencing and T cell and B cell receptor sequencing of matched, treatment-naive and post-GP chemotherapy NPC samples (n = 15 pairs), we show that GP chemotherapy activated an innate-like B cell (ILB)-dominant antitumor immune response. DNA fragments induced by chemotherapy activated the STING type-I-interferon-dependent pathway to increase major histocompatibility complex class I expression in cancer cells, and simultaneously induced ILB via Toll-like receptor 9 signaling. ILB further expanded follicular helper and helper type 1 T cells via the ICOSL-ICOS axis and subsequently enhanced cytotoxic T cells in tertiary lymphoid organ-like structures after chemotherapy that were deficient for germinal centers. ILB frequency was positively associated with overall and disease-free survival in a phase 3 trial of patients with NPC receiving GP chemotherapy ( NCT01872962 , n = 139). It also served as a predictor for favorable outcomes in patients with NPC treated with GP and immunotherapy combined treatment (n = 380). Collectively, our study provides a high-resolution map of the tumor immune microenvironment after GP chemotherapy and uncovers a role for B cell-centered antitumor immunity. We also identify and validate ILB as a potential biomarker for GP-based treatment in NPC, which could improve patient management.
Assuntos
Cisplatino , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/patologia , Cisplatino/uso terapêutico , Gencitabina , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/etiologia , Neoplasias Nasofaríngeas/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Desoxicitidina/uso terapêutico , Microambiente TumoralRESUMO
Doxorubicin is one of the most widely used antitumor drugs and is currently produced via the chemical conversion method, which suffers from high production costs, complex product separation processes, and serious environmental pollution. Biocatalysis is considered a more efficient and environment-friendly method for drug production. The cytochrome daunorubicin C-14 hydroxylase (DoxA) is the essential enzyme catalyzing the conversion of daunorubicin to doxorubicin. Herein, the DoxA from Streptomyces peucetius subsp. caesius ATCC 27952 was expressed in Escherichia coli, and the rational design strategy was further applied to improve the enzyme activity. Eight amino acid residues were identified as the key sites via molecular docking. Using a constructed screening library, we obtained the mutant DoxA(P88Y) with a more rational protein conformation, and a 56% increase in bioconversion efficiency was achieved by the mutant compared to the wild-type DoxA. Molecular dynamics simulation was applied to understand the relationship between the enzyme's structural property and its substrate-binding efficiency. It was demonstrated that the mutant DoxA(P88Y) formed a new hydrophobic interaction with the substrate daunorubicin, which might have enhanced the binding stability and thus improved the catalytic activity. Our work lays a foundation for further exploration of DoxA and facilitates the industrial process of bio-production of doxorubicin.
Assuntos
Sistema Enzimático do Citocromo P-450 , Daunorrubicina , Daunorrubicina/metabolismo , Simulação de Acoplamento Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Doxorrubicina/química , Conformação ProteicaRESUMO
A novel exopolysaccharide (EPS) from Paenibacillus polymyxa PYQ1 was extracted, well purified and characterized. This EPS was homogeneous glucomannan-type polysaccharide with the average molecular weight of 4.38 × 106 Da. Structural characterization indicated that the monosaccharides of EPS were pyranoses connected by ß-glycosidic linkages. Furthermore, our results showed the protective benefits of EPS against UVC induced cytotoxicity in HaCaT cells through scavenging excessive reactive oxygen species, mitigating the decrease of mitochondrial membrane potential, improving catalase activity and maintaining membrane integrity. Taken together, this study qualified EPS from P. polymyxa PYQ1 was a promising natural polymer which worth further investigation as a skin-care agent.
Assuntos
Citoproteção/efeitos dos fármacos , Paenibacillus polymyxa/metabolismo , Polissacarídeos Bacterianos/isolamento & purificação , Polissacarídeos Bacterianos/farmacologia , Raios Ultravioleta/efeitos adversos , Catalase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos da radiação , Peso Molecular , Monossacarídeos/análise , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Head and neck squamous cell carcinomas (HNSCCs) harbor a subset of cells that are CD44(+) and present with malignancy and radiotherapy resistance. As a key regulator of self-renewal, Nanog expression not only determines cell fate in pluripotent cells but also mediates tumorigenesis in cancer cells; thus, we examined the role of Nanog in CD44(+) HNSCC. Three HNSCC cell lines, tumor xenografts, and patient tumors were examined. Nanog levels were significantly higher in CD44(+) HNSCC spheroids than in CD44(-) spheroids, and further increased when grown as spheroids to enrich for CSCs. CD44(+) spheroids showed a 3.4-7.5-fold increase in migration and invasion compared with CD44(-) spheroids and were resistant to radiation therapy, which was reversed by inhibiting Nanog. Nanog knockdown also decreased spheroid formation by 66.5-68.8%. Moreover, a phosphokinase array identified upregulated ERK1/2 signaling in CD44(+) HNSCC cells compared with that in CD44(-) cells. ERK1/2 signaling was found to regulate Nanog expression, aiding tumor progression, metastasis, and radiotherapy resistance. In xenograft models, the combination of radiation and Nanog or ERK1/2 inhibition inhibited tumor growth by 75.6% and 79.1%, respectively. In lung metastasis models, CD44(+) cells injected into the tail vein of mice led to significantly more lung metastases and higher Nanog expression level compared with that by ERK1/2-knockdown CD44(+) cells. Finally, in tumor tissues, CD44 and Nanog expression levels were correlated with tumorigenesis in HNSCC patients. Thus, targeting Nanog and the ERK1/2 signaling pathway may prevent or reverse CSC phenotypes and epithelial-mesenchymal transition that drive tumor progression, metastasis, and radiotherapy resistance in HNSCC.
Assuntos
Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Animais , Transição Epitelial-Mesenquimal , Humanos , Masculino , Camundongos , Camundongos Nus , Fenótipo , Transdução de SinaisRESUMO
The grain contamination by Aspergillus spp. has been a serious issue. This study exhibited the excellent antifungal effects of the essential oil compounds (EOCs) geraniol and citral against common grain pathogens (A. flavus and A. ochraceus) in vitro and in situ. The inhibitory mechanisms were also evaluated from the perspective of cell membrane permeability, reactive oxygen species (ROS) generation, and Aspergillus spp. growth-related gene expression. Meanwhile, the combined effects of EOCs in the vapor phase and modified atmosphere packaging (MAP) were examined to find an alternative preservation method for controlling Aspergillus spp. The results indicated that citral exhibited the antifungal activity mainly by downregulating the sporulation- and growth-related genes for both pathogens. Geraniol displayed inhibitory effectiveness against A. flavus predominantly by inducing the intracellular ROS accumulation and showed toxicity against A. ochraceus principally by changing cell membrane permeability. Furthermore, the synthetic effects of EOCs and MAP (75% CO2 and 25% N2) induced better grain quality than the current commercial fumigant AlP. These findings reveal that EOCs have potential to be a novel grain preservative for further application.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Aspergillus flavus/efeitos dos fármacos , Aspergillus ochraceus/efeitos dos fármacos , Monoterpenos/química , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Terpenos/química , Monoterpenos Acíclicos , Aspergillus flavus/genética , Aspergillus flavus/ultraestrutura , Aspergillus ochraceus/genética , Aspergillus ochraceus/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Grão Comestível/microbiologia , Perfilação da Expressão Gênica , Testes de Sensibilidade Microbiana , Espécies Reativas de Oxigênio/metabolismoRESUMO
Class three semaphorins were originally identified as mediators of axon guidance, which repelled axons and collapsed growth cones. As a member of class three semaphorins, semaphorin3F (Sema3F) has been found to have similar effects on tumor cells and endothelial cells and also is implicated in the signaling of tumor metastasis by forming a complex with neuropilins and plexins. In this study, our laboratory produced a monoclonal antibody against the C-terminal domain of Sema3F (Sema3Fc mAb) using the hybridoma method, expecting to explore the potential role of the antibody and its application in the detection of Sema3F. The capture enzyme-linked immunosorbent assay (ELISA) method indicated that mAb belonged to the IgM subclass and purified Sema3Fc mAb had a titer of 5.12 × 105 against Sema3Fc by indirect ELISA. In addition, results showed that the Sema3Fc mAb could be applied in such experiments as Western blotting, flow cytometry, immunofluorescence, and immunocytochemical staining. It indicates the Sema3Fc mAb is available in the detection of Sema3F with specificity and will help further study the role and mechanism of Sema3F among tumor cells.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Anticorpos Monoclonais/isolamento & purificação , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Células Hep G2 , Humanos , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Domínios ProteicosRESUMO
OBJECTIVE: To investigate the effect of DNA methyhransferase l (DNMT1) gene silencing on methylation of suppressor of cytokine signaling (SOCS-1) in multiple myeloma RPMI 8226 cells. METHODS: Recombinant plasmid pshRNA-DNMTl was transfected into multiple myeloma RPMI 8226 cells by lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of DNMTl in RPMI 8226 cells respectively before and after transfection. Methylation-specific polymerase chain reaction was used to detect the methylation level change of SOCS-1 gene in RPMI8226 cells transfected. RESULTS: DNMTl targeted short hairpin RNA(shRNA) was successfully inserted into the plasmid vector pshRNA. RT-PCR results showed that the relative mRNA expression level of DNMTI gene in RPMI 8226 cells transfected with pshRNA was 0.176±0.004 which was significantly lower than that in cells transfected by empty vector (0.956±0.033, P<0.01). Western blot analysis showed that the relative expression level of DNMT1 protein of RPMI 8226 cells transfected by pshRNA was 0.065±0.014, which was significantly lower than that in transfected cells by empty vector(0.415±0.027) (P<0.05). These results indicated that the recombinant plasmid pshRNA could effectively knock down the expression of DNMT1 gene in RPMI 8226 cells. Methylation analysis showed that the methylation level of SOCS-1 gene was obviously reduced after transfection. CONCLUSION: DNMT1 gene in RPMI 8226 cell can be silenced by shRNA. DNMT1 gene silencing can significantly induce SOCS-1 gene hypomethylation, which indicates that DNMT1 may play an important role in the process of SOCS-1 hypermethylation.
Assuntos
Metilação de DNA , Inativação Gênica , Mieloma Múltiplo , Linhagem Celular Tumoral , Vetores Genéticos , Humanos , RNA Mensageiro , RNA Interferente Pequeno , Proteínas Repressoras , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina , TransfecçãoRESUMO
The aim of the present study was to assess the effects of sprouty homolog 2 (SPRY2) gene regulation by miR-21 on the occurrence, development and tumor metastasis in multiple myeloma (MM). The miR-21 expression lentiviral vector (LV)-anti-miR-21 and a liposome transfection method were used to screen MM cell lines with stable silent SPRY2. Real-time quantitative polymerase chain reaction (PCR) and western blot analyses were used to detect SPRY2 expression and miR-21 protein expression levels. An MTT assay was used to assess cell proliferation. Flow cytometry was used for analysis of cell cycle. A scratch test/wound healing assay was used to detect the cell migration ability. A Transwell assay was used to detect the cell invasion ability. Real-time quantitative PCR and western blot analysis showed that in the MM cell lines with high endogenous miR-21 expression (RPMI8226 and KM3), SPRY2 expression was significantly lower. Conversely, in the U266 cell line with low endogenous miR-21 expression, SPRY2 expression was significantly higher, and the gray values of miR-21 and SPRY2 protein in the respective cell lines showed statistically significant differences (P<0.01). Following transfection of U266 cells, the expression of miR-21 in the U266/LV-anti-miR21 lentiviral multiplicity of infection (MOI) 20 group and -MOI 40 group decreased significantly compared with that in the untransfected U266 group (P<0.05). SPRY2 protein expression in U266 cells transfected with miR-21 mimics was significantly reduced compared with that in the non-transfected (untreated) group and the negative control-transfected group (P<0.01). An MTT assay showed that compared with the non-transfected and negative control groups, the cell growth rate as well as the proliferation rate were significantly decreased in the transfection group 48, 72 and 96 h after transfection (P<0.01). Flow cytometric analysis showed that 48 and 72 h after transfection of U266 cells with miR-21 mimics, the apoptotic rates were (24.7 ± 1.97 and 38.6 ± 1.56%) in the U266 group, (27.3 ± 1.72 and 37.3 ± 1.59%) in the siRNA group and (12.7 ± 1.27 and 22.1 ± 1.63%) in the U266/miR-21 group. Compared with the two control groups, the apoptotic rate in the U266/miR-21 group was significantly decreased and the G0/G1 phase cell population was significantly reduced (P<0.05). Scratch experiments showed that the cell migration ability was significantly reduced in the transfection group 24 and 48 h after transfection (P<0.05). A Transwell invasion assay confirmed that the number of U266 cells which migrated through a Matrigel-covered polyphosphate membrane significantly decreased in the transfection group 24 and 48 h after transfection. The cell-penetrating ability was also significantly decreased (P<0.05). In conclusion, the downregulation of SPRY2 gene expression mediated by miR-21 promotes the proliferation and invasion of MM cells in vitro, suggesting that miR-21 may be a novel potential molecular therapeutic target in the treatment of MM.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Oligonucleotídeos Antissenso/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
OBJECTIVE: To evaluate the effects of neonatal exposure to different doses of bisphenol A (BPA) on the vaginal opening day (VOD), hypothalamic Kiss-1 mRNA expression, and ovarian estrogen receptor (ER) mRNA expression in female rats. METHODS: Neonatal female Sprague-Dawley (SD) rats were randomly divided into six groups: blank control, vehicle, 17ß-estradiol (17ß-estradiol, E2, 10â µg/d), low-dose BPA [25â µg(kg·d)], medium-dose BPA [50â µg(kg·d)], and high-dose BPA groups [250â µg(kg·d)]. The rats were subcutaneously injected with respective agents on postnatal days 0-6. The VOD was recorded, and each rat was sacrificed on the same day. The hypothalamus and ovary were taken and weighed, and the organ coefficients of hypothalamus and ovary were calculated. The hypothalamic Kiss-1 mRNA expression and ovarian ERα and ERß mRNA expression were measured by real-time PCR. RESULTS: Compared with the control group, the E2 and medium- and high-dose BPA groups had advanced VOD, and the E2 group had significantly reduced hypothalamic Kiss-1 mRNA expression and ovarian ERß mRNA expression (P<0.05). CONCLUSIONS: Neonatal exposure to medium- and high-dose BPA[50 and 250 µg/(kg·d)] can induce precocious puberty in rats, but it may not result from the change in hypothalamic Kiss-1 mRNA expression. Neonatal exposure to low-dose BPA [25â µg/(kg·d)] does not induce precocious puberty in rats.
Assuntos
Compostos Benzidrílicos/toxicidade , Fenóis/toxicidade , Maturidade Sexual/efeitos dos fármacos , Envelhecimento , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Kisspeptinas/genética , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/genéticaRESUMO
CYP450 3A4 (CYP3A4), encoded by the CYP3A4 gene, is a major enzyme catalyzing the metabolism of both endogenous and exogenous agents that may play a role in the etiology of carcinogenesis. Several potentially functional polymorphisms of the CYP3A4 gene have been implicated in cancer risk, but individually published studies have shown inconclusive results. The aim of this Human Genome Epidemiology (HuGE) review and meta-analysis was to investigate the association between CYP3A4*1B (rs2740574 A > G) polymorphism and cancer risk. Eleven studies were included with a total of 3,810 cancer patients and 3,173 healthy controls. We found that the G allele and GG genotype of CYP3A4*1B polymorphism were associated with increased risk of cancers using the fixed effects model (allele model: odds ratio (OR) = 1.24, 95 %CI: 1.09-1.42, P = 0.001; recessive model: OR = 1.77, 95 %CI: 1.30-2.41, P < 0.001; homozygous model: OR = 1.72, 95 %CI: 1.19-2.47, P = 0.004). Subgroup analyses by cancer type showed that the G allele and G carrier (AG + GG) of CYP3A4*1B polymorphism had significant associations with increased risk of prostate cancer, but not with breast cancer, leukemia, or other cancers. With further subgroup analysis based on different ethnicities, the results indicated that the GG genotype of CYP3A4*1B polymorphism might increase the risk of cancer among African populations. However, similar associations were not observed among Caucasian and Asian populations. Results from the current meta-analysis indicate that the G allele and GG genotype of CYP3A4*1B polymorphism might be associated with increased cancer risk, especially for prostate cancer among African populations.
Assuntos
Citocromo P-450 CYP3A/genética , Neoplasias/etiologia , Polimorfismo Genético/genética , Estudos de Casos e Controles , Predisposição Genética para Doença , Humanos , Literatura de Revisão como Assunto , Fatores de RiscoRESUMO
OBJECTIVE: To investigate the clinicopathologic features, immunophenotyping, differential diagnoses and prognosis of histiocytic sarcoma (HS). METHODS: The clinical and pathologic findings of 4 cases of HS were reviewed. The samples were used for paraffin section, HE stain, immunohistochemistry stain by EnVision method, electron microscope observation. Follow-up information was available in all patients. RESULTS: The age of patients, 2 males and 2 females, ranged from 22 to 65 years old (median, 43.25 years). The sites of involvement included lymph node (2 cases), skin or soft tissue (1 case) and colon (1 case). The tumor cells were widespread infiltration, diffused distribution, no adhesion to each other. Tumor cells were middling and large, round, orbicular-ovate, polygon, epithelium appearance, plentiful cytoplasm and acidophilia, cystose. Nucelus was round, orbicular-ovate, dissymmetry. Nuclear chromatin was vacuole appearance, basophilia nucleolus, caryocinesia and pathological mitotic figure. Three of the cases showed conjugate nuclei, increased pleomorphism with multinucleated tumor giant cell formation. Focal cytoplasmic with foamy appearance was identified in 2 cases. One case demonstrated foci of spindly sarcomatoid appearance. Hemophagocytosis was identified in 2 cases. The tumor cells of 4 cases were often accompanied by various numbers of inflammatory cells. Immunohistochemical study showed that all cases were diffusely positive for α-1-ACT, CD68, CDl63 and lysozyme. Three of 4 cases also expressed CD45, CD45RO. The electron microscope results of 4 cases showed that the tumor cells were plentiful cytoplasm and a few cytolysosome in the cytoplasm, and no birbeck cytorrhyctes, cell-cell junction and digitation. Amongst the 4 patients with follow-up information available, three died of the disease 6-13 months after diagnosis. One patient, whose lesion was localized at the skin and soft tissue, survived at the present time. CONCLUSION: HS was a scarce malignant tumor with mature histiocyte morphology and immunophenotype character. The diagnosis should be based on tissue morphology, immunohistochemistry and electron microscope observation to exclude other disorders.
Assuntos
Sarcoma Histiocítico/diagnóstico , Sarcoma Histiocítico/patologia , Adulto , Idoso , Diagnóstico Diferencial , Feminino , Histiócitos/patologia , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Adulto JovemRESUMO
Mesenchymal stem sells (MSCs) have received much attention in the field of bone tissue engineering due to their biological capability to differentiate into osteogenic lineage cells. Hypoxia-inducible factor 1alpha (HIF-1α) plays an important role in the MSC-related bone regeneration during hypoxia, while core binding factor alpha 1 (Cbfα1) is a transcription regulator that is involved in the chondrocyte differentiation and ossification. In the present study, we investigated the effects of hypoxia on biological capability of MSCs. MSCs were isolated from adult rabbit bone marrow, and were cultured in vitro under normoxia (air with 5% CO(2)) or hypoxia (5% CO(2) and 95% N(2)). The proliferation of MSCs, alkaline phosphatase (ALP) activity, and production of collagens type I and type III (Col I/III) were examined. The expression levels of HIF-1α and Cbfα1 were measured by real-time PCR and western blot analyses. We found that hypoxia significantly induced the proliferation of MSCs and increased ALP activity and the production of Col I/III. Moreover, hypoxia increased the expression of Cbfα1 mRNA after 12 h, whereas the expression of HIF-1α mRNA was increased after 1 h of hypoxia. Knockdown of HIF-1α expression with a small interfering RNA significantly increased the expression levels of Cbfα1 protein either under the normoxia or hypoxia condition. Our results indicate that hypoxia enhances MSCs to differentiate into osteogenic lineage cells and suggest that Cbfα1 may be negatively regulated by HIF-1α.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Animais , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Osteogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , CoelhosRESUMO
Bacillus cereus ZH14 was previously found to produce a new type of antiviral ribonuclease, which was secreted into medium and active against tobacco mosaic virus. In order to enhance the ribonuclease production, in this study the optimization of culture conditions using response surface methodology was done. The fermentation variables including culture temperature, initial pH, inoculum size, sucrose, yeast extract, MgSO(4).7H(2)O, and KNO(3) were considered for selection of significant ones by using the Plackett-Burman design, and four significant variables (sucrose, yeast extract, MgSO(4).7H(2)O, and KNO(3)) were further optimized by a 2(4) factorial central composite design. The optimal combination of the medium constituents for maximum ribonuclease production was determined as 8.50 g/l sucrose, 9.30 g/l yeast extract, 2.00 g/l MgSO(4).7H(2)O, and 0.62 g/l KNO(3). The enzyme activity was increased by 60%. This study will be helpful to the future commercial development of the new bacteria-based antiviral ribonuclease fermentation process.
Assuntos
Bacillus cereus/enzimologia , Proteínas de Bactérias/biossíntese , Técnicas de Cultura de Células/métodos , Fermentação , Ribonucleases/biossíntese , Bacillus cereus/crescimento & desenvolvimento , Meios de Cultura , Análise Multivariada , Análise de Regressão , Ribonucleases/isolamento & purificação , Vírus do Mosaico do TabacoRESUMO
A new ribonuclease (RNase) with tobacco mosaic virus inhibition was isolated and purified from Bacillus cereus ZH14 through ammonium sulfate precipitation, ultrafiltration, ion-exchange chromatography of DEAE-Sephadex A-50 column, and gel chromatography of Sephacryl S-200HR column. The enzyme was purified approximately 134-fold with a recovery of 9.2%. The RNase had an MW of 75.6 kDa in SDS-PAGE, which differed from RNases reported previously. The inhibitory activity of the RNase in the purification process against tobacco mosaic virus was tested, and the percentage inhibition of the purified RNase (48 U/ml) reached 90%. The protein could tolerate 90 degrees C and pH 4.0.
Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Bacillus cereus/enzimologia , Espaço Extracelular/enzimologia , Ribonucleases/isolamento & purificação , Ribonucleases/farmacologia , Vírus do Mosaico do Tabaco/efeitos dos fármacos , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Espaço Extracelular/efeitos dos fármacos , Metais/farmacologia , Peptídeo Hidrolases/metabolismo , TemperaturaRESUMO
BACKGROUND & OBJECTIVE: MASPIN gene is closely associated with carcinogengsis and plays a key role in cell proliferation, adhesion, migration and apoptosis. This study was to construct a recombinant eukaryotic vector expressing MASPIN, and explore the effect of MASPIN overexpression on the apoptosis in human gastric carcinoma cell line SGC7901. METHODS: A eukaryotic expression vector MASIPIN/PCR2.1 was constructed and transfected into SGC7901 cells. RT-PCR and Western blot were used to detect the expression changes of MASPIN and Bax/Bcl-2. TNF-related apoptosis inducing ligand (TRAIL) (50ng/ml) was used to induce apoptosis at different time courses. DNA apoptotic ladders were determined using agarose gel electrophoresis (AGE). Cell apoptosis was measured by flow cytometry (FCM). RESULTS: Recombinant plasmid MASIPIN/PCR2.1 was successfully constructed and transfected into SGC7901 cells. The mRNA and protein levels of MASPIN were significantly higher in the MASPIN/PCR2.1 group (33.6+/-1.2, 23.4+/-1.6) than in the PCR2.1(15.0+/-1.5, 12.3+/-1.5)and the untreated group (13.7+/-2.0, 12.0+/-1.3) (P<0.05). After transfection of MASIPIN/PCR2.1, DNA apoptotic ladders appeared in SGC7901 cells and the induction of apoptosis was in a time-dependent manner. The apoptosis rates were 8.0%, 16.3% and 25.8%in the MASPIN/PCR2.1 plus TRAIL group, 3.0%, 8.2%, 14.4% in the MASPIN/PCR2.1 group, and 4.1%, 9.8%,15.9% in the TRAIL group at 12, 24, and 48 h(P<0.05). The expression levels of Bax mRNA and protein at 48 h after MASIPIN/PCR2.1 transfection were significantly higher in MASPIN/PCR2.1 plus TRAIL group(55.3+/-2.1, 75.4+/-1.3) than in the PCR2.1 group (34.3+/-1.2, 40.7+/-1.8) and the TRAIL group (43.2 +/-1.8,36.2+/-1.3)(P<0.05). The expression of Bcl-2 mRNA in the MASPIN/PCR2.1 plus TRAIL group, PCR2.1 group and TRAIL group were 28.3+/-2.5, 34.3+/-1.2, 32.8+/-2.1, respectively (P<0.05), and those of Bcl-2 protein were 17.4+/-1.5, 45.1+/-2.1, 42.8+/-1.5 in the three groups, respectively (P<0.05). CONCLUSIONS: upregulation of MASPIN/PCR2.1 can significantly enhance the sensibility of gastric cancer cell line SGC7901 to the apoptosis inducer. This maybe related to the upregulation of Bax and downregulation of Bcl-2.
Assuntos
Apoptose , Serpinas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Vetores Genéticos , Humanos , Plasmídeos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Serpinas/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transfecção , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
A large number of bacteria were isolated from plant samples and screened for antiviral activity against the Tobacco mosaic virus (TMV). The bacterium ZH14, which was isolated from Chinese Anxi oolong tea, secreted the antiviral substances, having 94.2% virus inhibition when the bacterial culture filtrate and TMV extract were mixed at a ratio of 1:1. The ZH14 strain is a gram-positive, spore-forming rod and has the ability to degrade ribonucleic acid. Based on its effectiveness on virus inhibition, ZH14 was selected for characterization and was identified as a strain of the Bacillus cereus group based on phenotypic tests and comparative analysis of its 16S rDNA sequence. At the same time, we determined the antiviral product of ZH14 as an extracellular protein with high molecular mass, having an optimum temperature of 15-60 degrees C and an optimum pH of 6-10. Hence, the ZH14 strain and its culture filtrate have potential application in controlling plant diseases caused by TMV.