RESUMO
Olmutinib, the third-generation tyrosine kinase inhibitor, is applied in treating non-small cell lung cancer (NSCLC). The aim of this study is to elucidate the interaction mechanism of olmutinib with human α-1 acid glycoprotein (HAG), an important carrier protein, by mean of multi-spectroscopic and molecular simulation techniques. Fluorescence spectral results confirmed that the fluorescence of this carrier protein can be quenched by olmutinib in the static quenching mode, and this anticancer drug possesses a moderate binding affinity on HAG. The evidence from thermodynamic analysis, replacement interaction with ANS and sucrose, and computational simulation results showed that hydrogen bonding, hydrophobic interactions, and van der Waals forces involved the olmutinib-HAG complexation process. The results from UV-vis, 3D fluorescence and synchronous fluorescence spectroscopy proved that binding anticancer drug olmutinib caused the alteration in the microenvironment around Trp residues. And, circular dichroism spectral results provided the support for the conformational alterations in the carrier protein. The data also proved that olmutinib preferably bound to the hydrophobic cavity of HAG and the binding distance between the two was 2.21 nm. In addition, it can be found that the presence of some metal ions such as Zn2+, Ca2+, Ni2+ and Cu2+ would exert a certain extent effect on the olmutinib-HAG complexation process.Communicated by Ramaswamy H. Sarma.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Sítios de Ligação , Ligação Proteica , Orosomucoide/metabolismo , Simulação de Acoplamento Molecular , Espectrometria de Fluorescência/métodos , Dicroísmo Circular , Termodinâmica , Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Microambiente TumoralRESUMO
Four rhizobial strains, designated CCBAU 85046(T), CCBAU 85051, CCBAU 85048 and CCBAU 85049, isolated from root nodules of Oxytropis glabra grown in Tibet, China, were previously defined, using amplified 16S rRNA gene restriction analysis, as a novel group within the genus Rhizobium. To clarify their taxonomic position, these strains were further analysed and compared with reference strains of related bacteria using a polyphasic approach. The 16S rRNA gene analysis showed that the four isolates formed a distinct phylogenetic lineage in the genus Rhizobium. The isolates showed highest sequence similarity (97.8â %) to Rhizobium indigoferae CCBAU 71042(T). Phenotypic and physiological tests, DNA-DNA hybridization, phylogenetic analyses of housekeeping genes recA, atpD and glnII and fatty acid profiles also indicated that these four strains constitute a novel group distinct from recognized species of the genus Rhizobium. Based on this evidence, strains CCBAU 85046(T), CCBAU 85051, CCBAU 85048 and CCBAU 85049 represent a novel species in the genus Rhizobium, for which the name Rhizobium tubonense sp. nov. is proposed. The type strain is CCBAU 85046(T) (=LMG 25225(T) =HAMBI 3066(T)) and its DNA G+C content is 59.52 % (T(m)). Strain CCBAU 85046(T) could form effective nodules on plant species Vigna unguiculata and Medicago sativa but not on its host of origin Oxytropis glabra.
Assuntos
Oxytropis/microbiologia , Raízes de Plantas/microbiologia , Rhizobium/classificação , Rhizobium/isolamento & purificação , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Filogenia , RNA Ribossômico 16S/genética , Rhizobium/genética , Rhizobium/fisiologia , Análise de Sequência de DNA , TibetRESUMO
BACKGROUND AND AIM: Gene silence of IRX1 tumor suppressor by promoter CpG methylation combined with loss of heterozygosity (LOH) has been identified in human gastric cancer. This study investigated the association between methylation of IRX1 and Helicobacter pylori infection in gastric mucosa tissues and cell line. METHODS: IRX1 methylation was studied by methylation specific polymerase chain reaction (MSP) and bisulfate sequencing polymerase chain reaction (BSP) methods in gastric mucosa tissues from H. pylori-positive chronic gastritis patients or H. pylori-negative chronic gastritis patients. Promoter activity, methylation status and gene expressing level of IRX1 were evaluated by persistent infecting H. pylori on human gastric cells GES-1 in vitro. Electron microscopy was used to observe the effect of H. pylori infection on GES-1 gastric mucosa cells. RESULTS: The methylation level of IRX1 promoter in H. pylori positive chronic gastritis and H. pylori negative chronic gastritis was 55.30%±13.17 versus 5.20%±6.31, respectively (P<0.01). H. pylori infection stimulated increased microvillus, and mucous secretion on GES-1 cells. Infection of H. pylori induced IRX1 promoter methylation and downregulation of the promoter activity as well as gene expression significantly. CONCLUSIONS: This study firstly demonstrated that H. pylori infection contributes to IRX1 promoter methylation on gastric mucosa.
Assuntos
Metilação de DNA , Mucosa Gástrica/microbiologia , Gastrite/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Estudos de Casos e Controles , Linhagem Celular , Doença Crônica , Regulação para Baixo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestrutura , Gastrite/genética , Gastrite/metabolismo , Genes Reporter , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/patologia , Proteínas de Homeodomínio/metabolismo , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
BACKGROUND: To investigate the effects of valproic acid (VPA) on autophagic flux in multiple myeloma (MM) cells. METHODS AND RESULTS: Cell proliferation was assayed by the Cell Counting Kit-8 assay. The qRT-PCR was used to measure the expressions of LC3-II at mRNA level. Autophagic flux was measured by LC3-II turnover using western blot analysis and flow cytometry using the fluorescent dye Cyto-ID. An assay using the RFP-GFP-LC3 tandem construct was performed to monitor autophagic flux. Cell proliferation assay showed that VPA could inhibit the proliferation of MM cells and the inhibitory effects were enhanced with the extension of time. The qRT-PCR and western blot showed that the expression level of LC3-II in the VPA plus CQ group was significantly higher than that in CQ group. Cyto-ID autophagy test showed that the intracellular average fluorescence intensity in VPA plus CQ group was significantly higher than that in control and VPA group (all p < 0.001). The results of RFP-GFP-LC3 tandem construct showed that the numbers of yellow puncta and red puncta in VPA group was higher than that in control group. CONCLUSIONS: VPA could inhibit the proliferation of MM cells and the inhibitory effects were enhanced with the extension of time. VPA could enhance autophagic flux in MM cells, and the increase of autophagosomes was caused by autophagy enhancement rather than inhibition. These findings provided rationale for the treatment of MM with VPA.
Assuntos
Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Ácido Valproico/farmacologia , Antineoplásicos/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Associadas aos Microtúbulos/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologiaRESUMO
Mass spectrometry (MS) has been widely used for specific, sensitive and rapid analysis of proteins and has shown a high potential for bacterial identification and characterization. Type strains of four species of rhizobia and Escherichia coli DH5α were employed as reference bacteria to optimize various parameters for identification and classification of species of rhizobia by matrix-assisted laser desorption/ionization time-of-flight MS (MALDI TOF MS). The parameters optimized included culture medium states (liquid or solid), bacterial growth phases, colony storage temperature and duration, and protein data processing to enhance the bacterial identification resolution, accuracy and reliability. The medium state had little effects on the mass spectra of protein profiles. A suitable sampling time was between the exponential phase and the stationary phase. Consistent protein mass spectral profiles were observed for E. coli colonies pre-grown for 14 days and rhizobia for 21 days at 4°C or 21°C. A dendrogram of 75 rhizobial strains of 4 genera was constructed based on MALDI TOF mass spectra and the topological patterns agreed well with those in the 16S rDNA phylogenetic tree. The potential of developing a mass spectral database for all rhizobia species was assessed with blind samples. The entire process from sample preparation to accurate identification and classification of species required approximately one hour.