RESUMO
We presented a strategy utilizing 2D NMR-based metabolomic analysis of crude extracts, categorized by different pharmacological activities, to rapidly identify the primary bioactive components of TCM. It was applied to identify the potential bioactive components from Scutellaria crude extracts that exhibit anti-non-small cell lung cancer (anti-NSCLC) activity. Four Scutellaria species were chosen as the study subjects because of their close phylogenetic relationship, but their crude extracts exhibit significantly different anti-NSCLC activity. Cell proliferation assay was used to assess the anti-NSCLC activity of four species of Scutellaria. 1H-13C HSQC spectra were acquired for the chemical profiling of these crude extracts. Based on the pharmacological classification (PCA, OPLS-DA and univariate hypothesis test) were performed to identify the bioactive constituents in Scutellaria associated with the anti-NSCLC activity. As a result, three compounds, baicalein, wogonin and scutellarin were identified as bioactive compounds. The anti-NSCLC activity of the three potential active compounds were further confirmed via cell proliferation assay. The mechanism of the anti-NSCLC activity by these active constituents was further explored via flow cytometry and western blot analyses. This study demonstrated 2D NMR-based metabolomic analysis of pharmacologically classified crude extracts to be an efficient approach to the identification of active components of herbal medicine.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Espectroscopia de Ressonância Magnética , Metabolômica , Extratos Vegetais , Scutellaria , Scutellaria/química , Humanos , Proliferação de Células/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Apigenina/farmacologia , Apigenina/química , Apigenina/isolamento & purificação , Apigenina/análise , Flavanonas/farmacologia , Flavanonas/química , Flavanonas/isolamento & purificação , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Glucuronatos/farmacologia , Glucuronatos/isolamento & purificação , Glucuronatos/química , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos AntitumoraisRESUMO
Two-dimensional (2D) 1H-13C heteronuclear single quantum coherence (HSQC) has been increasingly applied to metabolomics studies because it can greatly improve the resolving capability compared with one-dimensional (1D) 1H NMR. However, preprocessing methods such as peak matching and alignment tools for 2D NMR-based metabolomics have lagged behind similar methods for 1D 1H NMR-based metabolomics. Correct matching and alignment of 2D NMR spectral features across multiple samples are particularly important for subsequent multivariate data analysis. Considering different intensity dynamic ranges of a variety of metabolites and the chemical shift variation across the spectra of multiple samples, here, we developed an efficient peak matching and alignment algorithm for 2D 1H-13C HSQC-based metabolomics, called global intensity-guided peak matching and alignment (GIPMA). In GIPMA, peaks identified in all spectra are pooled together and sorted by intensity. Chemical shift of a stronger peak is regarded to be more accurate and reliable than that of a weaker peak. The strongest undesignated peak is chosen as the reference of a new cluster if it is not located within the chemical shift tolerance of any existing peak cluster (PC), or otherwise it is matched to an existing PC and the aligned chemical shift of the PC is updated as the intensity-weighted average of the chemical shifts of all peaks in the cluster. Setting an optimum chemical shift tolerance (Δδo) is critical for the peak matching and alignment across multiple samples. GIPMA dynamically searches for and intelligently selects the Δδo for peak matching to maximize the number of valid peak clusters (vPC), that is, spectral features, among multiple samples. By GIPMA, fully automatic peakwise matching and alignment do not require any spectrum as initial reference, while the chemical shift of each PC is updated as the intensity-weighted average of the chemical shifts of all peaks in the same PC, which is warranted to be statistically more accurate. Accurate chemical shifts for each representative spectral feature will facilitate subsequent peak assignment and are essential for correct metabolite identification and result interpretation. The proposed method was demonstrated successfully on the spectra of six model mixtures consisting of seven typical metabolites, yielding correct matching of all known spectral features. The performance of GIPMA was also demonstrated on 2D 1H-13C HSQC spectra of 87 real extracts of 29 samples of five Dendrobium species. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of the 87 matched and aligned spectra by GIPMA generates correct classification of the 29 samples into five groups. In summary, the proposed algorithm of GIPMA provided a practical peak matching and alignment method to facilitate 2D NMR-based metabolomics studies.
Assuntos
Algoritmos , Metabolômica , Metabolômica/métodos , Espectroscopia de Ressonância Magnética/métodos , Análise por ConglomeradosRESUMO
BACKGROUND: Cytochrome P450 2E1 (CYP2E1) plays a key role in the metabolism of xenobiotic and endogenous low-molecular-weight compounds. This study aimed to determine if the genetic variations of 96-bp insertion/deletion (I/D) and C-1054T (rs2031920) in CYP2E1 were associated with the risk of gestational diabetes mellitus (GDM). METHODS: CYP2E1 polymorphisms were genotyped in a case-control study of 1,134 women with uncomplicated pregnancies and 723 women with GDM. The effects of genotype on the clinical, metabolic, and oxidative stress indices were assessed. RESULTS: The CYP2E1 C-1054T variant was associated with an increased risk of GDM based on the genotype, recessive, dominant, and allele genetic models (P < 0.05). The TT + CT genotype remained a significant predictive factor for GDM risk after correcting for maternal age and pre-pregnancy body mass index (OR = 1.277, 95% CI: 1.042-1.563, P = 0.018). Moreover, fasting insulin concentrations and homeostatic model assessment of insulin resistance were significantly higher in GDM patients carrying the T allele than in those with the CC genotype (P < 0.05). Furthermore, the combined genotype II + ID/TT + CT of the 96-bp I/D and C-1054T polymorphisms further increased the risk of GDM when the combined genotype DD/CC was set as the reference category (OR = 1.676, 95% CI: 1.182-2.376, P = 0.004). CONCLUSIONS: The T allele of the C-1054T polymorphism and its combination with the I allele of the 96-bp I/D variation in CYP2E1 are associated with an increased risk of GDM in the Chinese population. The - 1054T allele may be associated with more serious insulin resistance in patients.
Assuntos
Diabetes Gestacional , Resistência à Insulina , Gravidez , Humanos , Feminino , Diabetes Gestacional/genética , Diabetes Gestacional/metabolismo , Citocromo P-450 CYP2E1/genética , Resistência à Insulina/genética , Estudos de Casos e Controles , População do Leste Asiático , Predisposição Genética para Doença , Polimorfismo Genético , Genótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The formation of death-inducing signaling complex (DISC) and death effector domain (DED) filament initiates extrinsic apoptosis. Recruitment and activation of procaspase-8 at the DISC are regulated by c-FLIP. The interaction between c-FLIP and procaspase-8 is mediated by their tandem DEDs (tDED). However, the structure of c-FLIPtDED and how c-FLIP interferes with procaspase-8 activation at the DISC remain elusive. Here, we solved the monomeric structure of c-FLIPtDED (F114G) at near physiological pH by solution nuclear magnetic resonance (NMR). Structural superimposition reveals c-FLIPtDED (F114G) adopts a structural topology similar to that of procaspase-8tDED. Our results provide a structural basis for understanding how c-FLIP interacts with procaspase-8 and the molecular mechanisms of c-FLIP in regulating cell death.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Domínio Efetor de Morte , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Transdução de SinaisRESUMO
Cellular FLICE-like inhibitory protein (c-FLIP) regulates extrinsic apoptosis by controlling procaspase-8 activation through its tandem N-terminal death effector domains (DEDs). Here, we present the expression and purification of c-FLIP tandem DEDs (tDED) from Escherichia coli. We observed that the c-FLIPtDED maintains monomeric form under near-physiological pH condition in vitro. Our results also reveal a significant correlation between the pH conditions and the structure of c-FLIPtDED (F114A). The described methods and results would be helpful for follow-up study on the structural and functional of c-FLIP.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Domínio Efetor de Morte , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , SeguimentosRESUMO
Scrophulariae Radix (SR) is one of the oldest and most frequently used Chinese herbs for oriental medicine in China. Before clinical use, the SR should be processed using different methods after harvest, such as steaming, "sweating", and traditional fire-drying. In order to investigate the difference in chemical constituents using different processing methods, the two-dimensional (2D) 1H-13C heteronuclear single quantum correlation (1H-13C HSQC)-based metabolomics approach was applied to extensively characterize the difference in the chemical components in the extracts of SR processed using different processing methods. In total, 20 compounds were identified as potential chemical markers that changed significantly with different steaming durations. Seven compounds can be used as potential chemical markers to differentiate processing by sweating, hot-air drying, and steaming for 4 h. These findings could elucidate the change of chemical constituents of the processed SR and provide a guide for the processing. In addition, our protocol may represent a general approach to characterizing chemical compounds of traditional Chinese medicine (TCM) and therefore might be considered as a promising approach to exploring the scientific basis of traditional processing of TCM.
Assuntos
Medicamentos de Ervas Chinesas , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Medicina Tradicional Chinesa , Metabolômica/métodos , Raízes de Plantas/químicaRESUMO
BACKGROUND: Oxidative stress plays a pivotal role in the pathogenesis of polycystic ovary syndrome (PCOS). Genetic variations in myeloperoxidase (MPO; G-463A) and NADPH oxidase p22phox subunit (CYBA; C242T) cause inter-individual variability in enzyme activities. Here, we investigated the associations between MPO activity and the MPO G-463A and CYBA C242T polymorphisms in Chinese women with PCOS. METHODS: This case-control study included 1003 patients with PCOS and 810 controls. The G-463A and C242T polymorphisms were detected by polymerase chain reaction and restriction analysis, and clinical, hormonal, metabolic and oxidative stress parameters and MPO activity were analysed. RESULTS: The frequencies of the GA + AA genotype and A allele frequency of the MPO G-463A polymorphism were significantly higher in the PCOS group than in the control group. Logistic regression analysis showed that the MPO-463A allele is a risk factor for PCOS (OR = 1.261, 95% CI: 1.042-1.526, P = .017). Patients with the AA genotype tended to have higher plasma MPO activity than those with the GG genotype. No statistical significance was found in the genotype and allele frequencies of the CYBA C242T polymorphism between the PCOS and control groups. However, we demonstrated that the coexistence of the MPO A allele (GA + AA genotypes) and the CYBA CC genotype was associated with an increased risk of PCOS when compared with the wild-type GG/CC genotypes (OR = 1.302, 95% CI: 1.030-1.646, P = .027). CONCLUSION: The MPO G-463A variant, but not CYBA C242T variant, is associated with a risk of PCOS in Chinese women.
Assuntos
NADPH Oxidases/genética , Peroxidase/genética , Síndrome do Ovário Policístico/genética , Adulto , Povo Asiático , Estudos de Casos e Controles , China , Feminino , Predisposição Genética para Doença , Humanos , Estresse Oxidativo/genética , Polimorfismo Genético , Adulto JovemRESUMO
Glycosaminoglycans (GAGs) are conserved polysaccharides composed of linear repeating disaccharides and play crucial roles in multiple biological processes in animal kingdom. However, saccharide-branched GAGs are rarely found, except the fucose-branched one from sea cucumbers. There was conjecture about the presence of disaccharide-branched GAG since 30 years ago, though not yet confirmed. Here, we report a GAG containing galactose-fucose branches from Thelenota ananas. This unique branch was confirmed as d-Gal4S(6S)-α1,2-l-Fuc3S by structural elucidation of oligosaccharides prepared from T. ananas GAG. Bioassays indicated that oligomers with a larger degree of polymerization exhibited a potent anticoagulation by targeting the intrinsic tenase. Heptasaccharide was proven as the minimum fragment retaining the anticoagulant potential and showed 92.6% inhibition of venous thrombosis in vivo at sc. of 8 mg/kg with no obvious bleeding risks. These results not only solve a long-standing question about the presence of disaccharide-branched GAG in Holothuroidea, but open up new opportunities to develop safer anticoagulants.
Assuntos
Glicosaminoglicanos , Pepinos-do-Mar , Animais , Anticoagulantes/farmacologia , Coagulação Sanguínea , Dissacarídeos/farmacologiaRESUMO
OBJECTIVE: To investigate the relationship between angiotensin â -converting enzyme (ACE) insertion/deletion (I/D) gene polymorphism and the genetic risks for polycystic ovary syndrome (PCOS) and to evaluate the impact of ACE I/D genotypes on clinical, hormonal, metabolic and oxidative stress parameters in patients with PCOS. METHODS: This was a retrospective case-control study involving a total of 1 020 PCOS patients and 825 female controls who visited the outpatient clinic of the Department of Reproductive Endocrinology, West China Second Hospital of Sichuan University between 2006 and 2019. The ages of the subjects ranged between 17 and 44. The ACE I/D genotypes were determined by polymerase chain reaction (PCR) and gel electrophoresis. 667 PCOS patients and 527 controls were selected for an analysis of their genotypes and the hormonal, metabolic and oxidative stress parameters. RESULTS: The genotype distributions of the ACE I/D single nucleotide polymorphism was in Hardy-Weinberg equilibrium in both the PCOS group and the control group (all P>0.05), which was representative of the population. There were no statistically significant differences in genotype and allele frequencies between the PCOS and the control groups ( P>0.05). After adjusting for both age and body mass index (BMI), there was no statistically significant difference in clinical characteristics among all genotypes in either the PCOS group or the control group. In the PCOS group, compared with the II genotype subgroup, the ID genotype subgroup had lower luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, while the DD genotype subgroup had higher homeostatic model assessment of insulin resistance (HOMA-IR) and malondialdehyde (MDA) levels. Compared with the ID genotype subgroup, the DD genotype subgroup had lower serum sex hormone binding globulin (SHBG) level, but higher total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels ( P<0.05). In the control group, II genotype subgroup had a higher level of total oxidant status (TOS) than that of the DD genotype subgroup. CONCLUSION: ACE I/D genetic polymorphism is not associated with risks for PCOS. The I/D variation of ACE gene may be related to insulin resistance, dyslipidaemia, hyperandrogenemia and oxidative stress in PCOS patients.
Assuntos
Peptidil Dipeptidase A/genética , Síndrome do Ovário Policístico , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Humanos , Síndrome do Ovário Policístico/genética , Polimorfismo de Nucleotídeo Único , Estudos RetrospectivosRESUMO
An automatic approach to identification of natural products (NPid) in complex extracts by exploring pure shift HSQC (psHSQC) and H2BC spectra of the mixture is developed, which integrated information on chemical shifts (CS), adjacent relationships (AR) and peak intensities (PI) of 1H-13C groups for identification of candidate natural product in a customized NMR database. A weighted comprehensive score is calculated for each candidate from the values of CS, AR and PI to rate the likelihood of its existence in the complex mixture. Using the crude extract of crabapple (Malus fusca) as an example, a customized NMR database of natural products from plants of the genus Malus was constructed. The performance of NPid was first evaluated using simulated data in four scenarios, that is, for identification of structurally similar natural products, identification of natural products with part of peaks missing in psHSQC due to low concentration, without available adjacent relationship information, or without useful peak intensity information. The false positive and false negative rates of the natural products identified by NPid were estimated by Monte Carlo simulation. It shows that AR and PI can effectively reduce the false positive rate of identification. Proof of concept of the proposed method was elucidated on a model mixture consisting of 10 known natural products. Application of this method was then demonstrated on an authentic sample of crude extract of crabapple and 19 known natural products were successfully identified and confirmed by standard spiking.
Assuntos
Produtos Biológicos/análise , Extratos Vegetais/análise , Algoritmos , Produtos Biológicos/química , Bases de Dados de Compostos Químicos/estatística & dados numéricos , Espectroscopia de Ressonância Magnética/estatística & dados numéricos , Malus/química , Estrutura Molecular , Extratos Vegetais/química , Estudo de Prova de ConceitoRESUMO
Objective: To evaluate the feasibility and efficacy of the modified reverse puncture device (mRPD) technique for transanal anastomosis in total laparoscopic colorectal resection with natural orifice specimen extraction surgery (NOSES).Methods: From August 2015 to September 2017, 34 patients underwent laparoscopic colorectal resection using the mRPD technique to place the anvil in the abdominal cavity and complete transanal anastomosis.Results: All patients who underwent total laparoscopic colorectal resection with NOSES were analyzed. The anvil placement time was 5-14 min, with an average of 6.7 min. The postoperative pain visual analogue scale (VAS) score was 1-4 points, with an average of 2.2 points. The postoperative hospital stay was 7-13 days, with an average of 8.7 days. No complications, such as anastomotic bleeding or stenosis, occurred. During a 14- to 28-month follow-up period (average, 19.5 months), no cases of long-term complications were observed.Conclusion: Total laparoscopic colorectal resection using mRPD is a technically feasible and safe procedure with satisfactory short-term efficacy.
Assuntos
Neoplasias Colorretais/cirurgia , Laparoscopia/métodos , Cirurgia Endoscópica por Orifício Natural/efeitos adversos , Punções/efeitos adversos , Adulto , Idoso , Fístula Anastomótica , Estudos de Viabilidade , Feminino , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Complicações Pós-Operatórias , Estudos RetrospectivosRESUMO
Diverse derivatives of amino acids with different steric configurations are important biosynthetic building blocks. In biology, epimerization is an important way to generate steric diversity. MarH catalyzes the epimerization of the ß-position of (3R)-ß-methyl-indolepyruvate (MeInPy), forming (3S)-ß-MeInPy. Both compounds are derivatives of l-tryptophan (l-Trp) and are important precursors of bioactive natural products. Here, we report the crystal structures of MarH and the NMR structure of its complex with l-Trp, an analogue of its native substrate, (3R)-ß-MeInPy. Structural analysis and mutagenesis studies indicated that His25 acts as a base to remove Hß and generate a planar carbanion intermediate, which is then putatively reprotonated on the opposite face by a water molecule to form (3S)-ß-MeInPy in a stereospecific manner. The details of ß-site isomerization at the atomic level provide deeper insights into the epimerization mechanism of MarH and will facilitate further enzyme design to extend the substrate scope.
Assuntos
Racemases e Epimerases/química , Indóis/química , Indóis/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Piruvatos/química , Piruvatos/metabolismo , Racemases e Epimerases/metabolismoRESUMO
1H and 2H NMR spectra of 4 natural and synthetic nicotine samples were collected in a non-quantitative way and site-specific 2H/1H peak intensity ratio (SPIR) was calculated for 12 distinct sites of nicotine. Experimental results illustrated that the SPIRs at sites of 6, 2', and 5'ß of natural nicotine were significantly different from those of the synthetic nicotine, and could be used for nicotine authentication as the measured SPIRs were indicative of the site-specific natural isotope fractionation. We demonstrated that this method could be applied to detect adulteration of natural nicotine with as low as 20% synthetic nicotine, without the need to measure the site-specific δD values, which usually required time-consuming quantitative 2H NMR and additional IRMS for the overall 2H/1H isotopic ratio determination. The distinguishable 2H/1H SPIRs of nicotine, which can be quickly measured by NMR in non-quantitative way, can serve as an attractive alternative tool for tobacco authentication. Graphical abstract.
Assuntos
Contaminação de Medicamentos , Nicotina/análise , Espectroscopia de Prótons por Ressonância Magnética/métodos , Espectrometria de Massas/métodosRESUMO
NMR and LC-MS are two powerful techniques for metabolomics studies. In NMR spectra and LC-MS data collected on a series of metabolite mixtures, signals of the same individual metabolite are quantitatively correlated, based on the fact that NMR and LC-MS signals are derived from the same metabolite covary. Deconvoluting NMR spectra and LC-MS data of the mixtures through this kind of statistical correlation, NMR and LC-MS spectra of individual metabolites can be obtained as if the specific metabolite is virtually isolated from the mixture. Integrating NMR and LC-MS spectra, more abundant and orthogonal information on the same compound can significantly facilitate the identification of individual metabolites in the mixture. This strategy was demonstrated by deconvoluting 1D 13C, DEPT, HSQC, TOCSY, and LC-MS spectra acquired on 10 mixtures consisting of 6 typical metabolites with varying concentration. Based on statistical correlation analysis, NMR and LC-MS signals of individual metabolites in the mixtures can be extracted as if their spectra are acquired on the purified metabolite, which notably facilitates structure identification. Statistically correlating NMR spectra and LC-MS data (CoNaM) may represent a novel approach to identification of individual compounds in a mixture. The success of this strategy on the synthetic metabolite mixtures encourages application of the proposed strategy of CoNaM to biological samples (such as serum and cell extracts) in metabolomics studies to facilitate identification of potential biomarkers.
Assuntos
Cromatografia Líquida/métodos , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Metaboloma , Fluxo de TrabalhoRESUMO
Aim: To evaluate the feasibility, safety, short- and long-term efficacy of a reverse puncture device (RPD) technique for esophagojejunostomy in laparoscopic-assisted total gastrectomy. Patients & methods: This retrospective study analyzed outcome data of 104 patients in propensity score matching whom were divided into the RPD and the purse-string suture technique group. Results: The RPD group had a shorter anvil placement time, shorter operative time, longer resected esophageal length, shorter incision length, shorter postoperative drainage time, shorter postoperative hospital stay and anastomotic complications than the purse-string suture technique group (p < 0.05). Multivariate analysis showed that BMI (odds ratio: 6.285, 1.446-27.322) and anvil placement time (odds ratio: 5.645, 1.089-29.321) were independent risk factors for anastomotic complications (p < 0.05). Conclusion: Laparoscopic-assisted total gastrectomy using an RPD technique is feasible, safe and effective.
Assuntos
Esôfago/cirurgia , Gastrectomia/métodos , Jejunostomia/métodos , Jejuno/cirurgia , Laparoscopia/métodos , Anastomose Cirúrgica/métodos , Feminino , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Complicações Pós-Operatórias/etiologia , Pontuação de Propensão , Punções/métodos , Estudos Retrospectivos , Fatores de Risco , Técnicas de SuturaRESUMO
OBJECTIVE: To study the effect of methylation level of microRNA promoter on the expression of microRNAs (miRNA34a, miRNA34b, miRNA148a, miRNA203a) and on the proliferation, migration and invasion of lung cancer A549 cells. METHODS: The proliferation of A549 cells treated with different concentrations of demethylated drug 5-aza-2'-deoxycytidine (5-Aza-CdR) was measured by CCK8 assay and calculated the inhibitory rate in 24 h, 48 h and 72 h, respectively. After 72 h of treatment with 20 µmol/L 5-Aza-CdR, methylation-specific PCR (MSP) was used to detect the methylation level of A549 cells in miRNAs gene promoter regions, and real-time quantitative PCR (real-time PCR) was used to test the expression of miRNAs. The migration abilities of A549 cells treated with 20 µmol/L 5-Aza-CdR in 24 h and 48 h were performed with wound healing assay, while the invasion abilities in 48 h were evaluated by Transwell assay, respectively. RESULTS: The proliferation inhibition rate of A549 cells gradually increased with the treatment concentration of 5-Aza-CdR increased and the treatment time prolonged. Compared with the control group, the methylated band of the experimental group was weaker and the unmethylated band was stronger, and the miRNAs gene promoter regions methylation level of the experimental group was lower than that of the control group. The expression level of miRNAs was significantly increased in the experimental group (P<0.05) . The migration and invasion of the experimental group of A549 cells were inhibited compared with the control group (P<0.05) . CONCLUSION: 5-Aza-CdR can reverse methylation levels of miRNAs promoter regions and upregulate the expression level of miRNA34a, miRNA34b, miRNA148a, miRNA203a, resulting in significantly inhibiting the proliferation, migration and invasion of lung cancer cells.
Assuntos
Azacitidina/farmacologia , Metilação de DNA , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Regiões Promotoras Genéticas , Células A549 , Movimento Celular , Proliferação de Células , Humanos , Invasividade NeoplásicaRESUMO
FR900452 and spirocyclic maremycins, including F and G components, are structurally related indole alkaloids, previously identified from different Streptomyces species. These alkaloids feature an indole diketopiperazine motif linked with a cyclopentenone moiety, but the linkage differs in FR900452 and the spirocyclic maremycins. Here, FR900452 and its two new analogues were identified from the fermentation broth of Streptomyces sp. B9173, the producer of maremycins. Gene inactivation and heterologous expression of the mar gene cluster confirmed that production of FR900452 shares the same biosynthetic machinery that produces maremycins. FR900452 was identified as the precursor of maremycin A/B by feeding studies. MarP, a SnoaL-like protein, was demonstrated to differentiate the biosynthesis of FR900452 from that of spiro-form maremycin G.
Assuntos
Ciclopentanos/metabolismo , Dicetopiperazinas/metabolismo , Alcaloides Indólicos/metabolismo , Indóis/metabolismo , Compostos de Espiro/metabolismo , Streptomyces/metabolismo , Vias Biossintéticas , Ciclopentanos/química , Dicetopiperazinas/química , Fermentação , Alcaloides Indólicos/química , Indóis/química , Família Multigênica , Compostos de Espiro/química , Streptomyces/química , Streptomyces/genéticaRESUMO
A wide variety of methods, such as enzymatic methods, LC-MS, and LC-MS/MS, are currently available for the concentration determination of plasma glucose in studies of diabetes, obesity, exercise, etc. However, these methods rarely discriminate endogenous and exogenous glucose in plasma. A novel NMR strategy for discriminative quantification of the endogenous and exogenous glucose in plasma has been developed using an adapted isotope dilution 1H-13C heteronuclear single-quantum correlation (ID-HSQC) with uniformly 13C-labeled glucose as a tracer of exogenous glucose. This method takes advantage of the distinct 1H-13C chemical shifts of the hemiacetal group of the α-D-glucopyranose and makes use of the 13C-13C one-bond J-coupling (1JCC) in uniformly 13C-labeled glucose to differentiate the 1H-13C HSQC signal of labeled glucose from that of its natural counterpart when data are acquired in high-resolution mode. The molar ratio between the endogenous and exogenous plasma glucose can then be calculated from the peak intensities of the natural and labeled glucose. The accuracy and precision of the method were evaluated using a series of standard mixtures of natural and uniformly 13C-labeled glucose with varied but known concentrations. Application of this method is demonstrated for the quantification of endogenous and exogenous glucose in plasma derived from healthy and diabetic cynomolgus monkeys. The results nicely agree with our previous LC-MS/MS results. Considering the natural abundance of 13C isotope at the level of 1.1% in endogenous glucose, comparable peak intensities of quantitatively measurable signals derived from natural and labeled glucose imply that the ID-HSQC can tolerate a significantly high ratio of isotope dilution, with labeled/natural glucose at ~ 1%. We expect that the ID-HSQC method can serve as an alternative approach to the biomedical or clinical studies of glucose metabolism.
Assuntos
Glicemia/análise , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Espectroscopia de Prótons por Ressonância Magnética/métodos , Animais , Glicemia/metabolismo , Estudos de Casos e Controles , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Técnicas de Diluição do Indicador , Macaca fascicularis , Camundongos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Quantification of endogenous and exogenous plasma glucose can help more comprehensively evaluate the glucose metabolic status. A ratio-based approach using isotope dilution liquid chromatography tandem mass spectrometry (ID LC-MS/MS) with indirect multiple reaction monitoring (MRM) of the derivative tag was developed to simultaneously quantify endo-/exogenous plasma glucose. Using diluted D-[13C6] glucose as tracer of exogenous glucose, 12C6/13C6 glucoses were first derivatized and then data were acquired in MRM mode. The metabolism of exogenous glucose can be tracked and the concentration ratio of endo/exo-genous glucose can be measured by calculating the endo-/exo-genous glucose concentrations from peak area ratio of specific daughter ions. Joint application of selective derivatization and MRM analysis not only improves the sensitivity but also minimizes the interference from the background of plasma, which warrants the accuracy and reproducibility. Good agreement between the theoretical and calculated concentration ratios was obtained with a linear correlation coefficient (R) of 0.9969 in the range of D-glucose from 0.5 to 20.0 mM, which covers the healthy and diabetic physiological scenarios. Satisfactory reproducibility was obtained by evaluation of the intra- and inter-day precisions with relative standard deviations (RSDs) less than 5.16%, and relative recoveries of 85.96 to 95.92% were obtained at low, medium, and high concentration, respectively. The method was successfully applied to simultaneous determination of the endo-/exogenous glucose concentration in plasma of non-diabetic and type II diabetic cynomolgus monkeys. Graphical Abstract The scheme of the proposed ratio-based approach using isotope dilution LC-MS/MS with indirect MRM of the derivative tag for simultaneous quantification of endogenous and exogenous plasma glucose.
Assuntos
Glicemia/análise , Diabetes Mellitus/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Glicemia/metabolismo , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Cromatografia Líquida/métodos , Diabetes Mellitus/metabolismo , Humanos , Técnicas de Diluição do Indicador , Limite de Detecção , Macaca fascicularis , Reprodutibilidade dos TestesRESUMO
Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks.