RESUMO
Protein arginine methyltransferase CARM1 has been shown to methylate a large number of non-histone proteins, and play important roles in gene transcriptional activation, cell cycle progress, and tumorigenesis. However, the critical substrates through which CARM1 exerts its functions remain to be fully characterized. Here, we reported that CARM1 directly interacts with the GATAD2A/2B subunit in the nucleosome remodeling and deacetylase (NuRD) complex, expanding the activities of NuRD to include protein arginine methylation. CARM1 and NuRD bind and activate a large cohort of genes with implications in cell cycle control to facilitate the G1 to S phase transition. This gene activation process requires CARM1 to hypermethylate GATAD2A/2B at a cluster of arginines, which is critical for the recruitment of the NuRD complex. The clinical significance of this gene activation mechanism is underscored by the high expression of CARM1 and NuRD in breast cancers, and the fact that knockdown CARM1 and NuRD inhibits cancer cell growth in vitro and tumorigenesis in vivo. Targeting CARM1-mediated GATAD2A/2B methylation with CARM1 specific inhibitors potently inhibit breast cancer cell growth in vitro and tumorigenesis in vivo. These findings reveal a gene activation program that requires arginine methylation established by CARM1 on a key chromatin remodeler, and targeting such methylation might represent a promising therapeutic avenue in the clinic.
Assuntos
Neoplasias da Mama , Montagem e Desmontagem da Cromatina , Regulação Neoplásica da Expressão Gênica , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Proteína-Arginina N-Metiltransferases , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Humanos , Feminino , Animais , Linhagem Celular Tumoral , Ciclo Celular/genética , Camundongos , Metilação , Arginina/metabolismo , Carcinogênese/genética , Ativação TranscricionalRESUMO
Two ionic hydrogen-bonded organic frameworks (iHOF-10, iHOF-11) were prepared using 1,1'-diamino-4,4'-bipyridine diiodide (Dbpy â 2I) and tetrakis(4-sulfophenyl)ethylene (H4 TPE). With increasing RH and temperature, water molecules induce single crystal to single crystal (SCSC) transformation of iHOF-10, resulting in the formation of iHOF-11. At 90 °C, 98 % RH, the proton conductivity of iHOF-11 (7.03×10-3 â S cm-1 ) is 2.09 times higher than iHOF-10 (3.37×10-3 â S cm-1 ). At 50 °C, 98 % RH, iHOF-11 (9.49×10-4 â S cm-1 ) is 19.06 times higher than iHOF-10 (4.98×10-5 â S cm-1 ). The proton conductivity shows water molecules enter the crystal and induce crystal transformation and reorganization of the hydrogen bonding structure, thus increasing the proton conductivity and stability.
RESUMO
BACKGROUND: Involvement of transverse mesocolon (TM) during acute necrotizing pancreatitis(ANP) indicates that inflammation has spread from retroperitoneal space to peritoneum. Nevertheless, the impact of TM involvement, as confirmed by contrast-enhanced computed tomography (CECT), on local complications and clinical outcomes was poorly investigated. PURPOSE: This study aimed to explore the association between CECT-diagnosed TM involvement and the development of colonic fistula in a cohort of ANP patients. METHODS: This is a single-center, retrospective cohort study involving ANP patients admitted from January 2020 to December 2020. TM involvement was diagnosed by two experienced radiologists. The study subjects were enrolled consecutively and divided into two groups: TM involvement and non-TM involvement. The primary outcome was colonic fistula during the index admission. Clinical outcomes were compared between the two groups, and the association between the TM involvement and the development of colonic fistula was assessed using multivariable analysis to adjust for baseline unbalances. RESULTS: A total of 180 patients with ANP were enrolled, and 86 (47.8%) patients had TM involvement. The incidence of the colonic fistula is significantly higher in patients with TM involvement (16.3% vs. 5.3%;p = 0.017). Moreover, the length of hospital stay was 24(13,68) days in patients with TM involvement and 15(7,31) days in those not (p = 0.001). Analysis of multivariable logistic regression revealed that TM involvement is an independent risk factor for the development of colonic fistula (odds ratio: 10.253, 95% CI: 2.206-47.650, p = 0.003). CONCLUSION: TM involvement in ANP patients is associated with development of colonic fistula in ANP patients.
Assuntos
Fístula , Mesocolo , Pancreatite Necrosante Aguda , Humanos , Pancreatite Necrosante Aguda/complicações , Pancreatite Necrosante Aguda/diagnóstico por imagem , Estudos Retrospectivos , Inflamação , Fístula/complicaçõesRESUMO
JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes.
Assuntos
Processamento Alternativo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fator de Processamento U2AF/metabolismo , Animais , Células HEK293 , Humanos , Hidroxilação , Lisina/metabolismo , Camundongos , Camundongos Knockout , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fator de Processamento U2AF/químicaRESUMO
Although "histone" methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain-containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor ß2 (RARß2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70's function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control.
Assuntos
Arginina/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Receptores do Ácido Retinoico/genética , Tretinoína/farmacologia , Sequência de Aminoácidos , Cromatina/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Humanos , Metilação , Dados de Sequência Molecular , Fator de Transcrição TFIIH/metabolismo , Transcrição GênicaRESUMO
Glycoconjugates are ubiquitously present and play a critical role in various biological processes. Due to their low stability and incredibly high degree of structural diversity, the structural characterization of glycan generally requires chemical derivatization and sophisticated instrumentation. Herein, we report a method for complicated glycan characterization in a single assay by employing 2,5-dihydroxybenzoic acid functionalized mercury telluride nanoparticles (HgTe@DHB NPs) as a dual ionization-dissociation element in matrix-assisted laser desorption/ionization mass spectrometry. Using a linear glycan, HgTe@DHB NPs promote laser-induced extensive and intense dissociation of the glycan, superior to HgTe microparticles and other inorganic nanoparticles (TiO2, ZnO, and Mn2O3 NPs). Abundant generation of diagnostic glycosidic (Y-, and B-type ions) and cross-ring cleavage (A-type ions) ions permits unambiguous determination of the composition, sequence, branching, and linkage of labile sialylated glycans. The general utility of this approach was demonstrated by the characterization of labile sialylated glycans and two sets of complicated isomeric glycans. This phenomenon was delineated further by investigating the NP's physico-chemical characteristics, revealing that their nanoscale-dependent thermodynamic properties, including UV absorption, photoelectron release dynamics and thermal energy, were the key to levitate temperature synergistically, thus inducing spontaneous glycan decomposition during the nanoparticle-assisted laser desorption-ionization process. Our results show that this "pseudo-MS/MS" obtained by HgTe@DHB can be beneficial for the analysis of biologically relevant and more complicated carbohydrates, without the need for chemical pre-derivatization and conventional tandem mass spectrometry.
Assuntos
Compostos de Mercúrio , Nanopartículas Metálicas , Polissacarídeos/análise , Telúrio , Lasers , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Two novel ionic hydrogen-bonded organic frameworks (iHOF-17 and iHOF-18) were obtained by integrating organosulfonic acids with amidine salts. Among them, iHOF-18 exhibits fast, reversible, and high-contrast UV-induced photochromic properties, and this property is solvent-controlled. This work provides valuable insights for designing advanced anti-counterfeiting techniques and encryption applications.
RESUMO
Cysteine sulfinic acid decarboxylase (Csad) is the rate-limiting enzyme in the de novo biosynthesis of taurine. There are a number of physiological roles of taurine, such as bile salt synthesis, osmoregulation, lipid metabolism, and oxidative stress inhibition. To investigate the role of de novo synthesis of taurine during embryonic development, zebrafish csad was cloned and functionally analyzed. Semi-quantitative RT-PCR showed that csad transcripts are maternally deposited, while whole-mount in situ hybridization demonstrated that csad is expressed in yolk syncytial layer and various embryonic tissues such as notochord, brain, retina, pronephric duct, liver, and pancreas. Knockdown of csad significantly reduced the embryonic taurine level, and the affected embryos had increased early mortality and cardiac anomalies. mRNA coinjection and taurine supplementation rescued the cardiac phenotypes suggesting that taurine originating from the de novo synthesis pathway plays a role in cardiac development. Our findings indicated that the de novo synthesis pathway via Csad plays a critical role in taurine homeostasis and cardiac development in zebrafish early embryos.
Assuntos
Carboxiliases/metabolismo , Desenvolvimento Embrionário , Proteínas de Peixes/metabolismo , Taurina/biossíntese , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Carboxiliases/genética , Feminino , Proteínas de Peixes/genética , Homeostase , Masculino , Peixe-Zebra/genéticaRESUMO
Coronary heart disease (CHD) related genes and targets, as well as drug targets for preventing and treating CHD were taken as the study objects to build a CHD disease network and a drug action network preventing and treating CHD. Such topological characteristic parameters of the networks as degree distribution, characteristic path length, connectivity and heterogeneity were analyzed to verify the reliability of the networks. On that basis, the intersection calculation was conducted for both networks to analyze the drug action mechanism of their sub-networks. The disease network are composed of 15,221 nodes and 31,177 sides, while the drug action network preventing and treating CHD has 15,073 nodes and 32,376 sides. Both of their topological characteristic parameters showed scale-free small world structural characteristics. Two reaction pathways in the sub-networks-calcitonin gene-related peptide and IL-6 activated JAK/STAT were taken as examples to discuss the indirect action mechanism for preventing and treating CHD. The results showed that the biological network analysis method combining the disease network and the drug action network is helpful to further studies on the action mechanism of the drugs, and significant to the prevention and treatment of diseases.
Assuntos
Biologia Computacional , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/prevenção & controle , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Bases de Dados Genéticas , Humanos , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacosRESUMO
To analyze the intersections among the western medicine action network for preventing and treating coronary heart disease (CHD), as well as traditional Chinese medicine (TCM) action network for activating blood and dissolving stasis. In this article, 11 characteristic parameter values of network nodes, including connectivity, bottleneck, betweenness, were calculated. The target identification model was established based on key node characteristic parameters in the CHD-western medicine intersection network with support vector machine. Its C and y parameters were 5.14 and--1.11, respectively, with the predicted accuracies for positive and negative samples of 81.6% and 79.2%. The predicted sensitivity, specificity and accuracy of the test set samples were 81.5%, 78.3% and 79.6%, respectively. Besides, the model was applied to predict potential action targets of the CHD-activating blood and dissolving stasis TCM intersection network, and 180 positive nodes and 42 negative nodes were obtained. In this article, 9 positive nodes, including calnexin, interleukin-1, tumor necrosis factor, were taken as examples to analyze the action mechanism of TCM for preventing and treating CHD. The results showed that the network potential key target analysis method was helpful to explore the potential action targets of activating blood and dissolving stasis TCMs for preventing and treating CHD, methodologically supportive to reveal the action mechanism of TCMs at molecular and systematic levels, and significant in guiding the research and development of new drugs.
Assuntos
Doença da Artéria Coronariana/terapia , Medicina Tradicional Chinesa/métodos , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/prevenção & controle , Sistemas de Liberação de Medicamentos , Medicamentos de Ervas Chinesas , Humanos , Transdução de SinaisRESUMO
Numerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.
Assuntos
Neoplasias da Mama/genética , Neoplasias Colorretais/genética , Ribonucleoproteína Nuclear Heterogênea A1/genética , Neoplasias da Próstata/genética , Proteína-Arginina N-Metiltransferases/genética , Processamento Alternativo , Sequência de Aminoácidos , Arginina/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea A1/antagonistas & inibidores , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Masculino , Metilação/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Spliceossomos/efeitos dos fármacos , Spliceossomos/genética , Spliceossomos/metabolismo , Especificidade por SubstratoRESUMO
In this article, we describe the analysis of aptamers for Hg(2+) ions through CE with LIF (CE-LIF) detection using 2% poly(ethylene oxide) solutions containing OliGreen (fluorophore). In the presence of an EOF, DNA strands migrating against the EOF were detected at the cathode end. Four DNA strands - T(33), T(5)C(28), T(5)C(5)T(23), and T(15)C(5)T(13) - could not be separated through CE-LIF in the absence of Hg(2+). At 0.3 mM Hg(2+), however, all four were partially separated within 20 min, with SDs of the migration times all being less than 2.5%. From the CE, fluorescence, and ellipticity data, we concluded that the conformations of these four DNA strands all changed from random-coil to folded structures as a result of T-Hg(2+)-T bonding. In addition, we found that this CE approach provided different electropherograms patterns for T(7), T(15), and T(33) in the absence and presence of Hg(2+), indicating various interactions of the DNA strands with Hg(2+). Using this simple, high-resolution CE approach, we also demonstrated that adenosine triphosphate has a stronger interaction with the adenosine triphosphate aptamer than with either the platelet-derived growth factor aptamer or T(33). This CE approach holds great potential for screening aptamers for small solutes, studying the catalytic activity of DNAzymes, and evaluating the biological functions of microRNA.
Assuntos
Aptâmeros de Nucleotídeos/análise , DNA/análise , Eletroforese Capilar/métodos , Dicroísmo Circular , DNA de Cadeia Simples/isolamento & purificação , Eletro-Osmose , Mercúrio/química , Conformação de Ácido Nucleico , Espectrometria de FluorescênciaRESUMO
We have demonstrated the analysis of aristolochic acids (AAs) that are naturally occurring nephrotoxin and carcinogen by capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs (OAAs), reduction of the analytes by iron powder in 10.0 mM HCl is required prior to CE analysis. The reduced AAs (RAAs) exhibit fluorescence at 477 nm when excited at 405 nm using a solid-state blue laser. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS, the determination of AA-I and AA-II by CE-LIF has been achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The simple CE-LIF method has been validated by the analysis of 61 Chinese herbal samples. Prior to CE analysis, OAAs were extracted by using 5.0 mL MeOH, and then the extracts were subjected to centrifugation at 3,000 rpm for 5 min. After reduction, extraction, and centrifugation, the supernatants were collected and subjected to CE analysis. Of the 61 samples, 14 samples contain AA-I and AA-II, as well as 10 samples contain either AAI or AAII. The relative standard deviation (RSD) values of the migration times for AA-I and AA-II are less than 2.5% and 2.1% for three consecutive measurements of each sample. The RSD values for the peak heights corresponding to AA-I and AA-II in most samples are about 8.0% and 10.0%, respectively. The result shows that the present CE-LIF approach is sensitive, simple, efficient, and accurate for the determination of AAs in real samples.
Assuntos
Ácidos Aristolóquicos/análise , Medicamentos de Ervas Chinesas/química , Eletroforese Capilar/métodos , Ácidos Aristolóquicos/isolamento & purificação , Suplementos Nutricionais/análise , Lasers , Oxirredução , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodosRESUMO
Yin Yang 1 (YY1) is a multifunctional transcription factor shown to be critical in a variety of biological processes. Although it is regulated by multiple types of post-translational modifications (PTMs), whether YY1 is methylated, which enzyme methylates YY1, and hence the functional significance of YY1 methylation remains completely unknown. Here we reported the first methyltransferase, SET7/9 (KMT7), capable of methylating YY1 at two highly conserved lysine (K) residues, K173 and K411, located in two distinct domains, one in the central glycine-rich region and the other in the very carboxyl-terminus. Functional studies revealed that SET7/9-mediated YY1 methylation regulated YY1 DNA-binding activity both in vitro and at specific genomic loci in cultured cells. Consistently, SET7/9-mediated YY1 methylation was shown to involve in YY1-regulated gene transcription and cell proliferation. Our findings revealed a novel regulatory strategy, methylation by lysine methyltransferase, imposed on YY1 protein, and linked YY1 methylation with its biological functions.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Fator de Transcrição YY1/metabolismo , Sistemas CRISPR-Cas , Proliferação de Células/genética , Células HEK293 , Células HeLa , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Plasmídeos/química , Plasmídeos/metabolismo , Domínios Proteicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Fator de Transcrição YY1/antagonistas & inibidores , Fator de Transcrição YY1/genéticaRESUMO
In this study, we employed HgTe nanostructure-based matrices (nanomartrices; NMs) for surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) for the analyses of polyethylene glycol (PEG) derivatives as well as thiol-PEG-modified gold nanoparticles (PEG-Au NPs). Relative to common organic matrices, the use of HgTe NMs as the matrix for SALDI-MS resulted in more highly efficient analyses of PEG derivatives, in terms of sensitivity and reproducibility. The symmetric MS profiles of PEG (Mw: ca. 8000 Da) obtained through HgTe NMs/SALDI-MS analysis revealed the absence of polymer degradation during this process. Under optimal conditions, the HgTe NMs/SALDI-MS system enabled the detection of PEG sample as low as 100 pg and with molecular weights of up to approximately 42,000 Da. We also used this approach for the analyses of PEG-Au NPs in which various functional groups (carboxymethyl, amine, biotin) were present at the PEG termini, revealing that the combination of SALDI-MS and HgTe NMs have great potential for use in the characterization of modified polymer-ligands on nanomaterials. We also demonstrated the PEG-Au NPs can be coupled with HgTe NMs/SALDI-MS for characterization of biorecognition events. After avidin, the target protein, had been selectively captured by the biotin-PEG-Au NPs, we found that the desorption/ionization efficiency of biotin-PEG from the Au NP surface was suppressed; accordingly, this novel SALDI-MS approach allows rapid detection of avidin with high specificity and sensitivity. Au NP surfaces functionalized with other functional-PEG ligands might also allow amplification of signals from other biological interactions.
Assuntos
Nanopartículas/química , Nanoestruturas/química , Polímeros/química , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ouro/química , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Nanoestruturas/ultraestrutura , Polietilenoglicóis/química , Reprodutibilidade dos Testes , Espectrometria por Raios X , EspectrofotometriaRESUMO
The association between vinyl chloride monomer (VCM) exposure and DNA damage has been established. However, the relationship between individual exposure and DNA single strand breaks was limited. Since environmental monitoring may not reflect the actual exposure, a useful marker of exposure is needed to assess the individual exposure. In our previous study, we have found a high correlation between air VCM level and urinary thiodiglycolic acid (TdGA) at the commencement of the next shift. Here, we further used comet assay to evaluate the relationship between urinary TdGA levels and DNA single strand breaks in polyvinyl chloride monomer (PVC) workers. Urinary TdGA levels (n=26) at the commencement of the following shift were analyzed. Ten of the 26 workers also had personal air sampling for air VCM exposure. Questionnaires were administered to obtain epidemiological information including detailed history of occupation and lifestyles. Workers experiencing air VCM level greater than 5 ppm had higher tail moment and tail intensity (%) than those experiencing VCM exposure between 1 and 5, or <1 ppm, respectively (P < 0.05). The results also revealed that level of DNA single strand breaks, including tail moment and tail intensity, were increased with urinary TdGA level. The dose-response relationship of urinary TdGA level and DNA single strand breaks was particularly significant among the workers with 4 mg/g Cr of urinary TdGA level, which is equivalent to 5 ppm air VCM level. We concluded that air VCM exposure greater than 5 ppm could induce DNA damage. Further sensitive assay should be developed for the diction of DNA damage when air VCM exposure below 5 ppm.
Assuntos
Poluentes Ocupacionais do Ar/análise , Indústria Química , Dano ao DNA/efeitos dos fármacos , DNA de Cadeia Simples/efeitos dos fármacos , Exposição Ocupacional , Tioglicolatos/urina , Cloreto de Vinil/análise , Poluentes Ocupacionais do Ar/toxicidade , Ensaio Cometa , Relação Dose-Resposta a Droga , Humanos , Linfócitos/química , Masculino , Inquéritos e Questionários , Taiwan , Tioglicolatos/toxicidadeRESUMO
We applied surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures as the matrix for the detection of single- and double-stranded oligodeoxynucleotides (ss-ODNs and ds-ODNs). The concentrations of surfactant and additives (metal ions, an amine) and the pH and ionic strength of the sample matrix played significantly different roles in the detection of ss- and ds-ODNs with various sequences. In the presence of Brij 76 (1.5 %), Hg(2+) (7.5 µM), and cadaverine (10 µM) at pH 5.0, this SALDI-MS approach allowed the simultaneous detection of T15, T20, T33, and T40, with limits of detection at the femtomole-to-picomole level and sample-to-sample intensity variation <23 %. In the presence of Ag(+) (1 µM) and cadaverine (10 µM) at pH 7.0, this technique allowed the detection of randomly sequenced ss- and ds-ODNs at concentrations down to the femtomole level. To the best of our knowledge, this paper is the first to report the detection of ss-ODNs (up to 50-mer) and ds-ODNs (up to 30 base pairs) through the combination of SALDI-MS with HgTe nanostructures as matrices. We demonstrated the practicality of this approach through analysis of a single nucleotide polymorphism that determines the fate of the valine residue in the ß-globin of sickle cell megaloblasts.
Assuntos
Oligodesoxirribonucleotídeos/análise , Oligodesoxirribonucleotídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cadaverina/química , Ácido Cítrico/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Modelos Lineares , Metais Pesados/química , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Compostos de Amônio Quaternário/químicaRESUMO
We describe simultaneous analysis of naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and NDA-biogenic amine derivatives by CE in conjunction with light-emitting diode-induced fluorescence detection using poly(ethylene oxide) (PEO) solutions containing sodium dodecyl sulfate (SDS). After sample injection, via EOF 0.1% PEO prepared in 100mM TB solution (pH 9.0) containing 30 mM SDS entered a capillary filled with 0.5M TB solution (pH 10.2) containing 40 mM SDS. Under this condition, 14 NDA-amino acid and NDA-amine derivatives were separated within 16 min, with high efficiency ((1.0-3.2)x10(5) theoretical plates) and sensitivity (LODs at S/N=3 ranging from 2.06 to 19.17 nM). In the presence of SDS and PEO, analytes adsorption on the capillary wall was suppressed, leading to high efficiency and reproducibility. The intraday analysis RSD values (n=3) of the mobilities for the analytes are less than 0.52%. We have validated the practicality of this approach by quantitative determination of 9 amino acids in breast cancer cells (MCF-7) and 10 amino acids in normal epithelial cells (H184B5F5/M10). The concentrations of Tau and Gln in the MCF-7 cells were different than those in the H184B5F5/M10 cells, respectively. Our results show the potential of this approach for cancer study.
Assuntos
Aminoácidos/análise , Aminas Biogênicas/análise , Neoplasias da Mama/química , Eletroforese Capilar/métodos , Naftalenos/análise , Aminoácidos/química , Aminas Biogênicas/química , Linhagem Celular Tumoral , Feminino , Fluorescência , Humanos , Limite de Detecção , Naftalenos/química , Polietilenoglicóis/química , Reprodutibilidade dos Testes , Dodecilsulfato de Sódio/químicaRESUMO
Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.