RESUMO
OBJECTIVE: The aim of this study was to explore the role of microRNA-579-3p (miR-579-3p) in the development of squamous cell lung carcinoma (SCLC). Our findings might provide new insights into the treatment of SCLC. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression level of miR-579-3p in 30 pairs of CRC tissues and para-cancerous tissues. The relation between miR-579-3p expression and clinical features of SCLC patients was analyzed. MiR-579-3p expression in SCLC cells was further verified by qRT-PCR as well. In addition, the effects of miR-579-3p on the migration and invasion of SK-MES-1 cells were examined through the transwell assay. RESULTS: QRT-PCR results revealed for the first time that miR-579-3p was significantly down-regulated in SCLC tissues. This indicated that miR-579-3p was possibly involved in the development of SCLC. According to bioinformatics prediction websites and the luciferase activity assay, macrophage scavenger receptor 1 (MSR1) was predicted and verified as the target gene of miR-579-3p. Transfection of miR-579-3p mimics significantly reduced the protein expression level of MSR1 in cells, eventually inhibiting the proliferation, invasion, and migration of SCLC cells. CONCLUSIONS: The miR-579-3p/MSR1 axis might be a novel regulatory pathway of apoptosis, which could be used as potential therapeutic sites in SCLC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Apoptose , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/genéticaRESUMO
Gas-phase conformations of the sodium-cationized forms of the 2'-deoxycytidine and cytidine mononucleotides, [pdCyd+Na]+ and [pCyd+Na]+, are examined by infrared multiple photon dissociation action spectroscopy. Complimentary electronic structure calculations at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory provide candidate conformations and their respective predicted IR spectra for comparison across the IR fingerprint and hydrogen-stretching regions. Comparisons of the predicted IR spectra and the measured infrared multiple photon dissociation action spectra provide insight into the impact of sodium cationization on intrinsic mononucleotide structure. Further, comparison of present results with those reported for the sodium-cationized cytidine nucleoside analogues elucidates the impact of the phosphate moiety on gas-phase structure. Across the neutral, protonated, and sodium-cationized cytidine mononucleotides, a preference for stabilization of the phosphate moiety and nucleobase orientation is observed, although the details of this stabilization differ with the state of cationization. Several low-energy conformations of [pdCyd+Na]+ and [pCyd+Na]+ involving several different orientations of the phosphate moiety and sugar puckering modes are observed experimentally.
Assuntos
Citidina/química , DNA/química , RNA/química , Sódio/química , Espectrofotometria Infravermelho/métodos , Cátions Monovalentes/química , Monofosfato de Citidina/química , Desoxicitidina Monofosfato/química , Gases/química , Conformação de Ácido NucleicoRESUMO
The gas-phase conformations of transition metal cation-uracil complexes, [Ura+Cu]+ and [Ura+Ag]+, were examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra were measured over the IR fingerprint and hydrogen-stretching regions. Structures and linear IR spectra of the stable tautomeric conformations of these complexes were initially determined at the B3LYP/6-31G(d) level. The four most stable structures computed were also examined at the B3LYP/def2-TZVPPD level to improve the accuracy of the predicted IR spectra. Two very favorable modes of binding are found for [Ura+Cu]+ and [Ura+Ag]+ that involve O2N3 bidentate binding to the 2-keto-4-hydroxy minor tautomer and O4 monodentate binding to the canonical 2,4-diketo tautomer of Ura. Comparisons between the measured IRMPD and calculated IR spectra enable elucidation of the conformers present in the experiments. These comparisons indicate that both favorable binding modes are represented in the experimental tautomeric conformations of [Ura+Cu]+ and [Ura+Ag]+. B3LYP suggests that Cu+ exhibits a slight preference for O4 binding, whereas Ag+ exhibits a slight preference for O2N3 binding. In contrast, MP2 suggests that both Cu+ and Ag+ exhibit a more significant preference for O2N3 binding. The relative band intensities suggest that O4 binding conformers comprise a larger portion of the population for [Ura+Ag]+ than [Ura+Cu]+. The dissociation behavior and relative stabilities of the [Ura+M]+ complexes, M+ = Cu+, Ag+, H+, and Na+) are examined via energy-resolved collision-induced dissociation experiments. The IRMPD spectra, dissociation behaviors, and binding preferences of Cu+ and Ag+ are compared with previous and present results for those of H+ and Na+. Graphical Abstract á .
RESUMO
The gas-phase conformations of the protonated forms of the DNA and RNA cytosine mononucleotides, [pdCyd+H]+ and [pCyd+H]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy over the IR fingerprint and hydrogen-stretching regions complemented by electronic structure calculations. The low-energy conformations of [pdCyd+H]+ and [pCyd+H]+ and their relative stabilities are computed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers allow the conformers present in the experiments to be determined. Similar to that found in previous IRMPD action spectroscopy studies of the protonated forms of the cytosine nucleosides, [dCyd+H]+ and [Cyd+H]+, both N3 and O2 protonated cytosine mononucleotides exhibiting an anti orientation of cytosine are found to coexist in the experimental population. The 2'-hydroxyl substituent does not significantly influence the most stable conformations of [pCyd+H]+ versus those of [pdCyd+H]+, as the IRMPD spectral profiles of [pdCyd+H]+ and [pCyd+H]+ are similar. However, the presence of the 2'-hydroxyl substituent does influence the relative intensities of the measured IRMPD bands. Comparisons to IRMPD spectroscopy studies of the deprotonated forms of the cytosine mononucleotides, [pdCyd-H]- and [pCyd-H]-, provide insight into the effects of protonation versus deprotonation on the conformational features of the nucleobase and sugar moieties. Likewise, comparisons to results of IRMPD spectroscopy studies of the protonated cytosine nucleosides provide insight into the influence of the phosphate moiety on structure. Comparison with previous ion mobility results shows the superiority of IRMPD spectroscopy for distinguishing various protonation sites. Graphical Abstract á .
Assuntos
Nucleotídeos de Citosina/química , Prótons , Espectrofotometria Infravermelho , Citosina/química , Gases/química , Modelos Moleculares , Conformação Molecular , Nitrogênio/química , Oxigênio/química , Fótons , Espectrofotometria Infravermelho/métodosRESUMO
Human colorectal cancer (CRC) is a major worldwide health concern, and its development has been shown to be associated with alcohol intake. We carried out a study to investigate the effect of the ADH1B Arg47His and ALDH2 Glu487Lys genetic polymorphisms and their interaction with alcohol consumption on development of CRC. Between March 2013 and May 2015, a total of 274 CRC patients and 358 healthy controls were recruited. Genotyping of sequence variations was performed using the polymerase chain reaction-restriction fragment length polymorphism method. Under a co-dominant model, individuals with the ADH1B Arg47His AA genotype showed increased CRC risk compared to those carrying the GG genotype, with an adjusted odds ratio (and 95% confidence interval) of 3.37 (2.00-5.70). Moreover, under dominant and recessive models, ADH1B Arg47His variant genotypes were associated with greater susceptibility to CRC when compared with the wild-type sequence. Both polymorphisms examined were positively associated with alcohol consumption in a Spearman correlation analysis of CRC risk. In conclusion, our study suggests that the ADH1B Arg47His polymorphism, but not the ALDH2 Glu487Lys variation, may influence development of CRC in the Chinese population.
Assuntos
Álcool Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial/genética , Povo Asiático/genética , Neoplasias Colorretais/genética , Interação Gene-Ambiente , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Estudos de Casos e Controles , Demografia , Feminino , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Our previous gas-phase infrared multiple photon dissociation action spectroscopy study of protonated 2'-deoxyadenosine and adenosine, [dAdo+H](+) and [Ado+H](+), found that both N3 and N1 protonated conformers are populated with the N3 protonated ground-state conformers predominant in the experiments. Therefore, N-glycosidic bond dissociation mechanisms of N3 and N1 protonated [dAdo+H](+) and [Ado+H](+) and the associated quantitative thermochemical values are investigated here using both experimental and theoretical approaches. Threshold collision-induced dissociation (TCID) of [dAdo+H](+) and [Ado+H](+) with Xe is studied using guided ion beam tandem mass spectrometry techniques. For both systems, N-glycosidic bond cleavage reactions are observed as the major dissociation pathways resulting in production of protonated adenine or elimination of neutral adenine. Electronic structure calculations are performed at the B3LYP/6-311+G(d,p) level of theory to probe the potential energy surfaces (PESs) for N-glycosidic bond cleavage of [dAdo+H](+) and [Ado+H](+). Relative energetics of the reactants, transition states, intermediates and products along the PESs for N-glycosidic bond cleavage are determined at the B3LYP/6-311+G(2d,2p), B3LYP-GD3BJ/6-311+G(2d,2p), and MP2(full)/6-311+G(2d,2p) levels of theory. The predicted N-glycosidic bond dissociation mechanisms for the N3 and N1 protonated species differ. Base rotation of the adenine residue enables formation of a strong N3H(+)O5' hydrogen-bonding interaction that stabilizes the N3 protonated species and its glycosidic bond. Comparison between experiment and theory indicates that the N3 protonated species determine the threshold energies, as excellent agreement between the measured and B3LYP computed activation energies (AEs) and reaction enthalpies (ΔHrxns) for N-glycosidic bond cleavage of the N3 protonated species is found.
RESUMO
Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments combined with theoretical calculations are performed to investigate the stable gas-phase conformations of the protonated adenine mononucleotides, [pdAdo+H](+) and [pAdo+H](+). Conformations that are present in the experiments are elucidated via comparative analyses of the experimental IRMPD spectra and the B3LYP/6-311+G(d,p) IR spectra predicted for the conformers optimized at this level of theory. N3 protonation is preferred as it induces base rotation, which allows a strong hydrogen bond to be formed between the excess proton of adenine and the phosphate moiety. In contrast, both N1 and N7 protonation are predicted to be >35 kJ/mol less favorable than N3 protonation. Only N3 protonated conformers are present in the experiments in measurable abundance. Both the low-energy conformers computed and the experimental IRMPD spectra of [pdAdo+H](+) and [pAdo+H](+) indicate that the 2'-hydroxyl moiety does not significantly impact the structure of the most stable conformer or the IRMPD spectral profile of [pAdo+H](+) vs that of [pdAdo+H](+). However, the 2'-hydroxyl leads to a 3-fold enhancement in the IRMPD yield of [pAdo+H](+) in the fingerprint region. Comparison of present results to those reported in a previous IRMPD study of the analogous protonated adenine nucleosides allows the effects of the phosphate moiety on the gas-phase conformations to be elucidated.
Assuntos
Monofosfato de Adenosina/química , Nucleotídeos de Desoxiadenina/química , Ligação de Hidrogênio , Nitrogênio/química , Prótons , Espectrofotometria InfravermelhoRESUMO
Non-small cell lung cancer (NSCLC) is the most common cancer globally. The XRCC1 protein interacts with ligase and poly(ADP-ribose) polymerase to repair cisplatin-induced DNA damage. The authors of previous studies have reported XRCC1 Arg399Gln, Arg280His, and Arg194Trp polymorphisms and advanced NSCLC prognosis, but the results are inconclusive. We investigated the association between clinical outcome and XRCC1 Arg399Gln, Arg280His, and Arg194Trp polymorphisms in advanced NSCLC patients treated with cisplatin. We recruited 252 patients with advanced NSCLC (TNM stages: IIIB and IV) and used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to genotype the polymorphisms. Patients with the TT genotype of XRCC1 Arg194Trp showed a significantly better response to chemotherapy than those with the CC genotype. The GA+AA genotype of Arg194Trp was correlated with better response to chemotherapy than the wild-type form. The TT genotype of Arg194Trp was associated with longer survival time than the CC genotype. The TT genotype of Arg194Trp was correlated with lower risk of death from all causes than the CC genotype. The Arg194Trp polymorphisms interacted with squamous cell carcinoma and affected overall survival of advanced NSCLC. However, there was no association between Arg399Gln and Arg280His polymorphisms and response to cisplatin-based chemotherapy and overall survival in advanced NSCLC. The results suggest that the TT genotype of Arg194Trp is significantly associated with better response to chemotherapy and longer overall survival of advanced NSCLC patients than the wild-type form. Our investigation offers insight into the influence of XRCC1 gene polymorphisms on the treatment outcome of advanced NSCLC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Polimorfismo Genético , Alelos , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/administração & dosagem , Genótipo , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Estadiamento de Neoplasias , Resultado do Tratamento , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
The gas-phase structures of protonated thymidine, [dThd + H](+), and its modified form, protonated 5-methyluridine, [Thd + H](+), are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy combined with electronic structure calculations. IRMPD action spectra are measured over the ranges extending from ~600 to 1900 cm(-1) and ~2800 to 3800 cm(-1) using the FELIX free electron laser and an optical parametric oscillator/amplifier (OPO/OPA) laser system, respectively. Comparisons between the B3LYP/6-311+G(d,p) linear IR spectra calculated for the stable low-energy conformers and the measured IRMPD spectra are used to determine the most favorable tautomeric conformations of [dThd + H](+) and [Thd + H](+) and to identify those populated in the experiments. Both B3LYP and MP2 levels of theory predict a minor 2,4-dihydroxy tautomer as the ground-state conformer of [dThd + H](+) and [Thd + H](+) indicating that the 2'-hydroxyl substituent of Thd does not exert a significant impact on the structural features. [dThd + H](+) and [Thd + H](+) share parallel IRMPD spectral profiles and yields in both the FELIX and OPO regions. Comparisons between the measured IRMPD and calculated IR spectra suggest that minor 2,4-dihydroxy tautomers and O2 protonated conformers of [dThd + H](+) and [Thd + H](+) are populated in the experiments. Comparison of this work to our previous IRMPD spectroscopy study of protonated 2'-deoxyuridine and uridine suggests that the 5-methyl substituent alters the preferences of O2 versus O4 protonation.
Assuntos
Oxigênio/química , Prótons , Timidina/química , Uridina/análogos & derivados , Gases/química , Isomerismo , Espectrometria de Massas , Metilação , Modelos Moleculares , Uridina/químicaRESUMO
OBJECTIVES: To explore whether sex hormone-binding globulin (SHBG) and free androgen index (FAI) can be seen as therapeutic effect indexes of women with polycystic ovarian syndrome (PCOS). MATERIALS AND METHODS: The body mass index (BMI), basal sexual hormones, SHBG, fasting blood glucose (FBG), and fasting insulin (FINS) were collected from 579 women with PCOS, were divided into two groups according to BMI: obese group (n = 145) and non-obese group (n = 434), according to homeostasis model assessment of insulin status (HOMA-IR). Patients were then divided into four groups: A: non-obese without insulin resistance (n = 174), B: non-obese with insulin resistance (n = 260), C: obese without insulin resistance (n = 34), D: obese with insulin resistance (n = 111). A and B groups received Diane-35 alone, C and D groups received Diane-35 plus metformin for three months. Then clomiphene citrate and HMAG were used to induce ovulation then compared ovulation rate and pregnancy outcome. RESULTS: FAI decreased significantly and SHBG increased significantly in all groups. In A group FINS and HOMA-IR increased significantly (p < 0.05), but in B and D groups FINS and HOMA-IR decreased significantly (p < 0.05). After treatment the ovulation rate in non-obese group was higher than obese group (p < 0.01). Compared with non-ovulation patients, SHBG increased significantly and FAI decreased significantly in the patient with ovulation. Regarding the pregnancy outcome, FAI decreased significantly in delivery patients than spontaneous abortion patients. Furthermore, SHBG increased significantly. CONCLUSION: It was important to check SHBG and FAI during the treatment of PCOS patient. They could be used to assess whether the treatment was effective and as a guidance of clinical medication.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Glicemia/metabolismo , Acetato de Ciproterona/uso terapêutico , Etinilestradiol/uso terapêutico , Hipoglicemiantes/uso terapêutico , Insulina/sangue , Metformina/uso terapêutico , Síndrome do Ovário Policístico/tratamento farmacológico , Globulina de Ligação a Hormônio Sexual/metabolismo , Aborto Espontâneo , Adulto , Índice de Massa Corporal , Clomifeno/uso terapêutico , Combinação de Medicamentos , Quimioterapia Combinada , Estradiol/sangue , Feminino , Fármacos para a Fertilidade Feminina/uso terapêutico , Hormônio Foliculoestimulante/sangue , Humanos , Resistência à Insulina , Nascido Vivo , Hormônio Luteinizante/sangue , Obesidade/complicações , Obesidade/metabolismo , Indução da Ovulação/métodos , Síndrome do Ovário Policístico/complicações , Síndrome do Ovário Policístico/metabolismo , Prolactina/sangue , Testosterona/sangue , Resultado do Tratamento , Adulto JovemRESUMO
The gas-phase conformations of protonated uridine, [Urd+H](+), and its modified form, protonated 2'-deoxyuridine, [dUrd+H](+), generated by electrospray ionization are investigated using infrared multiple photon dissociation (IRMPD) action spectroscopy techniques. IRMPD action spectra of [Urd+H](+) and [dUrd+H](+) are measured over the IR fingerprint and hydrogen-stretching regions. [Urd+H](+) and [dUrd+H](+) exhibit very similar IRMPD spectral profiles. However, the IRMPD yields of [Urd+H](+) exceed those of [dUrd+H](+) in both the IR fingerprint and hydrogen-stretching regions. The measured spectra are compared to the linear IR spectra predicted for the stable low-energy structures of these species computed at the B3LYP/6-311+G(d,p) level of theory to determine the tautomeric conformations populated by electrospray ionization. Both B3LYP and MP2 methods find O4 and O2 protonated canonical as well as 2,4-dihydroxy tautomers among the stable low-energy structures of [Urd+H](+) and [dUrd+H](+). Comparison between the measured IRMPD and calculated linear IR spectra suggests that these species exist in their ring-closed forms and that both 2,4-dihydroxy tautomers as well as O4 protonated canonical conformers coexist in the population generated by electrospray ionization for both [Urd+H](+) and [dUrd+H](+). The 2'-deoxy modification of [dUrd+H](+) reduces the variety of 2,4-dihydroxy tautomers populated in the experiments vs. those of [Urd+H](+).
Assuntos
Desoxiuridina/química , Prótons , Uridina/química , Gases/química , Isomerismo , Modelos Moleculares , Conformação Molecular , Espectrofotometria InfravermelhoRESUMO
Infrared multiple photon dissociation action spectra of the protonated forms of the cytidyl nucleosides, 2'-deoxycytidine, [dCyd+H](+), and cytidine, [Cyd+H](+), are acquired over the IR fingerprint and hydrogen-stretching regions. Electronic structure calculations are performed at the B3LYP/6-311+G(d,p) level to determine the stable low-energy tautomeric conformations of these species generated upon electrospray ionization (ESI) and to generate the linear IR absorption spectra of these protonated nucleosides. Comparison between the experimental and theoretical spectra allows the tautomeric conformations of [dCyd+H](+) and [Cyd+H](+) populated by ESI to be determined. B3LYP predicts N3 as the preferred protonation site for both [dCyd+H](+) and [Cyd+H](+), whereas MP2 suggests that protonation at O2 is more favorable. The 2'-hydroxyl substituent does not significantly alter the structures of the B3LYP N3 and MP2 O2 protonated ground tautomeric conformations of [dCyd+H](+) vs [Cyd+H](+). [dCyd+H](+) and [Cyd+H](+) exhibit very similar spectral signatures in both regions. Nonetheless, the 2'-hydroxyl does affect the relative intensities of the IRMPD bands of [dCyd+H](+) vs [Cyd+H](+). The spectral features observed in the hydrogen-stretching region complement those of the fingerprint region and allow the N3 and O2 protonated tautomeric conformations to be readily distinguished. Comparison between the measured and computed spectra indicates that both N3 and O2 protonated tautomeric conformations coexist in the experiments, and the populations are dominated by the most stable N3 and O2 protonated tautomeric conformations. Least-squares fitting of the IRMPD spectra to the IR spectra for these most stable conformers suggests relative populations of â¼55% N3 vs 45% O2 protonated conformers of [dCyd+H](+), whereas â¼47% N3 vs 53% O2 protonated conformers of [Cyd+H](+). This change in the preferred site of protonation indicates that the 2'-hydroxyl substituent plays an important role in controlling the reactivity of the cytidyl nucleosides.
Assuntos
Citidina/química , Desoxicitidina/química , Gases/química , Nitrogênio/química , Oxigênio/química , Prótons , Hidrogênio/química , Análise dos Mínimos Quadrados , Modelos Químicos , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Infravermelho , VibraçãoRESUMO
BACKGROUND: The goal of this study was to assess the value of the 3-3 rule and the 3-3-1 rule in predicting difficult airways. METHODS: The authors conducted an observational study over a 6-month period. For each consenting adult patient undergoing general anesthesia, preoperative patient characteristics and data regarding difficult airway assessments and airway outcomes were collected. The 3-3-2 rule, 3-3-1 rule and 3-3 rule were included in preoperative difficult airway assessments. The 3-3-1 rule is defined as an interincisor distance (IID) less than three fingers, a hyoid-mental distance (HMD) less than three fingers, and a hyoid-thyroid cartilage distance (HTD) less than one finger. RESULTS: Among the 732 patients who were successfully recruited in this study, 67 patients had difficult laryngoscopy (DL) (9.2 %), and 25 patients had difficult intubation (DI) (3.4 % of the total). All of the DI patients were also DL patients (25/67, 37.3 %). The AUC of the 3-3-2, 3-3, and 3-3-1 rules for predicting difficult laryngoscopy were 0.702, 0.709, and 0.631, respectively. Significant differences between the 3-3-2 and 3-3-1 rules as well as between the 3-3 and 3-3-1 rules were evident. The AUC values for the 3-3-2, 3-3, and 3-3-1 rules for predicting DI were 0.830, 0.822, and 0.725, respectively. CONCLUSIONS: The 3-3 rule and the 3-3-2 rule are similar regarding their ability to predict difficult airways. A HTD less than two fingers or one finger is not predictive of DV or DI.
Assuntos
Anestesia Geral , Intubação Intratraqueal/métodos , Laringoscopia/métodos , Cuidados Pré-Operatórios/métodos , Cartilagem Tireóidea/patologia , Adolescente , Adulto , Idoso , Manuseio das Vias Aéreas/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de RiscoRESUMO
The gas-phase conformations of protonated 2'-deoxyadenosine, [dAdo+H](+), and its RNA analogue protonated adenosine, [Ado+H](+), generated upon electrospray ionization are examined using infrared multiple photon dissociation (IRMPD) action spectroscopy techniques and supported by complementary theoretical electronic structure calculations. IRMPD action spectra are measured over the IR fingerprint region using the FELIX free-electron laser and the hydrogen-stretching region using an optical parametric oscillator/amplifier laser system. The measured IRMPD spectra are compared to linear IR spectra predicted for the stable low-energy conformations of [dAdo+H](+) and [Ado+H](+) computed at the B3LYP/6-311+G(d,p) level of theory to determine the preferred site of protonation and to identify the structures populated in the experiments. N3 is found to be the most favorable site of protonation for both [dAdo+H](+) and [Ado+H](+), whereas conformers protonated at the N1 and N7 positions are much less stable by >25 kJ/mol. The 2'-hydroxyl substituent of Ado does not lead to a significant change in the structure of the ground-state conformer of [Ado+H](+) as compared to that of [dAdo+H](+), except that it provides additional stabilization via the formation of an O2'H···O3' hydrogen bond. Therefore, [dAdo+H](+) and [Ado+H](+) exhibit highly parallel IRMPD spectral features in both the fingerprint and hydrogen-stretching regions. However, the additional 2'-hydroxyl substituent markedly affects the IRMPD yield of the measured IR bands. The spectral signatures in the hydrogen-stretching region provide complementary information to that of the fingerprint region and enable facile differentiation of the conformers that arise from different protonation sites. In spite of the relative gas-phase stabilities of the N3 and N1 protonated conformers, present results suggest that both are accessed in the experiments and contribute to the measured IRMPD spectrum, indicating that the relative stabilities in solution also influence the populations generated by electrospray ionization.
Assuntos
Adenosina/química , Desoxiadenosinas/química , Gases/química , Prótons , Ligação de Hidrogênio , Modelos Químicos , Conformação Molecular , Espectrofotometria Infravermelho/métodos , Termodinâmica , VibraçãoRESUMO
The gas-phase structures of protonated 2'-deoxyguanosine, [dGuo+H](+), and its RNA analogue protonated guanosine, [Guo+H](+), are investigated by infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. IRMPD action spectra are measured over the range extending from â¼550 to 1900 cm(-1) using the FELIX free electron laser and from â¼2800 to 3800 cm(-1) using an optical parametric oscillator/amplifier (OPO/OPA) laser system. The measured IRMPD spectra of [dGuo+H](+) and [Guo+H](+) are compared to each other and to B3LYP/6-311+G(d,p) linear IR spectra predicted for the stable low-energy conformations computed for these species to determine the most favorable site of protonation, identify the structures accessed in the experiments, and elucidate the influence of the 2'-hydroxyl substituent on the structures and the IRMPD spectral features. Theoretical energetics and the measured IRMPD spectra find that N7 protonation is preferred for both [dGuo+H](+) and [Guo+H](+), whereas O6 and N3 protonated conformers are found to be much less stable. The 2'-hydroxyl substituent does not exert a significant influence on the structures and relative stabilities of the stable low-energy conformations of [dGuo+H](+) versus [Guo+H](+) but does provide additional opportunities for hydrogen bonding such that more low-energy structures are found for [Guo+H](+). [dGuo+H](+) and [Guo+H](+) share very parallel IRMPD spectral features in the FELIX and OPO regions, whereas the effect of the 2'-hydroxyl substituent is primarily seen in the relative intensities of the measured IR bands. The measured OPO/OPA spectral signatures, primarily reflecting the IR features associated with the O-H and N-H stretches, provide complementary information to that of the FELIX region and enable the conformers that arise from different protonation sites to be more readily distinguished. Insight gained from this and parallel studies of other DNA and RNA nucleosides and nucleotides should help better elucidate the effects of the local environment on the overall structures of DNA and RNA.
Assuntos
Desoxiguanosina/química , Guanosina/química , Modelos Teóricos , Espectrofotometria Infravermelho/métodos , Ligação de Hidrogênio , Espectrometria de Massas , PrótonsRESUMO
The gas-phase structures of proton-bound dimers of cytosine and modified cytosines and their d6-analogues generated by electrospray ionization are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. The modified cytosines examined include the 5-methyl-, 5-fluoro-, and 5-bromo-substituted species. IRMPD action spectra of seven proton-bound dimers exhibit both similar and distinctive spectral features over the range of â¼2600-3700 cm(-1). The IRMPD spectra of all of these proton-bound dimers are relatively simple, but exhibit obvious shifts in the positions of several bands that correlate with the properties of the substituent. The measured IRMPD spectra are compared to linear IR spectra calculated for the stable low-energy tautomeric conformations, determined at the B3LYP/6-31G* level of theory, to identify the conformations accessed in the experiments. Comparison of the measured IRMPD and calculated IR spectra indicates that only a single conformation, the ground-state structure, is accessed for all proton-bound homodimers, whereas the ground-state and a small population of the first-excited tautomeric conformations are accessed for all proton-bound heterodimers. In all cases, three hydrogen-bonding interactions in which the nucleobases are aligned in an antiparallel fashion analogous to that of the DNA i-motif are responsible for stabilizing the base pairing. Thus, base modifications such as 5-methyl- and 5-halo-substitution of cytosine should not alter the structure of the DNA i-motif.
Assuntos
Citosina/análogos & derivados , Pareamento de Bases , Dimerização , Gases/química , Ligação de Hidrogênio , Conformação de Ácido Nucleico , Fótons , Prótons , Espectrofotometria InfravermelhoRESUMO
The gas-phase structures of alkali metal cation-cytosine complexes generated by electrospray ionization are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra of five alkali metal cation-cytosine complexes exhibit both similar and distinctive spectral features over the range of ~1000-1900 cm(-1). The IRMPD spectra of the Li(+)(cytosine), Na(+)(cytosine), and K(+)(cytosine) complexes are relatively simple but exhibit changes in the shape and shifts in the positions of several bands that correlate with the size of the alkali metal cation. The IRMPD spectra of the Rb(+)(cytosine) and Cs(+)(cytosine) complexes are much richer as distinctive new IR bands are observed, and the positions of several bands continue to shift in relation to the size of the metal cation. The measured IRMPD spectra are compared to linear IR spectra of stable low-energy tautomeric conformations calculated at the B3LYP/def2-TZVPPD level of theory to identify the conformations accessed in the experiments. These comparisons suggest that the evolution in the features in the IRMPD action spectra with the size of the metal cation, and the appearance of new bands for the larger metal cations, are the result of the variations in the intensities at which these complexes can be generated and the strength of the alkali metal cation-cytosine binding interaction, not the presence of multiple tautomeric conformations. Only a single tautomeric conformation is accessed for all five alkali metal cation-cytosine complexes, where the alkali metal cation binds to the O2 and N3 atoms of the canonical amino-oxo tautomer of cytosine, M(+)(C1).
Assuntos
Citosina/química , Espectrometria de Massas/métodos , Metais Alcalinos/química , Cátions/química , Gases/química , Tamanho da Partícula , Estereoisomerismo , TermodinâmicaRESUMO
Five distinct organic anion transporter cDNAs, hOAT1-5, were isolated from human liver and kidney. hOAT1, 2, and 3 are homologous to their respective rat orthologues OAT1-3, whereas hOAT4 and 5 are novel clones that have not been identified in other species. hOAT1- and hOAT3-transfected cells showed uptake of p-aminohippurate and fluorescein. Cells expressing hOAT2 showed uptake of p-aminohippurate, methotrexate, cAMP, and alpha-ketoglutarate. Northern blot analysis indicated differential tissue distribution for the transporter transcripts. These results indicate the existence of a family of organic anion transporting proteins in humans distinct from the oatp-like family of transporters.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Rim/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes , Transportadores de Ânions Orgânicos , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Ânions , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonagem Molecular , AMP Cíclico/metabolismo , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Humanos , Técnicas In Vitro , Ácidos Cetoglutáricos/metabolismo , Metotrexato/metabolismo , Dados de Sequência Molecular , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transfecção , Xenopus laevis , Ácido p-Aminoipúrico/metabolismoRESUMO
Two new prenylflavans [kazinols Q (1) and R (2)] and five known compounds [kazinols D (3), K (4), and H, 7,4'-dihydroxyflavan (5), and oleanolic acid] were isolated from the root bark of Broussonetia kazinoki. The cytotoxic activity of 1-5 was evaluated against several different cell lines.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Flavonoides/isolamento & purificação , Plantas/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Flavonoides/química , Flavonoides/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Análise Espectral , Células Tumorais CultivadasRESUMO
PURPOSE: To uncover abnormalities of extracellular matrix (ECM) distribution in human corneas with pseudophakic and aphakic bullous keratopathy (PBK/ABK). METHODS: Indirect immunofluorescence with antibodies to 27 ECM components was used on frozen sections of 14 normal and 20 PBK/ABK corneas. RESULTS: Fibrillar deposits of an antiadhesive glycoprotein tenascin in the anterior and posterior stroma, epithelial basement membrane (BM), bullae and subepithelial fibrosis (SEF) areas, and posterior collagenous layer (PCL) were revealed in disease corneas. Tenascin in midstroma, which was observed in some cases, correlated with decreased visual acuity. In normal central corneas, tenascin was never found. Other major ECM abnormalities in PBK/ABK corneas compared to normals included: discontinuous epithelial BM straining for laminin-1 (alpha 1 beta 1 gamma 1), entactin/nidogen and fibronectin; accumulation of fibronectin and alpha 1-alpha 2 type IV collagen on the endothelial face of the Descemet's membrane; and abnormal deposition of stromal ECM (tenascin, fibronectin, decorin, types I, III, V, VI, VIII, XII, XIV collagen) and BM components (type IV, collagen, perlecan, bamacan, laminin-1, entactin-nidogen, fibronectin) in SEF areas and in PCL. CONCLUSIONS: The study provides a molecular description of an ongoing fibrosis on the epithelial, stomal, and endothelial levels in PBK/ABK corneas. These fibrotic changes may follow initial endothelial damage after cataract surgery, may be caused by the upregulation of fibrogenic cytokines, and may play a significant role in the progression of bullous keratopathy.