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1.
Nat Commun ; 11(1): 1098, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107375

RESUMO

The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.


Assuntos
Proteínas de Bactérias/genética , Reatores Biológicos , Caulobacter crescentus/genética , Engenharia Metabólica/métodos , Modelos Químicos , Proteínas de Bactérias/metabolismo , Biocombustíveis , Metabolismo dos Carboidratos/genética , Caulobacter crescentus/enzimologia , Simulação por Computador , Hidroliases/genética , Hidroliases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Redes e Vias Metabólicas/genética , NADP/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Xilose/metabolismo
2.
Biotechnol J ; 13(8): e1700529, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29697210

RESUMO

Shikimic acid 3-phosphate, as a central metabolite of the shikimate pathway, is of high interest as enzyme substrate for 5-enolpyruvoyl-shikimate 3-phosphate synthase, a drug target in infectious diseases and a prime enzyme target for the herbicide glyphosate. As the important substrate shikimic acid 3-phosphate is only accessible via a chemical multi-step route, a new straightforward preparative one-step enzymatic phosphorylation of shikimate using a stable recombinant shikimate kinase has been developed for the selective phosphorylation of shikimate in the 3-position. Highly active shikimate kinase is produced by straightforward expression of a synthetic aroL gene in Escherichia coli. The time course of the shikimate kinase-catalyzed phosphorylation is investigated by 1 H- and 31 P-NMR, using the phosphoenolpyruvate/pyruvate kinase system for the regeneration of the ATP cofactor. This enables the development of a quantitative biocatalytic 3-phosphorylation of shikimic acid. After a standard workup procedure, a good yield of shikimic acid 3-phosphate, with high HPLC- and NMR purity, is obtained. This efficient biocatalytic synthesis of shikimic acid 3-phosphate is superior to any other method and has been successfully scaled up to multi-gram scale.


Assuntos
Proteínas de Escherichia coli/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes/metabolismo , Ácido Chiquímico/análogos & derivados , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ácido Chiquímico/análise , Ácido Chiquímico/metabolismo
3.
Inorg Chem ; 53(13): 6684-97, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-24918934

RESUMO

The molecular structures of Li2[Ni(tmdta)]·5H2O (1a, tmdta = trimethylenediaminetetraacetate), {C(NH2)3}2[Ni(tmdta)]·6H2O (1b), and {Ni(H2O)6}[Ni(tmdta)]·2H2O (2a) have been determined. The central trimethylenediamine chelate ring shows half-chair (hc) geometries in 1a and 1b, while a twist-boat (tb) conformation is encountered in 2a. The coexistence of tb and hc forms in the solid state prompted us to elucidate the existence of a tb ⇌ hc equilibrium in aqueous solution. Evaluation of the data from solid state vibrational spectra (Raman and IR) for the hc and tb forms showed excellent agreement with simulated spectra obtained with DFT computations (TPSSh/TZVP). This outstanding matching between theory and experiment enabled us to build composite spectra with varying hc:tb ratios. Comparison of these results with Raman and IR spectra recorded for [Ni(tmdta)](2-) in aqueous solution revealed that simulated Raman and IR spectra with a hc:tb ratio = 2:3 match the solution spectra in an accurate way. This equilibrium ratio enabled us to compute (13)C NMR sifts for the paramagnetic solution spectrum of [Ni(tmdta)](2-) based on the relative contributions by hc and tb fractions. This leads to computed shifts that agree closely with the experimental ones. Also, the kinetics of the skeleton dynamics could be estimated quantitatively by temperature-dependent (13)C NMR spectroscopic measurements. An interesting effect encountered for the very first time here concerns a drastic intensity difference of the 10Dq band ((3)A2g → (3)T2g(F) transition) in solid state electronic spectra of tb vs hc isomers, where the intensity of this band in the case of the hc form is much lower than that of the tb conformer and thus more similar to the case of the usual Ni(II) chromophore in octahedral environment. The equilibrium constants for complex formation and protonation of Ni(II)-tmdta at low pH have been estimated by pH-dependent UV-vis titration experiments. Correlation of these data with those of Ni(II)-edta and related 3d M(II) edta and tmdta complexes allow important conclusions on the consequences resulting from extending the central diamine ring in the ligand by one methylene group in terms of both complex and protolytic stability for edta vs tmdta complexes.


Assuntos
Diaminas/química , Níquel/química , Quelantes/química , Cinética , Ligantes , Lítio/química , Conformação Molecular
4.
Schweiz Monatsschr Zahnmed ; 122(11): 1016-29, 2012.
Artigo em Inglês, Sueco | MEDLINE | ID: mdl-23184365

RESUMO

Panoramic radiographs are made routinely in dentistry and are regarded as a standard component of an initial dental examination. Often, these radiographs show opacities in the carotid artery territory (CAT), which frequently arise as a result of calcification in the internal (ICA) or external carotid artery (ECA). This study details the examination of patients with suspected calcifications in the carotid artery (CA), using a sonographic examination based on the panoramic radio graphs to confirm or rule out a possible stenosis in the cervical bloodvessels. Thirty-three patients were examined sonographically. Based on the ultrasound investigation in 4 patients, hemodynamic stenoses were detected. Eighteen patients had an atheroma in the ICA, but no hemodynamic stenosis, and 5 patients showed no sign of calcification. Three patients were not examined sonographically at the University Hospital in Basel and were therefore excluded from the evaluation. Three patients did not attend the sonographic examination. The diagnosis of panoramic radiographs should not be restricted to teeth and jaws; especially in patients over 50 years old and in those with health risk factors, greater attention should be paid to the lateral areas. Using the radiographs they already have, dentists can also contribute.


Assuntos
Calcinose/diagnóstico por imagem , Doenças das Artérias Carótidas/diagnóstico por imagem , Estenose das Carótidas/diagnóstico por imagem , Acidente Vascular Cerebral/prevenção & controle , Idoso , Aterosclerose/diagnóstico por imagem , Artéria Carótida Primitiva/diagnóstico por imagem , Artéria Carótida Externa/diagnóstico por imagem , Artéria Carótida Interna/diagnóstico por imagem , Diagnóstico Diferencial , Feminino , Humanos , Achados Incidentais , Masculino , Pessoa de Meia-Idade , Radiografia Dentária Digital , Radiografia Panorâmica , Estudos Retrospectivos , Ultrassonografia Doppler
5.
Dalton Trans ; 41(46): 14151-6, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23034568

RESUMO

The enantiomerization mechanism of the trigonal-prismatic [Zn(py)(3)(tach)](2+) complex and several derivatives has been studied by applying DFT calculations (B3LYP/LANL2DZp). The enantiomerization pathways of [Zn(py(3)tach-X)](2+) (X = C, Si, Ge, N, P, As, O, S and Se) start from a distorted trigonal-prismatic C(3) symmetric ground state via an ideal trigonal-prismatic C(3v) structure to end up in a C(3)' symmetric image of the ground state. The activation energy and structural data of the complexes depend on electronic and steric factors. The activation barriers of the complexes decrease in the order [Zn(py(3)tach-Ge)](2+) > [Zn(py(3)tach-Si)](2+) > [Zn(py(3)tach-As)](2+) > [Zn(py(3)tach-Se)](2+) > [Zn(py(3)tach-P)](2+) > [Zn(py(3)tach-S)](2+) > [Zn(py(3)tach-C)](2+) > [Zn(py(3)tach-N)](2+) > [Zn(py(3)tach-O)](2+).

6.
Schweiz Monatsschr Zahnmed ; 122(9): 714-24, 2012.
Artigo em Inglês, Alemão | MEDLINE | ID: mdl-23018773

RESUMO

Implantoplasty describes a method using rotating instruments to smoothen rough implant surfaces which are exposed to the oral cavity. The goal of this procedure is to reduce the adherence of plaque and to facilitate the cleaning of the implant surfaces. The aim of this study was to compare different rotary instruments for their effectiveness and efficiency to smoothen micro-rough implant surfaces. For this purpose, 22 implants were processed with 10 different cutters and one diamond bur under standardized conditions, and then analyzed by scanning electron microscopy. In addition, collection of roughness data (Ra values, arithmetic mean roughness, Rz values, and average roughness) was obtained by using tactile surface measurement. The time needed to reach a subjectively-assessed smooth surface was determined for each instrument. The statistical analysis included the calculation of the mean values (± SD) for the required time, Ra and Rz values and the examination of correlations between these parameters, taking the logarithm of the values obtained and comparing them with linear mixed models. Irrespective of the drill design (spherical or conical) all rotary instruments used in the study showed obvious variations in processing times as well as significant differences (p < 0.001) of Ra and Rz values. The processing time required did not correlate with the Ra-(p = 0.44) or the Rz values (p = 0.83). Compared to spherical carbide cutters with transversal grooves, the conical cutters had the lowest mean roughness values (<1 micron).


Assuntos
Equipamentos Odontológicos de Alta Rotação , Implantes Dentários , Polimento Dentário/instrumentação , Modelos Lineares , Microscopia Eletrônica de Varredura , Estatísticas não Paramétricas , Propriedades de Superfície , Fatores de Tempo
7.
J Clin Invest ; 122(9): 3211-20, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22886303

RESUMO

Breast cancers commonly become resistant to EGFR-tyrosine kinase inhibitors (EGFR-TKIs); however, the mechanisms of this resistance remain largely unknown. We hypothesized that resistance may originate, at least in part, from molecular alterations that activate signaling downstream of EGFR. Using a screen to measure reversion of malignant cells into phenotypically nonmalignant cells in 3D gels, we identified FAM83A as a candidate cancer-associated gene capable of conferring resistance to EGFR-TKIs. FAM83A overexpression in cancer cells increased proliferation and invasion and imparted EGFR-TKI resistance both in cultured cells and in animals. Tumor cells that survived EGFR-TKI treatment in vivo had upregulated FAM83A levels. Additionally, FAM83A overexpression dramatically increased the number and size of transformed foci in cultured cells and anchorage-independent growth in soft agar. Conversely, FAM83A depletion in cancer cells caused reversion of the malignant phenotype, delayed tumor growth in mice, and rendered cells more sensitive to EGFR-TKI. Analyses of published clinical data revealed a correlation between high FAM83A expression and breast cancer patients' poor prognosis. We found that FAM83A interacted with and caused phosphorylation of c-RAF and PI3K p85, upstream of MAPK and downstream of EGFR. These data provide an additional mechanism by which tumor cells can become EGFR-TKI resistant.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Quinazolinas/farmacologia , Tirfostinas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Lapatinib , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-raf/metabolismo , Quinazolinas/uso terapêutico , Interferência de RNA , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Neoplasia ; 13(10): 991-1004, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22028624

RESUMO

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.


Assuntos
Perfilação da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Neuroblastoma/genética , Esferoides Celulares/metabolismo , Antígeno AC133 , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Criança , Pré-Escolar , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/genética , Peptídeos/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Esferoides Celulares/patologia , Transplante Heterólogo , Células Tumorais Cultivadas , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
9.
Inorg Chem ; 50(3): 1005-13, 2011 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-21190377

RESUMO

Li[V(eddadp)]·3H(2)O (1a) and Cs[V(eddadp)]·2H(2)O (1b) were characterized by X-ray crystallography. 1a crystallizes in the monoclinic space group Cc with a = 11.467(7) Å, b = 13.398(8) Å, c = 12.529(8) Å, ß = 114.85(4)°; V = 1746.7(2) Å(3), and Z = 4; 1b crystallizes in the monoclinic space group P2(1)/n with a = 10.265(5) Å, b = 11.673(6) Å, c = 15.507(8) Å, ß = 104.29(2)°, V = 1800.6(2) Å(3), and Z = 4. The solution structure of 1 has been ascertained to be predominantly six-coordinated with a hexadentate eddadp which is based on a comparison of the electronic and Raman spectra of aqueous solutions of 1 with those in the solid state.

10.
Inorg Chem ; 48(16): 7864-84, 2009 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-19618946

RESUMO

The crystal structure of the as-yet-unknown salt K[Fe(III)(cydta)(H(2)O)].3H(2)O, where cydta = (+/-)-trans-1,2-cyclohexanediaminetetraacetate, has been resolved: orthorhombic space group Pbca with R1 = 0.0309, wR2 = 0.0700, and GOF = 0.99. There are two independent [Fe(III)(cydta)(H(2)O)](-) anions in the asymmetric unit, and the ligand is (R,R)-cydta in both cases. The coordination polyhedron is a seven-coordinate capped trigonal prism where the quadrilateral face formed by the four ligand donor oxygen atoms is capped by the coordinated water molecule. The speciation of [Fe(III)(cydta)(H(2)O)](-) in water was studied in detail by a combination of techniques: (i) Measurements of the pH dependence of the Fe(III/II)cydta redox potentials by cyclic voltammetry enabled the estimation of the stability constants (0.1 M KNO(3), 25 degrees C) of [Fe(III)(cydta)(H(2)O)](-) (log beta(III)(110) = 29.05 +/- 0.01) and [Fe(II)(cydta)(H(2)O)](2-) (log beta(II)(110) = 17.96 +/- 0.01) as well as pK(III)(a1OH) = 9.57 and pK(II)(a1H) = 2.69. The formation enthalpy of [Fe(III)(cydta)(H(2)O)](-) (DeltaH degrees = -23 +/- 1 kJ mol(-1)) was measured by direct calorimetry and is compared to the corresponding value for [Fe(III)(edta)(H(2)O)](-) (DeltaH degrees = -31 +/- 1 kJ mol(-1)). (ii) pH-dependent spectrophotometric titrations of Fe(III)cydta lead to pK(III)(a1OH) = 9.54 +/- 0.01 for deprotonation of the coordinated water and a dimerization constant of log K(d) = 1.07. These data are compared with those of Fe(III)pdta (pdta = 1,2-propanediaminetetraacetate; pK(III)(a1OH) = 7.70 +/- 0.01, log K(d) = 2.28) and Fe(III)edta (pK(III)(a1OH) = 7.52 +/- 0.01, log K(d) = 2.64). Temperature- and pressure-dependent (17)O NMR measurements lead to the following kinetic parameters for the water-exchange reaction at [Fe(III)(cydta)(H(2)O)](-) (at 298 K): k(ex) = (1.7 +/- 0.2) x 10(7) s(-1), DeltaH(++) = 40.2 +/- 1.3 kJ mol(-1), DeltaS(++) = +28.4 +/- 4.7 J mol(-1) K(-1), and DeltaV(++) = +2.3 +/- 0.1 cm(3) mol(-1). A detailed kinetic study of the effect of the buffer, temperature, and pressure on the reaction of hydrogen peroxide with [Fe(III)(cydta)(H(2)O)](-) was performed using stopped-flow techniques. The reaction was found to consist of two steps and resulted in the formation of a purple Fe(III) side-on-bound peroxo complex [Fe(III)(cydta)(eta(2)-O(2))](3-). The peroxo complex and its degradation products were characterized using Mossbauer spectroscopy. Formation of the purple peroxo complex is only observable above a pH of 9.5. Both reaction steps are affected by specific and general acid catalysis. Two different buffer systems were used to clarify the role of general acid catalysis in these reactions. Mechanistic descriptions and a comparison between the edta and cydta systems are presented. The first reaction step reveals an element of reversibility, which is evident over the whole studied pH range. The positive volume of activation for the forward reaction and the positive entropy of activation for the backward reaction suggest a dissociative interchange mechanism for the reversible end-on binding of hydrogen peroxide to [Fe(III)(cydta)(H(2)O)](-). Deprotonation of the end-on-bound hydroperoxo complex leads to the formation of a seven-coordinate side-on-bound peroxo complex [Fe(III)(cydta)(eta(2)-O(2))](3-), where one carboxylate arm is detached. [Fe(III)(cydta)(eta(2)-O(2))](3-) can be reached by two different pathways, of which one is catalyzed by a base and the other by deprotonated hydrogen peroxide. For both pathways, a small negative volume and entropy of activation was observed, suggesting an associative interchange mechanism for the ring-closure step to the side-on-bound peroxo complex. For the second reaction step, no element of reversibility was found.


Assuntos
Ácido Edético/análogos & derivados , Compostos Férricos/química , Peróxido de Hidrogênio/química , Termodinâmica , Dimerização , Ácido Edético/química , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Software , Espectrofotometria , Espectroscopia de Mossbauer , Temperatura , Titulometria , Água/química
11.
Am J Pathol ; 174(6): 1996-9, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19465642

RESUMO

This Commentary highlights two articles in this issue of the American Journal of Pathology, discussing the implications of stromal expression of caveolin-1 in breast cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Caveolina 1/metabolismo , Matriz Extracelular , Feminino , Humanos
12.
BMC Cancer ; 9: 97, 2009 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-19331667

RESUMO

BACKGROUND: Resistance of high-risk metastatic neuroblastoma (HR-NB) to high dose chemotherapy (HD-CT) raises a major therapeutic challenge in pediatric oncology. Patients are treated by maintenance CT. For some patients, an adjuvant retinoid therapy is proposed, such as the synthetic retinoid fenretinide (4-HPR), an apoptotic inducer. Recent studies demonstrated that NB metastasis process is enhanced by the loss of caspase-8 involved in the Integrin-Mediated Death (IMD) process. As the role of caspase-8 appears to be critical in preventing metastasis, we aimed at studying the effect of 4-HPR on caspase-8 expression in metastatic neuroblasts. METHODS: We used the human IGR-N-91 MYCN-amplified NB experimental model, able to disseminate in vivo from the primary nude mouse tumor xenograft (PTX) into myocardium (Myoc) and bone marrow (BM) of the animal. NB cell lines, i.e., IGR-N-91 and SH-EP, were treated with various doses of Fenretinide (4-HPR), then cytotoxicity was analyzed by MTS proliferation assay, apoptosis by the propidium staining method, gene or protein expressions by RT-PCR and immunoblotting and caspases activity by colorimetric protease assays. RESULTS: The IGR-N-91 parental cells do not express detectable caspase-8. However the PTX cells established from the primary tumor in the mouse, are caspase-8 positive. In contrast, metastatic BM and Myoc cells show a clear down-regulation of the caspase-8 expression. In parallel, the caspases -3, -9, -10, Bcl-2, or Bax expressions were unchanged. Our data show that in BM, compared to PTX cells, 4-HPR up-regulates caspase-8 expression that parallels a higher sensitivity to apoptotic cell death. Stable caspase-8-silenced SH-EP cells appear more resistant to 4-HPR-induced cell death compared to control SH-EP cells. Moreover, 4-HPR synergizes with drugs since apoptosis is restored in VP16- or TRAIL-resistant-BM cells. These results demonstrate that 4-HPR in up-regulating caspase-8 expression, restores and induces apoptotic cell death in metastatic neuroblasts through caspase-8 activation. CONCLUSION: This study provides basic clues for using fenretinide in clinical treatment of HR-NB patients. Moreover, since 4-HPR induces cell death in caspase-8 negative NB, it also challenges the concept of including 4-HPR in the induction of CT of these patients.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Fenretinida/farmacologia , Neuroblastoma/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antineoplásicos/farmacologia , Western Blotting , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Caspase 8/genética , Inibidores de Caspase , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Etoposídeo/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia
14.
Semin Cancer Biol ; 19(2): 103-10, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19015030

RESUMO

Although chemokines and their receptors were initially identified as regulators of cell trafficking during inflammation and immune response, they have emerged as crucial players in all stages of tumor development, primary growth, migration, angiogenesis, and establishment as metastases in distant target organs. Neuroectodermal tumors regroup neoplasms originating from the embryonic neural crest cells, which display clinical and biological similarities. These tumors are highly malignant and rapidly progressing diseases that disseminate to similar target organs such as bone marrow, bone, liver and lungs. There is increasing evidence that interaction of several chemokine receptors with corresponding chemokine ligands are implicated in the growth and invasive characteristics of these tumors. In this review we summarize the current knowledge on the role of CXCL12 chemokine and its CXCR4 and CXCR7 receptors in the progression and survival of neuroectodermal tumors, with particular emphasis on neuroblastoma, the most typical and enigmatic neuroectodermal childhood tumor.


Assuntos
Quimiocinas/imunologia , Tumores Neuroectodérmicos/imunologia , Receptores de Quimiocinas/imunologia , Animais , Quimiocina CXCL12/imunologia , Humanos , Receptores CXCR/imunologia , Receptores CXCR4/imunologia
15.
J Am Chem Soc ; 130(44): 14556-69, 2008 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-18839954

RESUMO

Paramagnetic effects on the relaxation rate and shift difference of the (17)O nucleus of bulk water enable the study of water exchange mechanisms on transition metal complexes by variable temperature and variable pressure NMR. The water exchange kinetics of [Mn(II)(edta)(H2O)](2-) (CN 7, hexacoordinated edta) was reinvestigated and complemented by variable pressure NMR data. The results revealed a rapid water exchange reaction for the [Mn(II)(edta)(H2O)](2-) complex with a rate constant of k(ex) = (4.1 +/- 0.4) x 10(8) s(-1) at 298.2 K and ambient pressure. The activation parameters DeltaH(double dagger), DeltaS(double dagger), and DeltaV(double dagger) are 36.6 +/- 0.8 kJ mol(-1), +43 +/- 3 J K(-1) mol(-1), and +3.4 +/- 0.2 cm(3) mol(-1), which are in line with a dissociatively activated interchange (I(d)) mechanism. To analyze the structural influence of the chelate, the investigation was complemented by studies on complexes of the edta-related tmdta (trimethylenediaminetetraacetate) chelate. The kinetic parameters for [Fe(II)(tmdta)(H2O)](2-) are k(ex) = (5.5 +/- 0.5) x 10(6) s(-1) at 298.2 K, DeltaH(double dagger) = 43 +/- 3 kJ mol(-1), DeltaS(double dagger) = +30 +/- 13 J K(-1) mol(-1), and DeltaV(double dagger) = +15.7 +/- 1.5 cm(3) mol(-1), and those for [Mn(II)(tmdta)(H2O)](2-) are k(ex) = (1.3 +/- 0.1) x 10(8) s(-1) at 298.2 K, DeltaH(double dagger) = 37.2 +/- 0.8 kJ mol(-1), DeltaS(double dagger) = +35 +/- 3 J K(-1) mol(-1), and DeltaV(double dagger) = +8.7 +/- 0.6 cm(3) mol(-1). The water containing species, [Fe(III)(tmdta)(H2O)](-) with a fraction of 0.2, is in equilibrium with the water-free hexa-coordinate form, [Fe(III)(tmdta)](-). The kinetic parameters for [Fe(III)(tmdta)(H2O)](-) are k(ex) = (1.9 +/- 0.8) x 10(7) s(-1) at 298.2 K, DeltaH(double dagger) = 42 +/- 3 kJ mol(-1), DeltaS(double dagger) = +36 +/- 10 J K(-1) mol(-1), and DeltaV(double dagger) = +7.2 +/- 2.7 cm(3) mol(-1). The data for the mentioned tmdta complexes indicate a dissociatively activated exchange mechanism in all cases with a clear relationship between the sterical hindrance that arises from the ligand architecture and mechanistic details of the exchange process for seven-coordinate complexes. The unexpected kinetic and mechanistic behavior of [Ni(II)(edta')(H2O)](2-) and [Ni(II)(tmdta')(H2O)](2-) is accounted for in terms of the different coordination number due to the strong preference for an octahedral coordination environment and thus a coordination equilibrium between the water-free, hexadentate [M(L)](n+) and the aqua-pentadentate forms [M(L')(H2O)](n+) of the Ni(II)-edta complex, which was studied in detail by variable temperature and pressure UV-vis experiments. For [Ni(II)(edta')(H2O)](2-) (CN 6, pentacoordinated edta) a water substitution rate constant of (2.6 +/- 0.2) x 10(5) s(-1) at 298.2 K and ambient pressure was measured, and the activation parameters DeltaH(double dagger), DeltaS(double dagger), and DeltaV(double dagger) were found to be 34 +/- 1 kJ mol(-1), -27 +/- 2 J K(-1) mol(-1), and +1.8 +/- 0.1 cm(3) mol(-1), respectively. For [Ni(II)(tmdta')(H2O)](2-), we found k = (6.4 +/- 1.4) x 10(5) s(-1) at 298.2 K, DeltaH(double dagger) = 22 +/- 4 kJ mol(-1), and DeltaS(double dagger) = -59 +/- 5 J K(-1) mol(-1). The process is referred to as a water substitution instead of a water exchange reaction, since these observations refer to the intramolecular displacement of coordinated water by the carboxylate moiety in a ring-closure reaction.

16.
Magn Reson Chem ; 46 Suppl 1: S94-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18855345

RESUMO

The effect of temperature and pressure on the water exchange reaction of [Fe(II)(NTA)(H2O)2](-) and [Fe(II)(BADA)(H2O)2](-) (NTA = nitrilotriacetate; BADA = beta-alanindiacetate) was studied by 17O NMR spectroscopy. The [Fe(II)(NTA)(H2O)2](-) complex showed a water exchange rate constant, k(ex), of (3.1 +/- 0.4) x 10(6) s(-1) at 298.2 K and ambient pressure. The activation parameters DeltaH( not equal), DeltaS( not equal) and DeltaV( not equal) for the observed reaction are 43.4 +/- 2.6 kJ mol(-1), + 25 +/- 9 J K(-1) mol(-1) and + 13.2 +/- 0.6 cm(3) mol(-1), respectively. For [Fe(II)(BADA)(H2O)2](-), the water exchange reaction is faster than for the [Fe(II)(NTA)(H2O)2](-) complex with k(ex) = (7.4 +/- 0.4) x 10(6) s(-1) at 298.2 K and ambient pressure. The activation parameters DeltaH( not equal), DeltaS( not equal) and DeltaV( not equal) for the water exchange reaction are 40.3 +/- 2.5 kJ mol(-1), + 22 +/- 9 J K(-1) mol(-1) and + 13.3 +/- 0.8 cm(3) mol(-1), respectively. The effect of pressure on the exchange rate constant is large and very similar for both systems, and the numerical values for DeltaV( not equal) suggest in both cases a limiting dissociative (D) mechanism for the water exchange process.


Assuntos
Compostos Ferrosos/química , Quelantes de Ferro/química , Espectroscopia de Ressonância Magnética/métodos , Água/química , Cinética , Isótopos de Oxigênio , Pressão , Relação Estrutura-Atividade , Temperatura
17.
Inorg Chem ; 47(14): 6314-21, 2008 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-18563876

RESUMO

The promising BioDeNO(x) process for NO removal from gaseous effluents suffers from an unsolved problem that results from the oxygen sensitivity of the Fe(II)-aminopolycarboxylate complexes used in the absorber unit to bind NO(g). The utilized [Fe(II)(EDTA)(H2O)](2-) complex is extremely oxygen sensitive and easily oxidized to give a totally inactive [Fe(III)(EDTA)(H2O)](-) species toward the binding of NO(g). We found that an in situ formed, less-oxygen-sensitive mixed-ligand complex, [Fe(II)(EDTA)(F)](3-), still reacts quantitatively with NO(g). The formation constant for the mixed ligand complex was determined spectrophotometrically. For [Fe(III)(EDTA)(F)](2-) we found log K(MLF)(F) = 1.7 +/- 0.1. The [Fe(II)(EDTA)(F)](3-) complex has a smaller value of log K(MLF)(F) = 1.3 +/- 0.2. The presence of fluoride does not affect the reversible binding of NO(g). Even over extended periods of time and fluoride concentrations of up to 1.0 M, the nitrosyl complex does not undergo any significant decomposition. The [Fe(III)(EDTA)(NO(-))](2-) complex releases bound NO on passing nitrogen through the solution to form [Fe(II)(EDTA)(H2O)](2-) almost completely. A reaction cycle is feasible in which fluoride inhibits the autoxidation of [Fe(II)(EDTA)(H2O)](2-) during the reversible binding of NO(g).

18.
Mol Cancer ; 7: 55, 2008 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-18549473

RESUMO

BACKGROUND: Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB) is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. RESULTS: We show here that the HDACi Sodium Butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA) strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. CONCLUSION: HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Neuroblastoma/enzimologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Butiratos/farmacologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Neuroblastoma/patologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vorinostat
19.
Inorg Chem ; 47(13): 5702-19, 2008 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-18510310

RESUMO

Because of our interest in evaluating a possible relationship between complex dynamics and water exchange reactivity, we performed (1)H NMR studies on the paramagnetic aminopolycarboxylate complexes Fe (II)-TMDTA and Fe (II)-CyDTA and their diamagnetic analogues Zn (II)-TMDTA and Zn (II)-CyDTA. Whereas a fast Delta-Lambda isomerization was observed for the TMDTA species, no acetate scrambling between in-plane and out-of-plane positions is accessible for any of the CyDTA complexes because the rigid ligand backbone prevents any configurational changes in the chelate system. In variable-temperature (1)H NMR studies, no evidence of spectral coalescence due to nitrogen inversion was found for any of the complexes in the available temperature range. The TMDTA complexes exhibit the known solution behavior of EDTA, whereas the CyDTA complexes adopt static solution structures. Comparing the exchange kinetics of flexible EDTA-type complexes and static CyDTA complexes appears to be a suitable method for evaluating the effect of ligand dynamics on the overall reactivity. In order to assess information concerning the rates and mechanism of water exchange, we performed variable-temperature and -pressure (17)O NMR studies of Ni (II)-CyDTA, Fe (II)-CyDTA, and Mn (II)-CyDTA. For Ni (II)-CyDTA, no significant effects on line widths or chemical shifts were apparent, indicating either the absence of any chemical exchange or the existence of a very small amount of the water-coordinated complex in solution. For [Fe (II)(CyDTA)(H 2O)] (2-) and [Mn (II)(CyDTA)(H 2O)] (2-), exchange rate constant values of (1.1 +/- 0.3) x 10 (6) and (1.4 +/- 0.2) x 10 (8) s (-1), respectively, at 298 K were determined from fits to resonance-shift and line-broadening data. A relationship between chelate dynamics and reactivity seems to be operative, since the CyDTA complexes exhibited significantly slower reactions than their EDTA counterparts. The variable-pressure (17)O NMR measurements for [Mn (II)(CyDTA)(H 2O)] (2-) yielded an activation volume of +9.4 +/- 0.9 cm (3) mol (-1). The mechanism is reliably assigned as a dissociative interchange (I d) mechanism with a pronounced dissociation of the leaving water molecule in the transition state. In the case of [Fe (II)(CyDTA)(H 2O)] (2-), no suitable experimental conditions for variable-pressure measurements were accessible.


Assuntos
Ácidos Carboxílicos/química , Compostos Ferrosos/química , Espectroscopia de Ressonância Magnética/métodos , Isótopos de Oxigênio , Água/química , Ácido Edético
20.
PLoS One ; 2(10): e1016, 2007 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-17925864

RESUMO

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Receptores CXCR4/fisiologia , Animais , Células da Medula Óssea , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Inativação Gênica , Humanos , Fígado/metabolismo , Camundongos , Camundongos Nus , Modelos Biológicos , Invasividade Neoplásica , Transplante de Neoplasias , Receptores CXCR4/metabolismo
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