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1.
J Am Chem Soc ; 142(13): 6268-6284, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32131594

RESUMO

Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.

2.
Angew Chem Int Ed Engl ; 57(7): 1831-1835, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29314482

RESUMO

AsqJ, an iron(II)- and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer are reported. Meanwhile, a detailed mechanistic study was carried out to decipher the desaturation mechanism. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived FeIV -oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate is preferred during AsqJ-catalyzed desaturation.


Assuntos
Compostos de Epóxi/metabolismo , Proteínas Fúngicas/metabolismo , Peptídeos/metabolismo , Aspergillus nidulans/enzimologia , Biocatálise , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/metabolismo , Compostos de Epóxi/química , Compostos Férricos/química , Proteínas Fúngicas/química , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Simulação de Dinâmica Molecular , Peptídeos/química , Teoria Quântica , Estereoisomerismo
3.
Dalton Trans ; 47(3): 726-733, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-29230458

RESUMO

By the combination of Pd-complexes and [V10O28]6-, three Pd-decavanadate compounds [Pd(NH3)4]3[V10O28]·8H2O (1), [Pd(deta)(H2O)]2(NH4)2[V10O28]·2H2O (2) (deta = diethylenetriamine) and [Pd(dpa)2](Hdpa)2(Et3NH)2[V10O28]·2H2O (3) (dpa = 2,2'-dipyridylamine) have been successfully synthesized and thoroughly characterized using single X-ray diffraction (SXRD), powder X-ray diffraction (PXRD), infrared spectroscopy (FT-IR) and elemental analyses (EA). Interestingly, in the three compounds, the molar ratios of Pd to decavanadate vary from 3 : 1 to 1 : 1 by changing N-ligands. The three Pd-decavanadates as heterogeneous catalysts are active in the aerobic oxidation of benzylic hydrocarbons under solvent-free conditions without adding any additives and co-catalysts. Moreover, compound 1 can be reused three times without losing its activity.

4.
Adv Exp Med Biol ; 1024: 195-212, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28921471

RESUMO

Toll-like receptors (TLRs) are one of the best characterised families of pattern recognition receptors (PRRs) and play a critical role in the host defence to infection. Accumulating evidence indicates that TLRs also participate in maintaining tissue homeostasis by controlling inflammation and tissue repair, as well as promoting antitumour effects via activation and modulation of adaptive immune responses. TLR agonists have successfully been exploited to ameliorate the efficacy of various cancer therapies. In this chapter, we will discuss the rationales of using TLR agonists as adjuvants to cancer treatments and summarise the recent findings of preclinical and clinical studies of TLR agonist-based cancer therapies.


Assuntos
Adjuvantes Imunológicos , Vacinas Anticâncer/imunologia , Neoplasias/prevenção & controle , Receptores Toll-Like/agonistas , Humanos , Receptores Toll-Like/imunologia
5.
J Org Chem ; 82(15): 8251-8257, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28664735

RESUMO

A new sequential approach for 2-vinylanilines utilizing aryl carboxylic acids as stable, inexpensive and widely available arylating reagents is described. Employing a Pd-POVs catalyst system, this protocol is not only overcoming the restriction barrier of decarboxylative coupling to ortho-substituted substrates, but also provides site-special to create new C(sp2)-N and C(sp2)-C(sp2) bonds. Mechanistic experiments suggest the cleavage of C(sp2)-COOH gives priority to C(sp2)-X bond in this reaction.

6.
Dalton Trans ; 46(25): 8245-8251, 2017 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-28612860

RESUMO

Two ionic organic-inorganic hybrid compounds, [CuII(C2N2H8)2]4[CuII(C2N2H8)2(H2O)2]2[PNb12O40VVVIVO2]·(OH)2·11H2O (1) and [CoIII(C2N2H8)3]2[CoIII(C2N2H8)2(H2O)2]0.5[H2.5PNb12O40 VVVIVO2]·20H2O (2), based on P-centered dicapped polyoxoniobates and organometallic cations were isolated and structurally characterized by routine techniques. The trivalent cobalt complex-containing compound exhibits a looser arrangement compared with its divalent copper complex-containing counterpart, with a space volume of 34.9% for the former and 17.0% for the latter. The two compounds were proved to be effective in facilitating the oxidation of benzyl-alkanes to ketone products in a heterogeneous manner, evidencing the feasiblity of the strategy of self-immobilization of catalytically active, readily soluble PNb12O40(VO)2 species in crystalline solids.

7.
J Org Chem ; 82(11): 5557-5565, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28497692

RESUMO

We previously reported an iterative synthesis of unsymmetrical 2,5-disubstituted pyrrolidines from pyrrolidine by two rounds of redox-triggered α-C-H functionalization. Although this approach can be used to introduce substituents at the 2- and 5-positions, it is lengthy because the redox auxiliary must be removed and then reinstalled. Therefore, we sought to develop a method to oxidize 2-functionalized pyrrolidine to cyclic N,O-acetal which could then react with a nucleophile for introduction of the 5-substituent. In this work, we found that molecular iodine can mediate the preferential oxidation of secondary over tertiary α-C-H bonds of α-substituted pyrrolidines to form cyclic N,O-acetals, improving the step economy of our previously reported method. With this strategy, (±)-preussin and its C(3) epimer were synthesized from (±)-pyrrolidin-3-ol.

8.
Inorg Chem ; 56(10): 5748-5756, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28430438

RESUMO

Six alkoxohexavanadate-based Cu- or Co-POVs [Cu(dpa)(acac)(H2O)]2[V6O13(OMe)6] (1), [Cu(phen)(acac)(MeOH)]2[V6O13(OMe)6] (2), [Co(dpa)(acac)2]2[V6O13(OMe)6]·2MeOH (3), [Co(phen)(acac)2]2[V6O13(OMe)6] (4), [Cu(dpa)(acac)]2[VIV2VV4O12(OMe)7] (5), and [Cu(dpa)(acac)(MeOH)]2[VIV2VV4O11(OMe)8] (6) (POV = polyoxovanadate; dpa = 2,2'-dipyridine amine; phen = 1,10-phenanthroline; acac = acetylacetone anion) have been synthesized by controlling the reaction conditions and characterized by single-crystal X-ray diffraction and powder X-ray diffraction analyses, FT-IR spectroscopy, element analyses, and X-ray photoelectron spectroscopy. In compounds 1-4 and 6, Cu or Co complexes and alkoxohexavanadate anions are assembled through electrostatic interactions. Differently, in compound 5, seven-methoxo-substituted Lindqvist-type [V6O12(OMe)7]2- are bridged to Cu complex via terminal O atoms by coordination bonds. All compounds 1-6 exhibit excellent heterogeneous catalytic performance in oxidative desulfurization and CEES ((2-chloroethyl) ethyl sulfide, a sulfur mustard simulant) abatement with H2O2 as oxidant. Among them, the catalytic activity of 6 [conv. of DBT (dibenzothiophene) up to 100% in 6 h; conv. of CEES reached 100% and selectivity of CEESO ((2-chloroethyl) ethyl sulfoxide) up to 85% after 4 h] outperforms others and can be reused without losing its activity.

9.
Oncotarget ; 7(37): 60593-60608, 2016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27533254

RESUMO

Barx2 is a Bar family homeodomain transcription factor shown to play a critical role in cell adhesion and cytoskeleton remodeling, key processes in carcinogenesis and metastasis. Using quantitative real-time PCR, Western blotting, and immunohistochemistry, we found that Barx2 is expressed at lower levels in human gastric cancer (GC) tissues than in adjacent normal mucosa. In a multivariate analysis, Barx2 expression emerged as an independent prognostic factor for disease-free and overall survival. Kaplan-Meier survival analysis showed a trend toward even shorter overall survival in the patient group with Barx2-negative tumors, independent of advanced UICC stage and tumor relapse. Using in vitro and in vivo assays, we demonstrated that under normal conditions Barx2 inhibited GC cell proliferation and invasiveness through inhibition of the Wnt/ß-catenin signaling pathway. These findings indicate that reduction or loss of Barx2 dis-inhibits GC cell proliferation and invasion, and that reduction in Barx2 could serve as an independent prognostic biomarker for poor outcome in GC patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias Gástricas/genética , Idoso , Animais , Biomarcadores Tumorais/genética , Carcinogênese , Adesão Celular , Proliferação de Células , Citoesqueleto/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/mortalidade , Análise de Sobrevida , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Am Chem Soc ; 138(33): 10390-3, 2016 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-27442345

RESUMO

Mechanisms have been proposed for α-KG-dependent non-heme iron enzyme catalyzed oxygen atom insertion into an olefinic moiety in various natural products, but they have not been examined in detail. Using a combination of methods including transient kinetics, Mössbauer spectroscopy, and mass spectrometry, we demonstrate that AsqJ-catalyzed (-)-4'-methoxycyclopenin formation uses a high-spin Fe(IV)-oxo intermediate to carry out epoxidation. Furthermore, product analysis on (16)O/(18)O isotope incorporation from the reactions using the native substrate, 4'-methoxydehydrocyclopeptin, and a mechanistic probe, dehydrocyclopeptin, reveals evidence supporting oxo↔hydroxo tautomerism of the Fe(IV)-oxo species in the non-heme iron enzyme catalysis.


Assuntos
Biocatálise , Enzimas/metabolismo , Compostos de Epóxi/química , Ferro , Alcenos/química , Aspergillus nidulans/enzimologia , Benzodiazepinonas/química , Benzodiazepinonas/metabolismo , Cinética , Oxigênio/química
11.
Inorg Chem ; 55(15): 7501-7, 2016 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-27442602

RESUMO

The controllable synthesis of two vanadium-containing polyoxoniobate-based three-dimensional organic-inorganic hybrid compounds, [Co(pn)2]4[HPNb10V(IV)2O40(V(IV)O)4]·17H2O (1) and [Co(pn)2]5[PNb12O40(V(IV)O)6](OH)7·15H2O (2), where pn = 1,2-diaminopropane, is realized by changing the hydrothermal temperature or adding N-(aminoethyl)piperazine as an additive. Both compounds 1 and 2 are structurally characterized by single-crystal/powder X-ray diffraction and IR and X-ray photoelectron spectroscopy. Compound 1 features a new divanadium-substituted Keggin polyoxoniobate capped by four vanadyl groups, and the polyanion in 2 exhibits the highest coordination number (10-connected) in polyoxoniobate chemistry. Moreover, the photocatalytic activities of 1 and 2 for hydrogen evolution are preliminarily assessed.

12.
Chemistry ; 22(1): 264-71, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26616530

RESUMO

Diamagnetic chemical exchange saturation transfer (CEST) contrast agents offer an alternative to Gd(3+) -based contrast agents for MRI. They are characterized by containing protons that can rapidly exchange with water and it is advantageous to have these protons resonate in a spectral window that is far removed from water. Herein, we report the first results of DFT calculations of the (1) H nuclear magnetic shieldings in 41 CEST agents, finding that the experimental shifts can be well predicted (R(2) =0.882). We tested a subset of compounds with the best MRI properties for toxicity and for activity as uncouplers, then obtained mice kidney CEST MRI images for three of the most promising leads finding 16 (2,4-dihydroxybenzoic acid) to be one of the most promising CEST MRI contrast agents to date. Overall, the results are of interest since they show that (1) H NMR shifts for CEST agents-charged species-can be well predicted, and that several leads have low toxicity and yield good in vivo MR images.


Assuntos
Meios de Contraste/química , Gadolínio/química , Hidroxibenzoatos/química , Imagem por Ressonância Magnética/métodos , Animais , Espectroscopia de Ressonância Magnética , Camundongos , Teoria Quântica
13.
Proc Natl Acad Sci U S A ; 112(51): E7073-82, 2015 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-26644565

RESUMO

There is a growing need for new antibiotics. Compounds that target the proton motive force (PMF), uncouplers, represent one possible class of compounds that might be developed because they are already used to treat parasitic infections, and there is interest in their use for the treatment of other diseases, such as diabetes. Here, we tested a series of compounds, most with known antiinfective activity, for uncoupler activity. Many cationic amphiphiles tested positive, and some targeted isoprenoid biosynthesis or affected lipid bilayer structure. As an example, we found that clomiphene, a recently discovered undecaprenyl diphosphate synthase inhibitor active against Staphylococcus aureus, is an uncoupler. Using in silico screening, we then found that the anti-glioblastoma multiforme drug lead vacquinol is an inhibitor of Mycobacterium tuberculosis tuberculosinyl adenosine synthase, as well as being an uncoupler. Because vacquinol is also an inhibitor of M. tuberculosis cell growth, we used similarity searches based on the vacquinol structure, finding analogs with potent (∼0.5-2 µg/mL) activity against M. tuberculosis and S. aureus. Our results give a logical explanation of the observation that most new tuberculosis drug leads discovered by phenotypic screens and genome sequencing are highly lipophilic (logP ∼5.7) bases with membrane targets because such species are expected to partition into hydrophobic membranes, inhibiting membrane proteins, in addition to collapsing the PMF. This multiple targeting is expected to be of importance in overcoming the development of drug resistance because targeting membrane physical properties is expected to be less susceptible to the development of resistance.


Assuntos
Anti-Infecciosos/farmacologia , Força Próton-Motriz/efeitos dos fármacos , Desacopladores/farmacologia , Alquil e Aril Transferases/antagonistas & inibidores , Anti-Infecciosos/química , Fenômenos Biofísicos , Clomifeno/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Piperidinas/farmacologia , Quinolinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Desacopladores/química
14.
Chemistry ; 21(40): 13894-9, 2015 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-26296038

RESUMO

Metal-organic frameworks (MOFs), as a class of microporous materials with well-defined channels and rich functionalities, hold great promise for various applications. Yet the formation and crystallization processes of various MOFs with distinct topology, connectivity, and properties remain largely unclear, and the control of such processes is rather challenging. Starting from a 0D Cu coordination polyhedron, MOP-1, we successfully unfolded it to give a new 1D-MOF by a single-crystal-to-single-crystal (SCSC) transformation process at room temperature as confirmed by SXRD. We also monitored the continuous transformation states by FTIR and PXRD. Cu MOFs with 2D and 3D networks were also obtained from this 1D-MOF by SCSC transformations. Furthermore, Cu MOFs with 0D, 1D, and 3D networks, MOP-1, 1D-MOF, and HKUST-1, show unique performances in the kinetics of the C-H bond catalytic oxidation reaction.

15.
Oncol Lett ; 9(5): 1997-2002, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26137001

RESUMO

Excessive activation of the hedgehog (Hh) signaling pathway is important in a variety of human cancer cell types, including gastric cancer. However, the underlying mechanisms of the Hh signaling pathway in inducing gastric tumorigenesis and its downstream target genes are largely unknown. In the present study, the inhibitory effect of cyclopamine on the Hh signaling pathway was investigated in the human gastric cancer AGS cell line. It was identified that cyclopamine treatment inhibited the proliferation, migration and invasion of the AGS cells in a dose- and time-dependent manner, and resulted in the downregulation of a number of key Hh signaling pathway-associated factors [glioma-associated oncogene homolog 1, C-X-C chemokine receptor type 4 and transforming growth factor (TGF)-ß1] at the RNA and protein levels. Furthermore, the secretion of TGF-ß1 was significantly reduced following the administration of cyclopamine to the AGS cells. The results of the present study provided insight into the mechanisms by which the Hh signaling pathway regulates gastric cancer formation and identified the Hh signaling pathway as a potential novel therapeutic target in human gastric cancer.

16.
J Mol Biol ; 427(12): 2220-8, 2015 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-25868383

RESUMO

IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent and an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG-catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate into (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate. In addition, the enzyme's structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism, which describes all stages from initial ring opening to formation of (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many prokaryotic and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.


Assuntos
Enzimas/química , Thermus thermophilus/enzimologia , Clonagem Molecular , Cristalografia por Raios X , Enzimas/genética , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Thermus thermophilus/genética
17.
Inorg Chem ; 54(4): 1454-61, 2015 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-25650591

RESUMO

Four alkoxohexavanadate-based Pd-POVs [Pd(dpa)(acac)]2[V6O13(OMe)6] (1), [Pd(dpa)(acac)]2[V6O11(OMe)8] (2), [Pd(dpa)(acac)]2[V6O11(OMe)8]·H2O (3), and [Pd(DMAP)2(acac)]2[V6O11(OMe)8]·H2O (4) (POV = polyoxovanadate; dpa = 2,2'-dipyridine amine; DMAP = 4-dimethylaminopyridine; acac = acetylacetone anion) have been synthesized and fully characterized by single crystal X-ray diffraction and powder X-ray diffraction analyses, Fourier transform infrared spectroscopy, element analyses, and X-ray photoelectron spectroscopy. In 1-4, Pd complexes and hexavanadate anions are assembled through electrostatic interactions. Interestingly, the [V6O11(OMe)8](2-) cores in 2 and 3 are a pair of isomers that can be isolated by controlling crystallization temperature. Moreover, to the best of our knowledge, the {V6} core in 3 represents a new octamethoxyhexavanadates cluster. It is notable that compounds 1-4 exhibit excellent heterogeneous catalytic performance in the oxidation of benzyl-alkanes with t-butylhydroperoxide as oxidant. Among them, the catalytic activity of 1 (conv. and selec. up to 99%, respectively) outperforms others and can be reused without losing its activity.

18.
Chem Sci ; 6(12): 6813-6822, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26865948

RESUMO

The protein IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMPPP) reductase, is an essential 4Fe-4S cluster-containing protein in the methylerythritol phosphate pathway for isoprenoid biosynthesis. Using a sequence similarity network we found that there are >400 IspH proteins that are about twice as large as most of the IspHs studied to date since their IspH domains are fused to either the ribosomal protein S1 (RPS1), or to a UbiA (4-hydroxybenzoate octaprenyltransferase)-like protein. Many of the IspH-RPS1 proteins are present in anaerobes found in the human gut and some, such as Clostridium botulinum, C. tetani and Fusobacterium nucleatum, are pathogens. The IspH-UbiAs are all found in sulfate-reducing anaerobes. The IspH domains in IspH-RPS1 are fused to 4 and in a few cases 6 tandem repeats in RPS1 that, in most organisms, bind to mRNA or form part of the bacterial ribosome. Mutants in which the four RPS1 domains were sequentially eliminated had similar IspH activity as wild-type protein, indicating they are not essential for IspH catalysis. Overall, the results are of interest since they represent the first isolation of a catalytically active IspH-RPS1, as well as the identification of IspH-UbiA hybrids, two "Rosetta stone" proteins that are likely to be functionally related-IspH producing the isoprenoids required for a UbiA-like prenyl transferase; the IspH-RPS1 hybrids, perhaps, being involved in the stringent response or as Fe/O2 sensors.

19.
Zhonghua Wei Chang Wai Ke Za Zhi ; 17(10): 1036-9, 2014 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-25341915

RESUMO

OBJECTIVE: To investigate the influence of miR-148a on cell proliferation of human gastric cancer cell lines MKN45. METHODS: Expression level of miR-148a was detected by qRT-PCR in the carcinoma tissues and the tissues adjacent to carcinoma of 60 patients with gastric cancer. Lentivirus packaging was used to establish MKN45 gastric cancer cell line expressing stable miR-148a as transfection group,and the untransfected MKN45 cell line as the control group. Gastric cancer MKN45 cell proliferation was detected by CCK8 method. The target gene of miR-148a was predicted by targetscan. Expression of target gene was examined by Western blotting. RESULT: Expression of miR-148a in gastric cancer tissues of 54 cases(90.0%, 54/60) decreased, and among them, 37 tissue samples(61.7%, 37/60) decreased by 2 times. mir-148a expression in cancer tissues of patients with lymph node metastasis, vascular invasion and TNM stage III(-IIII( was down-regulated more obviously(P<0.05). From the third day after transfection, the growth of MKN45 cells was significantly inhibited(P<0.01). Gene CDC25B might be the target gene of miR-148a according to the results of targetscan. Western blot showed that CDC25B expression in transfection group was lower as compared to control group. CONCLUSION: MiR-148a expression is down-regulated in gastric cancer tissues and inhibits gastric cancer cell proliferation. CDC25B may be the target gene of miR-148a that plays a role in tumor suppressor.


Assuntos
Proliferação de Células , MicroRNAs/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Metástase Linfática , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
20.
Chem Sci ; 5(4): 1642-1649, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24999381

RESUMO

IspG and IspH are proteins that are involved in isoprenoid biosynthesis in most bacteria as well as in malaria parasites and are important drug targets. They contain cubane-type 4Fe-4S clusters that are involved in unusual 2H+/2e- reductions. Here, we report the results of electron paramagnetic resonance spectroscopic investigations of the binding of amino- and thiolo-HMBPP (HMBPP=E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate) IspH substrate-analog inhibitors to both proteins, as well as the binding of HMBPP and an acetylene diphosphate inhibitor, to IspG. The results show that amino-HMBPP binds to reduced IspH by Fe-C π-bonding with the olefinic carbons interacting with the unique 4th Fe in the 4Fe-4S cluster, quite different to the direct Fe-N ligation seen with the oxidized protein. No such π-complex is observed when amino-HMBPP binds to reduced IspG. No EPR signal is observed with IspH in the presence of dithionite and thiolo-HMBPP, suggesting that the 4Fe-4S cluster is not reduced, consistent with the presence of a 420 nm feature in the absorption spectrum (characteristic of an oxidized cluster). However, with IspG, the EPR spectrum in the presence of dithionite and thiolo-HMBPP is very similar to that seen with HMBPP. The binding of HMBPP to IspG was studied using hyperfine sublevel correlation spectroscopy with 17O and 13C labeled samples: the results rule out direct Fe-O bonding and indicate π-bonding. Finally, the binding to IspG of a potent acetylene diphosphate inhibitor was studied by using electron-nuclear double resonance spectroscopy with 13C labeled ligands: the large hyperfine couplings indicate strong Fe-C π-bonding with the acetylenic group. These results illustrate a remarkable diversity in binding behavior for HMBPP-analog inhibitors, opening up new routes to inhibitor design of interest in the context of anti-bacterial and anti-malarial drug discovery, as well as "cubane-type" metallo-biochemistry, in general.

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