Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 513
Filtrar
1.
Nature ; 580(7803): 409-412, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296172

RESUMO

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis1-3. Although Mtb can synthesize vitamin B12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis2. Mtb does not encode any characterized cobalamin transporter4-6; however, the gene rv1819c was found to be essential for uptake of cobalamin1. This result is difficult to reconcile with the original annotation of Rv1819c as a protein implicated in the transport of antimicrobial peptides such as bleomycin7. In addition, uptake of cobalamin seems inconsistent with the amino acid sequence, which suggests that Rv1819c has a bacterial ATP-binding cassette (ABC)-exporter fold1. Here, we present structures of Rv1819c, which reveal that the protein indeed contains the ABC-exporter fold, as well as a large water-filled cavity of about 7,700 Å3, which enables the protein to transport the unrelated hydrophilic compounds bleomycin and cobalamin. On the basis of these structures, we propose that Rv1819c is a multi-solute transporter for hydrophilic molecules, analogous to the multidrug exporters of the ABC transporter family, which pump out structurally diverse hydrophobic compounds from cells8-11.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Bleomicina/metabolismo , Mycobacterium tuberculosis/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
2.
Mol Biol Rep ; 47(1): 533-544, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31724125

RESUMO

In the present investigation, we report cloning, expression, purification and characterization of a novel Bleomycin Resistance Dioxygenase (BRPD). His-tagged fusion protein was purified to homogeneity using Ni-NTA affinity chromatography, yielding 1.2 mg of BRPD with specific activity of 6.25 U mg-1 from 600 ml of E. coli culture. Purified enzyme was a dimer with molecular weight ~ 26 kDa in SDS-PAGE and ~ 73 kDa in native PAGE analysis. The protein catalyzed breakdown of hydrocarbon substrates, including catechol and hydroquinone, in the presence of metal ions, as characterized via spectrophotometric analysis of the enzymatic reactions. Bleomycin binding was proven using the EMSA gel retardation assay, and the putative bleomycin binding site was further determined by in silico analysis. Molecular dynamic simulations revealed that BRPD attains octahedral configuration in the presence of Fe2+ ion, forming six co-ordinate complexes to degrade hydroquinone-like molecules. In contrary, in the presence of Zn2+ ion BRPD adopts tetrahedral configuration, which enables degradation of catechol-like molecules.


Assuntos
Proteínas de Bactérias , Bleomicina , Dioxigenases , Hidrocarbonetos Aromáticos , Proteínas Recombinantes de Fusão , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Bleomicina/química , Bleomicina/metabolismo , Catecóis/química , Catecóis/metabolismo , Dioxigenases/química , Dioxigenases/genética , Dioxigenases/metabolismo , Farmacorresistência Bacteriana/genética , Hidrocarbonetos Aromáticos/química , Hidrocarbonetos Aromáticos/metabolismo , Hidroquinonas/química , Hidroquinonas/metabolismo , Metagenoma/genética , Simulação de Dinâmica Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Microbiologia do Solo
3.
Metabolomics ; 15(8): 111, 2019 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-31422500

RESUMO

INTRODUCTION: Polyhexamethylene guanidine phosphate (PHMG) has been used as a disinfectant and biocide, and was known to be harmless and non-toxic. However, in 2011, PHMG used as a humidifier disinfectant was reported to be associated with lung diseases, such as, fibrosis in the toxicant studies on pulmonary fibrosis by PHMG. However, no metabolomics study has been performed in PHMG-induced mouse models of pulmonary fibrosis. OBJECTIVES: We performed a metabolomic study to understand the biochemical events that occur in bleomycin (BLM)- and PHMG-induced mouse models of pulmonary fibrosis using gas chromatography-mass spectrometry (GC-MS), LC-tandem MS, and GC-tandem MS. RESULTS: The levels of 61 metabolites of 30 amino acids, 13 organic acids, 12 fatty acids, 5 polyamines, and oxidized glutathione were determined in the pulmonary tissues of mice with BLM- and PHMG-induced pulmonary fibrosis and in normal controls. Principal component analysis and partial least squares discriminant analysis used to compare level of these 61 metabolites in pulmonary tissues. Levels of metabolites were significantly different in the BLM and PHMG groups as compared with the control group. In particular, the BLM- and PHMG-induced pulmonary fibrosis models showed elevated collagen synthesis and oxidative stress and metabolic disturbance of TCA related organic acids including fumaric acid by NADPH oxidase. In addition, polyamine metabolism showed severe alteration in the PHMG group than that of the BLM group. CONCLUSION: This result suggests PHMG will be able to induce pulmonary fibrosis by arginine metabolism and NADPH oxidase signaling.


Assuntos
Bleomicina/metabolismo , Modelos Animais de Doenças , Guanidinas/metabolismo , Metabolômica , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Animais , Bleomicina/administração & dosagem , Bleomicina/análise , Cromatografia Gasosa , Cromatografia Líquida , Guanidinas/administração & dosagem , Guanidinas/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/patologia , Espectrometria de Massas em Tandem
4.
J Vis Exp ; (147)2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31157786

RESUMO

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.  However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.  Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.  It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.


Assuntos
Bleomicina/administração & dosagem , Pulmão/metabolismo , Administração por Inalação , Animais , Bleomicina/metabolismo , Brônquios/anatomia & histologia , Cateterismo/instrumentação , Cateterismo/métodos , Modelos Animais de Doenças , Pulmão/anatomia & histologia , Pulmão/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente
5.
Development ; 146(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30890567

RESUMO

The sensory nervous system of C. elegans comprises cells with varied molecular and functional characteristics, and is, therefore, a powerful model for understanding mechanisms that generate neuronal diversity. We report here that VAB-3, a C. elegans homolog of the homeodomain-containing protein Pax6, has opposing functions in regulating expression of a specific chemosensory fate. A homeodomain-only short isoform of VAB-3 is expressed in BAG chemosensory neurons, where it promotes gene expression and cell function. In other cells, a long isoform of VAB-3, comprising a Paired homology domain and a homeodomain, represses expression of ETS-5, a transcription factor required for expression of BAG fate. Repression of ets-5 requires the Eyes Absent homolog EYA-1 and the Six-class homeodomain protein CEH-32. We determined sequences that mediate high-affinity binding of ETS-5, VAB-3 and CEH-32. The ets-5 locus is enriched for ETS-5-binding sites but lacks sequences that bind VAB-3 and CEH-32, suggesting that these factors do not directly repress ets-5 expression. We propose that a promoter-selection system together with lineage-specific expression of accessory factors allows VAB-3/Pax6 to either promote or repress expression of specific cell fates in a context-dependent manner. This article has an associated 'The people behind the papers' interview.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Bleomicina/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Clorambucila/metabolismo , Cisplatino/metabolismo , Ciclofosfamida/metabolismo , Dactinomicina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/genética , Vimblastina/metabolismo
6.
Bioorg Med Chem ; 27(9): 1767-1775, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30885568

RESUMO

Bleomycin is considered to exert its antitumor activity via DNA cleavage mediated by activated oxygen generated from the iron complex in its chelator moiety. Spin-offs from this moiety, HPH-1Trt and HPH-2Trt, with anti-cancer activities were recently synthesized. In this paper, we developed inhibitors of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of Sirtuin protein (SIRT2), based on HPH-1Trt/HPH-2Trt, and aimed to generate new anti-cancer drugs. HPH-1Trt and HPH-2Trt had in vitro anti-SIRT2 inhibitory activity with 50% inhibitory concentration (IC50) values of 5.5 and 8.8 µM, respectively. A structural portion of HPH-1Trt/HPH-2Trt, a tritylhistidine derivative TH-1, had stronger activity (IC50 = 1.7 µM), and thus, fourteen derivatives of TH-1 were synthesized. Among them, TH-3 had the strongest activity (IC50 = 1.3 µM). Selective binding of TH-3 in the pocket of SIRT2 protein was confirmed with a molecular docking study. Furthermore, TH-3 strongly lowered viability of the breast cancer cell line MCF7 with an IC50 of 0.71 µM. A structure-activity relationship study using cell lines suggested that the mechanism of TH-3 to suppress MCF7 cells involves not only SIRT2 inhibition, but also another function. This compound may be a new candidate anti-cancer drug.


Assuntos
Bleomicina/química , Histidina/química , Inibidores de Histona Desacetilases/química , Sirtuína 2/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Bleomicina/metabolismo , Bleomicina/farmacologia , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Sirtuína 2/metabolismo , Relação Estrutura-Atividade
7.
Biochem J ; 475(9): 1553-1567, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29661915

RESUMO

Multidrug binding and transport by the ATP-binding cassette transporter ABCG2 is a factor in the clinical resistance to chemotherapy in leukaemia, and a contributory factor to the pharmacokinetic profiles of many other prescribed drugs. Despite its importance, the structural basis of multidrug transport, i.e. the ability to transport multiple distinct chemicals, has remained elusive. Previous research has shown that at least two residues positioned towards the cytoplasmic end of transmembrane helix 3 (TM3) of the transporter play a role in drug transport. We hypothesised that other residues, either in the longitudinal span of TM3, or a perpendicular slice through the intracellular end of other TM helices would also contribute to drug binding and transport by ABCG2. Single-point mutant isoforms of ABCG2 were made at ∼30 positions and were analysed for effects on protein expression, localisation (western blotting, confocal microscopy) and function (flow cytometry) in a mammalian stable cell line expression system. Our data were interpreted in terms of recent structural data on the ABCG protein subfamily and enabled us to propose a surface-binding site for the drug mitoxantrone (MX) as well as a second, buried site for the same drug. Further mutational analysis of residues that spatially separate these two sites prompts us to suggest a molecular and structural pathway for MX transport by ABCG2.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Bleomicina/metabolismo , Resistência a Múltiplos Medicamentos/genética , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Substituição de Aminoácidos , Transporte Biológico , Domínio Catalítico , Humanos , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/metabolismo , Conformação Proteica
8.
Chembiochem ; 19(1): 43-47, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29084369

RESUMO

Noncoding RNAs are pervasive in cells and contribute to diseases such as cancer. A question in biomedical research is whether noncoding RNAs are targets of medicines. Bleomycin is a natural product that cleaves DNA; however, it is known to cleave RNA in vitro. Herein, an in-depth analysis of the RNA cleavage preferences of bleomycin A5 is presented. Bleomycin A5 prefers to cleave RNAs with stretches of AU base pairs. Based on these preferences and bioinformatic analysis, the microRNA-10b hairpin precursor was identified as a potential substrate for bleomycin A5. Both in vitro and cellular experiments demonstrated cleavage. Importantly, chemical cleavage by bleomycin A5 in the microRNA-10b hairpin precursors occurred near the Drosha and Dicer enzymatic processing sites and led to destruction of the microRNA. Evidently, oncogenic noncoding RNAs can be considered targets of cancer medicines and might elicit their pharmacological effects by targeting noncoding RNA.


Assuntos
Bleomicina/metabolismo , RNA não Traduzido/metabolismo , Sequência de Bases , Bleomicina/química , Células HeLa , Humanos , MicroRNAs/química , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Clivagem do RNA , RNA não Traduzido/química , Ribonuclease III/metabolismo
9.
Bioorg Med Chem ; 25(17): 4595-4613, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28734664

RESUMO

Site specific drug delivery with desired therapeutic effect still remains challenging task due to suboptimal release, tissue toxicity, low selectivity and meager therapeutic efficacy in skin cancers. The aim of the current study was to fabricate pH responsive, self-assembled, chemically cross-linked biodegradable chitosan nanogel loaded with bleomycin to target the dermal area of the skin. The nanogel synthesized by ion gelation technique and was characterized for drug loading, swelling and thermal stability followed by in vitro analysis. HaCaT (Human Keratinocyte cell) and HDF (Human dermal fibroblast) cell line were used for the biocompatibility and cytocompatibility evaluation prior to the hemolysis assay and coagulation assessment. The nanogel had a size range of 150nm as determined by TEM and DLS. The nanogel possessed optimum thermal stability as analyzed by thermogravimetry (TG) and differential thermal analysis (DTA). Biodegradation was confirmed by lysozyme enzyme degradation assays. The drug entrapment efficacy was about 55% in the swollen state. The In vitro drug release profile revealed sustained release pattern. The hemolysis of 2.39% and prothrombin time (PT) and activated partial thromboplastin time (APTT) of 12.9 and 31s revealed the biocompatibility of nanogels. The cell uptake and localization profile was validated by fluorescence and confocal microscopy using HDF and HaCaT cell lines. Finally, the MTT assay demonstrated the cytocompatibility of nanogels. In conclusion, the present findings suggest that biodegradable chitosan nanogels with stimuli responsive nature can release the anticancer drug cargo in a sustained and controlled manner and offer promising potentials for treating skin cancers. STATEMENT OF SIGNIFICANCE: Drug delivery to the targeted site is a major challenge in clinical medicine. The newly constructed pH responsive biodegradable nanogel consisting of bleomycin revealed pH triggered drug release in a sustained manner to the dermal area offering novel approach against skin cancer. The nanogel system is biodegradable in nature possessing high drug entrapment efficiency and offers patient compliance with biocompatible and cytocompatible characteristics. This nanogel system can thus be highly useful for delivery of anticancer drugs to the skin in a controlled and sustained manner.


Assuntos
Bleomicina/química , Portadores de Fármacos/química , Polietilenoglicóis/química , Polietilenoimina/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Materiais Biocompatíveis/química , Bleomicina/metabolismo , Bleomicina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Portadores de Fármacos/farmacologia , Liberação Controlada de Fármacos , Difusão Dinâmica da Luz , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Nanogéis , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de Fourier
10.
Artigo em Inglês | MEDLINE | ID: mdl-28069656

RESUMO

The metallo-ß-lactamase NDM-1 is among the most worrisome resistance determinants and is spreading worldwide among Gram-negative bacilli. A bleomycin resistance gene, bleMBL, downstream of the blaNDM-1 gene has been associated with resistance almost systematically. Here, we characterized the corresponding protein, BRPMBL, conferring resistance to bleomycin, an antitumoral glycopeptide molecule. We have determined whether the expression of the blaNDM-1-bleMBL operon is inducible in the presence of carbapenems and/or bleomycin-like molecules using quantitative reverse transcription-PCR (qRT-PCR), determination of imipenem and zeocin MICs, and carbapenemase-specific activity assays. We showed that the blaNDM-1-bleMBL operon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathione S-transferase (GST)-BRPMBL, we demonstrated that BRPMBL is able to bind and sequester bleomycin-like molecules, thus preventing bleomycin-dependent DNA degradation. In silico modeling confirmed that the mechanism of action required the dimerization of the BRPMBL protein in order to sequester bleomycin and prevent DNA damage. BRPMBL acts specifically on bleomycin-like molecules since cloning and expression of bleMBL in Staphyloccoccus aureus did not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Bleomicina/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Bleomicina/química , Bleomicina/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Imipenem/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/metabolismo , Modelos Moleculares , Óperon , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , beta-Lactamases/química , beta-Lactamases/metabolismo
11.
J Biol Inorg Chem ; 22(1): 121-136, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27858165

RESUMO

The antibiotics known as bleomycins constitute a family of natural products clinically employed for the treatment of a wide spectrum of cancers. The drug acts as an antitumor agent by virtue of the ability of a metal complex of the antibiotic to cleave DNA. Bleomycins are differentiated by their C-terminal regions. Previous structural studies involving metal-bleomycin-DNA triads have allowed the identification of the bithiazole-(C-terminus substituent) segment in this molecule as the one that most closely interacts with DNA. Three different modes of binding of metallo-bleomycins to DNA (partial or total intercalation of the bithiazole unit between DNA bases, or binding to the minor groove) have been proposed in the literature. The therapeutic use of bleomycin is frequently associated with the development of pulmonary fibrosis. The severity of this side effect has been attributed to the C-terminus of the antibiotic by some researchers. The degree of pulmonary toxicity of bleomycin-A2 and -A5, were found to be higher than those of bleomycin-B2 and peplomycin. Since the introduction of Blenoxane to clinical medicine in 1972, attempts have been made at modifying the basic bleomycin structure at the C-terminus to improve its therapeutic index. However, the pharmacological and toxicological importance of particular C-termini on bleomycin remains unclear. The present study was designed to determine the effect of Zn(II)bleomycin-A2, -A5, -B2, and Zn(II)peplomycin on the structure of a DNA hairpin containing the 5'-GC-3' binding site. We provide evidence that different Zn(II)bleomycins affect the structure of the tested DNA segment in different fashions.


Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bleomicina/metabolismo , Bleomicina/farmacologia , DNA/genética , DNA/metabolismo , Sequências Repetidas Invertidas/efeitos dos fármacos , Sítios de Ligação , DNA/química , Espectroscopia de Ressonância Magnética
12.
Sci Rep ; 6: 33129, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27634406

RESUMO

Horizontal gene transfer (HGT) often has strong benefits for fungi. In a study of samples from apple canker in Shaanxi Province, China, diverse microbes, along with the necrotrophic pathogen Valsa mali, were found to colonize the apple bark, thus providing ample opportunity for HGT to occur. In the present study, we identified 32 HGT events in V. mali by combining phyletic distribution-based methods with phylogenetic analyses. Most of these HGTs were from bacteria, whereas several others were from eukaryotes. Three HGTs putatively functioned in competition with actinomycetes, some of which showed a significant inhibitory effect on V. mali. Three HGTs that were probably involved in nitrogen uptake were also identified. Ten HGTs were thought to be involved in pathogenicity because they were related to known virulence factors, including cell wall-degrading enzymes and candidate effector proteins. HGT14, together with HGT32, was shown to contribute to bleomycin resistance of V. mali.These results suggest that HGT drives the adaptive evolution of V. mali. The HGTs identified here provide new clues for unveiling the adaptation mechanisms and virulence determinants of V. mali.


Assuntos
Ascomicetos/genética , Ascomicetos/patogenicidade , Transferência Genética Horizontal/genética , Malus/genética , Malus/microbiologia , Actinobacteria/genética , Bactérias/genética , Bactérias/patogenicidade , Bleomicina/metabolismo , Parede Celular/genética , China , Eucariotos/genética , Nitrogênio/metabolismo , Filogenia , Virulência/genética , Fatores de Virulência/genética
13.
Physiol Rep ; 4(17)2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27624688

RESUMO

Although exertional dyspnea and worsening hypoxia are hallmark clinical features of idiopathic pulmonary fibrosis (IPF), no drug currently available could treat them. GBT1118 is a novel orally bioavailable small molecule that binds to hemoglobin and produces a concentration-dependent left shift of the oxygen-hemoglobin dissociation curve with subsequent increase in hemoglobin-oxygen affinity and arterial oxygen loading. To assess whether pharmacological modification of hemoglobin-oxygen affinity could ameliorate hypoxemia associated with lung fibrosis, we evaluated GBT1118 in a bleomycin-induced mouse model of hypoxemia and fibrosis. After pulmonary fibrosis and hypoxemia were induced, GBT1118 was administered for eight consecutive days. Hypoxemia was determined by monitoring arterial oxygen saturation, while the severity of pulmonary fibrosis was assessed by histopathological evaluation and determination of collagen and leukocyte levels in bronchoalveolar lavage fluid. We found that hemoglobin modification by GBT1118 had strong antihypoxemic therapeutic effects with improved arterial oxygen saturation to near normal level. Moreover, GBT1118 treatment significantly attenuated bleomycin-induced lung fibrosis, collagen accumulation, body weight loss, and leukocyte infiltration. This study is the first to suggest the beneficial effects of hemoglobin modification in fibrotic lungs and offers a promising and novel therapeutic strategy for the treatment of hypoxemia associated with chronic fibrotic lung disorders in human, including IPF.


Assuntos
Benzaldeídos/administração & dosagem , Bleomicina/administração & dosagem , Hipóxia/induzido quimicamente , Fibrose Pulmonar Idiopática/induzido quimicamente , Niacinamida/análogos & derivados , Oxigênio/metabolismo , Oxiemoglobinas/efeitos dos fármacos , Administração Oral , Animais , Benzaldeídos/metabolismo , Benzaldeídos/farmacocinética , Benzaldeídos/farmacologia , Bleomicina/efeitos adversos , Bleomicina/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Dispneia/diagnóstico , Dispneia/etiologia , Hipóxia/complicações , Hipóxia/tratamento farmacológico , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Niacinamida/administração & dosagem , Niacinamida/metabolismo , Niacinamida/farmacocinética , Niacinamida/farmacologia , Oxigênio/sangue , Oxiemoglobinas/metabolismo , Distribuição Aleatória
14.
Microb Cell Fact ; 15: 29, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26849882

RESUMO

BACKGROUND: Multiple cognate gene copy clones have often been used in order to increase the yield of recombinant protein expression in the yeast Pichia pastoris. The method of posttransformational vector amplification (PTVA) has allowed for the efficient generation of multi-copy clones in P. pastoris. However, despite its relative ease and success, this process can be expensive and time consuming. RESULTS: We have developed a modified version of PTVA, called Liquid PTVA, which allows for faster and cheaper selection of multi-copy clones. Cultures are grown in liquid medium with only a final selection carried out on agar plates, reducing overall antibiotic usage and increasing the speed of clone amplification. In addition, it was established that starting PTVA with a single copy clone resulted in higher copy number strains for both traditional plate PTVA and liquid PTVA. Furthermore, using the Zeocin selection marker in liquid PTVA results in strains with higher growth rates, which could be beneficial for recombinant protein production processes. CONCLUSIONS: We present a methodology for creating multi-copy clones that can be achieved over 12 days instead of the traditional 45 and at approximately half the cost.


Assuntos
Dosagem de Genes , Vetores Genéticos/metabolismo , Pichia/genética , Reação em Cadeia da Polimerase/métodos , Transformação Genética , Bleomicina/metabolismo , Células Clonais , Meios de Cultura , Proteínas de Fluorescência Verde/metabolismo , Indicadores e Reagentes , Pichia/crescimento & desenvolvimento , Fatores de Tempo
15.
Int Immunopharmacol ; 30: 188-193, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26604088

RESUMO

It has been shown that the expression of ST2, a receptor of interleukin (IL)-33, is elevated in the lungs of idiopathic pulmonary fibrosis patients and bleomycin-induced mouse models, however its contribution to the development of pulmonary fibrosis has yet to be tested. In the present study, we treated mice by intranasal instillation of lentivirus expressing soluble ST2 and evaluated lung inflammation and fibrosis in the bleomycin-induced pulmonary fibrosis mouse model. We found that ST2 lentivirus treatment significantly improved survival rate and reduced weight loss compared with controls treated with empty lentivirus. Furthermore, ST2 treatment profoundly attenuated the pulmonary inflammatory cell infiltration and fibrotic changes. Finally, ST2 treatment markedly lowered the levels of IL-4, IL-6, IL-13, IL-33, monocyte chemoattractant protein-1, and transforming growth factor-ß1, whereas it increased the levels of interferon-γ in bronchoalveolar lavage fluid. The results indicate that ST2 might prevent bleomycin-induced pulmonary fibrosis possibly through downregulating proinflammatory and profibrotic mediators. This study suggests that lentivirus expressing soluble ST2 might represent an effective therapeutic approach in the treatment of pulmonary fibrotic diseases.


Assuntos
Terapia Genética , Lentivirus/genética , Pneumonia/terapia , Fibrose Pulmonar/terapia , Receptores de Interleucina/metabolismo , Animais , Bleomicina/metabolismo , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Vetores Genéticos/genética , Humanos , Mediadores da Inflamação/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33/agonistas , Camundongos , Pneumonia/induzido quimicamente , Pneumonia/genética , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Receptores de Interleucina/genética
16.
Photochem Photobiol Sci ; 14(7): 1357-66, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26088711

RESUMO

Here we evaluate the photosensitizer meso-tetraphenyl chlorin disulphonate (TPCS2a) in survival studies of rat glioma cancer cells in combination with the novel photochemical internalization (PCI) technique. The tested anticancer drugs were bleomycin (BLM) and temozolomide (TMZ). Glioma cells were incubated with TPCS2a (0.2 µg ml(-1), 18 h, 37 °C) before BLM or TMZ stimulation (4 h) prior to red light illumination (652 nm, 50 mW cm(-2)). The cell survival after BLM (0.5 µm)-PCI (40 s light) quantified using the MTT assay was reduced to about 25% after 24 h relative to controls, and to 31% after TMZ-PCI. The supplementing quantification by clonogenic assays, using BLM (0.1 µm), indicated a long-term cytotoxic effect: the surviving fraction of clonogenic cells was reduced to 5% after light exposure (80 s) with PCI, compared to 70% in the case of PDT. In parallel, structural and morphological changes within the cells upon light treatment were examined using fluorescence microscopy techniques. The present study demonstrates that PCI of BLM is an effective method for killing F98 glioma cells, but smaller effects were observed using TMZ following the "light after" strategy. The results are the basis for further in vivo studies on our rat glioma cancer model using PDT and PCI.


Assuntos
Antineoplásicos/metabolismo , Bleomicina/metabolismo , Dacarbazina/análogos & derivados , Glioma/metabolismo , Processos Fotoquímicos , Animais , Antineoplásicos/química , Bleomicina/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dacarbazina/química , Dacarbazina/metabolismo , Luz , Estrutura Molecular , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Ratos , Temozolomida , Ensaio Tumoral de Célula-Tronco
17.
Biochemistry ; 54(19): 3100-9, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25905565

RESUMO

We have shown previously that the bleomycin (BLM) carbohydrate moiety can recapitulate the tumor cell targeting effects of the entire BLM molecule, that BLM itself is modular in nature consisting of a DNA-cleaving aglycone which is delivered selectively to the interior of tumor cells by its carbohydrate moiety, and that there are disaccharides structurally related to the BLM disaccharide which are more efficient than the natural disaccharide at tumor cell targeting/uptake. Because BLM sugars can deliver molecular cargoes selectively to tumor cells, and thus potentially form the basis for a novel antitumor strategy, it seemed important to consider additional structural features capable of affecting the efficiency of tumor cell recognition and delivery. These included the effects of sugar polyvalency and net charge (at physiological pH) on tumor cell recognition, internalization, and trafficking. Since these parameters have been shown to affect cell surface recognition, internalization, and distribution in other contexts, this study has sought to define the effects of these structural features on tumor cell recognition by bleomycin and its disaccharide. We demonstrate that both can have a significant effect on tumor cell binding/internalization, and present data which suggests that the metal ions normally bound by bleomycin following clinical administration may significantly contribute to the efficiency of tumor cell uptake, in addition to their characterized function in DNA cleavage. A BLM disaccharide-Cy5** conjugate incorporating the positively charged dipeptide d-Lys-d-Lys was found to associate with both the mitochondria and the nuclear envelope of DU145 cells, suggesting possible cellular targets for BLM disaccharide-cytotoxin conjugates.


Assuntos
Bleomicina/química , Bleomicina/metabolismo , Dissacarídeos/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência , Estrutura Molecular , Ratos , Relação Estrutura-Atividade
18.
Am J Respir Cell Mol Biol ; 52(2): 217-31, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25029475

RESUMO

The bleomycin-induced rodent lung fibrosis model is commonly used to study mechanisms of lung fibrosis and to test potential therapeutic interventions, despite the well recognized dissimilarities to human idiopathic pulmonary fibrosis (IPF). Therefore, in this study, we sought to identify genomic commonalities between the gene expression profiles from 100 IPF lungs and 108 control lungs that were obtained from the Lung Tissue Research Consortium, and rat lungs harvested at Days 3, 7, 14, 21, 28, 42, and 56 after bleomycin instillation. Surprisingly, the highest gene expression similarity between bleomycin-treated rat and IPF lungs was observed at Day 7. At this point of maximal rat-human commonality, we identified a novel set of 12 disease-relevant translational gene markers (C6, CTHRC1, CTSE, FHL2, GAL, GREM1, LCN2, MMP7, NELL1, PCSK1, PLA2G2A, and SLC2A5) that was able to separate almost all patients with IPF from control subjects in our cohort and in two additional IPF/control cohorts (GSE10667 and GSE24206). Furthermore, in combination with diffusing capacity of carbon monoxide measurements, four members of the translational gene marker set contributed to stratify patients with IPF according to disease severity. Significantly, pirfenidone attenuated the expression change of one (CTHRC1) translational gene marker in the bleomycin-induced lung fibrosis model, in transforming growth factor-ß1-treated primary human lung fibroblasts and transforming growth factor-ß1-treated human epithelial A549 cells. Our results suggest that a strategy focused on rodent model-human disease commonalities may identify genes that could be used to predict the pharmacological impact of therapeutic interventions, and thus facilitate the development of novel treatments for this devastating lung disease.


Assuntos
Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/genética , Pulmão/patologia , Transdução de Sinais/genética , Animais , Bleomicina/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Expressão Gênica/fisiologia , Genômica , Humanos , Pulmão/metabolismo , Biossíntese de Proteínas , Ratos Sprague-Dawley
19.
J Am Chem Soc ; 136(39): 13641-56, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25184545

RESUMO

The disaccharide moiety is responsible for the tumor cell targeting properties of bleomycin (BLM). While the aglycon (deglycobleomycin) mediates DNA cleavage in much the same fashion as bleomycin, it exhibits diminished cytotoxicity in comparison to BLM. These findings suggested that BLM might be modular in nature, composed of tumor-seeking and tumoricidal domains. To explore this possibility, BLM analogues were prepared in which the disaccharide moiety was attached to deglycobleomycin at novel positions, namely, via the threonine moiety or C-terminal substituent. The analogues were compared with BLM and deglycoBLM for DNA cleavage, cancer cell uptake, and cytotoxic activity. BLM is more potent than deglycoBLM in supercoiled plasmid DNA relaxation, while the analogue having the disaccharide on threonine was less active than deglycoBLM and the analogue containing the C-terminal disaccharide was slightly more potent. While having unexceptional DNA cleavage potencies, both glycosylated analogues were more cytotoxic to cultured DU145 prostate cancer cells than deglycoBLM. Dye-labeled conjugates of the cytotoxic BLM aglycons were used in imaging experiments to determine the extent of cell uptake. The rank order of internalization efficiencies was the same as their order of cytotoxicities toward DU145 cells. These findings establish a role for the BLM disaccharide in tumor targeting/uptake and suggest that the disaccharide moiety may be capable of delivering other cytotoxins to cancer cells. While the mechanism responsible for uptake of the BLM disaccharide selectively by tumor cells has not yet been established, data are presented which suggest that the metabolic shift to glycolysis in cancer cells may provide the vehicle for selective internalization.


Assuntos
Bleomicina/química , Bleomicina/metabolismo , Dissacarídeos/química , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Bleomicina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Clivagem do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Masculino , Estrutura Molecular , Relação Estrutura-Atividade
20.
Chem Commun (Camb) ; 50(56): 7485-7, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24881675

RESUMO

The preparation and characterisation of an oxoiron(IV) species with monoamido ligation are described. Reactivity studies revealed the important role of the amido ligand in enhancing the ability of oxoiron(IV) complexes to promote hydrogen atom transfer from external alkanes.


Assuntos
Bleomicina/análogos & derivados , Ferroproteínas não Heme/síntese química , Espécies Reativas de Oxigênio/síntese química , Bleomicina/síntese química , Bleomicina/metabolismo , Ferroproteínas não Heme/metabolismo , Estabilidade Proteica , Espécies Reativas de Oxigênio/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...