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1.
Oxid Med Cell Longev ; 2021: 6660193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777318

RESUMO

Palmatine (PAL), a natural isoquinoline alkaloid, possesses extensive biological and pharmaceutical activities, including antioxidative stress, anti-inflammatory, antitumor, neuroprotective, and gastroprotective activities. However, it is unknown whether PAL has a protective effect against ischemic stroke and cerebral ischemia/reperfusion (I/R) injury. In the present study, a transient middle cerebral artery occlusion (MCAO) mouse model was used to mimic ischemic stroke and cerebral I/R injury in mice. Our study demonstrated that PAL treatment ameliorated cerebral I/R injury by decreasing infarct volume, neurological scores, and brain water content. PAL administration attenuated oxidative stress, the inflammatory response, and neuronal apoptosis in mice after cerebral I/R injury. In addition, PAL treatment also decreases hypoxia and reperfusion- (H/R-) induced neuronal injury by reducing oxidative stress, the inflammatory response, and neuronal apoptosis. Moreover, the neuroprotective effects of PAL were associated with the activation of the AMP-activated protein kinase (AMPK)/nuclear factor E2-related factor 2 (Nrf2) pathway, and Nrf2 knockdown offsets PAL-mediated antioxidative stress and anti-inflammatory effects. Therefore, our results suggest that PAL may be a novel treatment strategy for ischemic stroke and cerebral I/R injury.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Transtornos Cerebrovasculares , Fator 2 Relacionado a NF-E2/metabolismo , Palmitatos/farmacologia , Traumatismo por Reperfusão , Transdução de Sinais/efeitos dos fármacos , Transtornos Cerebrovasculares/metabolismo , Transtornos Cerebrovasculares/patologia , Transtornos Cerebrovasculares/prevenção & controle , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , ômega-Cloroacetofenona
2.
Eur J Med Chem ; 214: 113225, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33550182

RESUMO

Pyruvate dehydrogenase kinases (PDKs) are promising therapeutic targets that have received increasing attentions in cancer metabolism. In this paper, we report the synthesis and biological evaluation of a series of novel dichloroacetophenones as potent PDKs inhibitors. Structure-activity relationship analysis enabled us to identify a potent compound 6u, which inhibited PDKs with an EC50 value of 0.09 µM, and reduced various cancer cells proliferation with IC50 values ranging from 1.1 to 3.8 µM, while show weak effect against non-cancerous L02 cell (IC50 > 10 µM). In the A375 xenograft model, 6u displayed an obvious antitumor activity at a dose of 5 mg/kg, but with no negative effect to the mice weight. Molecular docking suggested that 6u formed direct hydrogen bond interactions with Ser75 and Gln61 in PDK1, and meanwhile the aniline skeleton in 6u was sandwiched by the conserved hydrophobic residues Phe78 and Phe65, which contribute to the biochemical activity improvement. Moreover, 6u induced A375 cell apoptosis and cell arrest in G1 phase, and inhibited cancer cell migration. In addition, 6u altered glucose metabolic pathway in A375 cell by decreasing lactate formation and increasing ROS production and OCR consumption, which could serve as a potential modulator to reprogram the glycolysis pathway in cancer cell.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , ômega-Cloroacetofenona/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas , ômega-Cloroacetofenona/síntese química , ômega-Cloroacetofenona/química
3.
Int J Mol Med ; 47(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33448314

RESUMO

Mesenchymal stem cells (MSCs) have the function of repairing damaged tissue, which is known to be mediated by the secretome, the collection of secretory materials shed from MSCs. Adjusting the culture conditions of MSCs can lead to a significant difference in the composition of the secretome. It was hypothesized that pre­sensitization of MSCs with specific disease­causing agents could harness MSCs to release the therapeutic materials specialized for the disease. To validate this hypothesis, the present study aimed to generate a 'disease­specific secretome' for hepatitis caused by hepatitis B virus using hepatitis BX antigen (HBx) as a disease­causing material. Secretary materials (HBx­IS) were collected following the stimulation of adipose­derived stem cells (ASCs) with 100­fold diluted culture media of AML12 hepatocytes that had been transfected with pcDNA­HBx for 24 h. An animal model of hepatitis B was generated by injecting HBx into mice, and the mice were subsequently intravenously administered a control secretome (CS) or HBx­IS. Compared with the CS injection, the HBx­IS injection significantly reduced the serum levels of interleukin­6 and tumor necrosis factor­α (pro­inflammatory cytokines). Western blot analysis and immunohistochemistry of the liver specimens revealed that the HBx­IS injection led to a higher expression of liver regeneration­related markers, including hepatocyte growth factor and proliferating cell nuclear antigen, a lower expression of pro­apoptotic markers, such as cleaved caspase 3 and Bim in mouse livers, and a lower expression of pro­inflammatory markers (F4/80 and CD68) compared to the CS injection. HBx­IS exhibited higher liver regenerative, anti­inflammatory and anti­apoptotic properties, particularly in the mouse model of hepatitis B compared to CS. This suggests that the secretome obtained by stimulating ASCs with disease­causing agents may have a more prominent therapeutic effect on the specific disease than the naïve secretome.


Assuntos
Tecido Adiposo/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo/patologia , Animais , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Hepatite B/tratamento farmacológico , Hepatite B/metabolismo , Hepatite B/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos Endogâmicos BALB C , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , ômega-Cloroacetofenona
4.
J Biotechnol ; 325: 57-64, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-33220340

RESUMO

The asymmetric reduction of ketones is a frequently used synthesis route towards chiral alcohols. Amongst available chemo- and biocatalysts the latter stand out in terms of product enantiopurity. Their application is, however, restricted by low reaction output, often rooted in limited enzyme stability under operational conditions. Here, addition of 2-hydroxypropyl-ß-cyclodextrin to bioreductions of o-chloroacetophenone enabled product concentrations of up to 29 % w/v at full conversion and 99.97 % e.e. The catalyst was an E. coli strain co-expressing NADH-dependent Candida tenuis xylose reductase and a yeast formate dehydrogenase for coenzyme recycling. Analysis of the lyophilized biocatalyst showed that E. coli cells were leaky with catalytic activity found as free-floating enzymes and associated with the biomass. The biocatalyst was stabilized and activated in the reaction mixture by 2-hydroxypropyl-ß-cyclodextrin. Substitution of the wild-type xylose reductase by a D51A mutant further improved bioreductions. In previous optimization strategies, hexane was added as second phase to protect the labile catalyst from adverse effects of hydrophobic substrate and product. The addition of 2-hydroxypropyl-ß-cyclodextrin (11 % w/v) instead of hexane (20 % v/v) increased the yield on biocatalyst 6.3-fold. A literature survey suggests that bioreduction enhancement by addition of cyclodextrins is not restricted to specific enzyme classes, catalyst forms or substrates.


Assuntos
Ciclodextrinas , ômega-Cloroacetofenona , Escherichia coli/genética , Formiato Desidrogenases , Saccharomycetales
5.
J Immunol ; 205(8): 2077-2090, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32929040

RESUMO

Previously, we discovered that influenza-generated CD4 effectors must recognize cognate Ag at a defined effector checkpoint to become memory cells. Ag recognition was also required for efficient protection against lethal influenza infection. To extend these findings, we investigated if vaccine-generated effectors would have the same requirement. We compared live infection with influenza to an inactivated whole influenza vaccine. Live infection provided strong, long-lasting Ag presentation that persisted through the effector phase. It stimulated effector generation, long-lived CD4 memory generation, and robust generation of Ab-producing B cells. In contrast, immunization with an inactivated virus vaccine, even when enhanced by additional Ag-pulsed APC, presented Ag for 3 d or less and generated few CD4 memory cells or long-lived Ab-producing B cells. To test if checkpoint Ag addition would enhance this vaccine response, we immunized mice with inactivated vaccine and injected Ag-pulsed activated APC at the predicted effector checkpoint to provide Ag presentation to the effector CD4 T cells. This enhanced generation of CD4 memory, especially tissue-resident memory in the lung, long-lived bone marrow Ab-secreting cells, and influenza-specific IgG Ab. All responses increased as we increased the density of peptide Ag on the APC to high levels. This suggests that CD4 effectors induced by inactivated vaccine require high levels of cognate Ag recognition at the effector checkpoint to most efficiently become memory cells. Thus, we suggest that nonlive vaccines will need to provide high levels of Ag recognition throughout the effector checkpoint to optimize CD4 memory generation.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica , Vacinas contra Influenza/imunologia , Pulmão/imunologia , Animais , Anticorpos Antivirais/genética , Antígenos Virais/genética , Feminino , Vacinas contra Influenza/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , ômega-Cloroacetofenona
6.
Toxicol Lett ; 332: 36-41, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32629075

RESUMO

The study examined the degradation of riot control agents (RCAs): 2-chloroacetophenone (CN), 2-chlorobenzalmalononitrile (CS), and capsaicin, using the Reactive Skin Decontamination Lotion Kit (RSDL®) lotion and evaluated the the direct liquid phase reactivity of the RSDL lotion component with each RCA. RSDL lotion was mixed with the selected RCAs at different molar ratios. Reactivity of the active ingredient potassium 2,3-butanedione monoximate (KBDO) with the RCA was observed for one hour. Samples of 10 µL were taken and quenched, analyzed for residual RCA using LC-MS. CN, was degraded at molar ratios of two and above in less than 2 min. At a molar ratio of 1:1 KBDO:CN, ∼90 % of CN was degraded within 2 min, the remaining 10 % residual CN was observed for one hour without any change. CS, degradation of more than 68 % of CS was achieved at 20:1 M ratio of KBDO:CS within 1 h of reaction time. For capsaicin, no degradation was observed regardless of the higher molar ratios of up to 20:1 and longer reaction times of up to one hour. This study provides evaluation of neutralizing action of the RSDL lotion without assessment of the physical removal component by the RSDL Kit.


Assuntos
Capsaicina/química , Clorobenzenos/química , Descontaminação/métodos , Irritantes/química , Fármacos do Sistema Sensorial/química , Creme para a Pele/química , Gases Lacrimogênios/química , ômega-Cloroacetofenona/química , Calibragem , Capsaicina/análise , Clorobenzenos/análise , Cromatografia Líquida de Alta Pressão , Humanos , Irritantes/análise , Fármacos do Sistema Sensorial/análise , Pele , Gases Lacrimogênios/análise , ômega-Cloroacetofenona/análise
7.
Nanomedicine ; 21: 102076, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31394261

RESUMO

Pretargeting is an increasingly explored strategy to improve nanoparticle targeting, in which pretargeting molecules that bind both selected epitopes on target cells and nanocarriers are first administered, followed by the drug-loaded nanocarriers. Bispecific antibodies (bsAb) represent a promising class of pretargeting molecules, but how different bsAb formats may impact the efficiency of pretargeting remains poorly understood, in particular Fab valency and Fc receptor (FcR)-binding of bsAb. We found the tetravalent bsAb markedly enhanced PEGylated nanoparticle binding to target HER2+ cells relative to the bivalent bsAb in vitro. Pretargeting with tetravalent bsAb with abrogated FcR binding increased tumor accumulation of PEGylated liposomal doxorubicin (PLD) 3-fold compared to passively targeted PLD alone, and 5-fold vs pretargeting with tetravalent bsAb with normal FcR binding in vivo. Our work demonstrates that multivalency and elimination of FcRn recycling are both important features of pretargeting molecules, and further supports pretargeting as a promising nanoparticle delivery strategy.


Assuntos
Anticorpos Biespecíficos , Antineoplásicos Imunológicos , Portadores de Fármacos , Neoplasias Experimentais , Polietilenoglicóis , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Feminino , Humanos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , ômega-Cloroacetofenona
8.
Immunopharmacol Immunotoxicol ; 41(2): 277-284, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31084401

RESUMO

Objectives: Gouty arthritis is caused by the deposition of monosodium urate (MSU) crystals in joints, which is associated with the rise of serum urate content. This study aims to investigate the therapeutic effect of Madecassoside on gouty arthritis and hyperuricemia. Methods: DBA/1 mice were intradermally injected with MSU to stimulate joint inflammation or intraperitoneally injected with MSU to trigger peritonitis. Moreover, ICR mice were exposed to potassium oxonate to stimulate hyperuricemia. Results: Madecassoside repressed MSU-triggered pad swelling, joint 99mTc uptake, and joint inflammation in DBA/1 mice with gouty arthritis. Neutrophil infiltration and IL-1ß & IL-6 & MCP-1 secretion was also alleviated in lavage fluids from DBA/1 mice with peritonitis due to Madecassoside treatment. Furthermore, Madecassoside decreased MSU-induced neutrophil cytosolic factor 1, caspase-1 and NLRP3 expression in mice with peritoneal inflammation. In hyperuricemic mice, Madecassoside improved renal dysfunction. Serum uric acid, BUN, and creatinine were down-regulated by Madecassoside. Conclusion: These findings indicate that Madecassoside has potential to ameliorate inflammation in both acute gouty arthritis model and peritonitis model, probably via regulating IL-1ß and NLRP3 expression. Practical point: Madecassoside also exhibited a urate-lowering effect and a renal protective effect in hyperuricemic mice.


Assuntos
Anti-Inflamatórios/farmacologia , Artrite Gotosa/tratamento farmacológico , Hiperuricemia/tratamento farmacológico , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Triterpenos/farmacologia , Animais , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/imunologia , Citocinas/imunologia , Hiperuricemia/induzido quimicamente , Hiperuricemia/imunologia , Hiperuricemia/patologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos Endogâmicos ICR , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Peritonite/induzido quimicamente , Peritonite/imunologia , Peritonite/patologia , Ácido Úrico/toxicidade , ômega-Cloroacetofenona
9.
Front Immunol ; 9: 2824, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619247

RESUMO

A primary mechanism for activation of innate immunity is recognition of damage or pathogen associated molecular patterns by pattern recognition receptors (PRRs). Nucleic acid is a damage associated molecular pattern molecule that when internalized into a monocyte and recognized by intracellular nucleic acid sensing toll like receptors will cause production of type 1 interferon. The process by which DNA or RNA is delivered into the cytosol of monocytes in systemic lupus erythematosus remains incompletely understood, and therapeutic approaches to prevent DNA-mediated monocyte activation are needed. We identified two mechanisms for internalization of DNA by monocytes. IgG-bound DNA was internalized by interacting with Fc gamma receptor IIa, while high-mobility group box-1 protein-bound DNA was internalized by interacting with the receptor for advanced glycation end products. Both pathways contribute to an inflammatory phenotype in monocytes exposed to serum from patients with SLE. Moreover, both of these pathways can be inhibited by a pentapeptide, DWEYS, which is a DNA mimetope. In one instance DWEYS directly competes with DNA for antibody binding and in the other DWEYS binds high-mobility group box-1 and blocks its interaction with RAGE. Our data highlight distinct pathways involved in nucleic acid enters monocytes in SLE, and identify a potential therapeutic to prevent nucleic acid internalization in SLE.


Assuntos
Anticorpos Antinucleares , Ácidos Nucleicos Livres , Imunoglobulina G , Interferons , Lúpus Eritematoso Sistêmico , Monócitos , Animais , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/imunologia , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/imunologia , Feminino , Proteína HMGB1/sangue , Proteína HMGB1/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interferons/sangue , Interferons/imunologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Masculino , Camundongos Mutantes , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , ômega-Cloroacetofenona
11.
J Biotechnol ; 259: 120-125, 2017 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-28760442

RESUMO

Herein, we reported that Rhodobacter sphaeroides (R. sphaeroides) can be engineered by heterologous expression of an alcohol dehydrogenase (adh) from Leifsonia sp. to build a light-driven cofactor regeneration system for synthesis of chiral alcohol. The model substrate, 3'-chloroacetophenone, can be reduced by the engineered R. sphaeroides to (R)-1-(3-chlorophenyl) ethanol with an enantiomeric excess (e.e.) value of more than 99% in an n-hexane/aqueous biphasic media. This system, which is fully controlled by light, exhibited potential power to be an alternative cofactor regeneration platform for cheap synthesis of various chiral alcohols via the cloning other oxidoreductases with diverse characteristics.


Assuntos
Álcool Desidrogenase/metabolismo , Álcoois/química , Álcoois/metabolismo , Engenharia Metabólica/métodos , Rhodobacter sphaeroides/genética , Actinobacteria/enzimologia , Actinobacteria/genética , Álcool Desidrogenase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Luz , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodobacter sphaeroides/metabolismo , Estereoisomerismo , ômega-Cloroacetofenona
13.
J Cell Biochem ; 118(10): 3249-3259, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28262979

RESUMO

Saturated fatty acids (SFA) and their toxic metabolites contribute to hepatocyte lipotoxicity in nonalcoholic steatohepatitis (NASH). We previously reported that hepatocytes, under lipotoxic stress, express the potent macrophage chemotactic ligand C-X-C motif chemokine 10 (CXCL10), and release CXCL10-enriched extracellular vesicles (EV) by a mixed lineage kinase (MLK) 3-dependent mechanism. In the current study, we sought to examine the signaling pathway responsible for CXCL10 induction during hepatocyte lipotoxicity. Here, we demonstrate a role for signal transducer and activator of transcription (STAT) 1 in regulating CXCL10 expression. Huh7 and HepG2 cells were treated with lysophosphatidylcholine (LPC), the toxic metabolite of the SFA palmitate. In LPC-treated hepatocytes, CXCL10 induction is mediated by a mitogen activated protein kinase (MAPK) signaling cascade consisting of a relay kinase module of MLK3, MKK3/6, and p38. P38 in turn induces STAT1 Ser727 phosphorylation and CXCL10 upregulation in hepatocytes, which is reduced by genetic or pharmacological inhibition of this MAPK signaling cascade. The binding and activity of STAT1 at the CXCL10 gene promoter were identified by chromatin immunoprecipitation and luciferase gene expression assays. Promoter activation was attenuated by MLK3/STAT1 inhibition or by deletion of the consensus STAT1 binding sites within the CXCL10 promoter. In lipotoxic hepatocytes, MLK3 activates a MAPK signaling cascade, resulting in the activating phosphorylation of STAT1, and CXCL10 transcriptional upregulation. Hence, this kinase relay module and/or STAT1 inhibition may serve as a therapeutic target to reduce CXCL10 release, thereby attenuating NASH pathogenesis. J. Cell. Biochem. 118: 3249-3259, 2017. © 2017 Wiley Periodicals, Inc.


Assuntos
Quimiocina CXCL10/metabolismo , Hepatócitos/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fator de Transcrição STAT1/metabolismo , Células Hep G2 , Hepatócitos/patologia , Humanos , Lisofosfatidilcolinas/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/patologia , Ácido Palmítico/toxicidade , ômega-Cloroacetofenona
14.
J Biotechnol ; 257: 110-117, 2017 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-27913217

RESUMO

Product isolation from aqueous-organic reaction mixtures that contain high concentrations of whole cells constitutes a challenging task in bioprocessing. Stirring of the biphasic reaction media leads to the formation of solvent droplets coated by cells and other surface active components and an emulsion forms. We used an early focus on phase separation to simplify a whole-cell bioreduction. Octanol, heptanol, hexanol, hexane and dipropylether were tested as co-solvents in the E. coli catalyzed reduction of o-chloroacetophenone. All solvents showed very similar performance in bioreductions and highest yields were obtained with low organic-to-aqueous phase ratios. Reaction mixtures were directly investigated for organic-phase recovery. Phase separation was optimized in small-scale settling experiments and confirmed by the isolation of 20.4g (S)-1-(2-chlorophenyl)ethanol from a 0.5L batch reduction containing 40gCDW/L whole-cell catalyst. Solvent consumption during product isolation could be halved by the simple addition of sodium hydroxide prior to product extraction. Basification to pH 13.5 and three extraction steps with a total of 1.2v/v hexane led to an isolated yield of 87% (97% reduction yield). A general emulsion destabilizing effect under harsh conditions, as extreme pH values and presence of toxic reactants, was observed.


Assuntos
Biotecnologia/métodos , Biotransformação , Interações Hidrofóbicas e Hidrofílicas , Solventes/química , Biocatálise , Reatores Biológicos/microbiologia , Centrifugação , Emulsões/química , Escherichia coli/química , Filtração , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Compostos Orgânicos/química , Água/química , ômega-Cloroacetofenona/química
16.
Sci Rep ; 6: 32256, 2016 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-27558409

RESUMO

Candida albicans (C. albicans) is an important human commensal and opportunistic fungal pathogen. Secreted aspartyl proteinases (Saps) are a major virulence trait of C. albicans, and among these proteases Sap2 has the highest expression levels. It is possible that antibodies against Sap2 could provide an antifungal effect. In this study, two phages displaying anti-rSap2 single chain variable fragments (scFvs) were screened from human single fold scFv libraries, and their potential therapeutic roles were evaluated using a murine model infected by C. albicans. The in vivo efficacies were assessed by mortality rates, fungal burden and histological examination. Overall survival rates were significantly increased while the colony counts and infectious foci were significantly decreased after treatment with the scFv-phages relative to the control groups. In order to investigate the immune response provoked by scFv-phages, three kinds of cytokines (Th1, Th2 and Th17 types) were measured and a clear immune response was observed. These findings suggest that anti-rSap2 scFv-phages have potential in the therapy of systemic infection caused by C. albicans.


Assuntos
Anticorpos Antifúngicos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Candida albicans/imunologia , Candidíase/tratamento farmacológico , Proteínas Fúngicas/antagonistas & inibidores , Anticorpos de Domínio Único/farmacologia , Animais , Anticorpos Antifúngicos/química , Anticorpos Antifúngicos/genética , Anticorpos Antifúngicos/imunologia , Ácido Aspártico Endopeptidases/imunologia , Bacteriófago M13 , Candidíase/genética , Candidíase/imunologia , Candidíase/patologia , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/imunologia , Humanos , Camundongos Endogâmicos BALB C , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , ômega-Cloroacetofenona
17.
Biotechnol J ; 11(7): 890-8, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26901842

RESUMO

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes.


Assuntos
Álcool Desidrogenase/metabolismo , Formiato Desidrogenases/metabolismo , Corpos de Inclusão/enzimologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/metabolismo , Álcoois Benzílicos/química , Biocatálise , Candida/enzimologia , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Corpos de Inclusão/genética , NAD/metabolismo , Proteínas Recombinantes/genética , Rhodococcus/enzimologia , ômega-Cloroacetofenona/análogos & derivados , ômega-Cloroacetofenona/química
18.
Environ Sci Technol ; 50(4): 1868-76, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26814150

RESUMO

Increasing recognition that abiotic natural attenuation (NA) of chlorinated solvents can be important has created demand for improved methods to characterize the redox properties of the aquifer materials that are responsible for abiotic NA. This study explores one promising approach: using chemical reactivity probes (CRPs) to characterize the thermodynamic and kinetic aspects of contaminant reduction by reducing iron minerals. Assays of thermodynamic CRPs were developed to determine the reduction potentials (ECRP) of suspended minerals by spectrophotometric determination of equilibrium CRP speciation and calculations using the Nernst equation. ECRP varied as expected with mineral type, mineral loading, and Fe(II) concentration. Comparison of ECRP with reduction potentials measured potentiometrically using a Pt electrode (EPt) showed that ECRP was 100-150 mV more negative than EPt. When EPt was measured with small additions of CRPs, the systematic difference between EPt and ECRP was eliminated, suggesting that these CRPs are effective mediators of electron transfer between mineral and electrode surfaces. Model contaminants (4-chloronitrobenzene, 2-chloroacetophenone, and carbon tetrachloride) were used as kinetic CRPs. The reduction rate constants of kinetic CRPs correlated well with the ECRP for mineral suspensions. Using the rate constants compiled from literature for contaminants and relative mineral reduction potentials based on ECRP measurements, qualitatively consistent trends were obtained, suggesting that CRP-based assays may be useful for estimating abiotic NA rates of contaminants in groundwater.


Assuntos
Tetracloreto de Carbono/química , Monitoramento Ambiental/métodos , Compostos Ferrosos/química , Nitrobenzenos/química , Poluentes Químicos da Água/química , ômega-Cloroacetofenona/química , Ferro/química , Minerais/química , Oxirredução , Potenciometria , Espectrofotometria Ultravioleta
19.
Clin Toxicol (Phila) ; 54(2): 79-91, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26692048

RESUMO

INTRODUCTION: Chemical weapons dumped into the ocean for disposal in the twentieth century pose a continuing environmental and human health risk. OBJECTIVE: In this review we discuss locations, quantity, and types of sea-dumped chemical weapons, related environmental concerns, and human encounters with sea-dumped chemical weapons. METHODS: We utilized the Ovid (http://ovidsp.tx.ovid.com) and PubMed (http://www.pubmed.org) search engines to perform MEDLINE searches for the terms 'sea-dumped chemical weapons', 'chemical warfare agents', and 'chemical munitions'. The searches returned 5863 articles. Irrelevant and non-English articles were excluded. A review of the references for these articles yielded additional relevant sources, with a total of 64 peer-reviewed articles cited in this paper. History and geography of chemical weapons dumping at sea: Hundreds of thousands of tons of chemical munitions were disposed off at sea following World War II. European, Russian, Japanese, and United States coasts are the areas most affected worldwide. Several areas in the Baltic and North Seas suffered concentrated large levels of dumping, and these appear to be the world's most studied chemical warfare agent marine dumping areas. Chemical warfare agents: Sulfur mustard, Lewisite, and the nerve agents appear to be the chemical warfare agents most frequently disposed off at sea. Multiple other type of agents including organoarsenicals, blood agents, choking agents, and lacrimators were dumped at sea, although in lesser volumes. Environmental concerns: Numerous geohydrologic variables contribute to the rate of release of chemical agents from their original casings, leading to difficult and inexact modeling of risk of release into seawater. Sulfur mustard and the organoarsenicals are the most environmentally persistent dumped chemical agents. Sulfur mustard in particular has a propensity to form a solid or semi-solid lump with a polymer coating of breakdown products, and can persist in this state on the ocean floor for decades. Rates of solubility and hydrolysis and levels of innate toxicity of a chemical agent are used to predict the risk to the marine environments. The organoarsenicals eventually breakdown into arsenic, and thus present an indefinite timeline for contamination. Generally, studies assaying sediment and water levels of parent chemical agents and breakdown products at dumpsites have found minimal amounts of relevant chemicals, although arsenic levels are typically higher in dumpsites than reference areas. Studies of marine organisms have not shown concerning amounts of chemical agents or breakdown products in tissue, but have shown evidence of chronic toxicity. There is believed to be minimal risk posed by seafood consumption. Microbiota assays of dumpsites are significantly altered in species composition compared to reference sites, which may imply unseen but significant changes to ecosystems of dumpsites. Human health concerns: The major human health risk at this time appears to arise from acute exposure to an agent by either accidental recovery of a chemical weapon on a fishing vessel, or by munitions washed ashore onto beaches. CONCLUSIONS: Improving technology continues to make the deep sea more accessible, thus increasing the risk of disturbing munitions lying on or buried in the seabed. Pipe laying, cable burying, drilling, scuba diving, trawling, and undersea scientific research are the activities posing the most risk. The long-term threat to the benthic habitat via increased arsenic concentrations, shifts in microbiota speciation, and chronic toxicity to vertebrates and invertebrates is not currently understood. The risk to the environment of massive release via disturbance remains a distinct possibility. Terrorist recovery and re-weaponization of chemical agents is a remote possibility.


Assuntos
Substâncias para a Guerra Química/análise , Oceanos e Mares , Animais , Organismos Aquáticos/efeitos dos fármacos , Organismos Aquáticos/crescimento & desenvolvimento , Arsenicais/análise , Substâncias para a Guerra Química/toxicidade , Cianetos/análise , Cianetos/toxicidade , Bases de Dados Factuais , Monitoramento Ambiental , Europa (Continente) , Peixes/crescimento & desenvolvimento , Sedimentos Geológicos/análise , Humanos , Japão , Gás de Mostarda/análise , Gás de Mostarda/toxicidade , Agentes Neurotóxicos/análise , Agentes Neurotóxicos/toxicidade , Fosgênio/análise , Fosgênio/toxicidade , Medição de Risco , Estados Unidos , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidade , ômega-Cloroacetofenona/análise , ômega-Cloroacetofenona/toxicidade
20.
Biotechnol Adv ; 33(8): 1641-52, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26343336

RESUMO

Access to chiral alcohols of high optical purity is today frequently provided by the enzymatic reduction of precursor ketones. However, bioreductions are complicated by the need for reducing equivalents in the form of NAD(P)H. The high price and molecular weight of NAD(P)H necessitate in situ recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme recycling mode and reaction conditions represent design options that strongly affect bioreduction efficiency. In this paper, each option was critically scrutinized and decision rules formulated based on well-described literature examples. The development chain was visualized as a decision-tree that can be used to identify the most promising route towards the production of a specific chiral alcohol. General methods, applications and bottlenecks in the set-up are presented and key experiments required to "test" for decision-making attributes are defined. The reduction of o-chloroacetophenone to (S)-1-(2-chlorophenyl)ethanol was used as one example to demonstrate all the development steps. Detailed analysis of reported large scale bioreductions identified product isolation as a major bottleneck in process design.


Assuntos
Álcoois/química , Biocatálise , Engenharia Metabólica , Oxirredutases/química , Escherichia coli/química , Escherichia coli/genética , Cetonas/química , NADP/química , Oxirredução , Oxirredutases/genética , Especificidade por Substrato , ômega-Cloroacetofenona/química
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