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1.
Org Lett ; 21(22): 9198-9202, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31647672

RESUMO

This communication highlights the use of chiral sulfinamides as nitrogen nucleophiles in intermolecular aza-Michael reactions. When chiral sulfinamides are coupled to a chloroethyl group, the corresponding novel annulating reagents can be used to streamline the stereoselective synthesis of complex pyrrolidine-containing molecules. As a result, it has enabled a medicinal chemistry campaign for the synthesis of biologically active RORγt inverse agonists.

2.
J Org Chem ; 82(19): 10715-10721, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28809492

RESUMO

An addition of organozinc nucleophiles to N-acyl activated quinolines and isoquinolines is described. Simple transmetalation with the corresponding Grignard reagents using ZnCl2 forms organozinc compounds which are functional group tolerant and stable to reactive acyl chloride reagents for extended periods. A wide variety of substrates which include reactive electron-withdrawing groups are well tolerated to form 2-substituted dihydroquinolines and dihydroisoquinolines. This methodology has been applied toward an improved synthetic route of uncialamycin and its analogs.

3.
J Am Chem Soc ; 139(20): 6819-6822, 2017 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-28463562

RESUMO

Herein we describe concise enantioselective chemical syntheses of (-)-viridin and (-)-viridiol. Our convergent approach couples two achiral fragments of similar complexity and employs an enantioselective intramolecular Heck reaction to set the absolute stereochemical configuration of an all-carbon quaternary stereocenter. To complete the syntheses of these base- and nucleophile-sensitive natural products, we conduct carefully orchestrated site- and diastereoselective oxidations and other transformations. Our work is the first to generate these targets as single enantiomers.


Assuntos
Androstenodióis/síntese química , Androstenos/síntese química , Bacteriocinas/síntese química , Androstenodióis/química , Androstenos/química , Bacteriocinas/química , Estrutura Molecular , Estereoisomerismo
4.
J Mol Histol ; 48(3): 169-185, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28343338

RESUMO

Bone is a metabolically active organ subjected to continuous remodeling process that involves resorption by osteoclast and subsequent formation by osteoblasts. Osteoclast involvement in this physiological event is regulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Fusion of mono-nuclear pre-osteoclasts is a critical event for osteoclast differentiation and for bone resorption. Here we show that PBMCs can be successfully fused with polyethylenglicol (PEG) in order to generated viable osteoclast-like cells that exhibit tartrate-resistant acid phosphatase (TRAP) and bone resorptive activities. PEG-fused PBMCs expressed additional markers compatible with osteoclastogenic differentiation such as carbonic anhydrase II (CAII), calcitonin receptor (CR), cathepsin K (Cat K), vacuolar ATPase (V-ATPase) subunit C1 (V-ATPase), integrin ß3, RANK and cell surface aminopeptidase N/CD13. Actin redistribution in PEG-fused cells was found to be affected by cell cycle synchronization at G0/G1 or G2/M phases. PEG-induced fusion also led to expression of tyrosine kinases c-Src and Syk in their phosphorylated state. Scanning electron microscopy images showed morphological features typical of osteoclast-like cells. The results here shown allow concluding that PEG-induced fusion of PBMCs provides a suitable model system for understanding the mechanisms involved in osteoclastogenesis and for assaying new therapeutic strategies.


Assuntos
Diferenciação Celular , Fusão Celular , Leucócitos Mononucleares/metabolismo , Osteoclastos/citologia , Biomarcadores/análise , Biomarcadores/sangue , Reabsorção Óssea , Células Cultivadas , Humanos , Leucócitos Mononucleares/citologia , Modelos Biológicos , Polietilenoglicóis , Fosfatase Ácida Resistente a Tartarato/metabolismo
5.
J Bone Miner Metab ; 35(2): 127-141, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26969392

RESUMO

The bone remodeling process occurs through bone formation by osteoblasts and bone resorption by osteoclasts, a process involving the contribution of endocrine and nervous systems. The mechanisms associated to differentiation and proliferation of osteoclasts and osteoblasts are considered a potential therapeutic target for treating some erosive bone diseases. The aim of the present study is to explore the feasibility of generating active osteoclast-like cells from peripheral blood mononuclear cells (PBMCs) following polyethylene glycol (PEG)-induced fusion. PEG-fused PBMCs showed TRAP+-multinucleated cells and bone resorption activity, and were also positive for osteoclast markers such as carbonic anhydrase II, calcitonin receptor, vacuolar ATPase, and cathepsin K, when examined by reverse transcription-polymerase chain reaction, immunochemistry and Western blotting. TRAP expression and bone resorptive activity were higher in whole PEG-fused PBMCs than in separated T lymphocytes, B lymphocytes or monocytes. Both TRAP expression and bone resorptive activity were also higher in osteogenesis imperfecta patients compared to PEG-fused PBMCs from healthy individuals. PEG-induced fusion was more efficient in inducing TRAP and bone resorptive activities than macrophage colony-stimulating factor or dexamethasone treatment. Bone resorptive activity of PEG-fused PMBCs was inhibited by bisphosphonates. Evidence is provided that the use of PEG-based cell fusion is a straightforward and amenable method for studying human osteoclast differentiation and testing new therapeutic strategies.


Assuntos
Reabsorção Óssea/metabolismo , Leucócitos Mononucleares/citologia , Osteoclastos/citologia , Polietilenoglicóis/farmacologia , Adolescente , Linfócitos B/citologia , Linfócitos B/metabolismo , Técnicas de Cultura de Células , Fusão Celular , Células Cultivadas , Criança , Dexametasona/farmacologia , Difosfonatos/farmacologia , Humanos , Leucócitos Mononucleares/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Monócitos/citologia , Monócitos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese Imperfeita/fisiopatologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismo
6.
PPAR Res ; 2016: 4049373, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27382365

RESUMO

Rotavirus infection has been reported to induce an inflammatory response in the host cell accompanied by the increased expression or activation of some cellular molecules including ROS, NF-κB, and COX-2. PPARγ stimulation and N-acetylcysteine (NAC) treatment have been found to interfere with viral infections including rotavirus infection. Small intestinal villi isolated from in vivo infected mice with rotavirus ECwt were analyzed for the percentage of ECwt-infected cells, the presence of rotavirus antigens, and infectious virion yield following treatment with pioglitazone. Isolated villi were also infected in vitro and treated with PPARγ agonists (PGZ, TZD, RGZ, DHA, and ALA), all-trans retinoic acid (ATRA), and NAC. After treatments, the expression of cellular proteins including PPARγ, NF-κB, PDI, Hsc70, and COX-2 was analyzed using immunochemistry, ELISA, immunofluorescence, and Western blotting. The results showed that rotavirus infection led to an increased accumulation of the cellular proteins studied and ROS. The virus infection-induced accumulation of the cellular proteins studied and ROS was reduced upon pioglitazone treatment, causing also a concomitant reduction of the infectious virion yield. We hypothesized that rotavirus infection is benefiting from the induction of a host cell proinflammatory response and that the interference of the inflammatory pathways involved leads to decreased infection.

7.
World J Virol ; 5(2): 38-62, 2016 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-27175349

RESUMO

Rotaviruses are the single leading cause of life-threatening diarrhea affecting children under 5 years of age. Rotavirus entry into the host cell seems to occur by sequential interactions between virion proteins and various cell surface molecules. The entry mechanisms seem to involve the contribution of cellular molecules having binding, chaperoning and oxido-reducing activities. It appears to be that the receptor usage and tropism of rotaviruses is determined by the species, cell line and rotavirus strain. Rotaviruses have evolved functions which can antagonize the host innate immune response, whereas are able to induce endoplasmic reticulum (ER) stress, oxidative stress and inflammatory signaling. A networking between ER stress, inflammation and oxidative stress is suggested, in which release of calcium from the ER increases the generation of mitochondrial reactive oxygen species (ROS) leading to toxic accumulation of ROS within ER and mitochondria. Sustained ER stress potentially stimulates inflammatory response through unfolded protein response pathways. However, the detailed characterization of the molecular mechanisms underpinning these rotavirus-induced stressful conditions is still lacking. The signaling events triggered by host recognition of virus-associated molecular patterns offers an opportunity for the development of novel therapeutic strategies aimed at interfering with rotavirus infection. The use of N-acetylcysteine, non-steroidal anti-inflammatory drugs and PPARγ agonists to inhibit rotavirus infection opens a new way for treating the rotavirus-induced diarrhea and complementing vaccines.

8.
PLoS One ; 11(2): e0147666, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26828934

RESUMO

A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death.


Assuntos
Adaptação Fisiológica , Rotavirus/fisiologia , Antígenos Virais/imunologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular , Efeito Citopatogênico Viral , Quebras de DNA de Cadeia Dupla , Fragmentação do DNA , Reparo do DNA , Eletroforese em Gel de Ágar , Histonas/metabolismo , Humanos , Rotavirus/imunologia , Rotavirus/isolamento & purificação , Rotavirus/patogenicidade , Infecções por Rotavirus/virologia , Fatores de Tempo , Vírion/patogenicidade
9.
Rev. colomb. biotecnol ; 18(1): 33-48, ene.-jun. 2016. ilus
Artigo em Inglês | LILACS-Express | ID: lil-791230

RESUMO

Introduction. Rotavirus entry into cells seems to be mediated by sequential interactions between viral structural proteins and some cell surface molecules. However, the mechanisms by which rotavirus infects target cell are still not well understood. There is some evidence showing that rotavirus structural proteins VP5* and VP8* interact with some cell surface molecules. The availability of recombinant rotavirus structural proteins in sufficient quantity has become very important for the identification of the specific virus-cell receptor interactions during the early events of the infectious process. Objective. The aim of the present work is to perform an analysis of the interactions between recombinant rotavirus structural proteins VP5*, VP8* and VP6, and cellular proteins Hsc70 and PDI using their purified recombinant versions. Materials and methods. Rotavirus recombinant VP5* and VP8*, and cellular recombinant proteins Hsc70 and PDI were expressed in E. coli BL21(DE3) while VP6 was expressed in recombinant vaccinia virus-transfected MA104 cells. The interaction between rotavirus and cellular proteins was studied using ELISA, co-immunoprecipitation and SDS-PAGE/Western blotting analysis. Results. The optimal conditions for expression of recombinant proteins were determined and antibodies were raised against them. The findings suggested that viral proteins rVP5* and rVP6 interact with Hsc70 and PDI in vitro. These viral recombinant proteins were also found to interact with raft-associated Hsc70 in a cell culture system. The treatment of cells with either rVP6 or DLPs produced significantly inhibition of rotavirus infection. Conclusion. The results allow us to conclude that rVP5* and rVP6 interact with Hsc70 and PDI during the rotavirus infection process.


Introducción. La entrada de rotavirus a las células parece estar mediado por interacciones secuenciales entre las proteínas estructurales virales y algunas moléculas de la superficie celular. Sin embargo, los mecanismos por los cuales el rotavirus infecta la célula diana aún no se comprenden bien. Existe alguna evidencia que muestra que las proteínas estructurales de rotavirus VP5* y VP8* interactúan con algunas moléculas de la superficie celular. La disponibilidad de las proteínas estructurales de rotavirus recombinantes en cantidad suficiente se ha convertido en un aspecto importante para la identificación de las interacciones específicas de los receptores virus-célula durante los eventos tempranos del proceso infeccioso. Objetivo. El propósito del presente trabajo es realizar un análisis de las interacciones entre las proteínas estructurales de rotavirus recombinante VP5*, VP8* y VP6, y las proteínas celulares Hsc70 y PDI utilizando sus versiones recombinantes purificadas. Materiales y métodos. Las proteínas recombinantes de rotavirus VP5* y VP8* y las proteínas recombinantes celulares Hsc70 y PDI se expresaron en E. coli BL21 (DE3), mientras que VP6 se expresó en células MA104 con virus vaccinia recombinante transfectada. La interacción entre el rotavirus y las proteínas celulares se estudió mediante ELISA, co-inmunoprecipitación y SDS-PAGE/ Western. Resultados. Las condiciones óptimas para la expresión de proteínas recombinantes se determinaron y se generaron anticuerpos contra ellas. Los resultados sugirieron que las proteínas virales rVP5* y rVP6 interactúan con Hsc70 y PDI in vitro. También se encontró que éstas proteínas virales recombinantes interactúan con Hsc70 en las balsas lipídicas ("Rafts") en un cultivo celular. El tratamiento de las células, ya sea con DLP o rVP6 produjo significativamente la inhibición de la infección por rotavirus. Conclusión. Los resultados permiten concluir que rVP5 * y rVP6 interactúan con Hsc70 y PDI durante el proceso de la infección por rotavirus.

10.
J Org Chem ; 80(4): 2397-406, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-25648434

RESUMO

The mulinane class of diterpenoids is a set of tricyclic (5-6-7), biologically active natural products whose members exhibit a variety of oxidation states. Herein, we report the inaugural synthesis of four mulinanes employing a divergent approach that relies on a diastereoselective anionic oxy-Cope rearrangement to set the relative configuration of the C8 stereocenter and an unprecedented vinylogous Saegusa dehydrogenation reaction to address C-ring functionality.


Assuntos
Diterpenos/síntese química , Diterpenos/química , Conformação Molecular , Estereoisomerismo
11.
Pharmacotherapy ; 34(11): e333-40, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25251886

RESUMO

Rotaviruses are the leading cause of severe, acute, and dehydrating diarrhea affecting children under 5 years of age worldwide. Despite an important reduction in rotavirus-caused deaths as a consequence of the rotavirus vaccine, alternative or complementary strategies for preventing or treating rotavirus-associated diarrhea are needed mainly in the poorest countries. We describe the cases of four rotavirus-unvaccinated 12-13-month-old girls and a 5-year-old boy who developed rotavirus-associated diarrhea confirmed by enzyme-linked immunosorbent assay, Western blotting, and immunochemistry analyses. After the first day of diarrheal episodes, three of the five patients were immediately administered oral N-acetylcysteine (NAC) 60 mg/kg daily, divided into three equal doses every 8 hours. The other two patients did not receive NAC and served as controls. Administration of NAC resulted in a decreased number of diarrheal episodes, excretion of fecal rotavirus antigen, and resolution of symptoms after 2 days of treatment. Our results suggest that NAC treatment after the first diarrheal episode could be an efficient strategy for treating rotavirus-affected children and preventing the associated severe life-threatening accompanying dehydration.


Assuntos
Acetilcisteína/uso terapêutico , Antivirais/uso terapêutico , Diarreia Infantil/prevenção & controle , Diarreia/prevenção & controle , Gastroenterite/tratamento farmacológico , Infecções por Rotavirus/tratamento farmacológico , Acetilcisteína/administração & dosagem , Administração Oral , Antivirais/administração & dosagem , Pré-Escolar , Colômbia , Desidratação/etiologia , Desidratação/prevenção & controle , Diarreia/etiologia , Diarreia/fisiopatologia , Diarreia Infantil/etiologia , Diarreia Infantil/fisiopatologia , Feminino , Gastroenterite/fisiopatologia , Gastroenterite/virologia , Humanos , Lactente , Masculino , Recidiva , Infecções por Rotavirus/fisiopatologia , Infecções por Rotavirus/virologia , Índice de Gravidade de Doença , Resultado do Tratamento
12.
J Bacteriol ; 196(9): 1683-93, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24532776

RESUMO

In microbiology, gene disruption and subsequent experiments often center on phenotypic changes caused by one class of specialized metabolites (quorum sensors, virulence factors, or natural products), disregarding global downstream metabolic effects. With the recent development of mass spectrometry-based methods and technologies for microbial metabolomics investigations, it is now possible to visualize global production of diverse classes of microbial specialized metabolites simultaneously. Using imaging mass spectrometry (IMS) applied to the analysis of microbiology experiments, we can observe the effects of mutations, knockouts, insertions, and complementation on the interactive metabolome. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the impact on specialized metabolite production of a transposon insertion into a Pseudomonas aeruginosa phenazine biosynthetic gene, phzF2. The disruption of phenazine biosynthesis led to broad changes in specialized metabolite production, including loss of pyoverdine production. This shift in specialized metabolite production significantly alters the metabolic outcome of an interaction with Aspergillus fumigatus by influencing triacetylfusarinine production.


Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Mutagênese Insercional , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/metabolismo , Cromatografia Líquida , Fenazinas/metabolismo , Pseudomonas aeruginosa/química , Espectrometria de Massas em Tandem
13.
J Am Chem Soc ; 135(33): 12188-91, 2013 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-23855814

RESUMO

Herein, we describe a two-step method for the cyclopentannulation of conjugated enones using methyl 3-(tert-butyldimethylsilyloxy)-2-diazo-3-butenoate (1) as a bifunctional reagent. The enol silane and stabilized diazoalkane functionalities are exploited independently in sequential Mukaiyama-Michael and diastereoselective α,α'-diketone coupling. Di-, tri-, and tetrasubstituted enones are amenable to annulation under this protocol. Overall, this chemistry is an effective surrogate for a substituted "acetone 1,3-dipole".

14.
Arch Virol ; 158(6): 1323-36, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23404461

RESUMO

In the present study, a homologous rotavirus, ECwt, infecting small intestinal villi isolated from ICR and BALB/c mice were used as a model for identifying cell-surface molecules involved in rotavirus entry. Small-intestinal villi were treated with anti-Hsc70, anti-PDI, anti-integrin ß3 or anti-ERp57 antibodies or their corresponding F(ab')2 fragments before inoculation with rotavirus ECwt, RRV or Wa. Pretreatment of villi decreased virus infectivity by about 50-100 % depending of the rotavirus strain, antibody structure and detection assay used. Similar results were obtained by treating viral inocula with purified proteins Hsc70, PDI or integrin ß3 before inoculation of untreated villi. Rotavirus infection of villi proved to be sensitive to membrane-impermeant thiol/disulfide inhibitors such as DTNB and bacitracin, suggesting the involvement of a redox reaction in infection. The present results suggest that PDI, Hsc70 and integrin ß3 are used by both homologous and heterologous rotaviruses during infection of isolated mouse villi.


Assuntos
Proteínas de Choque Térmico HSC70/fisiologia , Integrina alfaVbeta3/fisiologia , Intestino Delgado/virologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Infecções por Rotavirus/virologia , Rotavirus/fisiologia , Internalização do Vírus , Animais , Animais Lactentes/virologia , Anticorpos/imunologia , Sobrevivência Celular , Feminino , Proteínas de Choque Térmico HSC70/imunologia , Proteínas de Choque Térmico HSC70/metabolismo , Integrina alfaVbeta3/imunologia , Integrina alfaVbeta3/metabolismo , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Isomerases de Dissulfetos de Proteínas/metabolismo , Rotavirus/isolamento & purificação , Infecções por Rotavirus/metabolismo
15.
Antiviral Res ; 96(1): 1-12, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22842004

RESUMO

Although the current rotavirus vaccines have shown good tolerance and significant efficacy, it would be useful to develop alternative or complementary strategies aimed at preventing or treating acute diarrhoeal disease caused by this viral agent. A variety of antiviral strategies other than vaccines have been assayed for rotavirus infection management. The recently demonstrated sensitivity of rotavirus infectivity to thiol/disulfide reagents prompted assays for screening drugs that potentially affect cellular redox reactions. MA104 or Caco-2 cells were inoculated with the rotavirus strains RRV, Wa, Wi or M69 and then incubated with different concentrations of drugs belonging to a selected group of 60 drugs that are currently used in humans for purposes other than rotavirus infection treatment. Eighteen of these drugs were able to inhibit rotavirus infectivity to different extents. A more systematic evaluation was performed with drugs that could be used in children such as N-acetylcysteine and ascorbic acid, in addition to ibuprofen, pioglitazone and rosiglitazone, all of which affecting cellular pathways potentially needed by the rotavirus infection process. Evidence is provided here that rotavirus infectivity is significantly inhibited by NAC in different cell-culture systems. These findings suggest that NAC has the potential to be used as a therapeutic tool for treatment and prevention of rotavirus disease in children.


Assuntos
Acetilcisteína/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , PPAR gama/antagonistas & inibidores , Rotavirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Haplorrinos , Testes de Sensibilidade Microbiana , Rotavirus/fisiologia
16.
Intervirology ; 55(6): 451-64, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22398681

RESUMO

OBJECTIVES: Determining the effect of membrane-impermeant thiol/disulfide exchange inhibitors on rhesus rotavirus infectivity in MA104 cells and investigating protein disulfide isomerase (PDI) as a potential target for these inhibitors. METHODS: Cells were treated with DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], bacitracin or anti-PDI antibodies and then infected with virus. Triple-layered particles (TLPs) were also pretreated with inhibitors before inoculation. The effects of these inhibitors on α-sarcin co-entry, virus binding to cells and PDI-TLP interaction were also examined. FACS analysis, cell-surface protein biotin-labeling, lipid-raft isolation and ELISA were performed to determine cell-surface PDI expression. RESULTS: Infectivity became reduced by 50% when cells or TLPs were treated with 1 or 6 mM DTNB, respectively; infectivity became reduced by 50% by 20 mM bacitracin treatment of cells whereas TLPs were insensitive to bacitracin treatment; anti-PDI antibodies decreased viral infectivity by about 45%. The presence of DTNB (2.5 mM) or bacitracin (20 mM) was unable to prevent virus binding to cells and rotavirus-induced α-sarcin co-entry. CONCLUSIONS: It was concluded that thiol/disulfide exchange was involved in rotavirus entry process and that cell-surface PDI was at least a potential target for DTNB and bacitracin-induced infectivity inhibition.


Assuntos
Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Infecções por Rotavirus/tratamento farmacológico , Rotavirus/efeitos dos fármacos , Rotavirus/fisiologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Anticorpos/imunologia , Bacitracina/farmacologia , Linhagem Celular , Dissulfetos/metabolismo , Ácido Ditionitrobenzoico/farmacologia , Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Macaca mulatta , Isomerases de Dissulfetos de Proteínas/imunologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Infecções por Rotavirus/metabolismo , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/farmacologia
17.
Biomedica ; 31(1): 70-81, 2011 Mar.
Artigo em Espanhol | MEDLINE | ID: mdl-22159485

RESUMO

INTRODUCTION: Rotavirus entry process involves a multi-step mechanism, the first of which is when the outermost viral proteins interact with four different integrins and Hsc70. Recently, rotavirus infection reportedly has been decreased after blocking cell surface protein disulfide isomerase (PDI). This suggested that this protein interacts with rotavirus during the entry process. OBJECTIVES: The aim was to establish the rotavirus-PDI interaction in an in vitro system using PDI isolated from bovine liver, and in a cell system consisting of MA104 cells and mouse small intestinal villi. MATERIALS AND METHODS: Protein disulfide isomerase was isolated from a bovine liver homogenate using anti-PDI antibodies coupled to agarose through hydrazone bonds. Purity of purified protein was assessed by SDS-PAGE and Western blot. The purified PDI was used to study its in vitro interaction with the rotavirus particles. This interaction was compared with that taking place in MA104 cells and small intestinal villi isolated from sucking mice ICR. RESULTS: The purified PDI showed an electrophoretic homogeneity and was able to bind rotavirus particles in vitro. Rotavirus-PDI interaction was detected by capture ELISA using purified protein and rotavirus strains RRV and wild-type ECwt. Interaction between rotavirus particles and cellular PDI was detected by ELISA using cell lysates after virus inoculation. CONCLUSIONS: Rotavirus-PDI interaction was demonstrated in vitro as well as inMA104 cells and intestinal villi from suckling mice.


Assuntos
Isomerases de Dissulfetos de Proteínas/metabolismo , Rotavirus/metabolismo , Animais , Animais Lactentes , Anticorpos/metabolismo , Bovinos , Linhagem Celular , Proteínas de Choque Térmico HSC70/metabolismo , Integrinas/metabolismo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/virologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Camundongos , Infecções por Rotavirus/virologia , Proteínas Virais/metabolismo
18.
Org Lett ; 13(19): 5164-7, 2011 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-21894952

RESUMO

A concise, stereocontrolled synthesis of the citrinadin B core architecture from scalemic, readily available starting materials is disclosed. Highlights include ready access to both cyclic tryptophan tautomer and trans-2,6-disubstituted piperidine fragments, an efficient, stereoretentive mixed Claisen acylation for the coupling of these halves, and further diastereoselective carbonyl addition and oxidative rearrangement for assembly of the core.


Assuntos
Alcaloides Indólicos/síntese química , Ésteres/química , Estrutura Molecular , Estereoisomerismo
19.
J Am Chem Soc ; 133(20): 8014-27, 2011 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-21539314

RESUMO

Full details are provided for an improved synthesis of cortistatin A and related structures as well as the underlying logic and evolution of strategy. The highly functionalized cortistatin A-ring embedded with a key heteroadamantane was synthesized by a simple and scalable five-step sequence. A chemoselective, tandem geminal dihalogenation of an unactivated methyl group, a reductive fragmentation/trapping/elimination of a bromocyclopropane, and a facile chemoselective etherification reaction afforded the cortistatin A core, dubbed "cortistatinone". A selective Δ(16)-alkene reduction with Raney Ni provided cortistatin A. With this scalable and practical route, copious quantities of cortistatinone, Δ(16)-cortistatin A (the equipotent direct precursor to cortistatin A), and its related analogues were prepared for further biological studies.


Assuntos
Compostos Policíclicos/síntese química , Células Cultivadas , Humanos , Compostos Policíclicos/química
20.
Biomédica (Bogotá) ; 31(1): 70-81, mar. 2011. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-617506

RESUMO

Introducción. La entrada del rotavirus a la célula implica un mecanismo de múltiples pasos; las proteínas virales externas interaccionan con cuatro diferentes integrinas y Hsc70. Recientemente reportamos que la infección por rotavirus disminuye cuando se bloquea la proteína disulfuro-isomerasa de la superficie celular, lo que sugiere su interacción con el rotavirus en el proceso de entrada. Objetivo. Establecer la interacción del rotavirus con la proteína disulfuro-isomerasa en un sistema in vitro utilizando la proteína aislada de hígado bovino y, en un sistema celular, utilizando vellosidades intestinales de ratón y células MA104. Materiales y métodos. Se aisló la proteína disulfuro-isomerasa a partir de un homogenizado de hígado bovino utilizando anticuerpos anti-proteína disulfuro-isomerasa acoplados a agarosa mediante enlace hidrazona. La proteína disulfuro-isomerasa purificada se examinó por SDS-PAGE y Western blot y se utilizó para estudiar su interacción in vitro con rotavirus. Esta interacción se comparó con aquella observada en células MA104 y en las vellosidades intestinales de ratón. Resultados. La proteína disulfuro-isomerasa purificada mostró homogeneidad electroforética y fue capaz de unirse a rotavirus en un sistema in vitro. La interacción proteína-rotavirus fue detectada por ELISA de captura usando la proteína disulfuro-isomerasa bovina purificada y rotavirus de las cepas RRV y silvestre ECwt. La interacción de partículas de rotavirus purificadas con la proteína disulfuro-isomerasa celular se evidenció con ELISA, usando lisado celular después de la inoculación viral. Conclusión. La interacción rotavirus-proteína disulfuro-isomerasa fue demostrada in vitro, en células MA104 y en vellosidades intestinales de ratón lactante.


Introduction. Rotavirus entry process involves a multi-step mechanism, the first of which is when the outermost viral proteins interact with four different integrins and Hsc70. Recently, rotavirus infection reportedly has been decreased after blocking cell surface protein disulfide isomerase (PDI). This suggested that this protein interacts with rotavirus during the entry process. Objectives. The aim was to establish the rotavirus-PDI interaction in an in vitro system using PDI isolated from bovine liver, and in a cell system consisting of MA104 cells and mouse small intestinal villi. Materials and methods. Protein disulfide isomerase was isolated from a bovine liver homogenate using anti-PDI antibodies coupled to agarose through hydrazone bonds. Purity of purified protein was assessed by SDS-PAGE and Western blot. The purified PDI was used to study its in vitro interaction with the rotavirus particles. This interaction was compared with that taking place in MA104 cells and small intestinal villi isolated from sucking mice ICR. Results. The purified PDI showed an electrophoretic homogeneity and was able to bind rotavirus particles in vitro. Rotavirus-PDI interaction was detected by capture ELISA using purified protein and rotavirus strains RRV and wild-type ECwt. Interaction between rotavirus particles and cellular PDI was detected by ELISA using cell lysates after virus inoculation. Conclusions. Rotavirus-PDI interaction was demonstrated in vitro as well as in MA104 cells and intestinal villi from suckling mice.


Assuntos
Intestino Delgado , Proteína Dissulfeto Redutase (Glutationa) , Receptores Virais , Rotavirus , Linhagem Celular , Cromatografia de Afinidade
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