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1.
J Toxicol Sci ; 48(10): 547-556, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37778983

RESUMEN

Pulmonary fibrosis is a lethal and progressive pulmonary disorder in human beings. Ephedrine is a compound isolated from Ephedra and plays a regulatory role in inflammatory response. This study focused on the anti-pulmonary fibrosis effect of ephedrine and its potential molecular mechanism. After a mouse model of pulmonary fibrosis was established through bleomycin (BLM) induction, the survival percentage, body weight, and pulmonary index were measured. Hematoxylin-eosin staining and Masson's trichrome staining for lung tissues were performed to observe the pathological alterations. The viability of lung epithelial BEAS-2B cells, intracellular production of reactive oxygen species, and the levels of pro-inflammatory cytokines were examined by cell counting kit-8 assays, 2',7'-dichlorofluorescein diacetate (DCF-DA) staining, and enzyme-linked immunosorbent assay, respectively. Immunofluorescence staining was performed to determine E-cadherin and vimentin expression after BLM or ephedrine treatment. The mRNA and protein levels of cytokeratin-8, E-cadherin, α-SMA, and vimentin were subjected to quantitative polymerase chain reaction and immunoblotting. Experimental results revealed that ephedrine treatment rescued the repressive impact of BLM on BEAS-2B cell viability, and ephedrine inhibited BLM-induced overproduction of reactive oxygen species and inflammatory response in BEAS-2B cells. Additionally, ephedrine suppressed epithelial-mesenchymal transition (EMT) process stimulated by BLM treatment, as demonstrated by the reduced α-SMA and vimentin levels together with the increased cytokeratin-8 and E-cadherin levels in BLM + Ephedrine group. In addition, ephedrine inhibited NF-κB and activated Nrf-2 signaling in BLM-treated BEAS-2B cells. Moreover, ephedrine ameliorated pulmonary fibrosis in BLM-induced mice and improved the survival of model mice. In conclusion, ephedrine attenuates BLM-evoked pulmonary fibrosis by repressing EMT process via blocking NF-κB signaling and activating Nrf-2 signaling, suggesting that ephedrine might become a potential anti-pulmonary fibrosis agent in the future.


Asunto(s)
Fibrosis Pulmonar , Ratones , Humanos , Animales , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/tratamiento farmacológico , FN-kappa B/metabolismo , Bleomicina/toxicidad , Efedrina/uso terapéutico , Efedrina/toxicidad , Queratina-8/metabolismo , Vimentina/metabolismo , Vimentina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transición Epitelial-Mesenquimal , Pulmón/metabolismo , Cadherinas/toxicidad , Cadherinas/metabolismo
2.
Insect Biochem Mol Biol ; 59: 1-17, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25662100

RESUMEN

Although many insect cell lines derived from various tissues are available, it is unclear whether endogenous receptors of Bacillus thuringiensis (Bt) crystal toxins are expressed in these cell lines. In the present study, we demonstrated that the ovaries-derived Spodoptera litura Sl-HP cell line was susceptible to activated Cry1Ac although larvae of S. litura are not susceptible to the toxin. Assays of the transcriptome revealed that thirteen ATP-binding cassette transporter genes (ABC) were expressed at different levels in this cell line. Of these, the SlABCC3 shared 52-55% amino acid sequence identity with the known Bt toxin receptor ABCC2. RNAi-mediated knockdown targeting SlABCC3 significantly decreased the susceptibility of Sl-HP cells to activated Cry1Ac. Over-expression of the gene strongly increased the susceptibility of Trichoplusia ni Hi5 cells to the toxin. Not only was SlABCC3 comparable to the heterologously expressed Helicoverpa armigera Hacadherin on the receptor-mediated cytotoxicity of activated Cry1Ac to Hi5 cells, but also SlABCC3 and Hacadherin had a strong synergistic effect on cytotoxicity of activated Cry1Ac. These results suggested that Bt toxin receptors-expressing insect cell lines can be used as an alternative model for evaluating cytotoxicity of Bt toxins and studying their mechanisms of action.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Botulínicas/metabolismo , Cadherinas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insectos/metabolismo , Receptores de Superficie Celular/metabolismo , Spodoptera/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Cadherinas/toxicidad , Línea Celular , Sinergismo Farmacológico , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Proteínas de Insectos/genética , Larva/efectos de los fármacos , Larva/metabolismo , Interferencia de ARN , Receptores de Superficie Celular/genética , Spodoptera/citología , Spodoptera/efectos de los fármacos , Transcriptoma
3.
Pest Manag Sci ; 67(9): 1076-81, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21495115

RESUMEN

BACKGROUND: Biopesticides containing Cry insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are effective against many lepidopteran pests, but there is a lack of Bt-based pesticides for efficient control of important coleopteran pests. Based on the reported increase in Bt toxin oligomerization by a polypeptide from the Cry3Aa receptor cadherin in Tenebrio molitor (Coleoptera: Tenebrionidae), it was hypothesized that this cadherin peptide, rTmCad1p, would enhance Cry3Aa toxicity towards coleopteran larvae. To test this hypothesis, the relative toxicity of Cry3Aa, with or without rTmCad1p, against damaging chrysomelid vegetable pests of China was evaluated. RESULTS: Cry3Aa toxicity was evaluated in the spotted asparagus beetle (Crioceris quatuordecimpunctata), cabbage leaf beetle (Colaphellus bowringi) and daikon leaf beetle (Phaedon brassicae). To assess the effect of rTmCad1p on Cry3Aa toxicity, neonate larvae were fed Cry3Aa toxin alone or in combination with increasing amounts of rTmCad1p. The data demonstrated that Cry3Aa toxicity was significantly increased in all three vegetable pests, resulting in as much as a 15.3-fold increase in larval mortality. CONCLUSION: The application of rTmCad1p to enhance Cry3Aa insecticidal activity has potential for use in increasing range and activity levels against coleopteran pests displaying low susceptibility to Bt-based biopesticides.


Asunto(s)
Proteínas Bacterianas/toxicidad , Cadherinas/toxicidad , Escarabajos/efectos de los fármacos , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Proteínas de Insectos/toxicidad , Tenebrio/genética , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Escarabajos/crecimiento & desarrollo , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad
4.
Appl Environ Microbiol ; 75(22): 7280-2, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19801487

RESUMEN

A peptide from cadherin AgCad1 of Anopheles gambiae larvae was reported as a synergist of Bacillus thuringiensis subsp. israelensis Cry4Ba's toxicity to the Anopheles mosquito (G. Hua, R. Zhang, M. A. Abdullah, and M. J. Adang, Biochemistry 47:5101-5110, 2008). We report that CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) and a longer peptide, CR9 to CR11 (CR9-11), from AgCad1 act as synergists of Cry4Ba's toxicity to Aedes aegypti larvae, but a Diabrotica virgifera virgifera cadherin-based synergist of Cry3 (Y. Park, M. A. F. Abdullah, M. D. Taylor, K. Rahman, and M. J. Adang, Appl. Environ. Microbiol. 75:3086-3092, 2009) did not affect Cry4Ba's toxicity. Peptides CR9-11 and CR11-MPED bound Cry4Ba with high affinity (13 nM and 23 nM, respectively) and inhibited Cry4Ba binding to the larval A. aegypti brush border membrane. The longer CR9-11 fragment was more potent than CR11-MPED in enhancing Cry4Ba against A. aegypti.


Asunto(s)
Aedes/efectos de los fármacos , Anopheles , Bacillus thuringiensis/química , Proteínas Bacterianas/toxicidad , Cadherinas/toxicidad , Endotoxinas/toxicidad , Proteínas Hemolisinas/toxicidad , Insecticidas , Control de Mosquitos , Animales , Anopheles/química , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacología , Larva/efectos de los fármacos , Unión Proteica
5.
Carcinogenesis ; 30(10): 1781-8, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19654099

RESUMEN

Cadherins belong to a family of Ca(2+)-dependent homophilic cell-cell adhesion proteins that are important for correct cellular localization and tissue integrity. They play a major role in the development and homeostasis of epithelial architecture. Recently, it has become more and more evident that P-cadherin contributes to the oncogenesis of many tumors. To analyze the role of P-cadherin in oral squamous cell carcinoma (OSCC), we used a cell line that was deficient of the classical cadherins, P-cadherin, E-cadherin and N-cadherin. This cell line was transfected with full-length P-cadherin (PCI52_PC). After overexpression of P-cadherin, PCI52_PC gained an epithelial-like brickstone morphology in contrast to the mock-transfected cells with a spindle-shaped mesenchymal morphology. Immunohistochemical analysis revealed a strong nuclear Snail staining in mock-transfected cells compared with a significantly reduced nuclear staining and translocation to the cytoplasm in P-cadherin-overexpressing cells. Interestingly, the effects triggered by P-cadherin overexpression could be reversed by transfecting the cells with an antisense P-cadherin plasmid construct. Additional investigations showed a reexpression of E-cadherin in all P-cadherin-transfected cell clones in contrast to the mock controls. Analyzing the signaling mechanism behind it, we found glycogen-synthase-kinase-3beta (GSK-3beta) bound to Snail in all cell clones. Furthermore, P-cadherin-overexpressing cell lines showed activated GSK-3beta that phosphorylated Snail leading to its cytoplasmic translocation. In summary, our results reveal P-cadherin as one major component in reconfiguring mesenchymal cells with epithelial features by triggering GSK-3beta-mediated inactivation and cytoplasmatic translocation of Snail in OSCC.


Asunto(s)
Cadherinas/toxicidad , Carcinoma de Células Escamosas/inducido químicamente , Glucógeno Sintasa Quinasa 3/metabolismo , Neoplasias de la Boca/inducido químicamente , Factores de Transcripción/metabolismo , Cadherinas/deficiencia , Cadherinas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Amplificación de Genes , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Células HeLa , Humanos , Inmunohistoquímica , Cinética , Mesodermo/efectos de los fármacos , Mesodermo/fisiología , Neoplasias de la Boca/patología , Invasividad Neoplásica , Regiones Promotoras Genéticas , Factores de Transcripción de la Familia Snail , Transfección
6.
Biochemistry ; 47(18): 5101-10, 2008 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-18407662

RESUMEN

A midgut cadherin AgCad1 cDNA was cloned from Anopheles gambiae larvae and analyzed for its possible role as a receptor for the Cry4Ba toxin of Bacillus thuringiensis strain israelensis. The AgCad1 cadherin encodes a putative 1735-residue protein organized into an extracellular region of 11 cadherin repeats (CR) and a membrane-proximal extracellular domain (MPED). AgCad1 mRNA was detected in midgut of larvae by polymerase chain reaction (PCR). The AgCad1 protein was localized, by immunochemistry of sectioned larvae, predominately to the microvilli in posterior midgut. The localization of Cry4Ba binding was determined by the same technique, and toxin bound microvilli in posterior midgut. The AgCad1 protein was present in brush border membrane fractions prepared from larvae, and Cry4Ba toxin bound the same-sized protein on blots of those fractions. The AgCad1 protein was expressed transiently in Drosophila melanogaster Schneider 2 (S2) cells. 125I-Cry4Ba toxin bound AgCad1 from S2 cells in a competitive manner. Cry4Ba bound to beads extracted 200 kDa AgCad1 and a 29 kDa fragment of AgCad1 from S2 cells. A peptide containing the AgCad1 region proximal to the cell (CR11-MPED) was expressed in Escherichia coli. Although Cry4Ba showed limited binding to CR11-MPED, the peptide synergized the toxicity of Cry4Ba to larvae. AgCad1 in the larval brush border is a binding protein for Cry4Ba toxin. On the basis of binding results and CR11-MPED synergism of Cry4Ba toxicity, AgCad1 is probably a Cry4Ba receptor.


Asunto(s)
Anopheles/metabolismo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Cadherinas/metabolismo , Cadherinas/toxicidad , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Secuencia de Aminoácidos , Animales , Anopheles/efectos de los fármacos , Anopheles/genética , Toxinas de Bacillus thuringiensis , Cadherinas/genética , Cadherinas/aislamiento & purificación , Clonación Molecular , Sistema Digestivo/metabolismo , Drosophila melanogaster , Regulación de la Expresión Génica , Larva/efectos de los fármacos , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica
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