RESUMEN
Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in â¼10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.
Asunto(s)
Escherichia coli Enterohemorrágica , Infecciones por Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Pinocitosis , Toxina Shiga I/metabolismo , Transcitosis , Actinas/metabolismo , Línea Celular , Colon/metabolismo , Colon/microbiología , Infecciones por Escherichia coli/microbiología , Humanos , Mucosa Intestinal/microbiología , Miosina Tipo II/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Enterocitos/efectos de los fármacos , Infecciones por Escherichia coli/fisiopatología , Galectina 3/metabolismo , Proteínas de Microfilamentos/metabolismo , Toxina Shiga I/farmacología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Enterocitos/metabolismo , Escherichia coli Enterohemorrágica/fisiología , Humanos , Mucosa Intestinal/fisiopatología , Modelos Biológicos , Transporte de Proteínas , Conejos , Proteínas Recombinantes/genética , Toxina Shiga I/genética , Toxina Shiga I/metabolismoRESUMEN
We developed a genetic mouse model of open-angle glaucoma by expression of mutated mouse myocilin (Myoc) in transgenic (Tg) mice. The Tyr423His point mutation, corresponding to the severe glaucoma-causing Tyr437His mutation in the human MYOC gene, was introduced into bacterial artificial chromosome DNA encoding the full-length mouse Myoc gene and long flanking regions. Both wild-type (Wt) and Tg animals expressed Myoc in tissues of the irido-corneal angle and the sclera. Expression of mutated Myoc induced the accumulation of Myoc in cell cytoplasm and prevented its secretion into the extracellular space. The levels of ATPase-1 were reduced in the irido-corneal angle of Tg mice compared with Wt animals. Tg mice demonstrated a moderate elevation of intraocular pressure, the loss of approximately 20% of the retinal ganglion cells (RGCs) in the peripheral retina, and axonal degeneration in the optic nerve. RGC depletion was associated with the shrinkage of their nuclei and DNA fragmentation in the peripheral retina. Pathological changes observed in the eyes of Tg mice are similar to those observed in glaucoma patients.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Ojo/metabolismo , Predisposición Genética a la Enfermedad/genética , Glaucoma de Ángulo Abierto/genética , Glicoproteínas/genética , Mutación Puntual/genética , Adenosina Trifosfatasas/metabolismo , Animales , Córnea/metabolismo , Córnea/fisiopatología , Citoplasma/metabolismo , Fragmentación del ADN , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Ojo/fisiopatología , Glaucoma de Ángulo Abierto/metabolismo , Glaucoma de Ángulo Abierto/fisiopatología , Iris/metabolismo , Iris/fisiopatología , Ratones , Ratones Transgénicos , Nervio Óptico/patología , Nervio Óptico/fisiopatología , Degeneración Retiniana/etiología , Degeneración Retiniana/fisiopatología , Células Ganglionares de la Retina/patologíaRESUMEN
PURPOSE: The present study compared properties of wild-type and mutated mouse and human myocilin (Myoc) proteins as a prerequisite for development of a mouse model of glaucoma. METHODS: cDNA encoding full-length mouse Myoc was cloned into the p3XFLAG-CMV-14 vector. Tyr423His and Ile463Ser mutations were introduced into the mouse Myoc protein by in vitro mutagenesis. Intracellular localization and secretion of wild-type and mutated mouse Myoc proteins were studied in immunostaining and Western blotting experiments, respectively, after transfection into COS-7 cells. RESULTS: Similar to human MYOC, wild-type and mutated mouse Myoc demonstrated vesicular staining in transfected cells. However, while wild-type human and mouse Myoc were preferentially located in both the endoplasmic reticulum and Golgi, mutated human and mouse Myoc were located mainly in the endoplasmic reticulum and were excluded from Golgi. Similar to mutations in human MYOC, mutations in mouse Myoc dramatically reduced its secretion from transfected cells. Secretion of mutated Myoc was partially restored by culturing cells at 30 degrees C instead of 37 degrees C. The presence of mutated human MYOC prevented secretion of wild-type mouse Myoc but did not dramatically affect secretion of alkaline phosphatase, thrombospondin, Timp3 or olfactomedin-1. CONCLUSIONS: Properties of the mouse Myoc protein are similar to those of the human MYOC. The presence of mutated mouse or human Myoc does not block a general secretory pathway. Expression of mutated Myoc in the eye in mice may mimic human glaucoma and lead to development of a genetic mouse model of glaucoma.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Proteínas del Ojo/fisiología , Vectores Genéticos , Glicoproteínas/fisiología , Transfección , Secuencia de Aminoácidos , Animales , Western Blotting , Células COS/metabolismo , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Proteínas de la Matriz Extracelular/genética , Glicoproteínas/genética , Aparato de Golgi/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/fisiología , PlásmidosRESUMEN
PURPOSE: To characterize the gene expression pattern in the human trabecular meshwork (TM) and identify candidate genes for glaucoma by expressed sequence tag (EST) analysis as part of the NEIBank project. METHODS: RNA was extracted from dissected human TM and used to construct unamplified, un-normalized cDNA libraries in the pSPORT1 vector. More than 4000 clones were sequenced from the 5' end. Clones were clustered and identified using GRIST software. In addition, the expression patterns of genes encoding olfactomedin-domain proteins were analyzed by RT-PCR. RESULTS: After non-mRNA contaminants were removed, 3459 independent TM-expressed clones were obtained. These were grouped in 1888 clusters, potentially representing individual expressed genes. Transcripts for the myocilin gene, a locus for inherited glaucoma, formed the third most abundant cluster in the TM collection, and several other genes implicated in glaucoma (PITX2, CYP1B1, and optineurin) were also represented. One abundant TM transcript was from the gene for the angiopoietin-like factor CTD6, which is located at on the long arm of chromosome 1, area 36.2-36.1 in the region of the glaucoma locus GLC3B, whereas other transcripts were from genes close to known glaucoma loci. The TM collection contains cDNAs for genes that are preferentially expressed in the lymphatic endothelium (matrix Gla protein, apolipoprotein D precursor, and selenoprotein P precursor). In addition to EST profiling, RT- PCR was used to detect transcripts of the olfactomedin-domain proteins latrotoxin receptor Lec3 and optimedin in the TM. CONCLUSIONS: The TM libraries are a good source of molecular markers for TM and candidate genes for glaucoma. The abundance of myocilin cDNAs corresponds to the critical role of this gene in glaucoma and contrasts with libraries derived from cultured tissue. The expression profile raises the possibility that cells of the TM and Schlemm's canal may be more similar to lymphatic, rather than blood vascular endothelium.
Asunto(s)
Etiquetas de Secuencia Expresada , Proteínas del Ojo/genética , Perfilación de la Expresión Génica , Glaucoma/genética , Malla Trabecular/metabolismo , Anciano , Anciano de 80 o más Años , ADN Complementario/genética , Bases de Datos Genéticas , Expresión Génica , Biblioteca de Genes , Humanos , Persona de Mediana Edad , National Institutes of Health (U.S.) , Oftalmología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia , Lugares Marcados de Secuencia , Estados UnidosRESUMEN
The application of film-forming organic polymers, which are in common use in membrane technology, as chromatographic adsorbents for packed and capillary columns has been suggested. The chromatographic characteristics of poly[1-(trimethylsilyl)-1-propine] (PTMSP) as an adsorbent were studied. The film-forming properties of PTMSP simplify manufacturing of capillary and packed gas-solid columns. It was shown that separation of C1-C4 hydrocarbon gases on the columns with PTMSP is of practical interest. In the authors' opinion, PTMSP is also promising for the separation of inorganic gases.
Asunto(s)
Cromatografía de Gases/instrumentación , Compuestos de Trimetilsililo/química , Adsorción , Hidrocarburos/aislamiento & purificaciónRESUMEN
Gamma-tocotrienol (γ-T3) is a member of the vitamin E family. Tocotrienols (T3s) are powerful antioxidants and possess anticancer, neuroprotective and cholesterol-lowering properties. Tocotrienols inhibit the growth of various cancer cell lines without affecting normal cells. Less is known about the exact mechanisms of action of T3s on cell death and other growth inhibitory pathways. In the present study, we demonstrate that γ-T3 induces apoptosis in MDA-MB 231 and MCF-7 breast cancer cells as evident by PARP cleavage and caspase-7 activation. Gene expression analysis of MCF-7 cells treated with γ-T3 revealed alterations in the expression of multiple genes involved in cell growth and proliferation, cell death, cell cycle, cellular development, cellular movement and gene expression. Further analysis of differentially modulated genes using Ingenuity Pathway Analysis software suggested modulation of canonical signal transduction or metabolic pathways such as NRF-2-mediated oxidative stress response, TGF-ß signaling and endoplasmic reticulum (ER) stress response. Analysis of ER-stress-related proteins in MCF-7 and MDA-MB 231 cells treated with γ-T3 demonstrated activation of PERK and pIRE1α pathway to induce ER stress. Activating transcription factor 3 (ATF3) was identified as the most up-regulated gene (16.8-fold) in response to γ-T3. Activating transcription factor 3 knockdown using siRNA suggested an essential role of ATF3 in γ-T3-induced apoptosis. In summary, we demonstrate that γ-T3 modulates ER stress signaling and have identified ATF3 as a molecular target for γ-T3 in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Cromanos/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Vitamina E/análogos & derivados , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Caspasa 7/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal , Vitamina E/farmacologíaRESUMEN
Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.
Asunto(s)
Membrana Celular/metabolismo , Endocitosis , Células Epiteliales/metabolismo , Infecciones por Escherichia coli/metabolismo , Escherichia coli O157/metabolismo , Mucosa Intestinal/metabolismo , Pinocitosis , Toxina Shiga I/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/microbiología , Clorpromazina/farmacología , Clatrina/metabolismo , Modelos Animales de Enfermedad , Endocitosis/efectos de los fármacos , Activación Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/patogenicidad , Etilmaleimida/farmacología , Humanos , Intestinos/efectos de los fármacos , Intestinos/microbiología , Masculino , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Pinocitosis/efectos de los fármacos , Conejos , Proteínas Recombinantes/metabolismo , Toxina Shiga II/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Trihexosilceramidas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Oxidized phospholipids may appear in the pulmonary circulation as a result of acute lung injury or inflammation. We have previously described barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on human pulmonary endothelial cells (EC) mediated by small GTPases Rac and Cdc42. This work examined OxPAPC-induced focal adhesion (FA) and adherens junction (AJ) remodeling and potential interactions between FA and AJ protein complexes involved in OxPAPC-induced EC barrier enhancement. Immunofluorescence analysis, subcellular fractionation, and coimmunoprecipitation assays have shown that OxPAPC induced translocation and peripheral accumulation of FA complexes containing paxillin, focal adhesion kinase, vinculin, GIT1, and GIT2, increased association of AJ proteins vascular endothelial-cadherin, p120-catenin, alpha-, beta-, and gamma-catenins, and dramatically enhanced cell junction areas covered by AJ. Coimmunoprecipitation, pulldown assays, and confocal microscopy studies have demonstrated that OxPAPC promoted novel interactions between FA and AJ complexes via paxillin and beta-catenin association, which was critically dependent on Rac and Cdc42 activities and was abolished by pharmacological or small interfering RNA (siRNA)-mediated inhibition of Rac and Cdc42. Depletion of beta-catenin using the siRNA approach attenuated OxPAPC-induced paxillin translocation to the cell periphery, but also significantly decreased interaction of paxillin with AJ protein complex. In turn, paxillin knockdown by specific siRNA attenuated AJ enhancement in response to OxPAPC. These results show for the first time the novel interactions between FA and AJ protein complexes critical for EC barrier regulation by OxPAPC.
Asunto(s)
Barrera Alveolocapilar/efectos de los fármacos , Células Endoteliales/metabolismo , Paxillin/metabolismo , Fosfatidilcolinas/farmacología , beta Catenina/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Uniones Adherentes/efectos de los fármacos , Barrera Alveolocapilar/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Adhesiones Focales/efectos de los fármacos , Humanos , Modelos Biológicos , Paxillin/deficiencia , Paxillin/genética , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , beta Catenina/deficiencia , beta Catenina/genética , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteínas de Unión al GTP rac/antagonistas & inhibidoresRESUMEN
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibits potent barrier protective effects on pulmonary endothelium, which are mediated by small GTPases Rac and Cdc42. However, upstream mechanisms of OxPAPC-induced small GTPase activation are not known. We studied involvement of Rac/Cdc42-specific guanine nucleotide exchange factors (GEFs) Tiam1 and betaPIX in OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective responses in the human pulmonary endothelial cells (EC). OxPAPC induced membrane translocation of Tiam1, betaPIX, Cdc42, and Rac, but did not affect intracellular distribution of Rho and Rho-specific GEF p115-RhoGEF. Protein depletion of Tiam1 and betaPIX using siRNA approach abolished OxPAPC-induced activation of Rac and its effector PAK1. EC transfection with Tiam1-, betaPIX-, or PAK1-specific siRNA dramatically attenuated OxPAPC-induced barrier enhancement, peripheral actin cytoskeletal enhancement, and translocation of actin-binding proteins cortactin and Arp3. These results show for the first time that Tiam1 and betaPIX mediate OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective response in pulmonary endothelium.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Células Endoteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Fosfatidilcolinas/farmacología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Proteínas de Ciclo Celular/genética , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Impedancia Eléctrica , Células Endoteliales/citología , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Oxidación-Reducción , Proteínas Serina-Treonina Quinasas/metabolismo , Arteria Pulmonar/citología , ARN Interferente Pequeño , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Quinasas p21 ActivadasRESUMEN
The role of endogenous luteinizing hormone-releasing hormone (LHRH) in the development of concanavalin A (ConA)-induced proliferative responses was studied in rat fetuses. Preliminary treatment of fetuses in utero with either the LHRH receptor antagonist or anti-LHRH antibodies resulted in the suppression of ConA-induced proliferative responses of thymocytes. LHRH and LHRH-immunopositive cells, morphologically similar to thymocytes, were detected in intact fetal thymus. A significant content of LHRH was also found in the peripheral blood of fetuses. The LHRH content in thymus and plasma was similar in males and females. Surgical ablation of the hypothalamus resulted in 2-fold decreases in thymus and plasma levels of LHRH in 21-day-old fetuses compared to sham-operated fetuses. It was concluded that LHRH regulates mitogen-induced proliferative responses of thymocytes during prenatal ontogenesis in the rat. The main source of plasma LHRH at that period is the hypothalamus. Moreover, LHRH is synthesized in the fetal thymus. Thus, LHRH is suggested to have not only a central effect but also to be involved in autocrine or paracrine regulation of proliferative immune responses.