RESUMEN
Enhanced signaling via RTKs in pulmonary hypertension (PH) impedes current treatment options because it perpetuates proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Here, we demonstrated hyperphosphorylation of multiple RTKs in diseased human vessels and increased activation of their common downstream effector phosphatidylinositol 3'-kinase (PI3K), which thus emerged as an attractive therapeutic target. Systematic characterization of class IA catalytic PI3K isoforms identified p110α as the key regulator of pathogenic signaling pathways and PASMC responses (proliferation, migration, survival) downstream of multiple RTKs. Smooth muscle cell-specific genetic ablation or pharmacological inhibition of p110α prevented onset and progression of pulmonary hypertension (PH) as well as right heart hypertrophy in vivo and even reversed established vascular remodeling and PH in various animal models. These effects were attributable to both inhibition of vascular proliferation and induction of apoptosis. Since this pathway is abundantly activated in human disease, p110α represents a central target in PH.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/fisiología , Hipertensión Pulmonar/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Adulto , Animales , Células Cultivadas , Humanos , Hipertensión Pulmonar/etiología , Lactante , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Interkingdom communication between bacteria and host organisms is one of the most interesting research topics in biology. Quorum sensing molecules produced by Gram-negative bacteria, such as acylated homoserine lactones and quinolones, have been shown to interact with host cell receptors, stimulating innate immunity and bacterial clearance. To our knowledge, there is no evidence that these molecules influence CNS function. Here, we have found that low micromolar concentrations of the Pseudomonas aeruginosa quorum sensing autoinducer, 2-heptyl-3-hydroxy-4-quinolone (PQS), inhibited polyphosphoinositide hydrolysis in mouse brain slices, whereas four selected acylated homoserine lactones were inactive. PQS also inhibited forskolin-stimulated cAMP formation in brain slices. We therefore focused on PQS in our study. Biochemical effects of PQS were not mediated by the bitter taste receptors, T2R4 and T2R16. Interestingly, submicromolar concentrations of PQS could be detected in the serum and brain tissue of adult mice under normal conditions. Levels increased in five selected brain regions after single i.p. injection of PQS (10 mg/kg), peaked after 15 min, and returned back to normal between 1 and 4 h. Systemically administered PQS reduced spontaneous locomotor activity, increased the immobility time in the forced swim test, and largely attenuated motor response to the psychostimulant, methamphetamine. These findings offer the first demonstration that a quorum sensing molecule specifically produced by Pseudomonas aeruginosa is centrally active and influences cell signaling and behavior. Quorum sensing autoinducers might represent new interkingdom signaling molecules between ecological communities of commensal, symbiotic, and pathogenic microorganisms and the host CNS.
Asunto(s)
Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , AMP Cíclico/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Pseudomonas aeruginosa/metabolismo , Quinolonas/farmacología , Percepción de Quorum , Transducción de Señal/efectos de los fármacos , Animales , Encéfalo/metabolismo , Interacciones Huésped-Patógeno , Hidrólisis , Técnicas In Vitro , Locomoción/efectos de los fármacos , Masculino , Ratones , Prueba del Laberinto Acuático de Morris/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Quinolonas/metabolismoRESUMEN
Prolyl 3-hydroxylation is a rare collagen type I post translational modification in fibrillar collagens. The primary 3Hyp substrate sites in type I collagen are targeted by an endoplasmic reticulum (ER) complex composed by cartilage associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and prolyl cis/trans isomerase B, whose mutations cause recessive forms of osteogenesis imperfecta with impaired levels of α1(I)3Hyp986. The absence of collagen type I 3Hyp in wild type zebrafish provides the unique opportunity to clarify the role of the complex in vertebrate. Zebrafish knock outs for crtap and p3h1 were generated by CRISPR/Cas9. Mutant fish have the typical OI patients' reduced size, body disproportion and altered mineralization. Vertebral body fusions, deformities and fractures are accompanied to reduced size, thickness and bone volume. Intracellularly, collagen type I is overmodified, and partially retained causing enlarged ER cisternae. In the extracellular matrix the abnormal collagen type I assembles in disorganized fibers characterized by altered diameter. The data support the defective chaperone role of the 3-hydroxylation complex as the primary cause of the skeletal phenotype.
Asunto(s)
Colágeno Tipo II/metabolismo , Colágeno Tipo I/metabolismo , Proteínas de la Matriz Extracelular/genética , Osteogénesis Imperfecta/genética , Prolil Hidroxilasas/genética , Animales , Sistemas CRISPR-Cas , Ciclofilinas/genética , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Hidroxilación , Osteogénesis Imperfecta/metabolismo , Fenotipo , Prolil Hidroxilasas/química , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Nutrients such as amino acids play key roles in shaping the metabolism of microorganisms in natural environments and in host-pathogen interactions. Beyond taking part to cellular metabolism and to protein synthesis, amino acids are also signaling molecules able to influence group behavior in microorganisms, such as biofilm formation. This lifestyle switch involves complex metabolic reprogramming controlled by local variation of the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP). The intracellular levels of this dinucleotide are finely tuned by the opposite activity of dedicated diguanylate cyclases (GGDEF signature) and phosphodiesterases (EAL and HD-GYP signatures), which are usually allosterically controlled by a plethora of environmental and metabolic clues. Among the genes putatively involved in controlling c-di-GMP levels in P. aeruginosa, we found that the multidomain transmembrane protein PA0575, bearing the tandem signature GGDEF-EAL, is an l-arginine sensor able to hydrolyse c-di-GMP. Here, we investigate the basis of arginine recognition by integrating bioinformatics, molecular biophysics and microbiology. Although the role of nutrients such as l-arginine in controlling the cellular fate in P. aeruginosa (including biofilm, pathogenicity and virulence) is already well established, we identified the first l-arginine sensor able to link environment sensing, c-di-GMP signaling and biofilm formation in this bacterium.
Asunto(s)
Arginina/metabolismo , Proteínas Bacterianas/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Pseudomonas aeruginosa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/química , Humanos , Hidrólisis , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/química , Liasas de Fósforo-Oxígeno/química , Unión Proteica , Dominios Proteicos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/química , Alineación de SecuenciaRESUMEN
Flucytosine (5-fluorocytosine, 5-FC) is a fluorinated analogue of cytosine currently approved for the systemic treatment of fungal infections, which has recently demonstrated a very promising antivirulence activity against the bacterial pathogen Pseudomonas aeruginosa. In this work, we propose novel inhalable hyaluronic acid (HA)/mannitol composite dry powders for repositioning 5-FC in the local treatment of lung infections, including those affecting cystic fibrosis (CF) patients. Different dry powders were produced in one-step by spray-drying. Powder composition and process conditions were selected after in depth formulation studies aimed at selecting the 5-FC/HA/mannitol formulation with convenient aerosolization properties and drug release profile in simulated lung fluids. The optimized 5-FC/HA/mannitol powder for inhalation (HyaMan_FC#3) was effectively delivered from different breath-activated dry powder inhalers (DPI) already available to CF patients. Nevertheless, the aerodynamic assessment of fine particles suggested that the developed formulation well fit with a low-resistance DPI. HyaMan_FC#3 inhibited the growth of the fungus Candida albicans and the production of the virulence factor pyoverdine by P. aeruginosa at 5-FC concentrations that did not affect the viability of both wild type (16HBE14o-) and CF (CFBE41o-) human bronchial epithelial cells. Finally, pharmacokinetics of HyaMan_FC#3 inhalation powder and 5-FC solution after intratracheal administration in rats were compared. In vivo results clearly demonstrated that, when formulated as dry powder, 5-FC levels in both bronchoalveolar lavage fluid and lung tissue were significantly higher and sustained over time as compared to those obtained with the 5-FC solution. Of note, when the same 5-FC amount was administered intravenously, no significant drug amount was found in the lung at each time point from the injection. To realize a 5-FC lung concentration similar to that obtained by using HyaMan_FC#3, a 6-fold higher dose of 5-FC should be administered intravenously. Taken together, our data demonstrate the feasibility to deliver 5-FC by the pulmonary route likely avoiding/reducing the well-known side effects associated to the high systemic 5-FC doses currently used in humans. Furthermore, our results highlight that an appropriate formulation design can improve the persistence of the drug at lungs, where microorganisms causing severe infections are located.
Asunto(s)
Antibacterianos/administración & dosificación , Antifúngicos/administración & dosificación , Reposicionamiento de Medicamentos , Inhaladores de Polvo Seco , Flucitosina/administración & dosificación , Ácido Hialurónico/química , Manitol/química , Administración por Inhalación , Aerosoles/química , Animales , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Antifúngicos/farmacocinética , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Flucitosina/farmacocinética , Flucitosina/farmacología , Humanos , Pulmón/microbiología , Enfermedades Pulmonares Fúngicas/tratamiento farmacológico , Masculino , Tamaño de la Partícula , Polvos , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Ratas WistarRESUMEN
AIMS: To develop new genetic tools for studying 3',5'-cyclic diguanylic acid (c-di-GMP) signalling in Pseudomonas aeruginosa. METHODS AND RESULTS: Plasmid pPcdrA::lux, carrying a transcriptional fusion between the c-di-GMP responsive promoter PcdrA and the luxCDABE reporter genes, has been generated and validated in purpose-built P. aeruginosa strains in which c-di-GMP levels can be increased or reduced upon arabinose-dependent induction of c-di-GMP synthetizing or degrading enzymes. CONCLUSIONS: The reporter systems described so far were able to detect a decrease in the c-di-GMP levels only in engineered strains overproducing c-di-GMP. Conversely, pPcdrA::lux could be used for studying any process or chemical compound expected to cause both an increase or a decrease with respect to the c-di-GMP levels produced by wild type P. aeruginosa. Another relevant aspect of this study has been the development of novel and improved genetic devices for the fine arabinose-dependent control of c-di-GMP levels in P. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic tools developed and validated in this study could facilitate investigations tackling the c-di-GMP signalling process on different fields, from cellular physiology to drug-discovery research.
Asunto(s)
GMP Cíclico/análogos & derivados , Técnicas Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transducción de Señal , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Plásmidos/genética , Plásmidos/metabolismo , Regiones Promotoras GenéticasRESUMEN
1. The aim of this study was to identify the most relevant welfare indicators for unloading, lairage, stunning, killing and post-mortem inspection in a poultry slaughter plant. Different indicators were unloading duration, lairage time, environmental variables in the lairage facilities, shackling time and electrical variables used in the water bath. 2. Lairage time did not correlate strongly with dead on arrival. Heat stress was limited by means of ventilation systems, correct cage placement and appropriate stocking density per crate. The acceptable shackling period was about 30 s. 3. The presence of a corneal reflex showed that an animal was alive, while spontaneous wing flapping, spontaneous eye blinking and response to a painful stimulus were regarded as indicators of stunning efficiency. 4. It was concluded that the presence of recent traumatic injuries during the post-mortem inspection could be a valid means to establish whether corrective measures concerning the handling, transport and loading procedures should be taken.
Asunto(s)
Mataderos , Bienestar del Animal , Pollos/fisiología , Animales , Calor , Italia , MasculinoRESUMEN
Photosynthetic activity in carbonate-rich benthic microbial mats located in saline, alkaline lakes on the Cariboo Plateau, B.C. resulted in pCO2 below equilibrium and δ(13) CDIC values up to +6.0 above predicted carbon dioxide (CO2 ) equilibrium values, representing a biosignature of photosynthesis. Mat-associated δ(13) Ccarb values ranged from ~4 to 8 within any individual lake, with observations of both enrichments (up to 3.8) and depletions (up to 11.6) relative to the concurrent dissolved inorganic carbon (DIC). Seasonal and annual variations in δ(13) C values reflected the balance between photosynthetic (13) C-enrichment and heterotrophic inputs of (13) C-depleted DIC. Mat microelectrode profiles identified oxic zones where δ(13) Ccarb was within 0.2 of surface DIC overlying anoxic zones associated with sulphate reduction where δ(13) Ccarb was depleted by up to 5 relative to surface DIC reflecting inputs of (13) C-depleted DIC. δ(13) C values of sulphate reducing bacteria biomarker phospholipid fatty acids (PLFA) were depleted relative to the bulk organic matter by ~4, consistent with heterotrophic synthesis, while the majority of PLFA had larger offsets consistent with autotrophy. Mean δ(13) Corg values ranged from -18.7 ± 0.1 to -25.3 ± 1.0 with mean Δ(13) Cinorg-org values ranging from 21.1 to 24.2, consistent with non-CO2 -limited photosynthesis, suggesting that Precambrian δ(13) Corg values of ~-26 do not necessitate higher atmospheric CO2 concentrations. Rather, it is likely that the high DIC and carbonate content of these systems provide a non-limiting carbon source allowing for expression of large photosynthetic offsets, in contrast to the smaller offsets observed in saline, organic-rich and hot spring microbial mats.
Asunto(s)
Biopelículas , Carbonatos/metabolismo , Cianobacterias/fisiología , Lagos/química , Lagos/microbiología , Biomarcadores/metabolismo , Colombia Británica , Isótopos de Carbono/metabolismo , Ácidos Grasos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Sedimentos Geológicos/química , Sedimentos Geológicos/microbiología , Fosfolípidos/metabolismo , Salinidad , Estaciones del AñoRESUMEN
Immunosuppressive drugs commonly used in the treatment of psoriatic arthritis make patients more susceptible to viral, bacterial, and fungal infections because of their mechanism of action. They not only increase the risk of new infections but also act altering the natural course of preexisting infections. While numerous data regarding the reactivation of tuberculosis infection are available in the literature, poor information about the risk of reactivation or exacerbation of hepatitis viruses B and C infections during treatment with biologics has been reported. Furthermore, reported series with biological therapy included short periods of followup, and therefore, they are not adequate to verify the risk of reactivation in the long-term treatment. Our study evaluated patients with a history of hepatitis B and psoriatic arthritis treated with adalimumab and monitored up to six years. During the observation period, treatment was effective and well tolerated in all patients, and liver function tests and viral load levels remained unchanged.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Artritis Psoriásica/tratamiento farmacológico , Anticuerpos contra la Hepatitis B/sangre , Anticuerpos contra la Hepatitis C/sangre , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adalimumab , Adulto , Anciano , Anticuerpos Monoclonales Humanizados/efectos adversos , Artritis Psoriásica/inmunología , Femenino , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/efectos adversos , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Tiempo , Carga Viral , Activación ViralRESUMEN
Primary aldosteronism (PA) is the most common endocrine form of hypertension and may carry an increased risk of atrial flutter or fibrillation (AFF). The primary goal of this multicentre cohort study is thus to prospectively establish the prevalence of PA in consecutive hypertensive patients referred for lone (non-valvular), paroxysmal or permanent AFF. Secondary objectives are to determine: (1) the predictors of AFF in patients with PA; (2) the rate of AFF recurrence at follow-up after specific treatment in the patients with PA; (3) the effect of AFF that can increase atrial natriuretic peptide via the atrial stretch and thereby blunt aldosterone secretion, on the aldosterone-to-renin ratio (ARR), and thus the case detection of PA; (4) the diagnostic accuracy of ARR based on plasma renin activity or on the measurement of active renin (DRA) for diagnosing PA in AFF patients. Case detection and subtyping of PA will be performed according to established criteria, including the 'four corners criteria' for diagnosing aldosterone-producing adenoma. Pharmacologic or direct current cardioversion will be undertaken whenever indicated following current guidelines. The hormonal values and ARR will be compared within patient between AFF and sinus rhythm. Organ damage, cardiovascular events and recurrence of AFF will also be assessed during follow-up in patients with PA.
Asunto(s)
Fibrilación Atrial/epidemiología , Aleteo Atrial/epidemiología , Hiperaldosteronismo/epidemiología , Proyectos de Investigación , Aldosterona/sangre , Antiarrítmicos/uso terapéutico , Fibrilación Atrial/diagnóstico , Fibrilación Atrial/terapia , Aleteo Atrial/diagnóstico , Aleteo Atrial/terapia , Biomarcadores/sangre , Distribución de Chi-Cuadrado , Cardioversión Eléctrica , Europa (Continente) , Humanos , Hiperaldosteronismo/sangre , Hiperaldosteronismo/diagnóstico , Hipertensión/diagnóstico , Hipertensión/epidemiología , Prevalencia , Estudios Prospectivos , Calidad de Vida , Recurrencia , Renina/sangre , Factores de Tiempo , Resultado del TratamientoRESUMEN
The success of molecular biology in identifying molecular pathways underlying chronic immune-mediated diseases and the rapid development of gene/cell engineering biotechnologies has resulted in the development of a number of targeted biological drugs, which have revolutionized the therapy of these diseases. Numerous data published over the last 10-15 years demonstrate a dramatic improvement in the clinical efficacy of biologics compared with conventional drugs. However, professional and public concern about serious biological drug-associated adverse events has also been growing steadily. We critically analyze recent literature on the efficacy and safety of biologics in the management of rheumatoid arthritis, psoriasis, psoriatic arthritis and immune thrombocytopenia. Our analysis of benefits, resistance to the therapy, risk of infections, tumors and other serious complications related to chronic administration of biologics is based on the molecular/cellular mechanisms of their interaction with the immune system. We also address whether it is feasible to attenuate the risks associated with biologics without limiting their benefits.
Asunto(s)
Productos Biológicos/uso terapéutico , Enfermedades del Sistema Inmune/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Productos Biológicos/efectos adversos , Productos Biológicos/inmunología , Ensayos Clínicos como Asunto , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Humanos , Enfermedades del Sistema Inmune/inmunología , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/inmunologíaRESUMEN
The increasing global incidence of diabetes and advancements in clinical pancreatic islet transplantation for the treatment of Type I diabetes have renewed the interest in understanding the variations of beta cell mass and function relative not only to transplant outcome but also to the onset and progression of diabetes. A deeper comprehension of the molecular and cellular processes involved in pancreatic islet inflammation and cytotoxicity is necessary to further improve efficacy of islet transplantation and to develop new therapies aimed at preserving beta cell function in pathological conditions. Available diagnostic methods based on metabolic response are unsuitable as they lack correlation to islet mass, viability and function. Great emphasis has been placed on developing noninvasive imaging technologies which enable the tracking of both endogenous and transplanted islet mass and potentially function overtime, the characterization of changes in islet vasculature and the degree of T-cell infiltration during insulitis. Among the more relevant modalities are magnetic resonance, positron emitted tomography, single photon emission computed tomography, bioluminescence and fluorescence optical imaging. This review focuses on the most recent advancements in magnetic resonance imaging (MRI) of pancreatic islets. In-vitro approaches aimed at characterizing the potency of isolated islets as well as in-vivo advancements in the assessment of transplanted beta cell mass are presented together with the significant progress made in the in-vivo imaging of the endocrine pancreas and islet vasculature and inflammation. Different experimental approaches are compared via their advantages and limitations with respect to their clinical implementation.
Asunto(s)
Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/patología , Animales , Humanos , Islotes Pancreáticos/fisiología , Imagen por Resonancia Magnética , Ratas , Ratas Endogámicas LewRESUMEN
BACKGROUND: Quality of life (QoL) in patients with vitiligo is strongly impaired. Therefore, it seems inadequate to describe the severity of the disease using only physical indicators. OBJECTIVES: To investigate the QoL of patients with vitiligo, identifying categories at risk for high impairment, also analysing single questions from a QoL instrument. METHODS: The Skindex-29 questionnaire, a QoL dermatology-specific instrument, was completed by 181 consecutive patients with vitiligo. Answers to the Skindex-29 items were given on a five-point scale, from 'never' to 'all the time'. Results The QoL problems more frequently experienced 'often' or 'all the time' were: worry of the disease getting worse (60%), anger (37%), embarrassment (34%), depression (31%), having social life affected (28%), and shame (28%). The prevalence of patients with probable depression or anxiety, evaluated using the 12-item General Health Questionnaire, was 39%, and the prevalence of patients with alexithymia, evaluated using the 20-item Toronto Alexithymia Scale, was 24%. The association of QoL impairment with psychological problems was very strong for all the items, and remained significant also when taking into account simultaneously gender, age, clinical severity, family history, and localization of vitiligo. CONCLUSIONS: Detailed information on QoL in patients with vitiligo may lead dermatologists to pay particular attention to patient categories at risk for a high QoL impairment.
Asunto(s)
Calidad de Vida , Vitíligo/psicología , Adolescente , Adulto , Factores de Edad , Actitud Frente a la Salud , Femenino , Humanos , Relaciones Interpersonales , Masculino , Psicometría , Factores de Riesgo , Índice de Severidad de la Enfermedad , Vitíligo/patología , Vitíligo/rehabilitaciónRESUMEN
This study investigates whether nanoporous micromachined biocapsules, with uniform membrane pore sizes of 24.5-nm, can be used to encapsulate insulin-secreting cells in vitro. This approach to cell encapsulation is based on microfabrication technology whereby immunoisolation membranes are bulk and surface micromachined to present uniform and well-controlled pore sizes as small as 10 nm, tailored surface chemistries, and precise microarchitectures. This study evaluates the behavior of insulinoma cells with micromachined membranes, the effect of matrix configurations within the biocapsule on cell behavior, as well as insulin and glucose transport through the biocapsule membranes.
Asunto(s)
Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Materiales Biocompatibles , Ingeniería Biomédica , Cápsulas , Difusión , Cámaras de Difusión de Cultivos , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Trasplante de Islotes Pancreáticos , Cinética , Ensayo de Materiales , Ratones , Tamaño de la Partícula , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
Indanocine is a potent tubulin-binding drug that is cytotoxic to multidrug-resistant cancer cell lines. We demonstrated that indanocine specifically induces apoptosis in malignant B cells from patients with chronic lymphocytic leukemia. To address the exact biochemical basis for indanocine toxicity, an indanocine-resistant clone was selected from mutagenized CEM human lymphoblastoid cells. The resistant cells displayed a stable indanocine-resistant phenotype for at least 9 months in drug-free culture. The cloned cells are cross-resistant to colchicine and vinblastine, but not to paclitaxel, and do not have increased expression of the multidrug-resistant p170 glycoprotein. In both parental cells and cell extracts, indanocine treatment caused tubulin depolymerization. In contrast, the tubulin in the resistant clone did not depolymerize under identical conditions. Both extract mixing and cell fusion experiments suggested that a stable structural change in microtubules, rather than a soluble factor, was responsible for indanocine resistance. Sequence analysis of parental and resistant cells revealed a single point mutation in the M40 isotype of beta-tubulin at nucleotide 1050 (G-->T, Lys(350)-->Asn) in the indanocine-resistant clone, in a region close to the putative colchicine binding site.
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Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos/genética , Indanos/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Fusión Celular , Sistema Libre de Células , Colchicina/farmacología , Resistencia a Antineoplásicos/genética , Humanos , Paclitaxel/farmacología , Mutación Puntual , Conformación Proteica , Isoformas de Proteínas , Electricidad Estática , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Vinblastina/farmacologíaRESUMEN
CD4(+) T-cell depletion is a characteristic of human immunodeficiency virus type 1 (HIV-1) infection. In this study, modulation of mRNA expression of 6800 genes was monitored simultaneously at eight time points in a CD4(+) T-cell line (CEM-GFP) during HIV infection. The responses to infection included: (1) >30% decrease at 72 h after infection in overall host-cell production of monitored mRNA synthesis, with the replacement of host-cell mRNA by viral mRNA, (2) suppression of the expression of selected mitochondrial and DNA repair gene transcripts, (3) increased expression of the proapoptotic gene and its gene p53-induced product Bax, and (4) activation of caspases 2, 3, and 9. The intense HIV-1 transcription resulted in the repression of much cellular RNA expression and was associated with the induction of apoptosis of infected cells but not bystander cells. This choreographed host gene response indicated that the subversion of the cell transcriptional machinery for the purpose of HIV-1 replication is akin to genotoxic stress and represents a major factor leading to HIV-induced apoptosis.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Regulación Viral de la Expresión Génica/inmunología , VIH-1/genética , Línea Celular Transformada , Fase G2/genética , Fase G2/inmunología , Proteínas Fluorescentes Verdes , VIH-1/metabolismo , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Recuento de Linfocitos , Mitosis/genética , Mitosis/inmunología , Transcripción Genética/inmunología , Virión/metabolismoRESUMEN
HIV-1 induces apoptosis and leads to CD4+ T-lymphocyte depletion in humans. It is still unclear whether HIV-1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV-1-induced apoptosis, we infected human CD4+ T cells with HIV-1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro-caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria was observed in HIV-infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. H IV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up-regulation of surface FasL expression, accompanied by a down-regulation of Fas-associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.
Asunto(s)
Apoptosis , VIH-1/fisiología , Mitocondrias/metabolismo , Mitocondrias/patología , Linfocitos T/patología , Linfocitos T/virología , Proteína p53 Supresora de Tumor/metabolismo , Factor Inductor de la Apoptosis , Caspasas/metabolismo , Células Cultivadas , Grupo Citocromo c/metabolismo , Activación Enzimática , Proteína Ligando Fas , Flavoproteínas/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Potenciales de la Membrana , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Modelos Biológicos , Permeabilidad , Fosforilación , Factores de TiempoRESUMEN
Group A rotavirus (RV) and coronavirus (CV) are common viral pathogens associated with neonatal diarrhoea in numerous animal species. The purpose of this work was to investigate the presence of these viral agents in two farm populations of captured guanacos (Lama guanicoe) in the Argentinean Patagonia region, that developed severe diarrhoea outbreaks. Stool and serum samples were analysed for RV and bovine CV antigen and antibody enzyme-linked immunosorbent assay. Rotavirus was detected in faeces from two new-born guanacos with acute diarrhoea, one in each farm. After electrophoretic analysis, each isolated strain, showed a distinctive long dsRNA electropherotype characteristic of group A rotaviruses (4:2:3:2). In addition, 95% (38 of 40) of the sampled animals were positive for RV antibodies, suggesting a high prevalence of RV infection in the populations tested. No evidence of CV circulation by antigen or antibody analysis was observed. To our knowledge, this is the first report of the detection and isolation of RV associated with neonatal diarrhoea in Lama guanicoe.
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Camélidos del Nuevo Mundo , Diarrea/veterinaria , Infecciones por Rotavirus/veterinaria , Rotavirus/aislamiento & purificación , Animales , Animales Recién Nacidos , Anticuerpos Antivirales/sangre , Argentina/epidemiología , Diarrea/epidemiología , Diarrea/virología , Heces/virología , Rotavirus/inmunología , Rotavirus/ultraestructura , Infecciones por Rotavirus/epidemiologíaRESUMEN
Thirty-four samples of roast and ground coffee, 14 samples of instant coffee and two samples of decaffeinated instant coffee were collected in markets and supermarkets in the city of Campinas, Brazil, and analysed for ochratoxin A using immunoaffinity columns for clean-up and HPLC with fluorescence detection for quantification. The limit of detection was 0.2 ng/g ochratoxin A. Twenty-three samples of ground and roast coffee were found to be contaminated with the toxin at levels ranging between 0.3 and 6.5 ng/g. The average concentration in all 34 samples was 0.9 ng/g. All samples of instant coffee contained ochratoxin A at levels ranging from 0.5 to 5.1 ng/g, with an average figure of 2.2 ng/g. Roast and ground coffee is the type of coffee most used by Brazilians for the preparation of the beverage. Considering that an average Brazilian adult takes five cups of coffee per day, which corresponds to 30 g of roast and ground coffee, the probable daily intake of ochratoxin A by a 70 kg adult would be 0.4 ng/kg bw, which is far below the current Provisional Tolerable Daily Intake of 14 ng/kg bw for ochratoxin A as set by the Codex Alimentarius. To study the transfer of ochratoxin A into coffee brew, the beverage was prepared by two methods: (a) the drip method and (b) the Brazilian country style method. No significant difference was observed between the two methods in terms of extraction of the toxin using five contaminated samples containing between 0.8 and 6.5 ng/g ochratoxin A. The drip method extracted 86 +/- 15% and the Brazilian country style 74 +/- 20% of the ochratoxin A initially present in the roast and ground coffee.
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Carcinógenos/análisis , Café/química , Contaminación de Alimentos/análisis , Micotoxinas/análisis , Ocratoxinas/análisis , Brasil , Cromatografía Líquida de Alta Presión , Manipulación de Alimentos/métodos , Calor , Concentración Máxima AdmisibleRESUMEN
Recent progress in molecular medicine has provided important tools to identify antigen-specific T cells. In most cases, the approach is based on oligomeric combinations of recombinant major histocompatibility complex-peptide complexes fixed to various rigid supports available for binding by the T-cell receptor. These tools have greatly increased our insight into mechanisms of immune responses mediated by CD8+ T cells. Examples of the diverse fields of application for this technology include immunization, viral infections and oral tolerance induction.