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1.
Immunity ; 54(11): 2650-2669.e14, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34592166

RESUMO

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.


Assuntos
COVID-19/imunologia , Interferon-alfa/imunologia , Células Matadoras Naturais/imunologia , SARS-CoV-2/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Bases , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Interferon-alfa/sangue , Fibrose Pulmonar/patologia , RNA-Seq , Índice de Gravidade de Doença , Transcriptoma/genética , Reino Unido , Estados Unidos
3.
Nat Commun ; 12(1): 1931, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33771993

RESUMO

The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1-7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/sangue , SARS-CoV-2/metabolismo , Saliva/virologia , COVID-19/epidemiologia , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Epidemias , Serviços de Assistência Domiciliar , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Curva ROC , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Manejo de Espécimes/métodos
4.
J Toxicol ; 2014: 291054, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25276124

RESUMO

High-throughput imaging-based hepatotoxicity studies capable of analyzing individual cells in situ hold enormous promise for drug safety testing but are frequently limited by a lack of sufficient metabolically competent human cells. This study examined cryopreserved HepaRG cells, a human liver cell line which differentiates into both hepatocytes and biliary epithelial cells, to determine if these cells may represent a suitable metabolically competent cellular model for novel High Content Analysis (HCA) applications. Characterization studies showed that these cells retain many features characteristic of primary human hepatocytes and display significant CYP3A4 and CYP1A2 induction, unlike the HepG2 cell line commonly utilized for HCA studies. Furthermore, this study demonstrates that CYP3A4 induction can be quantified via a simple image analysis-based method, using HepaRG cells as a model system. Additionally, data demonstrate that the hepatocyte and biliary epithelial subpopulations characteristic of HepaRG cultures can be separated during analysis simply on the basis of nuclear size measurements. Proof of concept studies with fluorescent cell function reagents indicated that further multiparametric image-based assessment is achievable with HepaRG. In summary, image-based screening of metabolically competent human hepatocyte models cells such as HepaRG offers novel approaches for hepatotoxicity assessment and improved drug screening tools.

5.
Neurotoxicology ; 42: 33-48, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24705302

RESUMO

The potential for adverse neurotoxic reactions in response to therapeutics and environmental hazards continues to prompt development of novel cell-based assays to determine neurotoxic risk. A challenge remains to characterize and understand differences between assays and between neuronal cellular models in their responses to neurotoxicants if scientists are to determine the optimal model, or combination of models, for neurotoxicity screening. Most studies to date have focused on developmental neurotoxicity applications. This study reports the development of a robust multiparameter High Content Analysis (HCA) assay for neurotoxicity screening in three differentiated neuronal cell models - SH-SY5Y, PC12 and human embryonic stem cell-derived hN2™ cells. Using a multiplexed detection reagent panel (Hoechst nuclear stain; antibodies against ßIII-Tubulin and phosphorylated neurofilament subunit H, and Mitotracker(®) Red CMXRos), a multiparametric HCA assay was developed and used to characterize a test set of 36 chemicals. HCA data generated were compared to data generated using MTT and LDH assays under the same assay conditions. Data showed that multiparametric High Content Analysis of differentiated neuronal cells is feasible, and represents a highly effective method for obtaining large quantities of robust data on the neurotoxic effects of compounds compared with cytotoxicity assays like MTT and LDH. Significant differences were observed between the responses to compounds across the three cellular models tested, illustrating the heterogeneity in responses to neurotoxicants across different cell types. This study provides data strongly supporting the use of cellular imaging as a tool for neurotoxicity assessment in differentiated neuronal cells, and provides novel insights into the neurotoxic effects of a test set of compounds upon differentiated neuronal cell lines and human embryonic stem cell-derived neurons.


Assuntos
Citotoxinas/toxicidade , Células-Tronco Embrionárias/citologia , Poluentes Ambientais/toxicidade , Ensaios de Triagem em Larga Escala/métodos , Células-Tronco Neurais/citologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neurônios/citologia , Células PC12 , Ratos
6.
Clin Cancer Res ; 17(9): 2734-43, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21364033

RESUMO

PURPOSE: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR). EXPERIMENTAL DESIGN: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors. RESULTS: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure. CONCLUSION: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile.


Assuntos
Ácidos Borônicos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Oligopeptídeos/efeitos adversos , Inibidores de Proteassoma , Pirazinas/efeitos adversos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Ácidos Borônicos/administração & dosagem , Bortezomib , Células Cultivadas , Cisteína Endopeptidases/administração & dosagem , Cisteína Endopeptidases/efeitos adversos , Sistemas de Liberação de Medicamentos , Células Hep G2 , Humanos , Masculino , Modelos Biológicos , Oligopeptídeos/administração & dosagem , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/administração & dosagem , Ratos , Ratos Sprague-Dawley
7.
J Vis Exp ; (27)2009 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-19417729

RESUMO

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with betaIII-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling betaIII-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.


Assuntos
Astrócitos/citologia , Técnicas de Cocultura/métodos , Neurônios/citologia , Testes de Toxicidade/métodos , Animais , Astrócitos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos
8.
Int Urol Nephrol ; 39(4): 1121-4, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17659448

RESUMO

INTRODUCTION: Transrectal ultrasound (TRUS) guided prostate biopsies are amongst the most common outpatient diagnostic procedures performed in urology practice. Of concern appear to be recent reports of infectious complications following this procedure in which contamination of the biopsy equipment was the likely source. This study looks at the rate of condom perforation during prostate biopsy and we look to highlight the potential problems, which may arise as a result of inadequate cleansing of the equipment between cases during a busy prostate biopsy clinic MATERIAL AND METHODS: All patients attending for prostate biopsies over a three-month period in our institution were included in the study. All condoms (latex) used were made by the same manufacturer and were checked prior to the procedure and found to have no leaks. The biopsy gun was inserted through an externally placed needle guide, as is standard practice in many departments in the UK. After the end of each procedure the condom was removed from the rectal probe and filled once again with water to assess for perforations. Two experienced surgeons carried out all the procedures. RESULTS: 10 out of 107 patients were found to have at least one perforation in the condom. In some of the condoms there were multiple perforations. DISCUSSION: We have demonstrated a significant condom perforation rate (9%) amongst patients undergoing prostate biopsies. This raises the serious issue of hygiene and cross infection, particularly with blood borne communicable diseases such as hepatitis and HIV unless strict disinfection and sterilization protocols are followed between patients. Perforation of the condoms used during TRUS guided prostate biopsy and hence faecal and blood contamination of the biopsy equipment could potentially have far-reaching implications for urologists and the infection control community. Although the risk of cross infection is probably small this serious issue needs addressing.


Assuntos
Biópsia/instrumentação , Preservativos , Infecção Hospitalar/etiologia , Endossonografia , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Infecção Hospitalar/prevenção & controle , Falha de Equipamento , Humanos , Masculino , Reto , Fatores de Risco
10.
Biochem Biophys Res Commun ; 355(2): 331-7, 2007 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-17300753

RESUMO

To examine the mechanism by which growth-stimulated pancreatic beta-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and betalox5, a cell line derived from human beta-cells which progressively dedifferentiated in culture. MIN6/betalox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that betalox5 expresses a dominant transacting factor(s) that represses beta-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary beta-cells as well as MIN6, but was repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in beta-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the beta-cell differentiated state.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Fusão Celular , Núcleo Celular/metabolismo , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Proteínas Repressoras/fisiologia , Animais , Linhagem Celular , Proteínas de Homeodomínio/metabolismo , Humanos , Células Híbridas , Camundongos , Reação em Cadeia da Polimerase , Transativadores/metabolismo
11.
Auton Neurosci ; 131(1-2): 1-8, 2007 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-16950660

RESUMO

Preganglionic neurons in the dorsal motor nucleus of the vagus (DMV) innervate most of the gastrointestinal tract; with the stomach and the cecum/proximal colon having a greater proportion of vagal input. Cecum-projecting neurons have been thought to be distinct from other preganglionic neurons due to their location within the DMV, but it is unknown whether these neurons innervate the cecum exclusively or what effect their activation has on cecal motor activity. Therefore, we investigated the extent of coinnervation of cecum and stomach by vagal neurons, their neurochemistry, and the effect of DMV stimulation on intracecal and intragastric volumes. Fluorescent retrograde tracers injected into the serosa of the cecum and stomach revealed that in the DMV 49+/-5% CTB-labeled cecum-projecting neurons also innervated the stomach. Immunocytochemical staining for nitric oxide (NO) synthase and tyrosine hydroxylase indicated that only 3+/-1% and 4+/-1% of cecum-projecting neurons contained these markers, respectively. In anesthetized rats gastric and cecal volumes were measured by prototypic miniaturized dual barostats that were developed for use in rodents. Microinjection of l-glutamate into the DMV increased gastric contractile activity and tone, and reduced on-going cecum contractile activity (2.6+/-0.7 contractions/2 min after injection versus 8.2+/-0.4 contractions/2 min before injection, N = 5). The barostat was able to detect decreases (-0.88+/-0.13 ml) and increases (0.25+/-0.05 ml) in cecum volume in response to carbachol and sodium nitroprusside, respectively. In summary, cecum-projecting neurons are not an entirely exclusive population within the DMV because a percentage of these also innervate the stomach. Central vagal stimulation can modulate both gastric and cecum contractile activity. Together, these data support a role of the vagus in neural reflexes involving gastric and large bowel motor function, such as the immediate phase of the gastrocolonic reflex.


Assuntos
Tronco Encefálico/citologia , Ceco/inervação , Neurônios Motores/fisiologia , Nervo Vago/fisiologia , Animais , Carbacol/farmacologia , Ceco/fisiologia , Contagem de Células/métodos , Toxina da Cólera/metabolismo , Agonistas Colinérgicos/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Motilidade Gastrointestinal/fisiologia , Ácido Glutâmico/farmacologia , Imuno-Histoquímica/métodos , Masculino , Microinjeções/métodos , Neurônios Motores/metabolismo , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Sprague-Dawley , Estômago/efeitos dos fármacos , Estômago/inervação , Estômago/fisiologia , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismo , Nervo Vago/citologia
12.
J Endocrinol ; 190(3): 889-96, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17003289

RESUMO

This study examined the effects of glucagon-like peptide-1 (GLP-1) on insulin secretion alone and in combination with sulphonylureas or nateglinide, with particular attention to K(ATP) channel-independent insulin secretion. In depolarised cells, GLP-1 significantly augmented glucose-induced K(ATP) channel-independent insulin secretion in a glucose concentration-dependent manner. GLP-1 similarly augmented the K(ATP) channel-independent insulin-releasing effects of tolbutamide, glibenclamide or nateglinide. Downregulation of protein kinase A (PKA)- or protein kinase C (PKC)-signalling pathways in culture revealed that the K(ATP) channel-independent effects of sulphonylureas or nateglinide were critically dependent upon intact PKA and PKC signalling. In contrast, GLP-1 exhibited a reduced but still significant insulin-releasing effect following PKA and PKC downregulation, indicating that GLP-1 can modulate K(ATP) channel-independent insulin secretion by protein kinase-dependent and -independent mechanisms. The synergistic insulin-releasing effects of combinatorial GLP-1 and sulphonylurea/nateglinide were lost following PKA- or PKC-desensitisation, despite GLP-1 retaining an insulin-releasing effect, demonstrating that GLP-1 can induce insulin release under conditions where sulphonylureas and nateglinide are no longer effective. Our results provide new insights into the mechanisms of action of GLP-1, and further highlight the promise of GLP-1 or similarly acting analogues alone or in combination with sulphonylureas or meglitinide drugs in type 2 diabetes therapy.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Canais de Potássio/metabolismo , Compostos de Sulfonilureia/farmacologia , Linhagem Celular , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Cicloexanos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Hipoglicemiantes/farmacologia , Insulina/análise , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Nateglinida , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Proteína Quinase C/antagonistas & inibidores , Estimulação Química , Acetato de Tetradecanoilforbol/farmacologia
13.
Nature ; 438(7069): 792-5, 2005 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-16319828

RESUMO

The surface of Saturn's largest satellite--Titan--is largely obscured by an optically thick atmospheric haze, and so its nature has been the subject of considerable speculation and discussion. The Huygens probe entered Titan's atmosphere on 14 January 2005 and descended to the surface using a parachute system. Here we report measurements made just above and on the surface of Titan by the Huygens Surface Science Package. Acoustic sounding over the last 90 m above the surface reveals a relatively smooth, but not completely flat, surface surrounding the landing site. Penetrometry and accelerometry measurements during the probe impact event reveal that the surface was neither hard (like solid ice) nor very compressible (like a blanket of fluffy aerosol); rather, the Huygens probe landed on a relatively soft solid surface whose properties are analogous to wet clay, lightly packed snow and wet or dry sand. The probe settled gradually by a few millimetres after landing.

14.
Acta Astronaut ; 56(3): 397-407, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15754476

RESUMO

We present a new European Mars mission proposal to build on the UK-led Beagle2 Mars mission and continue its astrobiology-focussed investigation of Mars. The small surface element to be delivered to the Martian surface--Vanguard--is designed to be carried by a Mars Express-type spacecraft bus to Mars and adopts a similar entry, descent and landing system as Beagle2. The surface element comprises a triad of robotic devices--a lander, a micro-rover of the Sojourner class for surface mobility, and three ground-penetrating moles mounted onto the rover for sub-surface penetration to 5 m depth. The major onboard instruments on the rover include a Raman spectrometer/imager, a laser plasma spectrometer, an infrared spectrometer--these laser instruments provide the basis for in situ "remote" sensing of the sub-surface Martian environment within a powerful scientific package. The moles carry the instruments' sensor head array to the sub-surface. The moles are thus required to undergo a one-way trip down the boreholes without the need for recovery of moles or samples, eliminating much of the robotic complexity invoked by such operations.


Assuntos
Geologia/instrumentação , Marte , Robótica , Voo Espacial/instrumentação , Astronave/instrumentação , Desenho de Equipamento , Exobiologia , Análise Espectral Raman
15.
Aging Cell ; 4(1): 21-30, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15659210

RESUMO

Fetal cardiomyocytes have been proposed as a potential source of cell-based therapy for heart failure. This study examined cellular senescence in cultured human fetal ventricular cardiomyocytes (HFCs). HFCs were isolated and identified by immunocytochemistry and RT-PCR. Cells were found to senesce after 20-25 population doublings, as determined by growth arrest, morphological changes and senescence-associated beta-galactosidase activity. Using the telomeric repeat amplification protocol assay, telomerase activity was undetectable in primary HFCs. Cells were transduced to express the human reverse transcriptase subunit (hTERT) of telomerase. This resulted in greatly increased telomerase activity, but no significant lifespan extension. Analysis of telomere length in primary HFCs revealed that the senescent phenotype was not accompanied by telomere shortening. Telomeres in hTERT-positive cells were elongated in comparison with primary cells, and elongation was retained in senescent cells. Levels of the tumor suppressor protein p16INK4A increased in all senescent cells whether telomerase-positive or -negative. Senescence was accompanied by a decline in transcript levels of the polycomb gene Bmi-1, Ets1 and Ets2 transcription factors, and Id1, Id2 and Id3 helix-loop-helix proteins, suggesting roles for these genes in maintenance of cardiomyocyte proliferative capacity. In addition to offering novel insights into the behavior of human fetal cardiomyocytes in culture, these findings have implications for the development of a cell-based therapy for cardiac injury using primary fetal heart tissue.


Assuntos
Senescência Celular/fisiologia , Miócitos Cardíacos/fisiologia , Telômero/fisiologia , Hipóxia Celular/fisiologia , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Feto/citologia , Expressão Gênica/genética , Humanos , Proteína 1 Inibidora de Diferenciação , Proteína 2 Inibidora de Diferenciação , Proteínas Inibidoras de Diferenciação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Proteína Proto-Oncogênica c-ets-1 , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Proteínas Repressoras/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , beta-Galactosidase/metabolismo
16.
Biochem Pharmacol ; 69(1): 59-63, 2005 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-15588714

RESUMO

Previous studies have shown that prolonged exposure to drugs, which act via blocking KATP channels, can desensitize the insulinotropic effects of drugs and nutrients acting via KATP channels. In this study, effects of prolonged exposure to diazoxide, a KATP channel opener, on beta cell function were examined using clonal BRIN-BD11 cells. The findings were compared to the long-term effects of KATP channel blockers nateglinide and tolbutamide. Following 18 h exposure to 200 microM diazoxide, the amounts of insulin secreted in response to glucose, amino acids and insulinotropic drugs were increased. Secretory responsiveness to a variety of agents acting via KATP channels was retained following prolonged diazoxide exposure. In contrast, 18 h exposure to 100 microM nateglinide significantly attenuated the insulin secretory responses to tolbutamide, nateglinide and BTS 67 582. Glucose- and L-alanine-stimulated insulin release were unaffected by prolonged nateglinide exposure, however responsiveness to L-leucine and L-arginine was diminished. Prolonged exposure to nateglinide had no effect on forskolin- and PMA-stimulated insulin release, and the overall pattern of desensitization was similar to that induced by 100 microM tolbutamide. We conclude that in contrast to chronic long-term KATP channel blockade, long-term diazoxide treatment is not harmful to KATP channel mediated insulin secretion and may have beneficial protective effects on beta cell function.


Assuntos
Trifosfato de Adenosina/fisiologia , Cicloexanos/farmacologia , Diazóxido/farmacologia , Insulina/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Canais de Potássio/fisiologia , Linhagem Celular , Esquema de Medicação , Humanos , Secreção de Insulina , Nateglinida , Bloqueadores dos Canais de Potássio/farmacologia
17.
Br J Pharmacol ; 142(2): 367-73, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15155541

RESUMO

Acute and chronic effects of the insulinotropic drug nateglinide upon insulin release were examined in the BRIN-BD11 cell line. Nateglinide (10-400 microm) stimulated a concentration-dependent increase (P<0.05-P<0.001) in insulin release at a non-stimulatory (1.1 mm) glucose concentration. The insulinotropic response to 200 microm nateglinide was increased at 30 mm (P<0.01), but not 5.6-16.7 mm glucose concentrations. In depolarized cells, nateglinide (50-200 microm) evoked K(ATP) channel-independent insulin secretion (P<0.05-P<0.001) in the absence and presence of 5.6-30.0 mm glucose (P<0.001). Exposure for 18 h to 100 microm nateglinide abolished the acute insulinotropic effects of 200 microm nateglinide, tolbutamide or glibenclamide, but had no effect upon the insulinotropic effect of 200 microm efaroxan. While 18 h exposure to 100 microm nateglinide did not affect basal insulin release or insulin release in the presence of 16.7 mm glucose, 25 microm forskolin or 10 nm PMA, significant inhibition of the insulinotropic effects of 20 mm leucine and 20 mm arginine were observed. These data show that nateglinide stimulates both K(ATP) channel-dependent and-independent insulin secretion. The maintained insulinotropic effects of this drug with increasing glucose concentrations support the antihyperglycaemic actions of nateglinide in Type II diabetes. Studies of the long-term effects of nateglinide indicate that nateglinide shares signalling pathways with sulphonylureas, but not the imidazoline efaroxan. This may be significant when considering a nateglinide treatment regimen, particularly in patients previously treated with sulphonylurea.


Assuntos
Cicloexanos/administração & dosagem , Insulina/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/administração & dosagem , Animais , Bovinos , Relação Dose-Resposta a Droga , Esquema de Medicação , Secreção de Insulina , Nateglinida , Ratos , Fatores de Tempo
18.
Pharmacol Res ; 50(1): 41-6, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15082027

RESUMO

Clonal insulin-secreting BRIN-BD11 cells were used to examine effects of chronic 72-144 h exposure to the sulphonylureas tolbutamide and glibenclamide on insulin release, cellular insulin content, and mRNA levels of the Kir6.2 and SUR1 subunits of the beta-cell K(ATP) channel. Chronic exposure for 72-144 h to 5-100 microM tolbutamide and glibenclamide resulted in a time- and concentration-dependent irreversible decline in sulphonylurea-induced insulin secretion. In contrast, the decline in cellular insulin content induced by chronic exposure to high concentrations of sulphonylureas was readily reversible. Chronic exposure to tolbutamide or glibenclamide had no effect upon transcription of the Kir6.2 or SUR1 subunits of the pancreatic beta-cell K(ATP) channel. Whilst further studies are required to understand the precise nature of the chronic interactions of sulphonylurea with the insulin exocytotic mechanism, these observations may partially explain the well-known progressive failure of sulphonylurea therapy in type 2 diabetes.


Assuntos
Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Pâncreas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização/genética , Tolbutamida/farmacologia , Transportadores de Cassetes de Ligação de ATP , Animais , Células Cultivadas , Células Clonais , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/análise , Secreção de Insulina , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Pâncreas/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Receptores de Droga , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Compostos de Sulfonilureia/farmacologia , Receptores de Sulfonilureias
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