RESUMEN
This paper reviews current understanding and presents new results on some of the nonlinear processes that underlie the function of the mammalian cochlea. These processes occur within mechano-sensory hair cells that form part of the organ of Corti. After a general overview of cochlear physiology, mathematical modelling results are presented in three parts. First, the dynamic interplay between ion channels within the sensory inner hair cells is used to explain some new electrophysiological recordings from early development. Next, the state of the art is reviewed in modelling the electro-motility present within the outer hair cells (OHCs), including the current debate concerning the role of cell body motility versus active hair bundle dynamics. A simplified model is introduced that combines both effects in order to explain observed amplification and compression in experiments. Finally, new modelling evidence is presented that structural longitudinal coupling between OHCs may be necessary in order to capture all features of the observed mechanical responses.
Asunto(s)
Cóclea/fisiología , Algoritmos , Animales , Biofisica/métodos , Calcio/metabolismo , Membrana Celular/metabolismo , Cóclea/embriología , Electroquímica , Células Ciliadas Auditivas/fisiología , Audición/fisiología , Humanos , Canales Iónicos , Modelos Biológicos , Modelos Teóricos , Dinámicas no Lineales , Rampa Timpánica/patologíaRESUMEN
It is generally accepted that the acute sensitivity and frequency discrimination of mammalian hearing requires active mechanical amplification of the sound stimulus within the cochlea. The prevailing hypothesis is that this amplification stems from somatic electromotility of the outer hair cells attributable to the motor protein prestin. Thus outer hair cells contract and elongate in synchrony with the sound-evoked receptor potential. But problems arise with this mechanism at high frequencies, where the periodic component of the receptor potential will be attenuated by the membrane time constant. On the basis of work in non-mammalian vertebrates, force generation by the hair bundles has been proposed as an alternative means of boosting the mechanical stimulus. Here we show that hair bundles of mammalian outer hair cells can also produce force on a submillisecond timescale linked to adaptation of the mechanotransducer channels. Because the bundle motor may ultimately be limited by the deactivation rate of the channels, it could theoretically operate at high frequencies. Our results show the existence of another force generator in outer hair cells that may participate in cochlear amplification.
Asunto(s)
Células Ciliadas Auditivas Externas/fisiología , Audición/fisiología , Mamíferos/fisiología , Animales , Animales Recién Nacidos , Fenómenos Biomecánicos , Vidrio , Movimiento , Estimulación Física , RatasRESUMEN
BACKGROUND: The aetiology of perforated colonic diverticular disease (PCDD) remains largely unknown. Perforation may result from a combination of high intracolonic pressures, secondary to excessive colonic segmentation, and impairment of the mucosal barrier. Calcium channel blockers and antimuscarinic drugs, which reduce colonic contractility and tone, could potentially protect against perforation. The aim of this study was to test this hypothesis using a case control design. METHODS: All cases of acute PCDD were identified over a five year period in two hospitals in Norfolk, UK. Each case was matched for age, sex, and date of admission to two controls groups: (1) patients undergoing cataract surgery and (2) patients with basal cell carcinoma. Data on drug use prior to hospital admission were obtained from medical and nursing records and compared between cases and controls. RESULTS: A total of 120 cases of PCDD were identified and matched to 240 controls in each group. A statistically significant protective association was seen between calcium channel blocker use and PCDD using both control groups. The odds ratios were 0.41 (95% confidence interval (CI) 0.18-0.93) using the ophthalmology control group and 0.36 (95% CI 0.16-0.82) using the dermatology control group. CONCLUSIONS: This study has shown for the first time that a protective association exists between calcium channel blockers and PCDD. The validity of this association is supported by the consistent finding in both control groups and the plausible biological mechanisms. Further studies are required to confirm this association but calcium channel blockers may represent a potential preventive therapy in PCDD.
Asunto(s)
Bloqueadores de los Canales de Calcio/uso terapéutico , Divertículo del Colon/prevención & control , Perforación Intestinal/prevención & control , Antagonistas Muscarínicos/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Acute perforated colonic diverticular disease has a mortality rate of up to 30 per cent, but little is known about its aetiology. The aim of this study was to test the hypothesis that three classes of drugs, namely non-steroidal anti-inflammatory drugs (NSAIDs), opioid analgesics and corticosteroids, are risk factors for perforated diverticular disease. METHODS: All patients with confirmed perforated colonic diverticular disease were identified over a 5-year period in two hospitals in Norfolk, UK. Two control groups were selected and matched for age, sex and hospital of admission. Data on medication use were obtained from hospital records. Odds ratios for each drug were calculated using conditional logistic regression. RESULTS: Opioid analgesics, NSAIDs and corticosteroids were all positively associated with perforated colonic diverticular disease. The odds ratio for opioid analgesics was 1.8 (95 per cent confidence interval (c.i.) 1.1 to 3.0) in the analysis with ophthalmology controls and 3.1 (95 per cent c.i. 1.8 to 5.5) in that with dermatology controls. Respective odds ratios for NSAIDs were 4.0 (95 per cent c.i. 2.1 to 7.6) and 3.7 (95 per cent c.i. 2.0 to 6.8), and those for corticosteroids were 5.7 (95 per cent c.i. 2.2 to 14.4) and 7.8 (95 per cent c.i. 2.6 to 23.3). CONCLUSION: Opioid analgesics, NSAIDs and corticosteroids are all positively associated with perforated colonic diverticular disease. The consistency of these associations, together with plausible biological mechanisms, suggests that these drugs may have a causative role in this condition.
Asunto(s)
Analgésicos Opioides/efectos adversos , Antiinflamatorios no Esteroideos/efectos adversos , Divertículo del Colon/inducido químicamente , Perforación Intestinal/inducido químicamente , Corticoesteroides/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Factores de RiesgoRESUMEN
Perforated colonic diverticular disease results in considerable mortality and morbidity. This review appraises existing evidence on the epidemiology and mechanisms of perforation, highlights areas of further study, and suggests an epidemiological approach towards preventing the condition. Computerised searches were used to identify published articles relating to the epidemiology, pathophysiology, and clinical features of perforated colonic diverticular disease. Several drug and dietary exposures have potential biological mechanisms for causing perforation. Of these only non-steroidal anti-inflammatory drugs have been consistently identified as risk factors in aetiological studies. The causes of perforated colonic diverticular disease remain largely unknown. Further aetiological studies, looking specifically at perforation, are required to investigate whether cause-effect relationships exist for both drug and dietary exposures. The identification of risk factors for perforation would allow primary public health prevention, secondary risk factor modification, and early prophylactic surgery to be aimed at people at high risk.
Asunto(s)
Diverticulitis del Colon/etiología , Consumo de Bebidas Alcohólicas/efectos adversos , Antiinflamatorios no Esteroideos/efectos adversos , Dieta/efectos adversos , Diverticulitis del Colon/epidemiología , Diverticulitis del Colon/prevención & control , Salud Global , Humanos , Incidencia , Factores de Riesgo , Fumar/efectos adversosRESUMEN
The mechanisms that regulate the concentration of ionized intracellular calcium (Ca(2+)(i)) in the base of neonatal mouse inner hair cells, close to synaptic sites, were investigated using confocal microscopy combined with conventional patch-clamp electrophysiology. Cells were depolarized under whole-cell voltage clamp to load the cell with C a(2+) through voltage-activated Ca(2+) channels. Repeated depolarizations produced Ca(2+)(i) increases with similar amplitudes and time-courses of recovery. The rate of recovery from depolarization-induced Ca(2+)(i) loads was used to assess the mechanisms responsible for Ca(2+)(i) regulation. Removal of extracellular sodium had no effect on resting Ca(2+)(i) or the rate of recovery of Ca(2+)(i) suggesting no role for Na:Ca exchange in these cells. Inhibitors of intracellular store uptake such as thapsigargin, 2,5-di(tert-butyl)hydroquinone (BHQ) and cyclopiazonic acid (CPA) caused an increase in resting Ca(2+)(i) and slowed the rate of recovery, indicating that Ca(2+) can be taken up intracellularly. However, 5mM caffeine failed to cause a detectable release of Ca(2+) from intracellular stores. FCCP, a mitochondrial inhibitor, slowed the rate of recovery from Ca(2+)(i) loads, indicating a role for mitochondrial Ca(2+) uptake. The largest effects were seen with intracellular vanadate (1mM) which caused an irreversible rise in resting Ca(2+)(i) and depolarization-induced increases in Ca(2+)(i) failed to recover fully. Together, these data indicate that both thapsigargin-sensitive stores and mitochondria can take up Ca(2+)(i), but that Ca(2+) efflux from the cell occurs solely via a plasma membrane Ca(2+)-ATPase.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Compuestos de Anilina/metabolismo , Animales , Animales Recién Nacidos , Cafeína/farmacología , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/metabolismo , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/efectos de los fármacos , Homeostasis , Hidroquinonas/farmacología , Indoles/farmacología , Transporte Iónico , Ratones , Microscopía Confocal , Mitocondrias/metabolismo , Técnicas de Placa-Clamp , Sodio/metabolismo , Tapsigargina/farmacología , Xantenos/metabolismoRESUMEN
Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.
Asunto(s)
Colorantes Fluorescentes/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Canales Iónicos/antagonistas & inhibidores , Mecanorreceptores/efectos de los fármacos , Compuestos de Piridinio/farmacología , Compuestos de Amonio Cuaternario/farmacología , Aminoglicósidos , Animales , Antibacterianos/farmacología , Calcio/metabolismo , Células Cultivadas , Quelantes/farmacología , Dineínas , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Espacio Extracelular/metabolismo , Colorantes Fluorescentes/farmacocinética , Células Ciliadas Auditivas/metabolismo , Heterocigoto , Homocigoto , Canales Iónicos/metabolismo , Cinética , Mecanorreceptores/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Mutantes , Miosina VIIa , Miosinas/deficiencia , Miosinas/genética , Técnicas de Placa-Clamp , Estimulación Física , Compuestos de Piridinio/farmacocinética , Compuestos de Amonio Cuaternario/farmacocinética , TemperaturaRESUMEN
The aetiology of perforation of large bowel diverticula is poorly understood and a case-control study is required to identify the causes. Before such a study can be attempted, the incidence must be determined and groups at particular risk identified. Cases of perforated large bowel diverticula living in the Norwich postal code region treated between 1995 and 1997 were identified. Fifty-eight cases presented in a population of 531 241. The incidence was 4.0 cases per 100,000 per year, increased with age and was higher in men than women (5.8 vs 3.1). The most frequently used drugs were non-steroidal anti-inflammatory drugs (NSAIDs) (29%) and opiate analgesics (26% of cases). This is the first report of the incidence of perforated diverticular disease and allows a calculation of the population size needed to recruit sufficient cases for an aetiological investigation. The differences in incidence between genders should prompt a search for factors which differ between the sexes such as diet. NSAIDs are a known risk factor, although the data show that opiate analgesics should be investigated.
Asunto(s)
Enfermedades del Ciego/complicaciones , Divertículo del Colon/complicaciones , Divertículo/complicaciones , Perforación Intestinal/etiología , Adulto , Anciano , Anciano de 80 o más Años , Analgésicos Opioides/efectos adversos , Antiinflamatorios no Esteroideos/efectos adversos , Enfermedades del Ciego/epidemiología , Estudios Transversales , Divertículo/epidemiología , Divertículo del Colon/epidemiología , Inglaterra/epidemiología , Femenino , Humanos , Incidencia , Perforación Intestinal/epidemiología , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
It has been suggested that N-nitroso compounds derived from meat may increase the risk of K-ras mutations in the human colon. We sought evidence of associations between red meat consumption, frequency and type of K-ras mutations in resected tumours, and the rate of crypt cell proliferation (CCP) in the normal mucosa of patients with left-sided colorectal carcinoma. Meat consumption was assessed by food frequency questionnaire, and CCP was determined in rectal biopsies obtained prior to surgery. K-ras mutations in the resected tumours were determined using a PCR-based oligonucleotide hybridization assay. Fifteen K-ras mutations were detected in tumours from 43 patients; 13/15 in codon 12, 3/15 in codon 13, and 1/15 in both codons 12 and 13. All mutations were G-->A or G-->T transitions. There was no statistically significant difference between intakes of red meat in patients with a K-ras mutation (92.4 +/- 9.7 g/day) and those without (82.3 +/- 7.7 g/day). Rectal CCP was significantly higher in patients than in healthy controls, but there was no correlation with meat consumption or K-ras mutation. These data do not support the hypothesis that meat consumption is a risk factor for acquisition of K-ras mutations during colorectal carcinogenesis.
Asunto(s)
Neoplasias Colorrectales/genética , Dieta , Genes ras/genética , Carne , Recto/citología , Anciano , División Celular , Transformación Celular Neoplásica , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Membrana Mucosa/citología , Reacción en Cadena de la Polimerasa , Medición de RiesgoRESUMEN
The current 6-month quarantine system for all cats and dogs entering the UK has kept the UK rabies-free since 1922. However, pressure is mounting for a change to a system of vaccination, microchip identification and serological testing. In response to the increasing controversy surrounding the quarantine system, the UK government recently set up an independent review panel to assess the alternatives. This paper quantifies public preferences for the current policy and three alternative rabies-prevention measures. A survey was used not only to assess the overall preferences for rabies-prevention policies but also to assess the importance of policy attributes and socio-economic characteristics in determining policy preferences. We interviewed a sample of pet-owners in North Yorkshire. The results showed that the existing system was the single most-preferred policy option. However, a large proportion of the sample preferred the vaccination-based policies. A logistic-regression model and ordered probit models were used to find that safety and animal welfare were the most-important factors determining policy preferences. The respondents' awareness of the rabies-policy review, a desire to take a pet abroad, the amount of foreign travel, occupation and previous experience of quarantine were all important factors in policy choice. Socio-economic characteristics such as income, pets owned and the number of children were not significant determinants of policy preference.
Asunto(s)
Enfermedades de los Gatos/prevención & control , Enfermedades de los Perros/prevención & control , Política Pública , Cuarentena/veterinaria , Rabia/veterinaria , Animales , Animales Domésticos , Gatos , Perros , Humanos , Entrevistas como Asunto , Modelos Logísticos , Modelos Biológicos , Proyectos Piloto , Salud Pública , Cuarentena/legislación & jurisprudencia , Rabia/prevención & control , Virus de la Rabia/crecimiento & desarrollo , Análisis de Regresión , Clase Social , Encuestas y Cuestionarios , Viaje , Reino Unido , Vacunación/veterinariaRESUMEN
Recent studies have suggested that glucose may activate insulin gene transcription through increases in intracellular Ca(2+) concentration, possibly acting via the release of stored insulin. We have investigated this question by dynamic photon-counting imaging of insulin- and c-fos-promoter-firefly luciferase reporter construct activity. Normalized to constitutive viral promoter activity, insulin promoter activity in MIN6 beta-cells was increased 1.6-fold after incubation at 30 mM compared with 3 mM glucose, but was unaltered at either glucose concentration by the presence of insulin (100 nM) or the Ca(2+) channel inhibitor, verapamil (100 microM). Increases in intracellular [Ca(2+)] achieved by plasma membrane depolarization with KCl failed to enhance either insulin or c-fos promoter activity in MIN6 cells, but increased c-fos promoter activity 5-fold in AtT20 cells. Together, these results demonstrate that glucose can exert a direct effect on insulin promoter activity in islet beta-cells, via a signalling pathway which does not require increases in intracellular [Ca(2+)] nor insulin release and insulin receptor activation.
Asunto(s)
Calcio/metabolismo , Glucosa/farmacología , Insulina/genética , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiología , Regiones Promotoras Genéticas/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Genes Reporteros , Genes fos , Humanos , Insulina/metabolismo , Insulina/farmacología , Secreción de Insulina , Luciferasas/genética , Ratones , Fosforilación , Transducción de Señal , Verapamilo/farmacologíaRESUMEN
Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.
Asunto(s)
Adenosina Trifosfato/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Animales , Secuencia de Bases , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Escarabajos/enzimología , Cartilla de ADN , Inmunohistoquímica , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Luciferasas/metabolismo , Mitocondrias/metabolismo , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
Recent advances in reporter gene technologies are now allowing us to image gene transcription at the single cell level, using either fluorescence or luminescence microscopy. Here, the basis of these techniques is outlined and their advantages and disadvantages in various biological systems are discussed.
Asunto(s)
Células/ultraestructura , Regulación de la Expresión Génica/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Animales , Células/metabolismo , Colorantes Fluorescentes , HumanosRESUMEN
Using laser-scanning confocal microscopy, we have monitored glucose-induced changes in the subcellular localization of insulin promoter factor-1 (IPF-1) labeled with a c-myc epitope tag. This construct trans-activated the insulin promoter in single living MIN6-beta-cells as assessed by luciferase-based promoter analysis. IPF-1.c-myc expression also enhanced the response of the insulin promoter to elevations in extracellular glucose concentration. In the majority (148/235, 63%) of cells maintained at low (3 mM) extracellular glucose concentration, IPF-1.c-myc immunoreactivity was confined to the nuclear periphery. Incubation of cells at stimulatory (30 mM) glucose concentrations caused a rapid redistribution of the chimera to the nucleoplasm (775/958, 81% of cells). By contrast, the irrelevant transcription factor c-Fos, tagged with either c-myc or as a chimera with luciferase, was localized exclusively to the nucleoplasm irrespective of the glucose concentration. Furthermore, IPF-1 extended with the bulky (27 kDa) enhanced green fluorescent protein (EGFP) group was confined largely to the nucleoplasm at all glucose concentrations tested and did not support trans-activation of the insulin promoter by glucose. Movement of endogenous IPF-1 from the nuclear periphery to the nucleoplasm may therefore increase the trans-activational capacity of this factor in native beta-cells exposed to high extracellular glucose concentrations.
Asunto(s)
Núcleo Celular/metabolismo , Glucosa/farmacología , Proteínas de Homeodominio , Insulina/biosíntesis , Islotes Pancreáticos/metabolismo , Transactivadores/metabolismo , Transporte Biológico , Biomarcadores , Compartimento Celular , Proteínas Fluorescentes Verdes , Procesamiento de Imagen Asistido por Computador , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genética , Activación TranscripcionalRESUMEN
The extrusion mechanism for intracellular Mg2+ was investigated in voltage-clamped snail neurones using Mg(2+)-sensitive microelectrodes and mag-fura-2. The intracellular free magnesium ion concentration ([Mg2+]1) of snail neurones voltage clamped to -60 mV was estimated to be 0.57 +/- 0.06 mM (mean +/- S.E.M.; n = 12) using Mg(2+)-sensitive microelectrodes and 0.62 +/- 0.05 mM (n = 15) using mag-fura-2. Raising extracellular MgCl2 from 5 to 20 mM caused an average increase in [Mg2+]1 of 0.25 +/- 0.04 mM (n = 7). In three experiments, removing extracellular Mg Cl2 caused an average decrease in [Mg2+]1 of 0.1 mM. Replacing extracellular Na+ with N-methyl-D-glucamine (NMDG) caused a rise in [Mg2+]1 of 1.8 +/- 0.5 mM (n = 7); [Mg2+]1 recovered to resting levels when extracellular Na+ was restored. Iontophoretic injections of MgCl2 were used to raise [Mg2+]1. The rate of recovery from such increases in [Mg2+]1 ¿calculated from the slope of the recovery was inhibited by 85-100% (n = 5) in the absence of extracellular Na2+ compared with control conditions. Raising extracellular Ca2+ from 7 to 35 mM caused a reversible rise in [Mg 2+]1 of 0.4 +/- 0.05 mM (mean +/- S.E.M., n = 7). It was concluded that in snail neurones the main mechanism for [Mg2+]1 extrusion is a Na(+)-Mg2+ exchanger which may be partially inhibited be high extracellular Ca2+ concentrations.
Asunto(s)
Caracoles Helix/fisiología , Líquido Intracelular/fisiología , Magnesio/metabolismo , Neuronas/fisiología , Animales , Calcio/farmacología , Electrofisiología , Colorantes Fluorescentes/metabolismo , Fura-2/análogos & derivados , Fura-2/metabolismo , Meglumina/análogos & derivados , Microelectrodos , Técnicas de Placa-Clamp , Sodio/farmacologíaRESUMEN
We have prepared recombinant cDNAs encoding chimaeras between human preproinsulin (sp.B.C.A., for B-, Connecting- and A-peptides) and a thermostable mutant of green fluorescent protein (GFPS65T,V163A, GFP*). The subcellular localization of the expressed chimaeras was monitored in living insulin-secreting INS-1 beta-cells by laser scanning confocal microscopy. When GFP* was fused at the immediate N-terminus of the B-chain (sp.[GFP*].B.C.A.myc) two distinct patterns of fluorescence were apparent. In 1530/1740 cells examined, fluorescence was confined to a reticular, exclusively extranuclear structure, and closely co-localized with the endoplasmic reticulum marker, calreticulin. However, 210/1740 (12.1%) of cells displayed punctate fluorescence, which partially co-localized with the trans-Golgi network marker, TGN 38, and with the dense core secretory granule marker, phogrin. Since secretion of GFP* fluorescence into the medium could not readily be measured, we prepared a chimaera in which firefly luciferase was fused at the C-terminus of proinsulin (sp.B.C.A.myc.[Luc]). This chimaera displayed a distribution closely similar to that of sp.[GFP*].B.C.A. myc, but with a lower proportion (15/310, 4.8%) of the cells showing clear punctate distribution. At substimulatory glucose concentrations (3 mM) secretion of sp.B.C.A.myc.[Luc] could not be detected (rate of release into the medium identical with that of the cytosolic Renilla reniformis luciferase), indicating that the chimaera did not enter the constitutive secretory pathway. However, elevated (30 mM) glucose stimulated the release of the sp.B.C.A.myc. [Luc] luciferase chimaera, without a detectable effect on R. reniformis luciferase release. These data suggest that fusion of insulin, and the much larger photoproteins GFP* and luciferase, leads predominantly to misfolding and retention in the endoplasmic reticulum. However, the properly folded chimaeras are apparently still correctly targeted to the regulated, rather than the constitutive, secretory pathway. These chimaeras should therefore be valuable tools to monitor the exocytosis of insulin in real time.
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Luciferasas/genética , Proteínas Luminiscentes/metabolismo , Proinsulina/genética , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica , Secreción de InsulinaRESUMEN
Elevated glucose concentrations stimulate L-pyruvate kinase (L-PK) gene transcription in liver and islet beta-cells. A glucose response element termed the L4 box (two noncanonical E-boxes located -165 and -154 base pairs upstream of the transcriptional start point) has previously been defined within the proximal promoter region of the gene. However, the identity of the transacting factor(s) which binds to this site remains unclear. We have used photon counting digital imaging of firefly luciferase activity to monitor promoter activity continuously in single living islet beta and derived INS-1 cells, and to analyze the molecular basis of the regulation by glucose. L-PK promoter activity, normalized to cytomegalovirus promoter activity using the distinct Renilla reniformis luciferase, was >/=6-fold higher in cells cultured at 16 mM glucose or above compared with cells cultured at 3 mM glucose. Microinjection of antibodies against the ubiquitous transcription factor USF2 inhibited L-PK promoter activity in beta- and INS-1 cells incubated at 30 mM glucose by 71-87%. Anti-USF2 antibodies had a much smaller effect on promoter activity in INS-1 cells cultured at 3 mM glucose, and on the activity of a modified promoter construct lacking an L4 box. These data support the view that glucose enhances L-PK gene transcription in beta-cells by modifying the transactivational capacity of USF2 bound to the upstream L4 box.
Asunto(s)
Proteínas de Unión al ADN , Glucosa/farmacología , Islotes Pancreáticos/enzimología , Regiones Promotoras Genéticas , Piruvato Quinasa/genética , Factores de Transcripción/fisiología , Animales , Masculino , Ratas , Transcripción Genética , Factores Estimuladores hacia 5'Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Colon/patología , Neoplasias del Colon/secundario , Seudoobstrucción Colónica/diagnóstico , Enfermedad de Crohn/diagnóstico , Neoplasias Pulmonares/diagnóstico , Síndromes Paraneoplásicos/diagnóstico , Biomarcadores/análisis , Enfermedad de Crohn/patología , Diagnóstico Diferencial , Resultado Fatal , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Síndromes Paraneoplásicos/patologíaRESUMEN
We assessed consumption of fruit and vegetables amongst patients with ulcerative colitis, colorectal polyps or previous carcinoma. The dietary intakes for 119 patients attending a gastroenterology clinic for colonoscopy were assessed using a questionnaire. A single age- and sex-matched control subject was recruited for each patient. The patients consumed 12.8% less energy than the controls (P < 0.02) and 28.9% less fruit and vegetables (P < 0.0001). Patients with neoplastic disease consumed 21% less fruit and vegetables than the controls (n = 60; P < 0.01). This group of patients at increased risk of colorectal cancer selected diets containing significantly less fruit and vegetables than symptomless controls.