RESUMO
It has recently been shown that optical reflection gratings fabricated directly into an atom chip provide a simple and effective way to trap and cool substantial clouds of atoms (Nshii et al. in Nat Nanotechnol 8:321-324, 2013; McGilligan et al. in Opt Express 23(7):8948-8959, 2015). In this article, we describe how the gratings are designed and microfabricated and we characterise their optical properties, which determine their effectiveness as a cold atom source. We use simple scalar diffraction theory to understand how the morphology of the gratings determines the power in the diffracted beams.
RESUMO
Organonitrates (ON) are important products of gas-phase oxidation of volatile organic compounds in the troposphere; some models predict, and laboratory studies show, the formation of large, multifunctional ON with vapor pressures low enough to partition to the particle phase. Organosulfates (OS) have also been recently detected in secondary organic aerosol. Despite their potential importance, ON and OS remain a nearly unexplored aspect of atmospheric chemistry because few studies have quantified particulate ON or OS in ambient air. We report the response of a high-resolution time-of-flight aerosol mass spectrometer (AMS) to aerosol ON and OS standards and mixtures. We quantify the potentially substantial underestimation of organic aerosol O/C, commonly used as a metric for aging, and N/C. Most of the ON-nitrogen appears as NO(x)+ ions in the AMS, which are typically dominated by inorganic nitrate. Minor organonitrogen ions are observed although their identity and intensity vary between standards. We evaluate the potential for using NO(x)+ fragment ratios, organonitrogen ions, HNO(3)+ ions, the ammonium balance of the nominally inorganic ions, and comparison to ion-chromatography instruments to constrain the concentrations of ON for ambient datasets, and apply these techniques to a field study in Riverside, CA. OS manifests as separate organic and sulfate components in the AMS with minimal organosulfur fragments and little difference in fragmentation from inorganic sulfate. The low thermal stability of ON and OS likely causes similar detection difficulties for other aerosol mass spectrometers using vaporization and/or ionization techniques with similar or larger energy, which has likely led to an underappreciation of these species.
Assuntos
Aerossóis , Química Orgânica/instrumentação , Química Orgânica/métodos , Química/métodos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Nitratos/química , Sulfatos/química , Poluentes Atmosféricos/análise , Poluentes Atmosféricos/química , Amônia/química , Cromatografia por Troca Iônica/métodos , Íons , Nitrogênio/química , Ácido Oleico/química , Tamanho da Partícula , Material Particulado/química , Fatores de TempoRESUMO
AIMS/HYPOTHESIS: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) offer unique opportunities for regenerative medicine and for the study of mammalian development. However, developing methods to differentiate hESCs/hIPSCs into specific cell types following a natural pathway of development remains a major challenge. METHODS: We used defined culture media to identify signalling pathways controlling the differentiation of hESCs/hIPSCs into pancreatic or hepatic progenitors. This approach avoids the use of feeders, stroma cells or serum, all of which can interfere with experimental outcomes and could preclude future clinical applications. RESULTS: This study reveals, for the first time, that activin/TGF-ß signalling blocks pancreatic specification induced by retinoic acid while promoting hepatic specification in combination with bone morphogenetic protein and fibroblast growth factor. Using this knowledge, we developed culture systems to differentiate human pluripotent stem cells into near homogenous population of pancreatic and hepatic progenitors displaying functional characteristics specific to their natural counterparts. Finally, functional experiments showed that activin/TGF-ß signalling achieves this essential function by controlling the levels of transcription factors necessary for liver and pancreatic development, such as HEX and HLXB9. CONCLUSION/INTERPRETATION: Our methods of differentiation provide an advantageous system to model early human endoderm development in vitro, and also represent an important step towards the generation of pancreatic and hepatic cells for clinical applications.
Assuntos
Ativinas/antagonistas & inibidores , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/farmacologia , Animais , Comunicação Celular , Diferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos SCID , Pâncreas/patologia , Medicina Regenerativa , Transdução de SinaisRESUMO
The transcription factor insulin promoter factor-1 (IPF-1) plays a central role in both the development of the pancreas and the regulation of insulin gene expression in the mature pancreatic beta cell. A dominant-negative frameshift mutation in the IPF-l gene was identified in a single family and shown to cause pancreatic agenesis when homozygous and maturity-onset diabetes of the young (MODY) when heterozygous. We studied the role of IPF-1 in Caucasian diabetic and nondiabetic subjects from the United Kingdom. Three novel IPF-1 missense mutations (C18R, D76N, and R197H) were identified in patients with type 2 diabetes. Functional analyses of these mutations demonstrated decreased binding activity to the human insulin gene promoter and reduced activation of the insulin gene in response to hyperglycemia in the human beta-cell line Nes2y. These mutations are present in 1% of the population and predisposed the subject to type 2 diabetes with a relative risk of 3.0. They were not highly penetrant MODY mutations, as there were nondiabetic mutation carriers 25-53 years of age. We conclude that mutations in the IPF-1 gene may predispose to type 2 diabetes and are a rare cause of MODY and pancreatic agenesis, with the phenotype depending upon the severity of the mutation.
Assuntos
Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodomínio , Transativadores/genética , Adulto , Idoso , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Glucose/metabolismo , Humanos , Insulina/genética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Fosforilação , Transcrição GênicaRESUMO
A radiolabelled peptide chloromethyl ketone (125I-tyrosyl-L-alanyl-L-lysyl-L-arginine chloromethyl ketone) was used to affinity-label proteinases in rat thyroid cells (FRTL5). Two major proteins of 34 kDa and 32 kDa were affinity-labelled. Inhibitor competition studies demonstrated that both proteins were cysteine proteinases. Over the range pH 5-8, they exhibited maximum activity against the affinity probe at pH 5. They were soluble rather than membrane-bound and were both glycosylated. The 32 kDa proteinase but not the 34 kDa proteinase was immunoprecipitated using an anti-rat liver cathepsin B antibody. The data suggested that these proteinases were molecular forms of cathepsin B. The affinity-labelled proteins in the thyroid were compared with those in an insulin-secreting cell line (HIT T15) and a liver cell line (Hep G2). Two molecular forms of cathepsin B of Mr 39,000 and 33,000 were identified in the insulin-secreting cell line and a single form of Mr 34,000 in the liver cell line. These molecular forms of cathepsin B may reflect the different functions and compartmentation of cathepsin B in these cells.
Assuntos
Catepsina B/isolamento & purificação , Isoenzimas/isolamento & purificação , Glândula Tireoide/enzimologia , Animais , Catepsina B/metabolismo , Linhagem Celular , Cricetinae , Epitélio/enzimologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/enzimologia , Isoenzimas/metabolismo , Fígado/enzimologia , Peso Molecular , RatosRESUMO
Prostacyclin and adenosine A2 receptors stimulate adenylate cyclase activity in the related somatic hybrid cell lines NG108-15 and NCB20. The role of cAMP in the desensitization of these receptors has been examined. Pretreatment for 17 h with forskolin or 8-bromo-cAMP had the same effect in both cell lines. There was no change in the response to sodium fluoride or forskolin, suggesting that the function of Gs and adenylate cyclase were unaffected by increased levels of cAMP. Receptor responses were affected however; the maximum response to N-ethylcarboxamidoadenosine (an A2 receptor agonist) was reduced by 30-40%, there was a small but consistent shift to the right of the dose-response curve for iloprost (a stable analogue of prostacyclin) and [3H]iloprost binding studies revealed a loss of prostacyclin receptors. However, the loss of receptor responsiveness was much smaller than that which occurs following pretreatment with prostacyclin or adenosine A2 receptor agonists (Keen et al. (1989) Biochem. Pharmacol. 38, 3827-3833; Kelly et al. (1990) Br. J. Pharmacol. 99, 309-316) suggesting that cAMP may not play a major role in agonist mediated desensitization.
Assuntos
AMP Cíclico/metabolismo , Epoprostenol/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores Purinérgicos/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenilil Ciclases/metabolismo , Linhagem Celular , Colforsina/farmacologia , Ativação Enzimática , Proteínas de Ligação ao GTP/genética , Iloprosta/metabolismo , Iloprosta/farmacologia , Magnésio/metabolismo , RNA Mensageiro/metabolismoRESUMO
Pancreatic duodenal homeobox-1 (PDX-1) is a homeodomain protein that plays an important role in the development of the pancreas and in maintaining the identity and function of the islets of Langerhans. It also regulates the expression of the insulin gene in response to changes in glucose and insulin concentrations. Glucose and insulin regulate PDX-1 by way of a signaling pathway involving phosphatidylinositol 3-kinase (PI 3-kinase) and SAPK2/p38. Activation of this pathway leads to phosphorylation of PDX-1 and its movement into the nucleus. To investigate the intracellular trafficking of PDX-1, immunocytochemistry was used to localize PDX-1 in the human beta-cell line NesPDX-1, in which PDX-1 is overexpressed, and in MIN6 beta-cells. In low-glucose conditions, PDX-1 localized predominantly to the nuclear periphery, with some staining in the cytoplasm. After stimulation with glucose, PDX-1 was present in the nucleoplasm. The translocation of PDX-1 to the nucleoplasm was complete within 15 min and occurred in 5-10 mmol/l glucose. Insulin and sodium arsenite, an activator of the stress-activated pathway, also stimulated PDX-1 movement from the nuclear periphery to the nucleoplasm. When cells were transferred between high glucose- and low glucose-containing medium, PDX-1 rapidly shuttled between the nuclear periphery and the nucleoplasm. Glucose- and insulin-stimulated translocation of PDX-1 to the nucleoplasm was inhibited by wortmannin and SB 203580, indicating that a pathway involving PI 3-kinase and SAPK2/p38 was involved; translocation was unaffected by PD 098959 and rapamycin, suggesting that neither mitogen-activated protein kinase nor p70(s6k) were involved. Arsenite-stimulated import of PDX-1 into the nucleus was inhibited by SB 203580 but not by wortmannin. Export from the nucleoplasm to the nuclear periphery was inhibited by calyculin A and okadaic acid, suggesting that dephosphorylation of PDX-1 was involved. These results demonstrated that PDX-1 shuttles between the nuclear periphery and nucleoplasm in response to changes in glucose and insulin concentrations and that these events are dependent on PI 3-kinase, SAPK2/p38, and a nuclear phosphatase(s).
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Homeodomínio , Transativadores/metabolismo , Androstadienos/farmacologia , Arsenitos/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Glucose/farmacologia , Humanos , Imidazóis/farmacologia , Imuno-Histoquímica , Insulina/farmacologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Piridinas/farmacologia , Compostos de Sódio/farmacologia , Wortmanina , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Glucose regulates insulin production in pancreatic beta-cells in the long term by stimulating insulin gene transcription. These effects are partially mediated through the activity of a homeodomain transcription factor, PDX-1, which binds to four sites within the human insulin gene promoter. The availability of a human beta-like cell line, NES2Y, which lacks PDX-1 but expresses the insulin gene, allowed us to determine whether PDX-1 was essential for the stimulatory effect of glucose on insulin mRNA levels. In NES2Y cells, glucose had no effect on the insulin gene promoter linked to a firefly luciferase reporter or on endogenous insulin mRNA levels. However, in NES2Y cells stably transfected with PDX-1 (NES-PDX-1), glucose exhibited a marked stimulatory effect on both the insulin promoter (5+/-0.2-fold, n = 6) and insulin mRNA levels (4.8+/-0.5-fold, n = 4). NES2Y cells were derived from a patient with persistent hyperinsulinemic hypoglycemia of infancy; the cells therefore lacked operational ATP-sensitive potassium channels, which results in the failure to control depolarization-dependent intracellular Ca2+ signaling. Despite the loss of control of Ca2+ channel activity, NES-PDX-1 cells maintained normal glucose-responsive insulin gene regulation. These results demonstrate that glucose modulation of insulin mRNA levels is dependent on the activity of PDX-1 and that these effects are independent of changes in intracellular Ca2+ concentrations.
Assuntos
Cálcio/metabolismo , Glucose/farmacologia , Proteínas de Homeodomínio , Insulina/genética , Membranas Intracelulares/metabolismo , RNA Mensageiro/metabolismo , Transativadores/fisiologia , Linhagem Celular , Humanos , Concentração Osmolar , Pancreatopatias/metabolismo , Pancreatopatias/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Transativadores/genética , TransfecçãoRESUMO
NES2Y is a proliferating human insulin-secreting cell line that we have derived from a patient with persistent hyperinsulinemic hypoglycemia of infancy. This disease is characterized by unregulated insulin release despite profound hypoglycemia. NES2Y cells, like beta-cells isolated from the patient of origin, lack functional ATP-sensitive potassium channels (KATP) and also carry a defect in the insulin gene-regulatory transcription factor PDX1. Here, we report that the NES2Y beta-cells that are transfected with the genes encoding the components of KATP channels in beta-cells, sulfonylurea receptor (SUR) 1 and Kir6.2, have operational KATP channels and show normal intracellular Ca2+ and secretory responses to glucose. However, these cells, designated NESK beta-cells, have impaired insulin gene transcription responses to glucose. NES2Y beta-cells that are transfected with either Kir6.2 or SUR1 alone do not express functional KATP channels and have impaired intracellular free Ca2+ concentration-signaling responses to depolarization-dependent beta-cell agonists. These findings document that in NES2Y beta-cells, coexpression of both subunits is critically required for fully operational KATP channels and KATP channel-dependent signaling events. This article further characterizes the properties of the novel human beta-cell line, NES2Y, and documents the usefulness of these cells in diabetes-related research.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Receptores de Droga/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Eletrofisiologia , Humanos , Insulina/genética , Secreção de Insulina , Membranas Intracelulares/metabolismo , Ilhotas Pancreáticas/fisiologia , Concentração Osmolar , Canais de Potássio/genética , Receptores de Sulfonilureias , Transcrição Gênica , TransfecçãoRESUMO
The principle of insulin delivery by ex-vivo somatic cell gene therapy involves the removal of non-B-cell somatic cells (e.g. fibroblasts) from a diabetic patient, and genetically altering them in vitro to produce and secrete insulin. The cells can be grown in culture and returned to the donor as a source of insulin replacement. Cells modified in this way could be evaluated before implantation, and reserve stocks could be cryopreserved. By using the patient's own cells, the procedure should obviate the need for immunosuppression and overcome the problem of tissue supply, while avoiding a recurrence of cell destruction. Ex-vivo somatic cell gene therapy requires an accessible and robust cell type that is amenable to multiple transfections and subject to controlled proliferation. Special problems associated with the use of non-B-cell somatic cells include the processing of proinsulin to insulin, and the conferment of sensitivity to glucose-stimulated proinsulin biosynthesis and regulated insulin release. Preliminary studies using fibroblasts, pituitary cells, kidney (COS) cells and ovarian (CHO) cells suggest that these challenges could be met, and that ex-vivo somatic cell gene therapy offers a feasible approach to insulin replacement therapy.
Assuntos
Diabetes Mellitus Experimental/terapia , Fibroblastos/metabolismo , Terapia Genética/métodos , Insulina/biossíntese , Insulina/genética , Animais , Fibroblastos/transplante , Engenharia Genética , Humanos , Camundongos , TransfecçãoRESUMO
In the past couple of years a number of major breakthroughs have been made in understanding the developmental biology of the islets of Langerhans. These include the involvement of the hedgehog signalling pathway in defining the region of the gut endoderm that will develop into the pancreas; the discovery that the transcription factor neurogenin3 and the Delta/Notch signalling pathway control endocrine cell differentiation through a lateral inhibition mechanism; and that alpha and beta cells are derived from an islet progenitor cell and follow independent lineage pathways rather than arising from a common mutihormonal progenitor cell as previously thought. This knowledge had been used in strategies to provide a replenishable supply of insulin-secreting cells for the treatment of diabetes mellitus. Thus, islet progenitor cells in adult pancreatic ducts or in isolated islets of Langerhans have been induced to grow in culture and their endocrine-like properties have been characterised. A proliferating beta-like cell line has been derived from tissue removed from a child with persistent hyperinsulinaemic hypoglycaemia of infancy and been engineered in culture to secrete insulin in response to glucose. And finally, embryonic stem cells have been shown to adopt islet-like characteristics under defined culture conditions.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Diabetes Mellitus Tipo 2/cirurgia , Ilhotas Pancreáticas/embriologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Transplante de Células , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Transplante das Ilhotas Pancreáticas , Pâncreas/citologia , Pâncreas/embriologia , Transdução de Sinais , Fatores de Transcrição/fisiologiaRESUMO
An RNA-arbitrarily primed PCR differential display strategy was used to identify candidate genes in the pituitary that are up-regulated by endogenously activated gamma-aminobutyric acid (GABA) systems that may also be involved in the control of reproduction. Goldfish were injected with the GABA metabolism inhibitor gamma-vinyl-GABA (GVG), known for its high efficiency to specifically increase endogenous brain and pituitary GABA levels in this species, resulting in higher levels of circulating gonadotropin-II (GTH-II). Several transcripts related to hormone secretion, signal transduction pathways, and messenger RNA (mRNA) editing were shown to be up-regulated after GVG injection. Among these transcripts we characterized an mRNA coding for the secretory vesicle protein secretogranin-II (SgII), a member of the chromogranin family, which is the precursor of a novel 34 amino acid neuropeptide, goldfish secretoneurin (SN). A semiquantitative PCR developed to measure pituitary SgII mRNA levels showed a 5-fold increase in GVG treated fish vs. control fish. Moreover, GVG treatment specifically increased SgII mRNA levels in gonadotrophs, concomitant with a decrease in GTH-II cell content. In addition, i.p. injection of synthetic goldfish SN increased GTH-II release in goldfish pretreated with the dopamine antagonist domperidone. Activation of GABAergic neurons has two effects, enhancing in vivo GTH-II release and up-regulating SgII mRNA specifically in goldfish gonadotrophs. Together with our SN bioactivity data, this suggests the existence in the pituitary of an autocrine or paracrine mechanism linked to the regulated secretory pathway in the gonadotrophs.
Assuntos
Carpa Dourada/metabolismo , Hipófise/metabolismo , Proteínas/genética , RNA Mensageiro/metabolismo , Ácido gama-Aminobutírico/farmacologia , Sequência de Aminoácidos , Animais , Northern Blotting , Cromograninas , DNA Complementar/genética , Inibidores Enzimáticos/farmacologia , Biblioteca Gênica , Dados de Sequência Molecular , Neuropeptídeos/fisiologia , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Secretogranina II , Vigabatrina , Ácido gama-Aminobutírico/análogos & derivadosRESUMO
Expression of the single mouse TSH beta gene gives rise to multiple mRNAs, and we have previously shown that in vitro, one of these mRNAs gives rise to a novel TSH beta-presubunit due to initiation of translation at an in-frame start site unique to this mRNA which is up-stream of the normal start site. The novel presubunit contains a 17-amino acid NH2-terminal extension sequence compared to the normal presubunit. Although this extension sequence does not have the characteristics of a normal signal sequence, the novel TSH beta-presubunit was processed in vitro by microsomal membranes. In this study we have examined the translation product of this mRNA in intact cells and whether in vivo it gives rise to a processed secreted TSH beta-subunit that has an NH2-terminal sequence different from that of the established TSH beta-subunit. Firstly, mRNAs encoding alpha-presubunit and either the normal or novel TSH beta-presubunit were microinjected into Xenopus oocytes, and it was found that immunoprecipitable TSH dimer was secreted into the medium regardless of the mRNA used for TSH beta-subunit synthesis. However, less TSH was obtained when the TSH beta-subunit was derived from the extended TSH beta-presubunit. Secondly, when COS cells were transiently transfected with plasmids expressing alpha-presubunit and either the normal or novel TSH beta-presubunit, secreted TSH was obtained when the TSH beta-subunit was derived from either presubunit. TSH dimer was also obtained when the TSH beta-presubunit was derived from a mRNA encoding the extended presubunit in which the down-stream AUG had been eliminated by site-specific mutagenesis. This demonstrated that the up-stream translation start site was used in the intact cell and that secreted TSH beta-subunit was derived from the extended presubunit and not from normal presubunit resulting from translational readthrough to the down-stream AUG. When secreted TSH beta-subunits derived from the normal and extended TSH beta-presubunits were digested with endoproteinase LysC, the NH2-terminal fragments were similar in size, suggesting that the NH2-terminal extension had little if any effect on the site of cleavage by signal peptidase. Our data, therefore, demonstrate that the longer TSH beta-presubunit is synthesized in vivo and strongly suggest that it is processed in the intact cell to give a mature secreted TSH beta-subunit indistinguishable from that derived from the normal TSH beta-presubunit.
Assuntos
Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Tireotropina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Camundongos , Sondas Moleculares/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Biossíntese de Proteínas , Tireotropina/química , Xenopus laevisRESUMO
We have studied the influence of hypothyroidism and thyroid hormone replacement in vivo on rat pituitary cytoplasmic concentrations of TSH beta and alpha-subunit mRNA, measured by cytoplasmic dot hybridization, as well as circulating TSH and pituitary TSH content. Cytoplasmic concentrations of GH, PRL, and LH beta-subunit mRNA were measured in parallel. Marked increases in serum TSH and TSH beta and alpha mRNA were found in hypothyroidism; these changes reversed 72 h after beginning T3 replacement. A more marked effect of hypothyroidism on TSH beta mRNA than on alpha mRNA was evident, suggesting differential control of regulation of the TSH genes. In contrast to a rapid fall in serum TSH after T3 administration, an increase in TSH beta and alpha mRNA was evident at 1 and 6 h, followed by a later fall in TSH mRNA to euthyroid values. Pituitary cytoplasmic concentrations of GH and PRL mRNA were reduced in hypothyroidism and increased after administration of T3; LH beta mRNA was unaffected by hypothyroidism or T3 replacement. The differential effects of thyroid status on TSH, GH, PRL, and LH mRNA indicate specificity of regulation of these anterior pituitary hormone genes.
Assuntos
Hipotireoidismo/genética , Fragmentos de Peptídeos/genética , Hipófise/fisiologia , Hormônios Adeno-Hipofisários/genética , Tireotropina/genética , Tri-Iodotironina/farmacologia , Animais , Citoplasma/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Subunidade alfa de Hormônios Glicoproteicos , Hormônio do Crescimento/genética , Hormônio Luteinizante/genética , Prolactina/genética , RNA Mensageiro/genética , Ratos , Tireotropina/sangue , Fatores de TempoRESUMO
Carboxypeptidase activity was studied in subcellular fractions from a transplantable rat insulinoma and found to be localised principally in the insulin secretory granule. The activity, which was specific for peptide substrates with C-terminal basic amino acids, appeared to be a single enzyme with Mr 54000. This enzyme differed with respect to size and pH optimum from other basic amino acid-specific carboxypeptidases, such as carboxypeptidases B and N, and may be a secretory granule-specific enzyme involved in propolypeptide processing.
Assuntos
Adenoma de Células das Ilhotas Pancreáticas/enzimologia , Carboxipeptidases/metabolismo , Grânulos Citoplasmáticos/enzimologia , Insulina/metabolismo , Insulinoma/enzimologia , Neoplasias Pancreáticas/enzimologia , Animais , Carboxipeptidases/antagonistas & inibidores , Concentração de Íons de Hidrogênio , RatosRESUMO
A hypervariable region consisting of repeats of a 14 base pair (bp) consensus sequence ACAGGGGT(G/C)(T/C)GGGG is located 363 bp upstream of the human insulin gene. Different repeat numbers of this oligonucleotide give rise to a polymorphism, and so this region is commonly known as the insulin gene linked polymorphic region (ILPR). Here we present evidence, based on the mobility in non-denaturing polyacrylamide gels of two dissimilarly sized oligonucleotides containing the ILPR consensus sequence, that this sequence can adopt a number of quadriplex DNA structures in vitro.
Assuntos
Sequência Consenso , DNA/química , Insulina/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Humanos , Modelos Químicos , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Polimorfismo GenéticoRESUMO
In the human insulin gene, a regulatory sequence upstream of the transcription start site at -229 to -258 (the E2 element) binds a ubiquitous factor USF. The present study led to the identification of a second factor, D0, that binds to an adjacent upstream site, the C2 element, that has previously not been described. The results demonstrate that D0 exhibits similar properties to RIPE3b1, a factor shown to be an important determinant of insulin gene beta-cell-specific expression. Binding of D0 to the C2 element was abolished by the oxidising agent diamide, and the alkylating agent N-ethylmaleimide. The results indicate that expression of the insulin gene may be regulated by a redox-dependent pathway involving RIPE3b1 or a RIPE3b1-like factor.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Insulina/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Sítios de Ligação , Humanos , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Oxirredução , Ratos , Alinhamento de Sequência , Transcrição GênicaRESUMO
A human insulinoma cDNA (PC2) that encodes a protein homologous to the Kex2/subtilisin-like proteinases has recently been described [1990, J. Biol. Chem. 265, 2997-3000]. In order to characterise the associated proteinase activity, mRNA encoding PC2 was synthesised in vitro and microinjected into Xenopus oocytes. The proteinase activity released into the media from oocytes microinjected with PC2 mRNA was assayed using small peptide fluorogenic substrates. Boc.Gln.Arg.Arg aminomethyl coumarin was hydrolysed in a Ca(2+)-dependent manner, but substrate analogues bearing a single basic aminoacid were not. The substrate specificity, inhibitor profile, and pH optimum of 5.5 were compatible with an involvement of PC2 in prohormone processing in mammalian cells.
Assuntos
DNA/genética , Expressão Gênica , Oócitos/metabolismo , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidases/genética , Subtilisinas , Animais , Cálcio/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Insulinoma , Microinjeções , Neoplasias Pancreáticas , Pró-Proteína Convertase 2 , Precursores de Proteínas/metabolismo , RNA Mensageiro/biossíntese , Inibidores de Serina Proteinase , Especificidade por Substrato , Transfecção , Xenopus laevisRESUMO
IUF1 is a pancreatic beta cell-specific factor which binds to the sequence 5'-CPyCTAATG-3' (CT box) within the human insulin gene enhancer. Here we show that IUF1 is composed of 2 binding activities that can be separated by DEAE ion exchange chromatography. South Western blot analysis indicates that these distinct binding activities have apparent molecular weights of 115 kDa and 46 kDa.