RESUMO
Although mut ENT departments in India are now departments of OtolaryHifniogy-Head and Neck surgery there is no uniform structured training programme to ensure that the postgraduates will receive a thorough grounding in the various subspecialties that make up modern otolaryngology. .4 model programme is presented which may serve as a basis for future discussion.
RESUMO
Regional and site-specific distribution of fifty cases of Laryngeal carcinoma is presented along with a review of the avialable literature.
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The mechanisms by which neuroendocrine stimulants regulate CCK gene transcription are unclear. We examined promoter activation by pituitary adenylate cyclase-activating polypeptide (PACAP), a known CCK secretagogue, in the enteroendocrine cell line STC-1. The promoter region from -70 to -87 bp, relative to the transcriptional start site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CRE binding protein (CREB) and AP1. PACAP (with IBMX) stimulated expression of an 87-bp construct 3.35+/-0.36-fold but had no effect on a -70 construct. The effect was blocked by the protein kinase A inhibitor H-89 and by a dominant-negative CREB plasmid. Mutation of the CRE/AP1 site to a canonical CRE site did not affect the response to PACAP, but mutation to a canonical AP1 site prevented it. CREB phosphorylation was increased after PACAP treatment. Electrophoretic mobility shift assay and supershift analysis revealed that CREB and not AP1 bound to the CRE/AP1 site and that PACAP increased the proportion of phosphorylated CREB that was bound. We conclude that PACAP increases CCK gene expression via a cAMP-mediated pathway involving CREB phosphorylation by protein kinase A and activation of a composite CRE/AP1 site.
Assuntos
Colecistocinina/genética , Neuropeptídeos/genética , Sulfonamidas , Transcrição Gênica/fisiologia , Animais , Cálcio/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletroforese , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes Reporter , Isoquinolinas/farmacologia , Luciferases/genética , Camundongos , Mutagênese/fisiologia , Tumores Neuroendócrinos , Sondas de Oligonucleotídeos , Fosforilação , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Regiões Promotoras Genéticas/fisiologia , Ratos , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: Hypergastrinemia occurs frequently in association with acid suppression and Helicobacter infection, but its role in the progression to gastric atrophy and gastric cancer has not been well defined. METHODS: The effects of hypergastrinemia, and possible synergy with Helicobacter felis infection, were investigated in insulin-gastrin (INS-GAS) transgenic mice. RESULTS: INS-GAS mice initially showed mild hypergastrinemia, increased maximal gastric acid secretion, and increased parietal cell number but later progressed to decreased parietal cell number and hypochlorhydria. Development of gastric atrophy was associated with increased expression of growth factors, heparin-binding epidermal growth factor and transforming growth factor alpha. At 20 months of age, INS-GAS mice showed no evidence of increased enterochromaffin-like cell number, but instead exhibited gastric metaplasia, dysplasia, carcinoma in situ, and gastric cancer with vascular invasion. Invasive gastric carcinoma was observed in 6 of 8 INS-GAS mice that were >20 months old. Helicobacter felis infection of INS-GAS mice led to accelerated (< or = 8 mo) development of intramucosal carcinoma (85%), with submucosal invasion (54%) and intravascular invasion (46%; P < or = 0.05). CONCLUSIONS: These findings support the unexpected conclusion that chronic hypergastrinemia in mice can synergize with Helicobacter infection and contribute to eventual parietal cell loss and progression to gastric cancer.
Assuntos
Gastrinas/sangue , Gastrite Atrófica/sangue , Infecções por Helicobacter/sangue , Neoplasias Gástricas/sangue , Neoplasias Gástricas/microbiologia , Animais , Contagem de Células , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/patologia , Ácido Gástrico/metabolismo , Gastrite Atrófica/microbiologia , Heparina/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Hiperplasia , Hipertrofia , Peptídeos e Proteínas de Sinalização Intercelular , Metaplasia , Camundongos , Camundongos Transgênicos , Neoplasias Gástricas/patologia , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The human histidine decarboxylase gene is regulated by gastrin through a cis-acting element known as the gastrin response element (GAS-RE) that was initially localized to a site (+2 to +24) downstream of the transcriptional start site. Electrophoretic mobility shift assays using sequentially deleted DNA probes and nuclear extracts from AGS-B gastric cancer cells showed that the GAS-RE is actually composed of two overlapping binding sites (GAS-RE1, +1 to +19; and GAS-RE2, +11 to +27) that bind distinct nuclear factors. Reporter gene assays demonstrated that each element alone could confer gastrin responsiveness, but the presence of both elements was required for complete gastrin response. Stimulation of AGS-B cells with gastrin for 10-20 min resulted in a >2-fold increase in factor binding. The binding was inhibited by pretreatment of AGS-B cells with cycloheximide and the MEK1 inhibitor PD98059, indicating a requirement for protein synthesis and also indicating that activation occurs through the MEK/mitogen-activated protein kinase pathway. UV cross-linking and Southwestern blot analysis showed that GAS-RE1 bound a 52-kDa protein, whereas GAS-RE2 bound a 35-kDa protein. Hence, activation of histidine decarboxylase gene promoter activity by gastrin is most likely mediated by two separate nuclear factors.
Assuntos
Gastrinas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Histidina Descarboxilase/genética , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Nucleares/genética , Sequência de Bases , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , MAP Quinase Quinase 1 , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Chromogranin A (CgA) is a multifunctional acidic protein that in the stomach is expressed predominantly in enterochromaffin-like cells (ECL cells) where it is regulated by gastrin. In order to investigate the transcriptional response of the mouse CgA (mCgA) promoter to gastrin stimulation, we studied a 4.8-kilobase mCgA promoter-luciferase reporter gene construct in transiently transfected AGS-B cells. 5'-Deletion analysis and scanning mutagenesis of mCgA 5'-flanking DNA showed that a Sp1/Egr-1 site spanning -88 to -77 base pairs (bp) and a cyclic AMP-responsive element (CRE) at -71 to -64 bp are essential for gastrin-dependent mCgA transactivation. Gastrin stimulation increased cellular Sp1 protein levels and Sp1-binding to the mCgA -88 to -77 bp element, as well as binding of CREB to its consensus motif at -71 to -64 bp. Gastrin also stimulated CREB Ser-133 phosphorylation, and abundance of cellular CREB protein levels. Overexpression of either Sp1 or phosphorylated CREB transactivated the mCgA promoter dose dependently, while coexpression of both transcription factors resulted in an additive mCgA promoter response. mCgA -92 to -64 bp, comprising the Sp1/Egr-1 site and the CRE motif, conferred gastrin responsiveness to a heterologous thymidine kinase promoter system, and therefore functions as a "true" enhancer element. This report demonstrates that Sp1 and CREB mediate CCK-B/gastrin receptor-dependent gene regulation, and that the effect of gastrin on the CgA gene is brought about by cooperative action of both transcription factors.
Assuntos
Cromograninas/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Gastrinas/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas Imediatamente Precoces , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Neoplasias Gástricas/genética , Animais , Sequência de Bases , Cromogranina A , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Gastrinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Cinética , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , Neoplasias Gástricas/metabolismo , Timidina Quinase/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células Tumorais CultivadasRESUMO
There is considerable evidence that glomerular deposits in Heymann nephritis, a rat model of membranous nephritis, result from shedding of immune complexes formed on podocytes and that the principal antigen is part of the extracellular domain of a cell surface glycoprotein receptor called gp330 or megalin. It has also been reported that the immunogen that induces Heymann nephritis is a complex formed between gp330 and the receptor-associated protein RAP. The recent elucidation of the primary structure of gp330 makes it possible to investigate the ability of defined portions of gp330, devoid of RAP, to induce Heymann nephritis. In the present study we show that a gp330-glutathione-S-transferase fusion protein, containing 137 amino acid residues (1114 to 1250) of the ectodomain, induces active Heymann nephritis and that heterologous antibodies against this fusion protein produce passive Heymann nephritis. By immunofluorescence, typical glomerular immunoglobulin deposits were found, but complement components were lacking and the rats did not develop proteinuria. In the active model, we obtained evidence indicating that the deposits contained portions of the ectodomain of gp330, including regions other than those of the fusion protein. Thus, the deposits were stained by polyclonal antibodies to gp330 and to the gp330 fusion protein, as well as by two monoclonal antibodies reactive with portions of the ectodomain of gp330, only one of which reacted with the fusion protein in vitro. Antibodies against the cytoplasmic domain of gp330 did not stain. Furthermore, we found that RAP was able to bind to gp330 in the glomerular deposits but not to the gp330 fusion protein in vitro. The results show that the region of gp330 spanning amino acid residues 1114 to 1250 contains peptides capable of inducing pathogenic antibodies of Heymann nephritis without a contributory role of RAP.
Assuntos
Glomerulonefrite/etiologia , Glicoproteínas de Membrana/fisiologia , Aminoácidos/análise , Animais , Anticorpos/análise , Anticorpos/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitopos/análise , Epitopos/imunologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Glomerulonefrite/imunologia , Glomerulonefrite/fisiopatologia , Complexo Antigênico da Nefrite de Heymann , Rim/química , Rim/fisiopatologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/imunologia , Ratos , Ratos Endogâmicos LewRESUMO
Bovine heart mitochondrial coupling factor B was isolated and purified to homogeneity in its active form. The amino-terminal amino acid sequence of the alkylated protein was determined. Two chains with exactly the same sequence except for the presence of an additional Phe at the amino-terminus on one of them were obtained. The 55 amino acid sequence appears to be largely hydrophilic with several charged amino acid residues. This sequence showed no homology with the E. coli unc operon, oligomycin sensitivity conferring protein, or coupling factor 6 or any protein in the data base.
Assuntos
Adenosina Trifosfatases/química , Mitocôndrias Cardíacas/química , ATPases Mitocondriais Próton-Translocadoras , Fatores Acopladores da Fosforilação Oxidativa , Sequência de Aminoácidos , Animais , Bovinos , Dados de Sequência Molecular , Peso MolecularRESUMO
The pathogenesis of Heymann nephritis, a rat model of human membranous glomerulonephritis, depends on the interaction of autoantibodies with a renal glycoprotein (GP330) on glomerular podocytes. Partial complementary DNAs coding for GP330 were isolated and sequenced. The deduced amino acid sequence from 4.3 kilobases of complementary DNA contains the sequences identical to two peptides derived from the isolated glycoprotein. The deduced amino acid sequence of this protein contains regions with homology to the human low density lipoprotein (LDL) receptor, an indication that GP330 and the LDL receptor may be members of the same gene family. Autoantibodies from the kidneys of rats with Heymann nephritis reacted with a nonglycosylated segment of GP330 that contains cysteine-rich 40-amino acid repeats, which are also features of the LDL receptor. GP330 is also similar in some regions to the mouse epidermal growth factor precursor.
Assuntos
Autoanticorpos/genética , Glomerulonefrite/imunologia , Glicoproteínas de Membrana/genética , Receptores de LDL/genética , Sequência de Aminoácidos , Animais , DNA/genética , Glomerulonefrite/genética , Complexo Antigênico da Nefrite de Heymann , Humanos , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Valores de Referência , Homologia de Sequência do Ácido NucleicoRESUMO
A modified procedure for the purification of E. coli galactose-1-phosphate uridyl transferase (E.C. 2.7.6.12) was developed which reproducibly gives pure enzyme. The purified enzyme was shown to be a dimeric protein with a subunit molecular weight of 41,000 and its amino acid composition and content of free sulfhydryl groups were determined. The N-terminal and C-terminal amino acid sequences were found to be NH2-thr-gln-phe-asn-pro-val-asp and -ser(val leu)-ala-COOH respectively. This N-terminal sequence allowed the identification of the start of the transferase gene in the DNA sequence determined by GRINDLEY. Furthermore it appears to define a nine base intercistronic region between the epimerase and transferase genes.