RESUMO
BACKGROUND: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions. METHODS: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches. RESULTS: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa. CONCLUSIONS: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract.
Assuntos
Microbiota , Animais , Cavalos/genética , Mucosa Intestinal/metabolismo , Metaboloma , Fezes/microbiologia , Anti-Inflamatórios não Esteroides/metabolismo , Inflamação/metabolismo , Fenilbutazona/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismoRESUMO
We have demonstrated that 0.45% quercetin added to a diet containing corn oil (15% w/w), as the lipid source, and cellulose (6% w/w), as the fiber source, was able to suppress the formation of high multiplicity aberrant crypt foci (ACF > 4 AC/focus), to lower proliferation and enhance apoptosis in a rat model of colon cancer. This experiment determined whether quercetin was acting as an antiinflammatory molecule in an in vivo model of colon cancer. We used weanling (21 d old) Sprague Dawley rats (n = 40) in a 2×2 factorial experiment to determine the influence of quercetin on iNOS, COX-1 and COX-2 expressions, all of which are elevated in colon cancer. Half of the rats received a diet containing either 0 or 0.45% quercetin, and within each diet group, half of the rats were injected with saline or azoxymethane (AOM, 15 mg/kg BW, sc, 2× during wk 3 and 4). The colon was resected 4 wk after the last AOM injection, and the mucosa scraped and processed for RNA isolation. Data from this experiment were analyzed using a mixed model in SAS for main effects and their interaction. AOM injection stimulated (P < 0.0001) iNOS expression. However there was an interaction such that, relative to rats injected with saline, AOM-injected rats consuming diets without quercetin had significantly elevated iNOS expression (5.29-fold), but the expression in AOM-injected rats consuming the diet with quercetin was not significantly elevated (1.68-fold). COX-1 expression was 20.2% lower (P < 0.06) in rats consuming diets containing quercetin. COX-2 expression was 24.3% higher (P < 0.058) in rats consuming diets without quercetin. These data suggest inflammatory processes are elevated in this early stage of colon carcinogenesis, yet quercetin may protect against colon carcinogenesis by down-regulating the expressions of COX-1 and COX-2.
RESUMO
We report the case of a young woman with Crohn's disease of the bowel who presented with a purulent tracheobronchitis and life-threatening upper airway obstruction. Fibreoptic bronchoscopy demonstrated severe tracheal and upper bronchial pseudotumours and stenosis. The role of recent discontinuation of corticosteroids, for quiescent inflammatory bowel disease, in the development of endobronchial disease and the dramatic response in airway patency after reintroduction of prednisolone in this rare complication of Crohn's disease are discussed.
Assuntos
Bronquite/etiologia , Doença de Crohn/complicações , Traqueíte/etiologia , Adulto , Anti-Inflamatórios/uso terapêutico , Bronquite/tratamento farmacológico , Bronquite/fisiopatologia , Broncoscopia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/fisiopatologia , Feminino , Humanos , Prednisolona/uso terapêutico , Testes de Função Respiratória , Traqueíte/tratamento farmacológico , Traqueíte/fisiopatologiaRESUMO
There is now general agreement that the etiology of proximal and distal colon cancers may differ, thus prompting renewed interest in understanding anatomical site-specific molecular mechanisms of tumor development. Using a 2x2x2 factorial design with male Sprague-Dawley rats (corn oil, fish oil; pectin, cellulose; plus or minus azoxymethane injection) we found a greater than 2-fold difference (P < 0.001) in tumor incidence proximally versus distally (prox/dist ratio: corn oil, 2.25; fish oil, 2.61). The purpose of the present study was to determine if the higher degree of proximal versus distal tumors in our model system could be accounted for by differences between these two sites in initial DNA damage, response to that damage or an effect of diet at one site but not the other. DNA damage was assessed by quantitative immunohistochemistry of O(6)-methylguanine adducts; repair by measurement of O(6)-methylguanine-DNA alkyltransferase and removal was determined by measurement of targeted apoptosis. Although overall initial DNA damage was similar at both sites, in the distal colon there was a greater expression of repair protein (P < 0.001) and a greater degree of targeted apoptosis (P < 0.0001). There was also a reduction in DNA damage in the distal colon of rats consuming fish oil. Together, these results suggest that the lower tumor incidence in the distal colon may be a result of the capacity to deal with initial DNA damage by the distal colon, as compared with the proximal colon. Therefore, the determination of site-specific mechanisms in tumor development is important because distinct strategies may be required to protect against cancer at different sites.
Assuntos
Adenocarcinoma/patologia , Azoximetano/farmacologia , Carcinógenos/farmacologia , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/enzimologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/enzimologia , Adutos de DNA , Reparo do DNA/efeitos dos fármacos , Técnicas Imunoenzimáticas , Metabolismo dos Lipídeos , Masculino , Metilação , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Ras proteins are critical regulators of cell function, including growth, differentiation, and apoptosis, with membrane localization of the protein being a prerequisite for malignant transformation. We have recently demonstrated that feeding fish oil, compared with corn oil, decreases colonic Ras membrane localization and reduces tumor formation in rats injected with a colon carcinogen. Because the biological activity of Ras is regulated by posttranslational lipid attachment and its interaction with stimulatory lipids, we investigated whether docosahexaenoic acid (DHA), found in fish oil, compared with linoleic acid (LA), found in corn oil, alters Ras posttranslational processing, activation, and effector protein function in young adult mouse colon cells overexpressing H-ras (YAMC-ras). We show here that the major n-3 polyunsaturated fatty acid (PUFA) constituent of fish oil, DHA, compared with LA (an n-6 PUFA), reduces Ras localization to the plasma membrane without affecting posttranslational lipidation and lowers GTP binding and downstream p42/44(ERK)-dependent signaling. In view of the central role of oncogenic Ras in the development of colon cancer, the finding that n-3 and n-6 PUFA differentially modulate Ras activation may partly explain why dietary fish oil protects against colon cancer development.
Assuntos
Alquil e Aril Transferases/metabolismo , Transformação Celular Neoplásica , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Insaturados/farmacologia , Genes ras/efeitos dos fármacos , Ácido Linoleico/farmacologia , Proteína Oncogênica p21(ras)/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Transformada , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colo , Óleo de Milho , Ativação Enzimática , Ácidos Graxos Ômega-6 , Óleos de Peixe , Lipídeos de Membrana/metabolismo , Camundongos , Proteína Oncogênica p21(ras)/genética , Ácido Palmítico/metabolismo , Fosfolipídeos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Evidence suggests that the majority of lung cancer patients have tumour-derived genetic alterations in circulating plasma DNA, and that this may be developed as a diagnostic tool. To this end, we have studied 60 individuals attending bronchoscopy clinic, with symptoms suspicious of lung cancer, for genetic alterations in bronchial mucosa biopsy (n = 47) and plasma (n = 40) DNA. Thirteen of 47 individuals from whom biopsies were taken displayed allelic loss of heterozygosity (LOH) in biopsy DNA for at least 1 of 4 markers. All 13 of these individuals had neoplastic tumour cells in their biopsies and were subsequently diagnosed with cancer. Thirteen of 40 individuals from whom plasma was taken displayed a plasma DNA LOH, and 12 of these 13 individuals were subsequently diagnosed with cancer. LOH in plasma was generally representative of LOH in the corresponding biopsy. In terms of sensitivity, using just 4 markers, biopsy LOH and plasma LOH were found in 13 of 44 (30%) and 12 of 29 (41%), respectively, of those patients subsequently diagnosed with cancer. Two patients were positive for LOH in plasma samples that pre-dated a diagnosis of cancer by several months. These data suggest that assay of genetic alterations in circulating plasma DNA may be developed as a useful addition to conventional techniques for the diagnosis of lung cancer.
Assuntos
DNA de Neoplasias/genética , Perda de Heterozigosidade , Neoplasias Pulmonares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/sangue , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , DNA de Neoplasias/sangue , Feminino , Marcadores Genéticos/genética , Humanos , Pulmão/patologia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Mucosa Respiratória/patologia , Sensibilidade e EspecificidadeRESUMO
There is epidemiological, clinical, and experimental evidence that dietary fish oil, containing n-3 polyunsaturated fatty acids, protects against colon tumor development. However, its effects on colonocytes in vivo remain poorly understood. Therefore, we investigated the ability of fish oil to modulate colonic methylation-induced DNA damage, repair, and deletion. Sprague Dawley rats were provided with complete diets containing either corn oil or fish oil (15% by weight). Animals were injected with azoxymethane, and the distal colon was removed 3, 6, 9, or 12 h later. Targeted apoptosis and DNA damage were assessed by cell position within the crypt using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay and quantitative immunohistochemical analysis of O6-methylguanine adducts, respectively. Localization and expression of the alkyl group acceptor, O6-methylguanine-DNA-methyltransferase, was also determined. Lower levels of adducts were detected at 6, 9, and 12 h in fish oil- versus corn oil-fed animals (P < 0.05). In addition, fish oil supplementation had the greatest effect on apoptosis in the top one-third of the crypt, increasing the apoptotic index compared with corn oil-fed rats (P < 0.05). In the top one-third of the crypt, fish oil feeding caused an incremental stimulation of apoptosis as adduct level increased. In contrast, a negative correlation between apoptosis and adduct incidence occurred with corn oil feeding (P < 0.05). Diet had no main effect (all tertiles combined) on O6-methylguanine-DNA-methyltransferase expression over the time frame of the experiment. The enhancement of targeted apoptosis combined with the reduced formation of O6-methylguanine adducts may account, in part, for the observed protective effect of n-3 polyunsaturated fatty acids against experimentally induced colon cancer.
Assuntos
Anticarcinógenos/farmacologia , Neoplasias do Colo/prevenção & controle , Adutos de DNA/efeitos dos fármacos , Óleos de Peixe/farmacologia , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Adutos de DNA/biossíntese , Adutos de DNA/química , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Guanina/análogos & derivados , Guanina/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Funções Verossimilhança , Masculino , Ratos , Ratos Sprague-Dawley , Estatísticas não Paramétricas , tRNA Metiltransferases/biossínteseRESUMO
We have recently demonstrated that overexpression of PKC beta(II) renders transgenic mice more susceptible to carcinogen-induced colonic hyperproliferation and aberrant crypt foci formation. In order to further investigate the ability of PKC beta(II) to modulate colonocyte cytokinetics, we determined the localization of PKC beta(II) with respect to cell proliferation and apoptosis along the entire colonic crypt axis following carcinogen and diet manipulation. Rats were provided diets containing either corn oil [containing n-6 polyunsaturated fatty acids (PUFA)] or fish oil (containing n-3 PUFA), cellulose (non-fermentable fiber) or pectin (fermentable fiber) and injected with azoxymethane (AOM) or saline. After 16 weeks, an intermediate time point when no macroscopic tumors are detected, colonic sections were utilized for immunohistochemical image analysis and immunoblotting. Cell proliferation was measured by incorporation of bromodeoxyuridine into DNA and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. In the distal colon, PKC beta(II) staining was localized to the upper portion of the crypt. In comparison, proximal crypts had more (P < 0.05) staining in the lower tertile. AOM enhanced (P < 0.05) PKC beta(II) expression in all regions of the distal colonic crypt (upper, middle and lower tertiles). There was also an interaction (P < 0.05) between dietary fat and fiber on PKC beta(II) expression (corn/pectin > fish/cellulose, fish/pectin > corn/cellulose) in all regions of the distal colonic crypt. With respect to colonic cell kinetics, proliferation paralleled the increase in PKC beta(II) expression in carcinogen-treated animals. In contrast, apoptosis at the lumenal surface was inversely proportional to PKC beta(II) expression in the upper tertile. These results suggest that an elevation in PKC beta(II) expression along the crypt axis in the distal colon is linked to enhancement of cell proliferation and suppression of apoptosis, predictive intermediate biomarkers of tumor development. Therefore, select dietary factors may confer protection against colon carcinogenesis in part by blocking carcinogen-induced PKC beta(II) expression.
Assuntos
Apoptose/fisiologia , Colo/citologia , Colo/enzimologia , Dieta , Isoenzimas/biossíntese , Proteína Quinase C/biossíntese , Animais , Apoptose/efeitos dos fármacos , Azoximetano , Carcinógenos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Celulose/farmacologia , Colo/efeitos dos fármacos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Óleo de Milho/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Imuno-Histoquímica , Masculino , Pectinas/farmacologia , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Proteína Quinase C beta , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/enzimologiaRESUMO
A case is reported of a 59 year old woman who presented with palpitations. Electrocardiographic studies revealed atrial fibrillation and atrioventricular block. Echocardiography and magnetic resonance imaging showed a right atrial cystic mass attached to the interatrial septum. The patient underwent surgical excision of the mass. Histopathological findings were of a cystic tumour of the atrioventricular nodal region. This is the second report of this condition diagnosed antemortem and treated successfully with surgical excision.
Assuntos
Arritmias Cardíacas/etiologia , Nó Atrioventricular , Bloqueio Cardíaco/etiologia , Neoplasias Cardíacas/cirurgia , Mesotelioma/cirurgia , Eletrocardiografia , Feminino , Neoplasias Cardíacas/patologia , Humanos , Mesotelioma/patologia , Pessoa de Meia-IdadeRESUMO
Polymorphic genes for the peroxide scavenger glutathione peroxidase I (GPX1) and 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase/apurinic (AP) lyase (hOGG1) map to loci on chromosome 3p which are subject to frequent loss of heterozygosity (LOH) in lung tumours. Levels of the pro-mutagenic, oxidative DNA lesion 8-OHdG, were measured in 37 paired normal and tumorous lung specimens using HPLC with electrochemical detection. Lung tumours were also analysed for 3p LOH by fluorescent PCR with Genescan analysis. No significant difference was observed between 8-OHdG levels in tumour [7.7 +/- 6.7 (mean +/- SE) 8-OHdG/10(6) 2'-deoxyguanosine (dG)] and normal (8.1 +/- 8.8 8-OHdG/10(6) dG) lung tissue. Adduct levels in normal lung tissue DNA were not associated with constitutive hOGG1 genotype although there was a trend towards lower 8-OHdG levels in individuals possessing the ALA6 GPX1 polymorphism. Lung tumours exhibiting 3p LOH (40%) contained higher levels of 8-OHdG adducts (10.9 +/- 2.6 8-OHdG/10(6) dG) (P = 0.05) and lower GPX1 enzyme activity [45.5 nmol glutathione (GSH)/min/mg] (P = 0.09) when compared with tumours without LOH at these sites (5.55 +/- 0.87 8-OHdG/10(6) dG and 63.6 nmol GSH/min/mg, respectively). In conclusion, tumours with 3p LOH at loci associated with hOGG1 and GPX1 appear to have compromised oxidative defence mechanisms as measured by reduced GPX1 enzyme activity and elevated 8-OHdG levels and this may affect the prognosis of lung cancer patients.
Assuntos
Adenocarcinoma/genética , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 3/genética , Desoxiguanosina/análogos & derivados , Glutationa Peroxidase/genética , Isoenzimas/genética , Neoplasias Pulmonares/genética , N-Glicosil Hidrolases/genética , Proteínas de Neoplasias/genética , 8-Hidroxi-2'-Desoxiguanosina , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Substituição de Aminoácidos , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Adutos de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , DNA-Formamidopirimidina Glicosilase , Desoxiguanosina/análise , Feminino , Genótipo , Glutationa Peroxidase/fisiologia , Humanos , Isoenzimas/fisiologia , Perda de Heterozigosidade , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , N-Glicosil Hidrolases/fisiologia , Proteínas de Neoplasias/fisiologia , Estresse Oxidativo , Polimorfismo Genético , Fumar/efeitos adversosRESUMO
We have characterized the cell movements and prospective cell identities as neural folds fuse during neural tube formation in Xenopus laevis. A newly developed whole-mount, two-color fluorescent RNA in situ hybridization method, visualized with confocal microscopy, shows that the dorsal neural tube gene xpax3 and the neural-crest-specific gene xslug are expressed far lateral to the medial site of neural fold fusion and that expression moves medially after fusion. To determine whether cell movements or dynamic changes in gene expression are responsible, we used low-light videomicroscopy followed by fluorescent in situ and confocal microscopy. These methods revealed that populations of prospective neural crest and dorsal neural tube cells near the lateral margin of the neural plate at the start of neurulation move to the dorsal midline using distinctive forms of motility. Before fold fusion, superficial neural cells apically contract, roll the neural plate into a trough and appear to pull the superficial epidermal cell sheet medially. After neural fold fusion, lateral deep neural cells move medially by radially intercalating between other neural cells using two types of motility. The neural crest cells migrate as individual cells toward the dorsal midline using medially directed monopolar protrusions. These movements combine the two lateral populations of neural crest into a single medial population that form the roof of the neural tube. The remaining cells of the dorsal neural tube extend protrusions both medially and laterally bringing about radial intercalation of deep and superficial cells to form a single-cell-layered, pseudostratified neural tube. While ours is the first description of medially directed cell migration during neural fold fusion and re-establishment of the neural tube, these complex cell behaviors may be involved during cavitation of the zebrafish neural keel and secondary neurulation in the posterior axis of chicken and mouse.
Assuntos
Sistema Nervoso Central/embriologia , Proteínas de Xenopus , Xenopus laevis/embriologia , Proteínas de Peixe-Zebra , Animais , Padronização Corporal/genética , Movimento Celular/genética , Sistema Nervoso Central/citologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , Camundongos , Microscopia de Vídeo , Crista Neural/citologia , Crista Neural/embriologia , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Xenopus laevis/genéticaRESUMO
Protein kinase C betaII (PKC betaII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC betaII function in vivo, we generated transgenic mice that overexpress PKC betaII in the intestinal epithelium. Transgenic PKC betaII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC betaII mice exhibit elevated colonic beta-catenin levels and decreased glycogen synthase kinase 3beta activity, indicating that PKC betaII stimulates the Wnt/adenomatous polyposis coli (APC)/beta-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC betaII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta-catenin signaling pathway.
Assuntos
Colo/patologia , Neoplasias do Colo/etiologia , Isoenzimas/fisiologia , Proteína Quinase C/fisiologia , Transativadores , Animais , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Mucosa Intestinal/citologia , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Transdução de Sinais , beta CateninaRESUMO
Computer simulations showed that the elastic modulus of the cell layer relative to the elastic modulus of the extracellular layers predicted the effectiveness of different force-generating mechanisms for sea urchin primary invagination [L. A. Davidson, M. A. R. Koehl, R. Keller, and G. F. Oster (1995) Development 121, 2005-2018]. Here, we measured the composite elastic modulus of the cellular and extracellular matrix layers in the blastula wall of Strongylocentrotus purpuratus embryos at the mesenchyme blastula stage. Combined, these two layers exhibit a viscoelastic response with an initial stiffness ranging from 600 to 2300 Pa. To identify the cellular structures responsible for this stiffness we disrupted these structures and correlated the resulting lesions to changes in the elastic modulus. We treated embryos with cytochalasin D to disrupt the actin-based cytoskeleton, nocodazole to disrupt the microtubule-based cytoskeleton, and a gentle glycine extraction to disrupt the apical extracellular matrix (ECM). Embryos treated less than 60 min in cytochalasin D showed no change in their time-dependent elastic modulus even though F-actin was severely disrupted. Similarly, nocodazole had no effect on the elastic modulus even as the microtubules were severely disrupted. However, glycine extraction resulted in a 40 to 50% decrease in the elastic modulus along with a dramatic reduction in the hyalin protein at the apical ECM, thus implicating the apical ECM as a major mechanical component of the blastula wall. This finding bears on the mechanical plausibility of several models for primary invagination.
Assuntos
Blastocisto/fisiologia , Actinas/fisiologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/ultraestrutura , Simulação por Computador , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Elasticidade , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/fisiologia , Embrião não Mamífero/ultraestrutura , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/ultraestrutura , Glicina/farmacologia , Matemática , Microtúbulos/efeitos dos fármacos , Modelos Biológicos , Morfogênese/fisiologia , Nocodazol/farmacologia , Ouriços-do-Mar/embriologia , ViscosidadeRESUMO
Epidemiological and experimental data suggest that dietary fiber and fat are major determinants of colorectal cancer. However, the mechanisms by which these dietary constituents alter the incidence of colon cancer have not been elucidated. Evidence indicates that dominant gain-of-function mutations short-circuit protooncogenes and contribute to the pathogenesis of cancer. Therefore, we began to dissect the mechanisms whereby dietary fat and fiber, fed during the initiation, promotion and progression stages of colon tumorigenesis, regulate ras p21 localization, expression and mutation frequency. Male Sprague-Dawley rats (140) were provided with corn oil or fish oil and pectin or cellulose plus or minus the carcinogen azoxymethane (AOM) in a 2 x 2 x 2 factorial design and killed after 34 weeks. We have previously shown adenocarcinoma incidence in these animals to be 70.3% (52/74) for corn oil + AOM and 56.1% (37/66) for fish oil + AOM (P < 0.05). Total ras expression as well as ras membrane:cytosol ratio was 4- to 6-fold higher in colon tumors than in mucosa from AOM- or saline-injected rats. Expression of ras in the mucosal membrane fraction was 13% higher for animals fed corn oil compared with fish oil feeding (P < 0.05), which is noteworthy since ras must be localized at the plasma membrane to function. The elevated ras membrane:cytosol ratio in tumors was not due to increased farnesyl protein transferase activity or prenylation state, as nearly all detectable ras was in the prenylated form. Phosphorylated p42 and p44 mitogen activated protein kinase (ERK) expression was two-fold higher in tumor extracts compared with uninvolved mucosa from AOM- and saline-injected rats (P < 0.05). The frequency of K-ras mutations was not significantly different between the various groups, but there was a trend toward a greater incidence of mutations in tumors from corn oil fed rats (85%) compared with fish oil fed rats (58%). Our results indicate that the carcinogen-induced changes in ras expression and membrane localization are associated with the in vivo activation of the ERK pathway. In addition, suppression of tumor development by dietary n-3 polyunsaturated fatty acids may be partly due to a combined effect on colonic ras expression, membrane localization, and mutation frequency.
Assuntos
Azoximetano/efeitos adversos , Carcinógenos/efeitos adversos , Colo/efeitos dos fármacos , Lipídeos/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/efeitos dos fármacos , Alquil e Aril Transferases/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Gorduras na Dieta/farmacologia , Fibras na Dieta/farmacologia , Ativação Enzimática/efeitos dos fármacos , Farnesiltranstransferase , Genes ras/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Mutação , Prenilação de Proteína/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
We report novel chemical properties of the ribozyme derived from the smallest group I intron (subgroup IC3) that comes from the pre-tRNA(Ile) of the bacterium Azoarcus sp. BH72. Despite the small size of the Azoarcus ribozyme (195 nucleotides (nt)), it binds tightly to the guanosine nucleophile (Kd = 15 +/- 3 microM) and exhibits activity at high temperatures (approximately 60-70 degrees C). These features may be due to the two GA3 tetraloop interactions postulated in the intron and the high GC content of the secondary structure. The second order rate constant for the Azoarcus ribozyme, ((k(cat)/Km)S = 8.4 +/- 2.1 x 10(-5) M(-1) min(-1)) is close to that found for the related ribozyme derived from the pre-tRNA(Ile) of the cyanobacterium Anabaena PCC7120. pH dependence studies and kinetic analyses of deoxy-substituted substrates suggest that the chemical cleavage step is the rate-determining process in the Azoarcus ribozyme. This may be due to the short 3-nt guide sequence-substrate pairing present in the Azoarcus ribozyme. Finally, the Azoarcus ribozyme shares features conserved in other group I ribozymes including the pH profile, the stereospecificity for the Rp-phosphorothioate at the cleavage site and the 1000-fold decrease in cleavage rate with a deoxyribonucleoside leaving group.
Assuntos
Azoarcus/enzimologia , Guanosina/metabolismo , RNA Catalítico/metabolismo , Azoarcus/genética , Sequência de Bases , Desoxirribonucleotídeos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese , Conformação de Ácido Nucleico , Compostos Organofosforados/metabolismo , RNA Catalítico/química , RNA Catalítico/genética , Especificidade por SubstratoRESUMO
AIMS: To describe a third case of congenital pulmonary acinar dysplasia, comparing its clinicopathological features with those of the two previous cases and with cystic adenomatoid malformations. METHODS AND RESULTS: An externally normal female infant was born to a 25-year-old after a normal pregnancy. Ventilation was not established. At autopsy the hypoplastic lungs showed bronchial development but no acinar development, resembling the pseudoglandular phase of 16 weeks gestation. As in the previous cases the infant was female, born at term with unaffected older sibling(s): however, no intrapulmonary haematopoietic tissue was identified in the present case. Cystic adenomatoid malformations also resemble microscopically the pseudoglandular phase but are focal pulmonary hyperplasias rather than generalized pulmonary hypoplasias. CONCLUSION: Congenital acinar dysplasia is a rare, apparently sporadic lethal developmental defect resulting in pulmonary hypoplasia due to failure of lung development beyond the pseudoglandular phase seen at 16 weeks gestation.
Assuntos
Alvéolos Pulmonares/anormalidades , Anormalidades Múltiplas/etiologia , Brônquios/anormalidades , Brônquios/patologia , Evolução Fatal , Feminino , Humanos , Recém-Nascido , Pulmão/anormalidades , Pneumopatias/congênito , Pneumopatias/patologia , Alvéolos Pulmonares/patologiaRESUMO
We have developed a non-invasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers. In this study, we used the rat colon carcinogenesis model to determine if changes in fecal protein kinase C (PKC) expression have predictive value in monitoring the neoplastic process. Weanling rats were injected with saline or azoxymethane (AOM) and 36 weeks later fecal samples and mucosa were collected, poly A+ RNA isolated, and quantitative RT-PCR performed using primers to PKC betaII and zeta. Fecal PKC betaII and zeta mRNA levels were altered by the presence of a tumor, with tumor-bearing animals having a 3-fold higher (P < 0.05) PKC betaII expression as compared with animals without tumors. In addition, AOM-injection increased mucosal PKC betaII mRNA expression compared with saline controls. No effect of tumor incidence on mucosal PKC betaII expression was observed. In contrast, fecal PKC zeta expression was 2.5-fold lower (P < 0.05) in animals injected with azoxymethane versus saline. Since tumor incidence exerts a reciprocal effect on fecal PKC betaII and zeta mRNA expression, data were also expressed as the ratio between PKC betaII and zeta. The isozyme ratio was strongly related to tumor incidence, i.e. ratio for animals with tumors was 2.18 +/- 1.25, animals without tumors was 0.50 +/- 0.16, P = 0.025. We demonstrate that the expression of fecal PKC betaII and zeta may serve as a noninvasive marker for development of colon tumors. A sensitive technique for the detection of colon cancer is of importance since early diagnosis can substantially reduce mortality.
Assuntos
Neoplasias do Colo/diagnóstico , Fezes/química , Isoenzimas/isolamento & purificação , Proteína Quinase C/isolamento & purificação , Animais , Azoximetano , Biomarcadores , Neoplasias do Colo/induzido quimicamente , Mucosa Intestinal/química , Isoenzimas/genética , Masculino , Reação em Cadeia da Polimerase , Proteína Quinase C/genética , Proteína Quinase C beta , RNA Mensageiro/análise , RNA Mensageiro/efeitos dos fármacos , RNA Neoplásico/análise , RNA Neoplásico/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
We investigated the utility of noninvasive technology utilizing feces containing exfoliated colonocytes to determine whether changes in fecal fatty acid-binding proteins have predictive value in monitoring the neoplastic process. Ninety male Sprague-Dawley rats were randomly divided into four groups in a 2 x 2 factorial design, with two dietary fiber sources (wheat bran or oat bran) and two treatment groups (injection with a carcinogen, azoxymethane, or saline). Fresh fecal samples were collected at Week 16 postinjection, and tumor frequency was determined at Week 36 of the study. Semiquantitative "mimic" reverse transcriptase polymerase chain reaction was used to quantitate the expression of liver fatty acid-binding protein (L-FABP), intestinal fatty acid-binding protein (i-FABP), and acyl CoA-binding protein (ACBP) mRNA in fecal samples to establish their prognostic value. Rats fed wheat bran diets had a lower incidence of tumors (p < 0.05). There was no effect of carcinogen injection or tumor incidence on the expression of L-FABP, i-FABP, or ACBP mRNA, L-FABP and i-FABP mRNA expression were significantly higher (p < 0.05) in feces from animals fed a wheat bran diet than in feces from animals fed an oat bran diet. In contrast, the expression of ACBP mRNA was significantly lower (p < 0.05) in animals fed a wheat bran diet than in animals fed an oat bran diet. Wheat bran also increased (p < 0.05) the total excretion of L-FABP, i-FABP, and ACBP over a 48-hour period. These data suggest that exfoliated colonocyte fatty acid-binding protein mRNA status may provide insight into the mechanisms by which diet influences colonic physiology.
Assuntos
Proteínas de Transporte/genética , Colo/metabolismo , Neoplasias do Colo/metabolismo , Fibras na Dieta/farmacologia , Expressão Gênica , Proteína P2 de Mielina/genética , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Animais , Avena , Inibidor da Ligação a Diazepam , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Fezes/química , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Masculino , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TriticumRESUMO
OBJECTIVES: To determine the incidence and case fatality of acute upper gastrointestinal haemorrhage in the west of Scotland and to identify associated factors. DESIGN: Case ascertainment study. SETTING: All hospitals treating adults with acute upper gastrointestinal haemorrhage in the west of Scotland. SUBJECTS: 1882 patients aged 15 years and over treated in hospitals for acute upper gastrointestinal haemorrhage during a six month period. MAIN OUTCOME MEASURES: Incidence of acute upper gastrointestinal haemorrhage per 100,000 population per year, and case fatality. RESULTS: The annual incidence was 172 per 100,000 people aged 15 and over. The annual population mortality was 14.0 per 100,000. Both were higher among elderly people, men, and patients resident in areas of greater social deprivation. Overall case fatality was 8.2%. This was higher among those who bled as inpatients after admission for other reasons (42%) and those admitted as tertiary referrals (16%). Factors associated with increased case fatality were age, uraemia, pre-existing malignancy, hepatic failure, hypotension, cardiac failure, and frank haematemesis or a history of syncope at presentation. Social deprivation, sex, and anaemia were not associated with increased case fatality after adjustment for other factors. CONCLUSIONS: The incidence of acute upper gastrointestinal haemorrhage was 67% greater than the highest previously reported incidence in the United Kingdom, which may be partially attributable to the greater social deprivation in the west of Scotland and may be related to the increased prevalence of Helicobacter pylori. Fatality after acute upper gastrointestinal haemorrhage was associated with age, comorbidity, hypotension, and raised blood urea concentrations on admission. Although deprivation was associated with increased incidence, it was not related to the risk of fatality.
Assuntos
Duodenopatias/epidemiologia , Doenças do Esôfago/epidemiologia , Hemorragia Gastrointestinal/epidemiologia , Gastropatias/epidemiologia , Adolescente , Adulto , Idoso , Duodenopatias/mortalidade , Doenças do Esôfago/mortalidade , Feminino , Hemorragia Gastrointestinal/mortalidade , Infecções por Helicobacter/epidemiologia , Helicobacter pylori , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Escócia/epidemiologia , Distribuição por Sexo , Fatores Socioeconômicos , Gastropatias/mortalidade , Taxa de SobrevidaRESUMO
Intrahepatic splenic tissue is uncommon being reported to date in three humans and one pig. This report is of a 54 year old man with chronic asthma who died from acute bronchial asthma. Twenty years previously he had undergone a splenectomy (the spleen was histologically normal). Necropsy revealed a well defined, smooth bordered, bilobed red mass on the left hepatic lobe; one lobe projected outwards the other was embedded in the liver. Histologically the mass was splenic tissue. This case of intrahepatic splenic tissue differing from the three human cases reported previously in that there was a common capsule beneath which splenic pulp directly abuts on hepatic tissue. This suggests that this case is more probably one of hyperplasia of congenitally ectopic splenic tissue following splenectomy rather than limited splenosis after implantation onto the serosal surface of splenic tissue released by trauma. Splenic ectopia should be considered in the differential diagnosis of hepatic lesions detected post-splenectomy and the liver should be considered as a possible site of residual splenic tissue if splenic function returns following splenectomy.