RESUMEN
Transforming growth factor alpha (TGF alpha) shares with epidermal growth factor (EGF) structural homology (35%), a common cell-surface membrane receptor (TGF alpha/EGF receptor), and a nearly identical spectrum of biological activity, including inhibition of gastric acid secretion. Herein, we report expression of TGF alpha mRNA in normal gastric mucosa of the adult guinea pig, rat, and dog. TGF alpha mRNA was also detected in matched surgically resected gastric mucosa and adjacent gastric carcinoma from 10 patients, and in gastric mucosa adjacent to a benign ulcer from an additional patient. TGF alpha protein was quantitated by radioimmunoassay and was present in tumor and adjacent mucosa. TGF alpha/EGF receptor mRNA was also detected in gastric mucosa from all species studied. Localization of TGF alpha and TGF alpha/EGF receptor mRNA expression was examined in samples of unfractionated guinea pig gastric mucosa and from chief cell-enriched and parietal cell-enriched fractions. All samples exhibited TGF alpha and TGF alpha/EGF receptor expression. The TGF alpha signal was greatest in the parietal cell fraction (5.8-fold increase), but was also enhanced in the chief cell fraction (1.9-fold increase) relative to the unfractionated gastric mucosa. Like TGF alpha expression, TGF alpha/EGF receptor mRNA expression was most intense in the parietal cell-enriched fraction (7.8-fold increase), but was also increased in the chief cell-enriched fraction (2.7-fold increase) relative to the unfractionated guinea pig gastric mucosa. We conclude that TGF alpha and TGF alpha/EGF receptor genes are expressed in normal adult mammalian gastric mucosa. These findings, when interpreted in light of described actions of TGF alpha and EGF, provide evidence that local production of TGF alpha could play an important role in the regulation of acid secretion and mucosal renewal in the stomach.
Asunto(s)
Receptores ErbB/aislamiento & purificación , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Factores de Crecimiento Transformadores/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Animales , Northern Blotting , Carcinoma/análisis , Carcinoma/metabolismo , Sondas de ADN , Perros , Receptores ErbB/fisiología , Mucosa Gástrica/fisiología , Cobayas , Humanos , Persona de Mediana Edad , ARN Mensajero/aislamiento & purificación , Ratas , Neoplasias Gástricas/análisis , Neoplasias Gástricas/metabolismo , Factores de Crecimiento Transformadores/metabolismo , Factores de Crecimiento Transformadores/fisiologíaRESUMEN
Transforming growth factor alpha and beta 1 (TGF alpha and TGF beta 1) are representative members of two distinct and expanding families of polypeptide growth factors. TGF alpha is an epithelial cell mitogen, whereas TGF beta 1 inhibits epithelial cell growth; the role of these factors in contributing to the transformed phenotype is uncertain. Steady state mRNA expression for these growth factors and their receptors in a panel of human colon cancers and adjacent normal mucosa is presented. Based in part on results from transgenic mice in which TGF alpha is selectively overproduced in the mammary gland, a possible role for TGF alpha as a tumor promoter in the process of transformation is discussed.
Asunto(s)
Neoplasias del Colon/genética , Neoplasias del Colon/patología , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/patología , Factor de Crecimiento Transformador alfa/análisis , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/genética , Animales , Transformación Celular Neoplásica , Colon/patología , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Familia de Multigenes , Proto-Oncogenes , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hydrolase [EC 3.2.1.25]) and alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C. The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts. The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C. The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.
Asunto(s)
Bacterias Anaerobias Gramnegativas/enzimología , Manosidasas/aislamiento & purificación , alfa-Galactosidasa/aislamiento & purificación , Secuencia de Carbohidratos , Estabilidad de Enzimas , Galactosa/análogos & derivados , Semivida , Calor , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Mananos/biosíntesis , Mananos/química , Manosidasas/química , Manosidasas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Especificidad por Sustrato , alfa-Galactosidasa/química , alfa-Galactosidasa/metabolismo , beta-ManosidasaRESUMEN
Exogenous TGF beta inhibits the proliferation of human breast cancer cells in vitro. These cells synthesize and secrete TGF beta into their medium predominantly in a latent form. With neutralizing antibodies against native, biologically active TGF beta (278ab and 282ab), we have examined whether HS578T and MDA-231 breast cancer cells utilize their endogenous TGF beta for growth regulation. Low levels of TGF beta activity were detectable in conditioned medium from confluent monolayers of both cell lines in the absence of acid or protease treatment as measured by radioreceptor assay. When added to subconfluent monolayers of the respective cell line, this untreated conditioned medium inhibited DNA synthesis and cell proliferation. This inhibition was blocked by anti-TGF beta antibodies, whereas nonimmune rabbit IgG had no effect. Similar to exogenous TGF beta 1, this conditioned medium induced a dose-dependent increase in steady-state TGF beta 1 mRNA levels when added to subconfluent HS578T cells; this increase was blocked by the 278ab. Consistent with the above, preincubation of either cell line with anti-TGF beta antibodies increased subsequent specific binding of 125I-TGF beta to cell surface receptors without changing binding affinity. Addition of 278ab to quiescent HS578T or MDA-231 cells induced a dose-dependent increase in [3H]thymidine incorporation. Both antibodies stimulated cell proliferation in serum-free medium and anchorage-independent growth of both cell lines. Finally, incubation of HS578T cells with 278ab under serum-free conditions decreased the basal level of TGF beta 1 message expression. These data indicate that cultured human breast cancer cells utilize endogenously produced TGF beta as an autocrine negative growth regulator.
Asunto(s)
Neoplasias de la Mama/patología , Factor de Crecimiento Transformador beta/fisiología , Anticuerpos/farmacología , Northern Blotting , Neoplasias de la Mama/metabolismo , División Celular/fisiología , Humanos , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales CultivadasRESUMEN
Bacteria that grow on p-xylene, p-toluic acid, and terephthalic acid (TPA) were isolated from a wastewater bioreactor that is used to treat a waste stream that contains all three of these compounds. Although previously described aerobic bacteria degrade p-xylene by initially oxidizing a single methyl group to form p-toluic acid and then cleaving the aromatic ring, some of the bacteria isolated during this study transformed p-xylene by oxidizing both methyl groups to produce TPA.
Asunto(s)
Residuos Industriales , Ácidos Ftálicos/metabolismo , Proteobacteria/aislamiento & purificación , Aguas del Alcantarillado/microbiología , Eliminación de Residuos Líquidos , Xilenos/metabolismo , Reactores Biológicos , Oxidación-Reducción , Proteobacteria/clasificación , Proteobacteria/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
An alpha-galactosidase and a beta-mannanase produced by the hyperthermophilic bacterium, Thermotoga neapolitana 5068 (TN5068), separately and together, were evaluated for their ability to hydrolyze guar gum in relation to viscosity reduction of guar-based hydraulic fracturing fluids used in oil and gas well stimulation. In such applications, premature guar gum hydrolysis at lower temperatures before the fracturing process is completed is undesirable, whereas thermostability and thermoactivity are advantageous. Hyperthermophilic enzymes presumably possess both characteristics. The purified alpha-galactosidase was found to have a temperature optimum of 100-105 degrees C with a half-life of 130 minutes at 90 degrees C and 3 min at 100 degrees C, while the purified beta-mannanase was found to have a temperature optimum of 91 degrees C and a half-life of 13h at this temperature and 35 min at 100 degrees C.These represent the most thermostable versions of these enzymes yet reported. At 25 degrees C, TN5068 culture supernatants, containing the two enzyme activities, reduced viscosity of a 0.7% (wt) guar gum solution by a factor of 1.4 after a 1.5-h incubation period and by a factor of 2.4 after 5 h. This is in contrast to a viscosity reduction of 100-fold after 1.5 h and 375-fold after 5 h for a commercial preparation of these enzymes from Aspergillus niger. In contrast, at 85 degrees C, the TN5068 enzymes reduced viscosity by 30-fold after 1.5 h and 100-fold after 5 h compared to a 2.5-fold reduction after 5 h for the control. The A. niger enzymes were less effective at 85 degrees C (1.6-fold reduction after 1.5 h and a 4.2-fold reduction after 5 h), presumably due to their thermal lability at this temperature. Furthermore, it was determined that the purified beta-mannanase alone can substantially reduce viscosity of guar solutions, while the alpha-galactosidase alone had limited viscosity reduction activity. However, the alpha-galactosidase appeared to minimize residual particulate matter when used in conjunction with the beta-mannanase. This could be the result of extensive hydrolysis of the alpha-1,6 linkages between mannose and galactose units in guar, allowing more extensive hydrolysis of the mannan chain by the beta-mannanase. The use of thermostable enzymatic breakers from hyperthermophiles in hydraulic fracturing could be used to improve well stimulation and oil and gas recovery.