RESUMEN
Aligned titanium dioxide nanotubes may be grown on the surface of titanium metal by electrochemical oxidation in the presence of fluoride ion. There are a number of salient parameters that have been reported to affect the nanotube growth i.e., the nature, pH and concentration of the fluoride electrolyte, the cell potential and process time for anodisation. Furthermore, it has been reported that the nanotubes as grown are amorphous and can be converted to a mixture of anatase and rutile crystalline phases by heat treatment at elevated temperatures. There have been no studies reported investigating the effect of surface roughness of the parent titanium metal on nanotube growth. In this work the electrochemical growth of titanium oxide nanotubes on titanium foil was investigated using an ammonium fluoride/ammonium sulphate electrolyte. The results confirm that the anodisation potential controls pore diameter. The surface coverage of nanotubes was dependent on the surface roughness of the parent titanium metal. AFM measurements on untreated titanium foil showed relatively high microscale roughness and low nanoscale roughness. SEM analysis of these samples showed nanotube growth to be confined to depressions or valleys on the surface and the nanotubes were of uniform pore diameter. Mechanically polishing the surface of the parent titanium decreased the microscale roughness and increased the nanoscale roughness which, resulted in more uniform surface coverage. However, this led to an increased variation in pore diameter and shape of the nanotubes. XRD was used to determine crystal structure before and after annealing at 460 degrees C.
RESUMEN
Human parathyroid glands obtained at autopsy were incubated with [(3)H]leucine and [(3)H]lysine. After incubation, nonradioactive parathyroid tissue of either human or bovine origin was added. Radioactive parathyroid hormone and proparathyroid hormone were isolated from the gland and medium by organic solvent and salt fractionation, trichloroacetic acid precipitation, Sephadex G-100 gel filtration, and carboxymethyl cellulose column chromatography. The human hormonal peptides were identified in the ion-exchange column eluates by their relatively high levels of radioactivity, their elution positions, and their immunoreactivity to anti-PTH antiserum. The time-course of radioactive amino acid incorporation into these peptides and a brief incubation of the gland with radioactive amino acids, followed by various lengths of incubation with nonradioactive amino acids, indicated that a precursor-product relationship exists for the two peptides. An alternate method for isolation of the hormone and prohormone, which involves separation of peptides by urea-polyacrylamide gel electrophoresis, confirmed the identities of the human parathyroid hormone and proparathyroid hormone.
Asunto(s)
Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/biosíntesis , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Cromatografía , Humanos , Cinética , Masculino , Glándulas Paratiroides/análisis , Hormona Paratiroidea/análisis , Precursores de Proteínas/análisis , Precursores de Proteínas/biosíntesis , Radioinmunoensayo , TritioRESUMEN
Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for over 20 years, yet little is known either qualitatively or quantitatively about MMC-induced DNA adduct formation and repair in vivo. As an initial means of investigating this, we used a recently developed 32P-postlabeling assay to examine the formation and loss of MMC-DNA adducts in the tissues of a simple in vivo model test system, the chick embryo, following treatment with a chemotherapeutic dose of MMC. As early as 15 min after MMC treatment, four adducts could be detected in the liver which were tentatively identified as the (CpG) N2G-MMC-N2G interstrand cross-link, the bifunctionally activated MMC-N2G monoadduct, and two isomers (alpha and beta) of the monofunctionally activated MMC-N2G monoadduct. The (GpG) N2G-MMC-N2G intrastrand cross-link appears to be a poor substrate for nuclease P1 and/or T4 kinase and was not evaluable by this assay. Levels of all four detectable adducts increased substantially within the first 2 h after MMC treatment, reached maximal levels by 6 h, and decreased progressively thereafter through 24 h, although low levels of certain adducts persisted beyond 24 h. Lung and kidney had comparable levels of total MMC adducts, which were approximately 60% those of the liver, and there were no significant differences in the proportion of specific adducts among the three tissues. The interstrand cross-link represented approximately 13-14% of the total MMC adducts, which is approximately 5-fold greater than the proportion of CpG sites in the genome. In addition, the interstrand cross-link was selectively decreased after 16 h relative to the three monoadducts, suggesting preferential repair. The effect of modulating different components of the Phase I and Phase II drug metabolism on MMC adduct formation, using either glutethimide, 3,4,3',4'-tetrachlorobiphenyl, dexamethasone, buthionine sulfoximine, ethacrynic acid, or N-acetylcysteine pretreatments, was examined to characterize the possible pathways of MMC metabolism and adduct formation in vivo. Surprisingly, none of these pretreatments had a significant effect on individual or total adducts with the exception of dexamethasone, which caused an almost 2-fold proportional increase in all four adducts in the liver.
Asunto(s)
Reactivos de Enlaces Cruzados/toxicidad , Aductos de ADN/análisis , ADN/efectos de los fármacos , Mitomicina/toxicidad , Radioisótopos de Fósforo , Acetilcisteína/farmacología , Animales , Biotransformación/efectos de los fármacos , Butionina Sulfoximina/farmacología , Embrión de Pollo , Daño del ADN , Reparación del ADN , Dexametasona/farmacología , Inducción Enzimática/efectos de los fármacos , Ácido Etacrínico/farmacología , Glutatión/metabolismo , Glutetimida/farmacología , Inactivación Metabólica , Riñón/química , Riñón/efectos de los fármacos , Riñón/embriología , Hígado/química , Hígado/efectos de los fármacos , Hígado/embriología , Pulmón/química , Pulmón/efectos de los fármacos , Pulmón/embriología , Bifenilos Policlorados/farmacología , Factores de TiempoRESUMEN
The tryptic hydrolysis of bivine intestinal calcium-binding protein in the presence and absence of excess calcium has been investigated. Calcium-binding activity and immunological reactivity of the protein were not significantly affected in the presence of 1.0 mM CaCl2 following 24 h incubation at 38 degrees C with trypsin at ratios of 1:9 of enzyme to calcium-binding protein. Some modification of the protein did occur under these conditions, however, since analysis by analytical acrylamide gel electrophoresis indicated the formation of a more rapidly-migrating species from the slower-moving original protein band. Omission of added calcium from the incubation medium resulted in rapid and essentially complete destruction of calcium-binding activity and immunological reactivity, and the formation of peptides of low molecular weight. This provides evidence that the conformation of the calcium-binding protein in the presence of calcium differs from that in its absence.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/metabolismo , Tripsina/metabolismo , Animales , Calcio/farmacología , Proteínas Portadoras/inmunología , Bovinos , IntestinosRESUMEN
Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for >20 years. However, little is known either qualitatively or quantitatively about the relationship between formation and repair of specific MMC-DNA adducts and specific biological outcomes. The goal of this study was to examine formation and removal of specific MMC-DNA adducts in breast cancer cells using a (32)P-postlabeling assay in relation to cytotoxicity and other biological end points. MMC-DNA adducts were measured in cultured human metastatic MDA-MB-435 cells, in the same cells xenografted as a mammary tumor in nude mice, and in metastatic tumor biopsies obtained from human breast cancer patients undergoing MMC-based therapy. MMC adducts corresponding to the CpG interstrand cross-link, the MMC-G bifunctional monoadduct, and two isomers of the MMC-G monofunctional monoadduct were detected in most samples. Despite similarities in the overall patterns of adduct formation, there were substantial differences between the cultured cells and the in vivo tumors in their adduct distribution profile, kinetics of adduct formation and removal, and relationship of specific adduct levels to cytotoxicity, suggesting that the in vivo microenvironment (e.g., degree of oxygenation, pH, activity of oxidoreductases, and other factors) of breast cancer cells may significantly modulate these parameters.
Asunto(s)
Neoplasias de la Mama/química , Aductos de ADN/análisis , ADN de Neoplasias/química , Mitomicina/análisis , Anciano , Animales , Biopsia , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ensayos Clínicos como Asunto , ADN de Neoplasias/análisis , Femenino , Genoma , Humanos , Ratones , Ratones Desnudos , Persona de Mediana Edad , Trasplante de Neoplasias , Radioisótopos de Fósforo , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis due to their ability to digest basement membrane and extracellular matrix components, thereby facilitating cell movement through connective tissues. At noncytotoxic concentrations, i.e., concentrations lower than those normally used in cancer chemotherapy, the anthracycline doxorubicin specifically inhibited collagenase 1 (MMP-1) gene expression in the highly invasive and metastatic human melanoma cell line A2058. This inhibition was specific for collagenase 1 because it did not affect the expression of two other MMPs, gelatinase A (MMP-2) and gelatinase B (MMP-9). The reduction in collagenase 1 expression correlated with a decrease in the invasive ability of tumor cells through a collagen type I matrix and was independent of the cytotoxic and antiproliferative effects usually associated with this anticancer drug. The selective modulation of collagenase 1 expression by nontoxic doses of doxorubicin suggests a novel application for this chemotherapeutic agent, perhaps in combination therapy, because it decreases the invasive/metastatic potential of melanoma cells that are otherwise unaffected by this drug.
Asunto(s)
Antineoplásicos/farmacología , Colagenasas/genética , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , División Celular/efectos de los fármacos , Colágeno , Colagenasas/biosíntesis , Gelatinasas/biosíntesis , Humanos , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Melanoma , Metaloendopeptidasas/biosíntesis , Invasividad Neoplásica , Metástasis de la Neoplasia , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Overexpression of the trans-membrane drug efflux pump P-glycoprotein is one of the major mechanisms by which cancer cells develop multidrug resistance. We demonstrated previously that noncytotoxic doses of various genotoxic chemicals, particularly DNA cross-linking agents, preferentially altered expression of inducible genes. These effects occurred principally at the transcriptional level and were closely correlated temporally with DNA damage. Because the mdr1 gene coding for P-glycoprotein has been reported to be highly inducible, we were interested in the effects of genotoxic cancer chemotherapy agents on its expression. We report that the DNA cross-linking agent mitomycin C significantly suppressed mRNA and protein expression of P-glycoprotein and decreased the rate of drug efflux. Mitomycin C pretreatment also significantly increased the sensitivity of cancer cells to subsequent killing by the P-glycoprotein substrate doxorubicin, decreasing the ED50 by 5- to 10-fold. Suppression of P-glycoprotein expression was also observed with subtoxic doses of the DNA cross-linking agents cisplatin, BMS181174, and chromium(VI). These effects occurred in both human and rodent cell lines; in cell lines derived from colon, breast, leukemia, neuroblastoma, and hepatoma tumors; and under both monolayer and "spheroid" culture conditions. These results suggest the basis for novel clinical cancer chemotherapy regimens aimed at drug-resistant tumors, in which a sub-chemotherapeutic dose of a DNA cross-linking agent is used to modulate the multidrug resistance phenotype prior to treatment with a second cytotoxic agent.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos/toxicidad , Reactivos de Enlaces Cruzados/toxicidad , Resistencia a Múltiples Medicamentos/genética , Mitomicinas , Transcripción Genética/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Animales , Antineoplásicos Alquilantes/toxicidad , Neoplasias de la Mama , Carcinoma Hepatocelular , Supervivencia Celular/efectos de los fármacos , Cisplatino/toxicidad , Neoplasias del Colon , Daño del ADN , ADN de Neoplasias/efectos de los fármacos , Doxorrubicina/toxicidad , Femenino , Humanos , Células K562 , Neoplasias Hepáticas , Mitomicina/toxicidad , Mitomicinas/toxicidad , Neuroblastoma , ARN Mensajero/biosíntesis , Ratas , Células Tumorales Cultivadas , Verapamilo/farmacologíaRESUMEN
We have studied the incorporation of radioactive P (32P) into lipids of bovine parathyroid tissue under conditions of stimulated and inhibited hormone secretion. Utilizing low (0.5 mM) and high (3.0 mM) concentrations of calcium to regulate parathyroid hormone secretion, we initially found that the labeling of the cellular phospholipids with 32P was greater in those tissues incubated in high-calcium medium. Thin-layer chromatography of lipid extracts prepared from tissue incubated in either low- or high-calcium media revealed that the increased incorporation of 32P (high or low) was localized primarily to two phospholipids. To determine whether the increases were due directly to the different calcium concentrations, the experiments were performed in media containing normal calcium concentrations (1.25 mM) and low (0.5) or high (3.0) magnesium concentrations to modulate hormone secretion. The results were identical to those obtained using low and high calcium, indicating that the increased 32P incorporation was not an effect of high calcium but rather correlated with the inhibition of hormone secretion. The use of other secretagogues confirmed this correlation. The identity of the two phospholipids was established, by two-dimensional thin-layer chromatography, to be phosphatidylinositol (PI) and lysophosphatidylinositol (LPI). The correlation of increased 32P incorporation with inhibition of secretion led us next to examine isolated secretory granules from tissues exposed to either high-or low-calcium conditions. Thin-layer chromatography of granule lipid extracts yielded chromatograms containing PI and LPI, and the radioactivity of each was greater in the high-calcium sample than in the low-calcium sample.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Lisofosfolípidos/metabolismo , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Calcio/metabolismo , Bovinos , Cromatografía en Capa Delgada , Gránulos Citoplasmáticos/metabolismo , Magnesio/metabolismo , Radioisótopos de Fósforo , RadioinmunoensayoRESUMEN
Chromogranin A (CgA) is a glycoprotein located in the secretory granules of multiple neuroendocrine tissues, including the parathyroid gland. Although the function of CgA is not known, a role has been proposed for CgA as a prohormone for biologically active peptides. Using 3H-CgA as a substrate for bovine parathyroid secretory granule extracts, we demonstrate a precursor product relationship between intact CgA and multiple N-terminal fragments of CgA. N-terminal CgA fragments of mol wt 24, 26, and 33 k are generated in a time dependent manner in the presence of bovine parathyroid secretory granule enzymes. The generation of the 33 kilodalton (kDa) N-terminal CgA fragment is calcium dependent. In the presence of EDTA, intermediate CgA fragments of mol wt 36 and 45 are generated. The effect of EDTA is reversible with added calcium. Based on immunodetection on Western blots, the 26 kDa N-terminal fragment of CgA is secreted by bovine parathyroid cells in a time and calcium-dependent manner in parallel with PTH and intact CgA. The secretion of the 26 kDa N-terminal fragment of CgA increases in response to low calcium incubation conditions and is suppressed by high calcium incubation conditions. We conclude that bovine parathyroid secretory granules contain enzymatic activity capable of processing CgA to multiple N-terminal fragments. The secretion of at least one N-terminal fragment (26 kDa) is calcium responsive. The physiological significance of CgA processing in parathyroid secretory granules is as yet unknown.
Asunto(s)
Cromograninas/metabolismo , Gránulos Citoplasmáticos/enzimología , Glándulas Paratiroides/ultraestructura , Fragmentos de Péptidos/metabolismo , Aminoácidos/análisis , Animales , Western Blotting , Calcio/farmacología , Bovinos , Cromogranina A , Cromograninas/análisis , Ácido Edético/farmacología , Peso Molecular , Glándulas Paratiroides/metabolismo , Precursores de Proteínas/metabolismoRESUMEN
Chromogranin-A (CgA) is a ubiquitous protein which colocalizes in secretory granules of multiple endocrine tissues and cosecretes with peptide hormones from these tissues. Although the function of CgA has remained unknown, there has been recent interest in its potential role as a prohormone for smaller, biologically active peptides. We isolated and characterized a 26-kDa N-terminal fragment of CgA which is a natural breakdown product of bovine parathyroid CgA in storage. A similar, if not identical, fragment of CgA is secreted by bovine parathyroid glands. The secreted fragment elutes on HPLC and migrates on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gels in the same position as the 26-kDa N-terminal fragment. When added to the incubation medium of dispersed bovine parathyroid cells, the 26-kDa N-terminal fragment of CgA inhibits the low calcium-stimulated secretion of both PTH and CgA. This N-terminal fragment is homologous to betagranin, which is a 21-kDa N-terminal fragment of CgA that is generated from CgA in rat insulin granules. Thus, a naturally occurring betagranin-like N-terminal fragment of bovine parathyroid CgA is not only secreted itself, but can inhibit the secretion of PTH and intact CgA by bovine parathyroid cells. The processing of intact CgA to fragments such as the N-terminal fragment that we describe may be important in the autocrine or paracrine regulation of secretion.
Asunto(s)
Cromograninas/metabolismo , Glándulas Paratiroides/metabolismo , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Bovinos , Cromatografía Líquida de Alta Presión , Cromogranina A , Cromograninas/química , Cromograninas/farmacología , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Peso Molecular , Glándulas Paratiroides/efectos de los fármacos , Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Homología de Secuencia de Ácido NucleicoRESUMEN
Bovine parathyroid gland slices were incubated in Krebs' buffer with [3-H] leucine in order to assess the biosynthesis and secretion of parathyroid hormone (PTH). After incubation the particulate structures of the tissue were extracted with sodium deoxycholate to yield primarily newly synthesized (radioactive) PTH in the extract and preexisting granule PTH in the residue. In pulse-chase experiments radioactive PTH entered the granule fraction at a time when total tissue radioactive hormone was declining, which indicates that some newly-synthesized PTH was packaged into these granules. The specific radioactivity of the PTH in the incubation medium was from 20-fold to more than 80-fold greater than that of the granule PTH of the tissue, but was similar in magnitude to that of the tissue's newly synthesized hormone. Secretion of newly synthesized PTH was greater during incubation in buffer with 1.25 mM Ca than it was at 2.5 mM Ca which indicated that the hormone release was subject to physiological control. The PTH content of the tissue granule fraction was lower following incubation at 1.25 mM Ca than at 2.5 mM Ca. The possibility was excluded that the higher specific radioactivity of PT1 of incubation medium compared to that of the secretory granule fraction resulted from a diffusion gradient of [3-H] leucine into the tissue slices. These data indicate that a major portion of the newly synthesized PTH was secreted without prior equilibration with the hormone in the pool of secretory granules.
Asunto(s)
Hormona Paratiroidea/metabolismo , Aminoácidos/metabolismo , Animales , Radioisótopos de Carbono , Bovinos , Cromatografía , Técnicas In Vitro , Leucina/metabolismo , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/biosíntesis , Precursores de Proteínas/biosíntesisRESUMEN
Fresh parathyroid gland homogenates and fractions thereof were analyzed for their content of PTH and carboxyl-terminal fragments of the hormone. The tissue proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then extracted from gel fractions for RIA. Native PTH and PTH-(37-84) were used as standards to mark the migration positions of these peptides in the gels. The RIA for carboxyl-terminal hormone fragments used PTH-(37-84) as radioiodinated tracer and responded equally on a molar basis to either PTH or PTH-(37-84), making possible quantitative evaluation of both peptides in one assay after their separation. The results indicated that tissue homogenates contain 0.3-0.5 PTH-(37-84) moleq for each mole of PTH. Particulate fractions of the homogenates contained 0.15-0.3 moleq of fragment/mol PTH, while the high speed supernatant fraction of the homogenate contained about 2 moleq of fragment/mol PTH. When the experiments were performed using homogenization and fractionation buffers that contained numerous protease inhibitors, the ratios of carboxyl-terminal PTH fragment to intact hormone were not decreased, indicating that the hormone fragments were not produced during tissue processing. In addition, PTH added to tissue homogenates was not degraded during subsequent manipulations. The results demonstrate that fresh bovine parathyroid tissue contains substantial levels of carboxyl-terminal PTH peptide fragments, which can be measured by RIA after separation from PTH and other hormonal species. The data support the hypothesis that hormone fragments reside in regions of the cell different from those that contain PTH.
Asunto(s)
Glándulas Paratiroides/análisis , Hormona Paratiroidea/análisis , Fragmentos de Péptidos/análisis , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Radioinmunoensayo , Fracciones Subcelulares/análisisRESUMEN
We have obtained a crude enzyme preparation from bovine parathyroid gland homogenates which when incubated with PTH, cleaves the hormone into two major fragments. Isolation and chemical analysis has led to the identification of these peptides, the 1-34 fragment and the 35-84 fragment. Digestion of PTH was totally inhibited by the inclusion of the cathepsin D inhibitor, pepstatin, in the enzyme digest. A comparison of the digest obtained using the crude enzyme fraction vs. digestion of PTH by purified bovine cathepsin D led to the findings that the same peptide products were formed in each case. The natural 1-34 hormone fragment derived from the procedure has been determined to be fully biologically active in a bone resorption system.
Asunto(s)
Catepsinas/metabolismo , Glándulas Paratiroides/enzimología , Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/metabolismo , Aminoácidos/análisis , Animales , Resorción Ósea/efectos de los fármacos , Catepsina D , Bovinos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Hormona Paratiroidea/farmacologíaRESUMEN
A series of compounds that increase the activity of delta-aminolevulinic acid synthase (ALAS) in chick embryo hepatocyte cultures were studied for their effects on steady-state levels of mRNA for ALAS and phenobarbital-inducible cytochrome PB1 P450. N-Ethylprotoporphyrin IX (N-EtPP), which is believed to lower heme levels by inhibition of ferrochelatase (FC), had little effect on steady-state ALAS mRNA levels. 3,5-Diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4- isobutylpyridine (4-isobutyl DDC), which is believed to lower heme levels by repetitive destruction of the heme moiety of cytochrome P450, increased steady-state levels of ALAS mRNA levels approximately 2-fold. 3,5-Diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DCC) which inhibits FC activity and destroys the heme moiety of cytochrome P450, increased ALAS mRNA levels approximately 4-fold. A combination of N-EtPP and 4-isobutyl DDC produced a synergistic increase in ALAS mRNA levels to approximately 6-fold over control levels. The synergistic increase in ALAS activity observed previously with this combination can be explained, at least in part, by a synergistic increase in ALAS mRNA levels. Other porphyrinogenic agents, which function as mechanism-based inactivators of cytochrome P450 and elevate ALAS activity, were found to elevate ALAS mRNA. These compounds included 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), 2,4-diethyl-2-methyl-1,2-dihydroquinoline (DMDQ), and 2,2,4-trimethyl-1,2,dihydroquinoline (TMDQ). The elevation of ALAS mRNA by these porphyrinogenic agents is probably due to their lowering of cellular heme levels by a combination of ferrochelatase inhibition and repetitive destruction of the heme moiety of cytochrome P450. The lowering of heme levels should result in an enhancement of ALAS mRNA half-life as it has been demonstrated by others that heme shortens the half-life of ALAS mRNA. It was of interest that some of these drug treatments also caused an elevation in steady-state levels of cytochrome PB1 P450 mRNA; the exception was TTMS, which along with its analogue 3-(2-phenylethyl)-4-methylsydnone (PEMS), did not alter cytochrome PB1 P450 mRNA levels. Increases in steady-state levels of cytochrome PB1 P450 mRNA subsequent to increases in steady-state levels of ALAS mRNA were observed with 4-ethyl DDC, 4-isobutyl DDC, DMDQ, and TMDQ. The data obtained with N-EtPP and a combination of N-EtPP and 4-isobutyl DDC on cytochrome PB1 P450 mRNA levels do not support the contention that heme functions as a positive regulator of cytochrome P450 gene expression.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Sistema Enzimático del Citocromo P-450/genética , Dihidropiridinas/farmacología , Hígado/efectos de los fármacos , Protoporfirinas/farmacología , Quinolinas/farmacología , ARN Mensajero/análisis , Sidnonas/farmacología , 5-Aminolevulinato Sintetasa/biosíntesis , Animales , Secuencia de Bases , Embrión de Pollo , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hemo/biosíntesis , Hígado/enzimología , Datos de Secuencia Molecular , Factores de Tiempo , Xenobióticos/farmacologíaRESUMEN
It previously has been shown that digestion of bovine parathormone (bPTH) with cathepsin-D results in rapid cleavage of the hormone between Phe34 and Val35 yielding PTH(1-34) and PTH(35-84). Since bPTH also contains a Phe at residue 7 we have conducted additional studies to determine whether cleavage at this position could occur. We have found that following longer incubation periods of hormone and enzyme, 2 additional peptides are generated; PTH(8-34) and PTH(1-7). Time course studies demonstrated that these 2 fragments are formed from the (1-34) peptide generated through the initial cleavage at Phe34-Val35 of PTH. The identification of the bPTH(8-34) was accomplished through amino acid analysis and N-terminal sequencing. bPTH(8-34) behaved as a PTH antagonist in an in vitro mouse calvarial bone resorption assay. Although bPTH(8-34) did not affect the PTH-stimulated cAMP response when added simultaneously with PTH, preincubation of bone cells with this peptide caused desensitization of the PTH-stimulated cAMP response.
Asunto(s)
Catepsina D/metabolismo , Antagonistas de Hormonas/aislamiento & purificación , Hormona Paratiroidea/aislamiento & purificación , Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Resorción Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , AMP Cíclico/metabolismo , Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacologíaRESUMEN
Tissue slices or dispersed cells of bovine parathyroid gland were incubated with [3H]leucine to label the intracellular proteins and then tested for their secretory response to isoproterenol and cycloheximide at different calcium concentrations. Secretion of the newly synthesized as well as the older PTH and SP-I was stimulated by isoproterenol at all calcium levels tested, even when it was maximally enhanced by low calcium. Cycloheximide interfered with neither the secretory process nor the secretory response to different stimuli, but decreased the amount of PTH and SP-I secreted. We conclude that the inhibitor decreased the secretion by reducing the supply of PTH and SP-I. Calculations derived from the data reveal that, under most secretory conditions, newly synthesized PTH contributed a major portion of the total hormone secretion in bovine parathyroid cells.
Asunto(s)
Cicloheximida/farmacología , Isoproterenol/farmacología , Glándulas Paratiroides/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Bovinos , Cromogranina A , Cromograninas , Matemática , Glándulas Paratiroides/efectos de los fármacos , Hormona Paratiroidea/metabolismoRESUMEN
Chronic human exposure to nonovertly toxic doses of arsenic is associated with an increased risk of cancer. Although its carcinogenic mechanism is still unknown, arsenic does not directly cause DNA damage or mutations and is therefore thought to act principally as a co-mutagen, co-carcinogen, and/or tumor promoter. Previous studies in our laboratory demonstrated that effects of low-dose arsenic (III) (arsenite) on expression of the hormone-regulated phosphoenolpyruvate carboxykinase (PEPCK) gene were strongly associated with the glucocorticoid receptor (GR)-mediated regulatory pathway. We therefore examined specifically the effects of arsenite on the biochemical function of GR in hormone-responsive H4IIE rat hepatoma cells. Completely noncytotoxic arsenite treatments (0.3-3.3 microM) significantly decreased dexamethasone-induced expression of transiently transfected luciferase constructs containing either an intact hormone-responsive promoter from the mammalian PEPCK gene or two tandem glucocorticoid response elements (GRE). Western blotting and confocal microscopy of a green fluorescent protein-tagged-GR fusion protein demonstrated that arsenite pretreatment did not block the normal dexamethasone-induced nuclear translocation of GR. These data indicate that nontoxic doses of arsenite can interact directly with GR complexes and selectively inhibit GR-mediated transcription, which is associated with altered nuclear function rather than a decrease in hormone-induced GR activation or nuclear translocation.
Asunto(s)
Arsenitos/farmacología , Carcinógenos/farmacología , Receptores de Glucocorticoides/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Animales , Intoxicación por Arsénico/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Pruebas de Precipitina , Ratas , Células Tumorales CultivadasRESUMEN
Certain forms of the heavy metals arsenic and chromium are considered human carcinogens, although they are believed to act through very different mechanisms. Chromium(VI) is believed to act as a classic and mutagenic agent, and DNA/chromatin appears to be the principal target for its effects. In contrast, arsenic(III) is considered nongenotoxic, but is able to target specific cellular proteins, principally through sulfhydryl interactions. We had previously shown that various genotoxic chemical carcinogens, including chromium (VI), preferentially altered expression of several inducible genes but had little or no effect on constitutive gene expression. We were therefore interested in whether these carcinogenic heavy metals might target specific but distinct sites within cells, leading to alterations in gene expression that might contribute to the carcinogenic process. Arsenic(III) and chromium(VI) each significantly altered both basal and hormone-inducible expression of a model inducible gene, phosphoenolpyruvate carboxykinase (PEPCK), at nonovertly toxic doses in the chick embryo in vivo and rat hepatoma H411E cells in culture. We have recently developed two parallel cell culture approaches for examining the molecular basis for these effects. First, we are examining the effects of heavy metals on expression and activation of specific transcription factors known to be involved in regulation of susceptible inducible genes, and have recently observed significant but different effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding. Second, we have developed cell lines with stably integrated PEPCK promoter-luciferase reporter gene constructs to examine effects of heavy metals on promoter function, and have also recently seen profound effects induced by both chromium(VI) and arsenic(III) in this system. These model systems should enable us to be able to identify the critical cis (DNA) and trans (protein) cellular targets of heavy metal exposure leading to alterations in expression of specific susceptible genes. It is anticipated that such information will provide valuable insight into the mechanistic basis for these effects as well as provide sensitive molecular biomarkers for evaluating human exposure.
Asunto(s)
Arsénico/toxicidad , Cromo/toxicidad , Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Neoplasias/etiología , Animales , Arsénico/farmacología , Transformación Celular Neoplásica , Embrión de Pollo , Cromo/farmacología , Exposición a Riesgos Ambientales , Humanos , Regiones Promotoras Genéticas , Ratas , Pruebas de Toxicidad/métodos , Factores de TranscripciónRESUMEN
In a retrospective analysis, we evaluated our results with endoscopic dilation of enterostomy stenoses that complicated gastric procedures performed for the treatment of morbid obesity. Of 541 patients who underwent a gastric procedure for treatment of morbid obesity, we found 19 patients in whom endoscopic dilations of stenoses had been attempted. We also include three patients who had surgery elsewhere but who underwent dilations at our institution. Fourteen had stenoses complicating gastric bypass with Roux-en-Y anastomoses, and eight had stenoses complicating a gastroplasty (gastrogastrostomy). Two different types of dilation were attempted during the interval reviewed--Fogarty balloon dilations and Grüntzig balloon dilations. None of the eight patients with gastroplasties benefited from the attempted dilation, but 10 of the 14 patients with stenoses complicating gastric bypasses have done well. We found no significant difference between Fogarty and Grüntzig balloon dilations. We conclude that balloon dilation is an effective means of treating stenosis that complicates gastric bypass performed with Roux-en-Y anastomoses in cases of morbid obesity.
Asunto(s)
Complicaciones Posoperatorias/cirugía , Gastropatías/cirugía , Estómago/cirugía , Adulto , Constricción Patológica/cirugía , Dilatación , Endoscopía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad/terapiaRESUMEN
This report analyzes experience with a modified four-limb W-shaped ileal pouch that has a larger initial capacity than do J- or S-shaped pouch designs. Fifteen patients (median age: 35 years) underwent W pouch reconstruction after proctocolectomy for ulcerative colitis and familial adenomatous polyposis. Follow-up on each patient averaged 14 months (range: 6 to 24 months). All procedures were performed without death and with minimal morbidity. Assessment of functional results showed 24-hour stool frequency (mean +/- SEM) decreasing from 6.0 +/- 0.39 initially to 4.8 +/- 0.43 at 1 year (p less than 0.005). Night evacuation decreased from 1.1 +/- 0.2 at 1 month after surgery to 0.25 +/- 0.12 at 1 year (p less than 0.025), with 10 of 15 patients having no nocturnal pouch evacuation. Continence was excellent in all patients with the exception of three of 15 patients who had occasional minimal nighttime seepage. Pouch volume determined at surgery by saline solution infusion was 200 +/- 21 ml. Pouch volume and compliance (pressure/volume) were measured before ileostomy closure and at 6 months after surgery via a special pressure-monitored balloon catheter. Maximal pouch volume increased from 190 +/- 21 ml (at time of ileostomy takedown) to 470 +/- 85 ml at 6 months. Ileal reservoir construction with a W pouch design resulted in a low 24-hour and nighttime stool frequency and excellent compliance and evacuation characteristics.