RESUMEN
Agriculture is a major nonpoint source of phosphorus (P) in the Midwest, but how surface runoff and tile drainage interact to affect temporal concentrations and fluxes of both dissolved and particulate P remains unclear. Our objective was to determine the dominant form of P in streams (dissolved or particulate) and identify the mode of transport of this P from fields to streams in tile-drained agricultural watersheds. We measured dissolved reactive P (DRP) and total P (TP) concentrations and loads in stream and tile water in the upper reaches of three watersheds in east-central Illinois (Embarras River, Lake Fork of the Kaskaskia River, and Big Ditch of the Sangamon River). For all 16 water year by watershed combinations examined, annual flow-weighted mean TP concentrations were >0.1 mg L(-1), and seven water year by watershed combinations exceeded 0.2 mg L(-1). Concentrations of DRP and particulate P (PP) increased with stream discharge; however, particulate P was the dominant form during overland runoff events, which greatly affected annual TP loads. Concentrations of DRP and PP in tiles increased with discharge, indicating tiles were a source of P to streams. Across watersheds, the greatest DRP concentrations (as high as 1.25 mg L(-1)) were associated with a precipitation event that followed widespread application of P fertilizer on frozen soils. Although eliminating this practice would reduce the potential for overland runoff of P, soil erosion and tile drainage would continue to be important transport pathways of P to streams in east-central Illinois.
Asunto(s)
Agricultura/métodos , Fósforo/análisis , Movimientos del Agua , Contaminantes Químicos del Agua/análisis , Lluvia , Ríos/química , Nieve , Abastecimiento de AguaRESUMEN
In coastal California nitrogen (N) in runoff from urban and agricultural land is suspected to impair surface water quality of creeks and rivers that discharge into the Monterey Bay Sanctuary. However, quantitative data on the impacts of land use activities on water quality are largely limited to unpublished reports and do not estimate N loading. We report on spatial and temporal patterns of N concentrations for several coastal creeks and rivers in central California. During the 2001 water year, we estimated that the Pajaro River at Chittenden exported 302.4 Mg of total N. Nitrate-N concentrations were typically <1 mg N l(-1) in grazing lands, oak woodlands, and forests, but increased to a range of 1 to 20 mg N l(-1) as surface waters passed through agricultural lands. Very high concentrations of nitrate (in excess of 80 mg N l(-1)) were found in selected agricultural ditches that received drainage from tiles (buried perforated pipes). Nitrate concentrations in these ditches remained high throughout the winter and spring, indicating nitrate was not being flushed out of the soil profile. We believe unused N fertilizer has accumulated in the shallow groundwater through many cropping cycles. Results are being used to organize landowners, resource managers, and growers to develop voluntary monitoring and water quality protection plans.
Asunto(s)
Agricultura/estadística & datos numéricos , Agua Dulce/análisis , Geografía/estadística & datos numéricos , Nitrógeno/análisis , Amoníaco/análisis , California , Nitratos/análisis , Compuestos de Nitrógeno/análisis , Estaciones del AñoRESUMEN
The Midwest has large riverine exports of nitrogen (N), with the largest flux per unit area to the Mississippi River system coming from Iowa and Illinois. We used historic and current data to estimate N inputs, outputs, and transformations for Illinois where human activity (principally agriculture and associated landscape drainage) have had a dominant impact. Presently, approximately 800,000 Mg of N is added each year as fertilizer and another 420,000 Mg is biologically fixed, primarily by soybean (Glycine max L. Merr.). These annual inputs are greater than exports in grain, which results in surplus N throughout the landscape. Rivers within the state export approximately 50% of this surplus N, mostly as nitrate, and the remainder appears to be denitrified or temporarily incorporated into the soil organic matter pool. The magnitude of N losses for 1880, 1910, 1950, and 1990 are compared. Initial cultivation of the prairies released large quantities of N (approximately 500,000 Mg N year(-1)), and resulted in riverine N transport during the late 19th century that appears to have been on the same order of magnitude as contemporary N losses. Riverine flux was estimated to have been at a minimum in about 1950, due to diminished net mineralization and low fertilizer inputs. Residual fertilizer N from corn (Zea mays L.), biological N fixed by soybean, short-circuiting of soil water through artificial drainage, and decreased cropping-system diversity appear to be the primary sources for current N export.
Asunto(s)
Agricultura , Fertilizantes/análisis , Agua Dulce/química , Nitrógeno/metabolismo , Agricultura/historia , Agricultura/estadística & datos numéricos , Animales , Animales Domésticos , Productos Agrícolas/metabolismo , Contaminantes Ambientales/análisis , Contaminantes Ambientales/metabolismo , Fertilizantes/historia , Fertilizantes/estadística & datos numéricos , Abastecimiento de Alimentos , Historia del Siglo XIX , Historia del Siglo XX , Illinois , Nitrógeno/análisis , Fijación del Nitrógeno , Suelo/análisis , Movimientos del AguaRESUMEN
Transforming growth factor alpha (TGFalpha) is widely expressed in malignant as well as normal cells and is involved in regulating cell growth and differentiation. Although processing of TGFalpha has been extensively studied in normal cells, there is little information regarding TGFalpha cleavage in malignant cells. Therefore, we compared the processing of TGFalpha in two human colon carcinoma cell lines. We found that there was a defective cleavage pattern for the TGFalpha precursor resulting in retention of partially processed TGFalpha on the cell surface of both the HCT116a2alphaS3 and CBS4alphaS2 cell lines. This raised the possibility that signaling from the resulting defective cleavage species could differ from that of soluble TGFalpha. The membrane-associated TGFalpha induced higher phosphorylation of EGFR on the cell surface of adjacent cells than equivalent levels of mature TGFalpha. The interaction of membrane bound TGFalpha precursor with the EGFR caused a slower internalization of activated EGFR relative to the internalization of the soluble TGFalpha/EGFR complexes. In addition, the tethered TGFalpha was resistant to the ability of protein-tyrosine phosphatases (PTPs) to reduce EGFR tyrosine phosphorylation, also contributing to higher activation of EGFR. The enhanced activation of EGFR by the tethered form of TGFalpha was reflected by higher activation of Grb2, SHC and Erk downstream mediators of EGF receptor signaling. The higher activation of EGFR by membrane tethered TGFalpha indicates that defective TGFalpha processing provides a mechanism whereby malignant cells can obtain a growth advantage over normal cells.
Asunto(s)
Neoplasias del Colon/metabolismo , Receptores ErbB/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Membrana Celular/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Pruebas de Neutralización , Fosforilación , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal , Transfección , Factor de Crecimiento Transformador alfa/inmunología , Células Tumorales CultivadasRESUMEN
Transforming growth factor beta1 (TGF-beta1) is a potent growth differentiation and morphogenesis factor. The amino-terminal 248 amino acid pro region of TGF-beta1, the beta1-latency-associated peptide (beta1-LAP), is noncovalently associated with TGF-beta1 in an inactive complex. Previous studies suggested that deglycosylated beta1-LAP can not form this latent complex with TGF-beta1. To study the role of the carbohydrate structures of beta1-LAP in its biological functions, we expressed simian beta1-LAP in Escherichia coli with a 10 histidine residue tag on the N-terminus. This polypeptide was solubilized from inclusion bodies with 6 M guanidine hydrochloride and purified by metal chelate affinity chromatography. Purified beta1-LAP was refolded to its dimeric form using a chaotrope-mediated folding procedure. The dimeric beta1-LAP forms 90 kDa complexes with TGF-beta1, TGF-beta2, and TGF-beta3, and reverses the inhibitory activity of TGF-beta1 on Mv1Lu cells. Solid phase binding assays demonstrate that refolded beta1-LAP binds to heparin and thrombospondin 1. FET cell adhesion promoted by refolded beta1-LAP was blocked by an RGD peptide. Purified beta1-LAP produced in Chinese hamster ovary cells, deglycosylated with N-glycosidase F, forms a 80-90 kDa complex with mature TGF-beta1. The carbohydrate structures of beta1-LAP are not required for binding to ligands or for its biological activity.
Asunto(s)
Carbohidratos/química , Fragmentos de Péptidos , Precursores de Proteínas , Proteínas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Sitios de Unión , Células CHO , Conformación de Carbohidratos , Adhesión Celular , Cricetinae , Matriz Extracelular/metabolismo , Glicosilación , Ligandos , Pliegue de Proteína , Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta1RESUMEN
We investigated TGF-beta response and the expression of TGF-beta receptors in clones of MOSER colon carcinoma cells (designated MOSER II and MOSER III-10) as a function of their growth state. TGF-beta 1 response as assessed by induction of fibronectin expression was higher (3.0-fold) in exponentially growing cells than in quiescent cells. The expression of type I receptor (RI) mRNA was greater (2.5 to 3.0-fold) in exponentially growing cells than in quiescent cells. In contrast, the expression of type II receptor (RII) mRNA was marginally increased in quiescent cells relative to exponential cells. Nuclear run-off assays, and actinomycin-D treatment indicated that the increased expression of RI mRNA in exponentially growing cells was primarily due to an increase in transcription, while a marginal increase in mRNA level for RII in quiescent cells was primarily due to an increase in mRNA stability. Affinity cross-linking with 125I-labeled TGF-beta 1, showed that the exponentially growing cells displayed greater amounts of 125I TGF-beta 1 binding to RI and RII than quiescent cells, indicating that increased cell surface expression of receptors was correlated with increased response in the exponential growth state. Immunoblot analysis also indicated greater amounts of RI protein in exponential compared to quiescent cells; however, no difference in RII protein was observed in the two growth states. These data indicate that expression of the receptors responsible for TGF-beta signal transduction are differentially controlled.
Asunto(s)
Receptores de Activinas Tipo I , Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Carcinoma/patología , División Celular , Neoplasias del Colon/patología , Fibronectinas/metabolismo , Humanos , ARN Mensajero/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The formation of a non-covalent complex between mature transforming growth factor beta 1 (TGF-beta 1) and its pro region, the beta 1-latency-associated peptide (beta 1-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian beta 1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line which secretes beta 1-LAP into the culture medium at > 1 mg/l, thus enabling structural studies of complex formation between beta 1-LAP and TGF-beta 1. The simian beta 1-LAP expressed in CHO cells reversed the growth inhibitory effect of exogenous TGF-beta 1 on Mv1Lu (mink lung epithelial) cells and was able to form a cross-linked complex with 125I-TGF-beta 1. Simian beta 1-LAP was purified to homogeneity by a combination of ammonium sulphate precipitation, gel filtration, dye ligand chromatography and anion-exchange chromatography, with a yield of 15%. The purified protein had an apparent molecular mass of 114 kDa as determined by SDS/PAGE, which is greater than that determined for the transient expression of simian beta 1-LAP in COS-1 and for the simian precursor of TGF-beta 1 (pro-TGF-beta 1) in CHO cells, this major difference being due to more extensive glycosylation of beta 1-LAP expressed by this CHO clone. Far-UV CD spectroscopy of simian beta 1-LAP indicates a mostly beta-sheet structure, with extensive structural rearrangements occurring upon formation of the latent complex between TGF-beta 1 and beta 1-LAP.
Asunto(s)
Fragmentos de Péptidos , Precursores de Proteínas , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células CHO/metabolismo , Carbohidratos/análisis , División Celular/efectos de los fármacos , Línea Celular , Precipitación Química , Cromatografía por Intercambio Iónico , Dicroismo Circular , Cricetinae , ADN Complementario/genética , Amplificación de Genes , Pulmón/citología , Pulmón/efectos de los fármacos , Manosafosfatos/análisis , Visón , Estructura Secundaria de Proteína , Proteínas/fisiología , Tetrahidrofolato Deshidrogenasa/genética , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
The interactions of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) with the epidermal growth factor receptor (EGFR) were examined by insertion mutagenesis of the receptor. Seventeen insertions were made throughout a construct containing only the extracellular domain. This truncated receptor (sEGFR) was secreted and had a dissociation constant similar to that of the full-length solubilized receptor. Receptors with insertions within subdomain III were not secreted. Two receptors with insertions at positions 291 and 474, which border subdomain III, have significantly decreased binding to both EGF and TGF alpha relative to wild type. This confirms previous work demonstrating that subdomain III forms the primary binding site for EGF and TGF alpha. Four of the mutants within subdomain II had a decreased binding to TGF alpha relative to wild type, but had wild type binding to EGF. These results suggest that a region within subdomain II may selectively regulate the binding of TGF alpha. Two receptors which contained insertions within subdomains II and IV, approximately equidistant from the center of subdomain III, bound twofold more ligand molecules than wild type receptor, with an affinity similar to that of wild type receptor. These findings suggest that insertion at these positions allows the access of more than one ligand molecule to the binding site.
Asunto(s)
Receptores ErbB/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Immunoblotting , Datos de Secuencia Molecular , Mutagénesis Insercional , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
Escape from negative growth regulation by transforming growth factor beta (TGF-beta) as a result of the loss of TGF-beta type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-beta 1 treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-beta 1-neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-beta autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.
Asunto(s)
Neoplasias del Colon/genética , Mutación , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Secuencia de Bases , Línea Celular , Células Clonales , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Cartilla de ADN , Expresión Génica , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptor Tipo II de Factor de Crecimiento Transformador beta , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Transfección , Factor de Crecimiento Transformador beta/análisis , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Proteolytic processing of the transforming growth factor beta precursor (pro-TGF beta) is an essential step in the formation of the biologically active TGF beta homodimeric protein (TGF beta). The 361-amino-acid precursor pro-TGF beta 1 has within its primary structure the R-H-R-R processing signal found in many constitutively secreted precursor proteins and potentially recognized by members of the mammalian convertase family of endoproteases. To determine whether cleavage of pro-TGF beta 1 can be achieved by the furin convertase in vitro, purified precursor was incubated in the presence of a truncated/secreted form of the enzyme. Immunoblots showed that the 55-kDa pro-TGF beta 1 was converted into the 44 and 12.5 kDa bands corresponding to the pro-region and the mature monomer, respectively. Treatment of pro-TGF beta 1 with furin resulted in a 5-fold increase in the production of biologically active TGF beta 1. Furthermore, when expressed in the furin-deficient LoVo cells, no processing of pro-TGF beta 1 was observed. In contrast, efficient processing was observed when pro-TGF beta was coexpressed with the furin convertase. Collectively, these results provide evidence that in our experimental systems the TGF beta 1 precursor is efficiently and correctly processed by human furin thus permitting release of the biologically active peptide.
Asunto(s)
Subtilisinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Secuencia de Aminoácidos , Secuencia de Consenso , Furina , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Peso Molecular , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Especificidad por Sustrato , Subtilisinas/antagonistas & inhibidoresRESUMEN
The role of transforming growth factor (TGF) beta type II receptor in reversing the malignant phenotype of human breast cancer MCF-7 cells was examined. MCF-7 cells were insensitive to TGF beta 1 and expressed undetectable levels of cell surface TGF beta type I receptor (RI) and type II receptor (RII) by cross-linking with 125I-TGF beta 1. Stable transfection of a RII expression vector yielded 3 transfectants with varying levels of exogenous RII mRNA and protein levels. Expression of RII also increased TGF beta 1 binding to RI in all 3 clones. Proliferation of RII-positive clones was inhibited by exogenous TGF beta 1 in a dose-dependent manner, whereas the control clones remained TGF beta-insensitive. The RII transfectants were growth arrested in monolayer culture at saturation densities which were 41-66% of that of the Neo controls. They also showed reduced clonogenicity in soft-agarose. Tumorigenicity in ovariectomized, estrogen-supplemented nude mice was delayed in transfectants with low RII levels. Transfectants expressing high levels of RII showed a large reduction in tumorigenicity as well as a longer delay in tumor formation. Tumor growth was associated with loss of exogenous RII expression in transfectants. The results indicate that when systems for TGF beta signal transduction are intact, reconstitution of the TGF beta receptor system can lead to reversion of malignancy in cells lacking RII.
Asunto(s)
Neoplasias de la Mama/patología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Animales , Secuencia de Bases , División Celular , Femenino , Humanos , Neoplasias Mamarias Experimentales/etiología , Ratones , Datos de Secuencia Molecular , Trasplante de Neoplasias , ARN Mensajero/análisis , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/farmacología , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Two membrane folate receptor (MFR) isoforms are present in human tissues i.e. MFR-1 (e.g. placenta) and MFR-2 (e.g. placenta, KB cells, CaCo-2 cells). MFR-1 was expressed in COS-1 cells and the resulting protein had the same polypeptide molecular weight as the native protein. The affinities of (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, and 5,10-dideazatetrahydrofolate as well as folic acid and methotrexate to MFR-1, MFR-2 and placental MFR (MFR-1 plus MFR-2) were determined in terms of the Ki values for their competitive inhibition of the binding of [3H]folic acid to these proteins. The results indicated a striking difference in the stereospecificity of MFR-1 and MFR-2 for reduced folate coenzymes; MFR-2 preferentially bound to the physiological (6S) diastereoisomers and MFR-1 bound preferentially to the unphysiological (6R) diastereoisomers, while dideazatetrahydrofolate did not show significant stereospecificity for MFR-1. Furthermore, MFR-2 displayed significantly (2- to 100-fold) greater affinities for all the compounds tested compared to MFR-1. Purified placental MFR, a natural source of MFR-1 which contains variable amounts of MFR-2, showed intermediate Ki values for the compounds tested compared with MFR-1 and MFR-2 and stereospecificities similar to MFR-1. These observations demonstrate striking differences in the ligand binding sites of MFR-1 and MFR-2 which could potentially be exploited in the design of MFR isoform specific antifolates.
Asunto(s)
Proteínas Portadoras/metabolismo , Antagonistas del Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Receptores de Superficie Celular , Animales , Unión Competitiva , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , ADN/genética , Femenino , Receptores de Folato Anclados a GPI , Haplorrinos , Humanos , Células KB , Cinética , Leucovorina/metabolismo , Metotrexato/metabolismo , Placenta/ultraestructura , Estereoisomerismo , Tetrahidrofolatos/metabolismoRESUMEN
Transforming growth factors-beta (TFG beta s) are multifunctional peptides that affect proliferation, differentiation, and many other functions in a variety of cell types. In this study we examined the effect of TGF beta 1 on aldosterone and adrenal renin production using cultured bovine adrenal zona glomerulosa cells. Collagenase-dispersed zona glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 72 h, and the medium was replaced with serum-free medium for the next 24 h. The cells during this 24-h period were exposed to TGF beta 1, ACTH, and (Bu)2cAMP (dbcAMP). It was observed that TGF beta 1 at 1 nM 1) inhibited basal aldosterone secretion from 680.0 +/- 40.0 to 270.0 +/- 10.0 pg/10(6) cells.h, 2) inhibited ACTH- and dbcAMP-stimulated aldosterone production, 3) increased levels of active renin in the cells from 17.8 +/- 2.5 to 70.7 +/- 4.4 pg angiotensin-I/10(6) cells.h and prorenin from 270.0 +/- 5.0 to 970.0 +/- 90 pg angiotensin-I/10(6) cells.h, 4) stimulated prorenin in the medium synergistically in combination with ACTH and dbcAMP, and 5) had no significant effect on basal cAMP production, but significantly inhibited the ACTH-stimulated production of cAMP. These observations show that TGF beta 1 is a potent inhibitor of basal and ACTH- and cAMP-stimulated aldosterone production and inhibits ACTH-stimulated cAMP production. Contrary to its effect on aldosterone, TGF beta 1 stimulates the synthesis and release of adrenal renin and prorenin. TGF beta 1 may act as an autocrine or paracrine regulator of aldosterone production.
Asunto(s)
Aldosterona/biosíntesis , Renina/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Zona Glomerular/metabolismo , Hormona Adrenocorticotrópica/farmacología , Animales , Bucladesina/farmacología , Bovinos , Células Cultivadas , AMP Cíclico/biosíntesis , Precursores Enzimáticos/biosíntesis , Antagonistas de Receptores de Mineralocorticoides/farmacologíaRESUMEN
Preprotransforming growth factor-beta 1 (TGF beta 1) is a 390-amino acid precursor polypeptide that undergoes a number of processing steps to yield mature TGF beta 1 (amino acid residues 279-390) and a pro portion (residues 30-278) termed beta 1-latency-associated peptide (beta 1LAP). The dimeric form of beta 1LAP has been shown to associate noncovalently with the mature growth factor, resulting in inactivation of biological activity. To further characterize this interaction, the mature TGF beta 1 was radioiodinated and used to determine dissociation constants. A cross-linking method using the bifunctional covalent cross-linker bis-(sulfosuccinimidyl)suberate was found to be the best approach for measuring the amount of bound growth factor. The efficiency of cross-linking was constant within each experiment and varied between 45-55%. Saturation plots and their associated Scatchard analyses indicate apparent Kd values between 1.1-1.8 nM. Competition of TGF beta 1 binding to beta 1LAP by TGF beta 2 and TGF beta 3 (two closely related growth factors) revealed that the latter also bind beta 1LAP tightly, with apparent Kd values of 1.9 and 0.4 nM, respectively.
Asunto(s)
Fragmentos de Péptidos , Proteínas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Cinética , Mamíferos , Visón , Unión Proteica , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
The existence of rat 18-kilodalton (kDa) prorelaxin, which has been postulated from the coding sequence of cloned cDNA and the results of cell-free translation studies, has been directly demonstrated in rat ovaries with antibodies against bacterially expressed rat prorelaxin. The peptide expressed in E. coli from a rat prorelaxin cDNA construct was comprised of the B- and A-chains of relaxin and a 105-amino acid connecting region. Immunoreactive bands of 18 and 16.5 kDa were shown in ovaries from day 20 pregnant rats. Partial amino acid sequence analysis of both peptides revealed that they had identical N-terminal sequences, corresponding to rat prorelaxin. Both 18- and 16.5-kDa bands were present only from midpregnancy until near term, when they declined sharply. These changes in the concentration of 18-kDa prorelaxin match changes in preprorelaxin mRNA levels, suggesting that relaxin synthesis is regulated at the transcriptional level and not by protein processing. Prorelaxin was transiently secreted by COS-1 cells transfected with preprorelaxin cDNA. Treatment of culture medium with trypsin resulted in the appearance of material corresponding in size to mature relaxin. Thus, correctly folded prorelaxin appears to be a suitable precursor for relaxin. The combined concentrations of 18- and 16.5-kDa peptides in ovaries on day 20 of pregnancy were considerably more than 30 times greater than that of relaxin, however, suggesting that prorelaxin might also be more than a precursor per se.
Asunto(s)
Bacterias/metabolismo , Sueros Inmunes/inmunología , Ovario/química , Preñez/metabolismo , Precursores de Proteínas/análisis , Relaxina/análisis , Secuencia de Aminoácidos , Animales , Femenino , Datos de Secuencia Molecular , Peso Molecular , Embarazo , Precursores de Proteínas/inmunología , Precursores de Proteínas/aislamiento & purificación , ARN Mensajero/análisis , Conejos , Ratas , Relaxina/inmunología , Relaxina/aislamiento & purificaciónRESUMEN
A series of site-specific insertion and deletion mutants was prepared in the pro domain of transforming growth factor beta 1 (TGF beta 1) encoded by simian TGF beta 1 cDNA. These mutants were transiently expressed in COS-1 cells and the ability of each to be properly processed, folded correctly, and secreted was determined by immunoblot analysis of cells and culture supernatants. Insertions in regions corresponding to amino acid residues 50, 154, and 170 blocked secretion; culture supernatants from COS-1 cells showed no immunologically reactive proteins, whereas intact cells contained high levels of the mutant polypeptides. Insertions in the middle portion of the pro domain at residues 81, 85, and 144 affected disulfide maturation of the mature TGF beta 1. An insertion at residue 110, on the other hand, appeared to destabilize the mature TGF beta 1 polypeptide, resulting in degraded growth factor. Relatively small (10 amino acids) to large (125 amino acids) deletion mutations in the pro domain of TGF beta 1, when expressed as the full-length pre-pro-TGF beta 1, appeared to block secretion. By contrast, if the pro domain (designated beta 1-latency-associated peptide [beta 1-LAP]) was expressed independently, deletion mutants in the region 40-110 were readily secreted by the COS-1 cells, whereas deletions in residues 110-210 either destabilized the structure of the protein or blocked its intracellular transport. Cross-linking assays employing radioiodinated TGF beta 1 and biological assays indicate that residues 50-85 of beta 1-LAP are required for association with mature TGF beta 1.
Asunto(s)
Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Factor de Crecimiento Transformador beta/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Deleción Cromosómica , Codón/genética , Humanos , Immunoblotting , Datos de Secuencia Molecular , Mutagénesis Insercional , Homología de Secuencia de Ácido Nucleico , Transfección , Factor de Crecimiento Transformador beta/análisisRESUMEN
Transforming growth factor beta 1 (TGF-beta 1) is proteolytically derived from the carboxyl terminus of a 390 amino acid precursor molecule termed pre-pro-TGF-beta 1. Previous studies have suggested that the pro piece of pre-pro-TGF-beta 1 may play an important role in the formation of an inactive, latent complex. These latent forms are thought to be important in the regulation of TGF-beta 1 activity. To understand this latent complex in more detail, we have expressed the pro domain of pre-pro-TGF-beta 1 in tissue culture cells independent of the mature growth factor. A stop codon was genetically engineered into the cDNA of pre-pro-TGF-beta 1 by changing the Arg-278 codon from CGA to the STOP codon TGA. The resulting protein is truncated just prior to the amino-terminal Ala residue of the mature growth factor. Transient expression studies and immunoblotting indicate that this pro piece is readily made and secreted by the COS-1 cells; the major form of the expressed pro piece, when analyzed by SDS-polyacrylamide gel electrophoresis, behaves as a disulfide-linked dimer (Mr 80,000). Bioassays, using mink lung indicator cells, reveal that the pro domain forms an inactive complex with exogenously added mature TGF-beta 1. Treatment of this complex with heat or acid results in the release of active TGF-beta 1, indicating an in vitro structure similar to natural, latent TGF-beta 1 complexes. The pro piece from TGF-beta 1 was also found to form latent structures with two closely related family members, TGF-beta 1.2 and TGF-beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Precursores de Proteínas , Proteínas , Factor de Crecimiento Transformador beta , Factores de Crecimiento Transformadores , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/genética , ADN/genética , Expresión Génica , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Precursores de Proteínas/genética , Proteínas/genética , Factores de Crecimiento Transformadores/genéticaRESUMEN
Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.
Asunto(s)
Fibrinolisina/metabolismo , Precursores de Proteínas , Factor de Crecimiento Transformador beta , Factores de Crecimiento Transformadores/genética , Animales , Línea Celular , Haplorrinos , Immunoblotting , Modelos Estructurales , Peso Molecular , Proteínas/genética , Ensayo de Unión Radioligante , Proteínas Recombinantes/metabolismo , Transfección , Factores de Crecimiento Transformadores/metabolismoRESUMEN
Transforming growth factor-alpha-like immunoreactivity (TGF-alpha-ir) was visualized in the adult rat forebrain using three antisera directed against carboxyterminal sequences in the TGF-alpha precursor. Using immunoperoxidase and immunofluorescence techniques with all three antisera, TGF-alpha-ir was found to be present in a subpopulation of astrocytes in the forebrain. Striatal and pallidal regions of the basal ganglia were studied in detail. In the striatum, there was an uneven distribution of astrocytes containing TGF-alpha-ir, with the greatest number in the dorsal medial third of the caudate-putamen and the overlying corpus callosum/external capsule. In addition, the region of the caudate-putamen bordering the globus pallidus contained numerous clusters of TGF-alpha-ir astrocytes. The globus pallidus itself contained numerous and more evenly distributed TGF-alpha-ir astrocytes. Other pallidal structures--including the ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata--contained moderate numbers of TGF-alpha-ir astrocytes. These results suggest that TGF-alpha precursor is present and, perhaps, synthesized in astrocytes. A related growth factor, epidermal growth factor (EGF), has also been reported to be present in pallidal regions of rat brain. Therefore, the TGF-alpha/EGF family of trophic factors may play a role in the function of the central nervous system.
Asunto(s)
Ganglios Basales/metabolismo , Precursores de Proteínas/metabolismo , Factor de Crecimiento Transformador alfa , Factores de Crecimiento Transformadores/metabolismo , Animales , Astrocitos/metabolismo , Cuerpo Estriado/metabolismo , Femenino , Globo Pálido/metabolismo , Inmunohistoquímica , Masculino , Ratas , Ratas Endogámicas , Distribución TisularRESUMEN
The role of glycosylation of the transforming growth factor-beta 1 (TGF-beta 1) precursor was investigated by treating a transfected Chinese hamster ovary (CHO) cell line expressing high levels of recombinant TGF-beta 1 (TGF-beta 3-2000 cells) with a series of glycosylational inhibitors. Tunicamycin, a nucleoside antibiotic which prevents the formation of the dolichol intermediate necessary for oligosaccharide addition of the nascent polypeptide chain, appeared to block secretory exit and led to an increase in the cellular associated, nonglycosylated pro-TGF-beta 1 form. 1-Deoxymannojirimycin and swainsonine, inhibitors of the mannosidases I and II, respectively, blocked complete glycoprotein processing of the TGF-beta 1 precursor as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by sensitivity to glycosidases. However, the abnormal TGF-beta 1 polypeptides containing the altered carbohydrate side chains were secreted readily by the CHO cells. In contrast, inhibitors of the glucosidases at the first step in glycoprotein remodeling, 1-deoxynojirimycin and castanospermine, markedly inhibited secretion of the TGF-beta 1 polypeptides from transfected CHO cells. In all cases, these inhibitors did not appear to affect proteolytic processing of the TGF-beta 1 polypeptides. Furthermore, inhibitor treatment did not affect mannose-6-phosphorylation of the TGF-beta 1 polypeptides. These results suggest that glycosylation and early stage remodeling of oligosaccharide side chains are necessary for secretion of TGF-beta 1. Treatment of the transfected CHO cells with weak bases (NH4Cl and chloroquine), or a monovalent ionophore (monensin), prevented proteolytic processing of the TGF-beta 1 precursor indicating that cleavage occurs by proteases in an acidic cellular compartment.