RESUMEN

OBJECTIVE: Several experimental and clinical studies indicate that the renin system may play a pivotal role in progressing renal disease. The combination of an angiotensin-converting enzyme inhibitor and an angiotensin receptor blocker could provide a higher degree of blockade of the renin-angiotensin system than either agent alone. Such enhanced suppression might be of benefit for patients exhibiting a progressive decline in renal function because of chronic renal disease. METHODS: A pilot multinational, multicentre, randomized, active-controlled, parallel group open-label study has been conducted in a group of patients with progressive chronic renal failure (creatinine clearance 20-45 ml/min) either with or without proteinuria and hypertension. The primary aim of the study was to investigate the safety and tolerability of the combination of valsartan and benazepril. Patients were randomly assigned to one of three groups: group 1 received valsartan 160 mg once daily (n = 22); group 2 received valsartan 80 mg once daily plus benazepril 5 or 10 mg once daily (n = 42); group 3 received valsartan 160 mg once daily plus benazepril 5 or 10 mg once daily (n = 44). The study lasted for 5 weeks, and in groups 2 and 3 benazepril was added on top of valsartan after the first week of therapy with the angiotensin receptor blocker. RESULTS: Serum creatinine increased in all three groups (mean change within a group: 11 micromol/l in group 1, P= 0.045; 9 micromol/l in group 2, P= 0.030; 15 micromol/l in group 3, P= 0.0006). Serum potassium also increased in all three groups of patients (mean change within a group: 0.28 mmol/l in group 1, P= 0.28; 0.48 mmol/l in group 2, P= 0.0008; 0.36 mmol/l in group 3, P= 0.02). After 5 weeks of treatment, the largest decrease in blood pressure was observed in group 3 (the mean change from baseline in seated diastolic blood pressure (SDBP) and seated systolic blood pressure (SSBP), respectively, were: -2.0 and -11.5 mmHg in group 1; -7.6 and -15.4 mmHg in group 2; -12.6 and -21.6 mmHg in group 3). In addition, both combination treatments resulted in the reduction of proteinuria. The total number of patients with adverse experiences were 10 (45.5%), 14 (33.3%) and 11 (25%) in groups 1,2 and 3, respectively. In six patients (5.6%) therapy was discontinued as a result of adverse experiences. Only one patient in each of the combined therapy groups withdrew from the study because of hyperkalaemia and no patients were forced to withdraw because of an increase in serum creatinine, acute renal failure or hospitalization. CONCLUSIONS: These results indicate that short-term combination of an angiotensin-converting enzyme inhibitor and an angiotensin receptor blocker is safe and well tolerated in patients with moderate chronic renal failure.

Asunto(s)

Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Antihipertensivos/uso terapéutico , Benzazepinas/uso terapéutico , Fallo Renal Crónico/tratamiento farmacológico , Tetrazoles/uso terapéutico , Valina/análogos & derivados , Anciano , Inhibidores de la Enzima Convertidora de Angiotensina/efectos adversos , Antihipertensivos/efectos adversos , Benzazepinas/efectos adversos , Presión Sanguínea/efectos de los fármacos , Creatinina/sangre , Combinación de Medicamentos , Femenino , Humanos , Hipertensión/complicaciones , Fallo Renal Crónico/sangre , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/fisiopatología , Masculino , Persona de Mediana Edad , Proyectos Piloto , Potasio/sangre , Proteinuria/orina , Seguridad , Tetrazoles/efectos adversos , Valina/efectos adversos , Valina/uso terapéutico , Valsartán

RESUMEN

Maspin is a unique member of the serpin family, which functions as a class II tumor suppressor gene. Despite its known activity against tumor invasion and motility, little is known about maspin's functions in normal mammary gland development. In this paper, we show that maspin does not act as a tPA inhibitor in the mammary gland. However, targeted expression of maspin by the whey acidic protein gene promoter inhibits the development of lobular-alveolar structures during pregnancy and disrupts mammary gland differentiation. Apoptosis was increased in alveolar cells from transgenic mammary glands at midpregnancy. However, the rate of proliferation was increased in early lactating glands to compensate for the retarded development during pregnancy. These findings demonstrate that maspin plays an important role in mammary development and that its effect is stage dependent.

Asunto(s)

Glándulas Mamarias Animales/embriología , Proteínas/fisiología , Inhibidores de Serina Proteinasa/fisiología , Serpinas/fisiología , Animales , Apoptosis , División Celular , Femenino , Genes Supresores de Tumor , Lactancia , Ratones , Ratones Transgénicos , Proteínas de la Leche/genética , Embarazo , Proteínas/genética , Serpinas/genética , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Transgenes

RESUMEN

The study compared valsartan 80 mg or 160 mg o.d. with captopril 25 mg t.i.d. or placebo on plasma lipids in normotensive and treated hypertensive patients with type II diabetes and microalbuminuria. One hundred and twenty-two adult outpatients were randomised to receive either valsartan 80 mg or 160 mg, captopril 25 mg or placebo for 360 days. Changes from baseline to endpoint in plasma lipid parameters were measured. The primary criterion for tolerability was the incidence of adverse events. All treatment groups showed minor changes in lipid parameters. Triglyceride increased by 2.7% (valsartan 160 mg) to 9.1% (placebo). Total cholesterol decreased under valsartan 80 mg, while other groups showed increases of up to 0.031 mmol/l. Decreases in total cholesterol (p = 0.018), apolipoprotein B (p = 0.042) and apolipoprotein A1 (p = 0.025), were significant for the comparison of 80 mg valsartan and captopril. Valsartan 80 mg or 160 mg o.d. does not cause deleterious changes in the diabetic lipid profile and, unlike captopril, is not associated with dry cough.

Asunto(s)

Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Antihipertensivos/administración & dosificación , Captopril/administración & dosificación , Lípidos/sangre , Tetrazoles/administración & dosificación , Valina/análogos & derivados , Adulto , Anciano , Albuminuria/sangre , Albuminuria/tratamiento farmacológico , Diabetes Mellitus Tipo 2/sangre , Método Doble Ciego , Femenino , Humanos , Hipertensión/sangre , Masculino , Persona de Mediana Edad , Valina/administración & dosificación , Valsartán

RESUMEN

The aim of this study was to evaluate the efficacy and tolerability of valsartan, a new angiotensin II receptor antagonist, versus atenolol in the treatment of severe primary hypertension. A total of 103 adult out-patients were randomised to receive either valsartan 160 mg or atenolol 100 mg once daily for 6 weeks. If necessary, additional blood pressure (BP) control could be provided as add-on therapy. Both valsartan and atenolol decreased mean sitting diastolic BP (DBP) and mean sitting systolic BP (SBP): least squares mean change from baseline in DBP; valsartan, -20.0 mm Hg; atenolol, -20.4 mm Hg: in SBP; valsartan, -30.0 mm Hg; atenolol, -25.5 mm Hg. There was no statistically significant difference between the treatment groups. Add-on hydrochlorothiazide (HCTZ) 25 mg was required by 97.2% of patients receiving atenolol and 83.6% of patients receiving valsartan; additional verapamil SR 240 mg was also required by 58.3% of patients receiving atenolol and 64.2% receiving valsartan. Valsartan was well tolerated, with a comparable incidence of treatment-related adverse experiences in both groups. In conclusion valsartan 160 mg is as well tolerated and effective as atenolol 100 mg in lowering BP in severely hypertensive patients.

Asunto(s)

Antihipertensivos/administración & dosificación , Atenolol/administración & dosificación , Hipertensión/tratamiento farmacológico , Tetrazoles/administración & dosificación , Valina/análogos & derivados , Adulto , Anciano , Antagonistas de Receptores de Angiotensina , Antihipertensivos/efectos adversos , Atenolol/efectos adversos , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , Tetrazoles/efectos adversos , Valina/administración & dosificación , Valina/efectos adversos , Valsartán

RESUMEN

Protease nexin-1 (PN-1), a member of the serpin superfamily, controls the activity of extracellular serine proteases and is expressed in the brain. Mutant mice overexpressing PN-1 in brain under the control of the Thy-1 promoter (Thy 1/PN-1) or lacking PN-1 (PN-1-/-) were found to develop epileptic activity in vivo and in vitro. Theta burst-induced long-term potentiation (LTP) and NMDA receptor-mediated synaptic transmission in the CA1 field of hippocampal slices were augmented in Thy 1/PN-1 mice and reduced in PN-1-/- mice. Compensatory changes in GABA-mediated inhibition in Thy 1/PN-1 mice suggest that altered brain PN-1 levels lead to an imbalance between excitatory and inhibitory synaptic transmission.

Asunto(s)

Proteínas Portadoras/fisiología , Epilepsia/fisiopatología , Hipocampo/fisiología , Potenciación a Largo Plazo , Neuronas/fisiología , Precursor de Proteína beta-Amiloide , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Epilepsia/genética , Hipocampo/fisiopatología , Cinética , Ratones , Ratones Noqueados , Ratones Mutantes Neurológicos , Ratones Transgénicos , Regiones Promotoras Genéticas , Nexinas de Proteasas , Receptores de Superficie Celular , Proteínas Recombinantes de Fusión/biosíntesis , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/fisiología , Transmisión Sináptica , Antígenos Thy-1/biosíntesis , Antígenos Thy-1/genética , Factores de Tiempo

RESUMEN

The rat protease nexin-1 (PN-1) promoter contains a GCGGGGGCG binding site for the transcription factors Krox-24, Krox-20 and NGFI-C. Mutations of this site abolished binding of Krox-24 in vitro. The wildtype protease nexin-1 promoter expressed beta-galactosidase similarity to the expression of protease nexin-1 mRNA. When the function of this Krox site was tested in vivo using transgenic F0 embryos, mutation had two opposite effects. beta-Galactosidase expression increased in cartilage and heart at both stages E11.5 and E13.5, but was abolished in nerves of the central and peripheral nervous system at stage E13.5. These results suggest that Krox factors are among the important transcription factors regulating protease nexin-1 expression and thereby intracellular proteolytic activity in embryonic heart, cartilage and parts of the nervous system.

Asunto(s)

Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces , Inhibidores de Serina Proteinasa/genética , Factores de Transcripción/metabolismo , Precursor de Proteína beta-Amiloide , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/metabolismo , Cartílago/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Proteína 2 de la Respuesta de Crecimiento Precoz , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Miocardio/metabolismo , Sistema Nervioso/metabolismo , Regiones Promotoras Genéticas/genética , Nexinas de Proteasas , Ratas , Receptores de Superficie Celular , Inhibidores de Serina Proteinasa/metabolismo , Factores de Tiempo , Factores de Transcripción/química , Transfección , Células Tumorales Cultivadas

RESUMEN

The B-lymphocyte-specific transcriptional factor called Oct binding factor (OBF)-1, OCA-B or Bob1 (refs 1-3) is thought to be involved in the transcription of immunoglobulin genes through recruitment to the highly conserved octamer site of immunoglobulin promoters, mediated by either Oct-1 or Oct-2. To define the in vivo role of OBF-1 we have used gene targeting in embryonic stem cells to generate mice lacking the coactivator OBF-1. Such OBF-1-/- mice are born normally, are fertile and seem healthy, and surprisingly, rearrangement and transcription of immunoglobulin genes are largely unaffected. However, mice deficient in OBF-1 have reduced numbers of mature B cells and a severe reduction in the number of recirculating B cells, but otherwise show normal B-cell differentiation. Serum IgA and particularly IgG levels are greatly reduced. If mutant mice are immunized with either a thymus-independent or a thymus-dependent antigen, their immune responses are dramatically weakened. Strikingly, germinal centres completely fail to develop after immunization with thymus-dependent antigen. Our results demonstrate that in vivo OBF-1 is not required for initial transcription of immunoglobulin genes or for B cell development, but instead is essential for the response of B cells to antigens, and is required for the formation of germinal centres.

Asunto(s)

Linfocitos B/inmunología , Centro Germinal/citología , Inmunidad/fisiología , Transactivadores/fisiología , Animales , Formación de Anticuerpos/fisiología , Linfocitos B/citología , Células de la Médula Ósea , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Marcación de Gen , Centro Germinal/inmunología , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Bazo/citología , Transactivadores/genética

RESUMEN

The major neuronal microtubule-associated protein MAP2 is selectively localized in dendrites, where its expression is under strong developmental regulation. To learn more about its potential effects on neuronal morphogenesis and its sorting within the neuronal cytoplasm, we have raised transgenic mice that express high levels of the embryonic form, MAP2c, in the adult brain. One transgenic line expressed higher levels of MAP2c than endogenous adult MAP2. This had no detectable effect on either the arrangement or morphology of neurons, suggesting that although MAP2c is necessary for neuronal morphogenesis it is not involved in its regulation. Like endogenous adult MAP2, transgenic MAP2c was present in dendrites but not axons, indicating that the signal responsible for its cytoplasmic sorting is contained within the 1.5 kb of its coding sequence. In situ hybridization with specific probes showed that transgenic MAP2c mRNA was limited to cell bodies. Thus, the dendritic localization of MAP2c protein cannot be the result of previous transport of its mRNA but must depend on a signal associated with the protein itself. Furthermore, because the amino acid sequence of MAP2c is present in all forms of MAP2, this signal is also contained within adult high-M(r) MAP2 protein. This raises the possibility that, rather than the conventional scheme of mRNA sorting preceding protein localization, the transport of adult MAP2 mRNA into dendrites could depend on it being part of a translation complex in which the targeting signal is on the nascent protein.

Asunto(s)

Encéfalo/metabolismo , Proteínas Asociadas a Microtúbulos/genética , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Hipocampo/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos

Asunto(s)

Proteínas Portadoras/genética , Mapeo Cromosómico , Proteínas de Microfilamentos/genética , Receptores Colinérgicos/genética , Precursor de Proteína beta-Amiloide , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Nexinas de Proteasas , Receptores de Superficie Celular , Serpina E2

RESUMEN

Regulation of neurite outgrowth and structural plasticity may involve the expression of intrinsic determinants controlling growth competence. We have tested this concept by targeting constitutive expression of the growth-associated protein GAP-43 to the neurons of adult transgenic mice. Such mice showed striking spontaneous nerve sprouting at the neuromuscular junction and in the terminal field of hippocampal mossy fibers. In control mice, these nerve fibers did not express GAP-43, and did not sprout spontaneously. Lesion-induced nerve sprouting and terminal arborization during reinnervation were greatly potentiated in GAP-43-overexpressing mice. A mutant GAP-43 that cannot be phosphorylated by PKC had reduced sprout-promoting activity. The results establish GAP-43 as an intrinsic presynaptic determinant for neurite outgrowth and plasticity.

Asunto(s)

Sustancias de Crecimiento/farmacología , Glicoproteínas de Membrana/farmacología , Proteínas del Tejido Nervioso/farmacología , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/crecimiento & desarrollo , Animales , Pollos/genética , Proteína GAP-43 , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Actividad Motora/genética , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Mutagénesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Unión Neuromuscular/citología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/crecimiento & desarrollo , Plasticidad Neuronal/genética , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Convulsiones/genética

RESUMEN

Protease Nexin-1 (PN-1) also known as Glia-Derived Nexin (GDN) inhibits the activity of several serine proteases including thrombin, tissue (tPA)- and urokinase (uPA)-type plasminogen activators. These and other serine proteases seem to play roles in development and tissue homeostasis. To gain insight into where and when PN-1 might counteract serine protease activities in vivo, we examined its mRNA and protein expression in the mouse embryo, postnatal developing nervous system and adult tissues. These analyses revealed distinct temporal and spatial PN-1 expression patterns in developing cartilage, lung, skin, urogenital tract, and central and peripheral nervous system. In the embryonic spinal cord, PN-1 expression occurs in cells lining the neural canal that are different from the cells previously shown to express tPA. In the developing postnatal brain, PN-1 expression appears transiently in many neuronal cell populations. These findings suggest a role for PN-1 in the maturation of the central nervous system, a phase that is accompanied by the appearance of different forms of PN-1. In adults, few distinct neuronal cell populations like pyramidal cells of the layer V in the neocortex retained detectable levels of PN-1 expression. Also, mRNA and protein levels did not correspond in adult spleen and muscle tissues. The widespread and complex regulation of PN-1 expression during embryonic development and, in particular, in the early postnatal nervous system as well as in adult tissues suggests multiple roles for this serine protease inhibitor in organogenesis and tissue homeostasis.

Asunto(s)

Proteínas Portadoras/genética , Desarrollo Embrionario y Fetal/genética , Sistema Nervioso/embriología , Inactivadores Plasminogénicos/genética , Precursor de Proteína beta-Amiloide , Animales , Northern Blotting , Expresión Génica/fisiología , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Nexinas de Proteasas , Receptores de Superficie Celular

RESUMEN

The pathogenicity of the human c-erbB-2 oncogene was evaluated in transgenic mice. A DNA sequence comprising the promoter-enhancer region of the MMTV LTR and a constitutively activated allele of the human c-erbB-2 growth factor receptor gene was introduced into the germ line of mice. Expression of the transgene was observed in kidney, lung, mammary gland, salivary gland, Harderian gland, and in epithelial cells of the male reproductive tract. All transgenic mice expressing the c-erbB-2 receptor died within four months of birth. Histopathological analysis suggests that preneoplastic lesions in kidney and lung most likely caused organ failure and the early death of the transgenic mice. Focal dilatation and atypical proliferation of the tubular epithelial cells was found in the kidney. These hyperplastic lesions were found adjacent to normal tubules. Immunohistochemistry showed that normal renal structures were completely negative for c-erbB-2 protein expression. Atypical pseudopapillary proliferation of bronchial and bronchiolar epithelial cells narrowed the bronchial lumen in lung. Alveoli appeared normal. The expression of c-erbB-2 protein was strictly limited to the proliferating epithelial cells and not detected in normal tissue. The mammary glands of two parous mice were underdeveloped, lacking lobular-alveolar structures and were lactation deficient. Only a few ducts were interspersed in the fat pad. A virgin mouse developed a focal adenocarcinoma infiltrating the mammary fat pad. Expression of the c-erbB-2 protein was enhanced in the proliferating epithelial cells. Transgenic males were sterile. Epithelial hyperplasia and hypertrophy in the epididymis, vas deferens and seminal vesicles was found. The transgene is not uniformly expressed in the tissues where the MMTV LTR is transcriptionally active. The scattered transgene expression invariably coincides with epithelial hyperplasia.

Asunto(s)

Regulación de la Expresión Génica , Riñón/patología , Pulmón/patología , Lesiones Precancerosas/patología , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Receptores de Superficie Celular/genética , Alelos , Animales , Muerte , Epidídimo/patología , Femenino , Vectores Genéticos , Humanos , Longevidad , Masculino , Glándulas Mamarias Animales/patología , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Transgénicos , Especificidad de Órganos , Lesiones Precancerosas/genética , Proteínas Proto-Oncogénicas/biosíntesis , Receptor ErbB-2 , Receptores de Superficie Celular/biosíntesis , Secuencias Repetitivas de Ácidos Nucleicos

RESUMEN

Transgenes encoding simian virus 40 (SV40) T antigen (Tag) can cause hyperplastic or tumorigenic lesions of desired but also of unforeseen cellular origin. Unexpectedly the human growth hormone-releasing factor (GRF) gene promoter directs expression of SV40 Tag specifically in thymic epithelial (TE) cells. Expression starts in the neonate, in which GRF-Tag+ cells display strict numerical and spatial constraints. Tag supersedes mechanisms that constrain these features and GRF-Tag mice develop thymic hyperplasia. To characterize GRF-Tag+ TE cells and their putative normal counterparts we compared phenotypic and functional effects caused by transgenes encoding mutant large T antigens. This strategy is applicable to any situation in which T antigen is used to alter development. One large Tag mutant (K1 + 5080) does not cause thymic hyperplasia. GRF-Tag (K1 + 5080)+ TE cells display strict temporal and spatial constraints throughout life. TE cells expressing other mutant large T antigens that cause thymic hyperplasia do not obey these rules and reveal that phenotypically distinct GRF-Tag+ TE-cell stages exist in vivo. Analysis of conditional immortal GRF-Tag(tsA58)+ TE cells expressing a temperature-sensitive large Tag shows that large Tag blocks differentiation in these cells. Phenotype and functions in these cells are regulated by cellular differentiation and interleukin 4 (IL-4).

Asunto(s)

Antígenos Transformadores de Poliomavirus/genética , Virus 40 de los Simios/inmunología , Timo/citología , Animales , Antígenos Transformadores de Poliomavirus/análisis , Diferenciación Celular , Células Epiteliales , Hormona Liberadora de Hormona del Crecimiento/genética , Interleucina-4/farmacología , Factor Estimulante de Colonias de Macrófagos/genética , Ratones , Ratones Endogámicos C57BL , Mutación , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-kit , Receptores de Interleucina-4 , Receptores Mitogénicos/genética , Temperatura

RESUMEN

To dissect mechanisms that co-ordinate specific events in thymopoiesis we have characterized alterations in thymic structure and function caused by expression of a transgene. This gene encodes SV40Tag and is specifically expressed in a subset of thymic epithelial (TE) cells around birth. As a result the number of immortal TE cells increases, thymic mass increases (up to 3 g), and thymopoiesis is expanded. The latter is reflected by a approximately 100-fold increase of the major thymocyte subsets and increased peripheral T cell counts. Grossly hyperplastic thymi retain many but not all morphological features of a normal thymus. Also in grafts, SV40Tag+ TE cells steer expansion (up to 8 g) and organize a tissue with mainly cortex-like features that includes mainly SV40Tag+ TE cells, thymocytes, and macrophages. To investigate expression of specialized gene functions in the immortal TE cells, a cell line was derived. The Epi-A1 cell line expresses the genes for major histocompatibility complex class I and II, Thy-1, interleukin (IL)-6, IL-7, macrophage-colony-stimulating factor, and transforming growth factor-beta 3. Most importantly, Epi-A1 cells also express the IL-4 receptor and the c-kit ligand (KL), a factor that, in concert with commitment factors, channels progenitors into hemopoietic lineages. The expression of low constitutive levels of KL mRNA does not require IL-4, but KL mRNA levels are increased dramatically in response to IL-4. Since constitutive expression of KL mRNA in vivo is restricted to a small subset of TE cells in the thymus, our findings reveal a novel specific interaction between thymocytes and a specialized subset of TE cells.

Asunto(s)

Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Superficie Celular/biosíntesis , Hiperplasia del Timo/metabolismo , Factores de Edad , Animales , Secuencia de Bases , Línea Celular , Factores Estimulantes de Colonias/análisis , Epitelio/metabolismo , Citometría de Flujo , Expresión Génica , Hormona Liberadora de Hormona del Crecimiento/biosíntesis , Inmunofenotipificación , Interleucina-4/farmacología , Interleucinas/análisis , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Proteínas Proto-Oncogénicas c-kit , ARN/análisis , Hiperplasia del Timo/inmunología , Hiperplasia del Timo/patología , Factor de Crecimiento Transformador beta/fisiología

RESUMEN

The 5' flanking regions of the mouse and pig urokinase plasminogen activator (uPA) genes were sequenced and sequence homology interrupted by repeat elements was found to extend to -4.6kb in pig and -6.6kb in mouse. A transient transfection procedure was devised for the murine macrophage cell line RAW264. Pig uPA promoter-CAT constructs were more active than mouse constructs in this assay. This contrast may involve sequence differences within 100 bp of the transcription start site. The selective deletion of distal regions of the promoter (greater than 2.6 kb upstream), and of a conserved element, 5'-AGGAGGAAATGAGG-TCA-3' around -2 kb greatly reduced the activity of reporter constructs in RAW264 cells. Electrophoretic mobility shift assays using the latter sequence identified a single nuclear protein complex. This element has been referred to as PEA3/AP1-like, but the complex did not comigrate with either AP1 or known proteins that bind polypurines (including the macrophage-specific factor PU-1) and was not competed by AP1 or polypurine oligonucleotides. uPA promoters contain multiple AP1 and AP2-like DNA sequences, which were recognised by nuclear proteins expressed constitutively in RAW264 cells. They also contain multiple binding sites for NF kappa B but activated NF kappa B was not expressed in RAW264 cells. The conserved, transcribed 5' non-coding sequences were also required for maximal gene expression. Hence, the uPA promoter contains multiple weak cis-acting elements distributed over 7.0 kb 5' to the translation start site.

Asunto(s)

Regulación Enzimológica de la Expresión Génica/genética , Macrófagos/enzimología , Regiones Promotoras Genéticas/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Clonación Molecular , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Porcinos , Transcripción Genética/genética , Transfección/genética

RESUMEN

Urokinase-type plasminogen activator (uPA) is expressed at higher levels in many transformed cells as compared with their non-transformed counterparts. The transformed phenotype is associated with changes in the cytoskeleton. Therefore, we have investigated whether alterations in the cytoskeleton can trigger changes in the expression of the uPA gene. To this end we analyzed the expression of the uPA gene following exposure of porcine kidney cells, LLC-PK1, to agents that modify the organization of specific components of the cytoskeleton. These cells exhibited increased uPA mRNA and protein after disruption of microtubules by colchicine or nocodazole treatment or after disruption of microfilaments by cytochalasin B treatment. Colchicine, nocodazole, and cytochalasin B did not cause alterations in the level of cAMP-dependent protein kinase in LLC-PK1 cells. In contrast, down-regulation of protein kinase C by phorbol myristate acetate, reduced, but did not fully prevent the induction of uPA mRNA when LLC-PK1 cells were subsequently exposed to colchicine, nocodazole, or cytochalasin B. Apparently, a signal transduction pathway in part involving protein kinase C but not cAMP-protein kinase mediates the regulatory changes at the transcriptional level of the uPA gene. Inhibition of protein synthesis by cycloheximide prior to the exposure of LLC-PK1 cells to colchicine, nocodazole, or cytochalasin B, largely prevented the induction of uPA mRNA.

Asunto(s)

Colchicina/farmacología , Citocalasina B/farmacología , Citoesqueleto/efectos de los fármacos , Precursores Enzimáticos/genética , Nocodazol/farmacología , Activadores Plasminogénicos/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Citoesqueleto de Actina/efectos de los fármacos , Animales , Línea Celular , Núcleo Celular , Citoesqueleto/ultraestructura , Inducción Enzimática , Precursores Enzimáticos/biosíntesis , Técnica del Anticuerpo Fluorescente , Expresión Génica , Microtúbulos/efectos de los fármacos , Activadores Plasminogénicos/biosíntesis , Proteínas Quinasas/metabolismo , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/genética , Mapeo Restrictivo , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis

RESUMEN

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.

Asunto(s)

Adenosina Desaminasa/genética , Genes , Nucleósido Desaminasas/genética , Regiones Promotoras Genéticas , Animales , Linfocitos B/inmunología , Trasplante de Médula Ósea , Cloranfenicol O-Acetiltransferasa/genética , Clonación Molecular , Activación de Linfocitos , Ratones , Ratones Endogámicos , Ratones Transgénicos , Especificidad de Órganos , Linfocitos T/inmunología

RESUMEN

The Thy-1 antigen is a cell-surface glycoprotein of unknown function expressed on mouse T lymphocytes, neurons, and hematopoietic stem cells. To alter the normal pattern of Thy-1 expression during hematopoietic differentiation, we created transgenic mice using a hybrid Thy-1 gene containing a transcriptional enhancer of the mouse immunoglobulin heavy chain gene (E mu). Strains of mice bearing the Thy-1.2/E mu gene express the Thy-1.2 antigen on mature B lymphocytes and their progenitors, and develop a heritable lymphoid hyperplasia characterized by massive expression of the Thy-1.2 antigen in the bone marrow and lymph nodes. The phenotype associated with inappropriate developmental regulation of the Thy-1 gene suggests that the Thy-1 antigen may play a role in inducing activation or differentiation events on early lymphocyte progenitor cells.

Asunto(s)

Antígenos de Superficie/genética , Regulación de la Expresión Génica , Trastornos Linfoproliferativos/inmunología , Animales , Antígenos de Superficie/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Médula Ósea/inmunología , Médula Ósea/patología , División Celular , Elementos de Facilitación Genéticos , Células Madre Hematopoyéticas/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Ganglios Linfáticos/inmunología , Tejido Linfoide/inmunología , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/patología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/genética , Recombinación Genética , Antígenos Thy-1

RESUMEN

The hypothalamic peptide growth hormone-releasing factor (GRF) regulates the secretion and production of growth hormone from the anterior pituitary (M. C. Gelato and G. R. Merriam, Annu. Rev. Physiol. 48:569-591). To study GRF gene regulation, transgenic mice were generated that harbor the human GRF promoter fused to the coding sequences from the simian virus 40 early region. These mice had normal hypothalamic functions but unexpectedly suffered from severe thymic hyperplasia. Immunohistochemical analysis revealed that large T antigen was expressed in the thymic epithelial cells. These cells have endocrine properties and are known to produce thymic hormones [corrected]. The thymic hyperplasia was the apparent consequence of inappropriate production of T-cell maturation factors by epithelial cells and could involve increased self renewal of apparently normal T stem cells in the thymus.

Asunto(s)

Antígenos Virales de Tumores/genética , Hormona Liberadora de Hormona del Crecimiento/genética , Ratones Transgénicos/fisiología , Regiones Promotoras Genéticas , Hiperplasia del Timo/genética , Animales , Regulación de la Expresión Génica , Genes ras , Ratones , Factores de Crecimiento Nervioso/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Distribución Tisular

RESUMEN

Proto-oncogene fos mRNA levels are rapidly and transiently elevated 12-fold in regenerating liver 10-60 min following partial hepatectomy. This response, and the induction of fos protein synthesis, has been simulated qualitatively and quantitatively in long term primary cultures of quiescent adult rat hepatocytes where proliferative transitions can be initiated directly in serum-free medium by known hepatocyte mitogens like epidermal growth factor. Expression of a second proto-oncogene, c-rasH, in proliferatively activated hepatocyte cultures between 6 and 24 h also simulates the delayed hepatic response that occurs in vivo following partial hepatectomy. These results suggest that sequential proto-oncogene expression during liver regeneration is caused directly by hepatocellular interactions with specific mitogens. In addition, a role for monovalent cations in the regulation of hepatocyte gene expression is implicated from findings that Na+ deprivation inhibits induction of fos expression in cultured hepatocytes by epidermal growth factor under chemically defined conditions.

Asunto(s)

Regeneración Hepática , Hígado/metabolismo , Proto-Oncogenes , ARN Mensajero/genética , Animales , División Celular , Transformación Celular Neoplásica , Células Cultivadas , Femenino , Cinética , Ratones , Ratones Endogámicos ICR , Ratas , Ratas Endogámicas F344