RESUMEN

Activation of the Wnt signaling pathway initiates the transformation of colorectal epithelial cells, although the transition to metastatic cancer requires angiogenesis. We have investigated the expression of the von Hippel-Lindau (VHL) tumor suppressor in the intestines from humans and mice. Here, we show that VHL expression is regulated by TCF4 and is restricted to the proliferative compartment at the bottom of intestinal crypts. Accordingly, VHL is completely absent from the proliferative intestinal pockets of Tcf4(-/-) perinatal mice. We observed complementary staining of the hypoxia-inducible factor (HIF) 1alpha to VHL in normal intestinal epithelium as well as in all stages of colorectal cancer (CRC). To the best of our knowledge, this is the first report demonstrating the presence of nuclear HIF1alpha in normoxic healthy adult tissue. Although we observed upregulated levels of VHL in very early CRC lesions from sporadic and familial adenomatous polyposis patients - presumably due to activated Wnt signaling - a clear reduction of VHL expression is observed in later stages of CRC progression, coinciding with stabilization of HIF1alpha. As loss of VHL in later stages of CRC progression results in stabilization of HIF, these data provide evidence that selection for VHL downregulation provides a proangiogenic impulse for CRC progression.

Asunto(s)

Adenocarcinoma/etiología , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/etiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Proteínas del Tejido Nervioso/fisiología , Factores de Transcripción TCF/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Proteínas Wnt/fisiología , beta Catenina/fisiología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/patología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Línea Celular , Colon/citología , Colon/metabolismo , Colon/patología , Pólipos del Colon/genética , Pólipos del Colon/metabolismo , Pólipos del Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Eritropoyetina/genética , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/análisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Riñón , Células L , Ratones , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transducción de Señal/fisiología , Factores de Transcripción TCF/deficiencia , Factores de Transcripción TCF/genética , Factor de Transcripción 4

RESUMEN

Loracarbef (LC) is the first clinically available carbacephem. In order to evaluate the antimicrobial activity of LC, a total of 593 clinical strains of H. influenzae, M. catarrhalis, S. pneumoniae and S. pyogenes were collected by seven participating study centres. Minimal inhibitory concentrations for loracarbef and amoxycillin-clavulanic acid combination (AC) were determined by a microdilution method. The MIC-90 of beta-lactamase-negative strains of H. influenzae was 2.0 (LC) and 0.5 (AC), and for beta-lactamase-positive strains 4.0 (LC) and 2.0 (AC) mg/l. For beta-lactamase-negative and beta-lactamase-positive strains of M. catarrhalis, the MIC-90 results were 0.5 and 2.0 (LC) and 0.13 and 0.5 (AC) mg/l. The MIC-90 for AC was

RESUMEN

Follicle-stimulating hormone (follitropin, FSH) belongs to a group of closely related glycoprotein hormones that contain two noncovalently linked dissimilar subunits designated alpha and beta. By using synthetic peptides, several receptor interaction sites in these hormones have been identified; however, the peptides have a reduced potency (lowest effective concentration of 10(-4) to 10(-5) M) relative to the hormone itself (10(-8) to 10(-11) M). This suggests that the peptides represent only a portion of a larger recognition site in the intact hormone that comprises parts of both the beta and the alpha chains. To develop peptides that exhibit FSH-antagonistic activity at low concentrations, we have constructed a three-dimensional model for FSH, which is based on an alignment of both the beta and the alpha chains of glycoprotein hormones with thioredoxin, for which x-ray diffraction data are available. This model resulted in the prediction of a conformational receptor-binding site in FSH, in which (parts of) three earlier proposed binding regions on the FSH molecule [namely, the regions FSH alpha-(34-37), with the amino acid sequence SRAY; FSH beta-(40-43), with the amino acid sequence TRDL; and FSH beta-(87-94), the "determinant loop" with the amino acid sequence CDSDSTDC] are located within 10 A of one another. On the basis of this model, peptides have been synthesized in which two of these binding regions are linked by a synthetic amino acid whose length was derived from the model, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH2 and Ac-SRAY-NH-(CH2)4-CO-TRDL-NH2. Both peptides inhibited FSH-induced cAMP production in Sertoli cells at 1000-fold lower concentrations (10(-7) M) than the peptides Ac-TRDL-NH2, Ac-SRAY-NH2, or Ac-TDSDS-NH2. In another peptide, Ac-TDSDS-NH-(CH2)5-CO-SRAY-NH-(CH2)4-CO-TRDL-NH2, all three binding regions have been linked. This peptide appeared to be a strong agonist of FSH action, as measured by the ability to stimulate cAMP production, at concentrations as low as 10(-7) M. The observation that a synthetic peptide, in which (parts of) three earlier described receptor interaction sites are combined according to the three-dimensional model, can mimic the action of FSH, at 10(-7) M, shows that this model is useful to predict a conformational receptor-binding site in FSH and that combination of only a few amino acid residues from the alpha and beta chains of FSH in a small synthetic peptide is sufficient to transduce a signal upon binding to the receptor.

Asunto(s)

Hormona Folículo Estimulante , Oxidorreductasas , Receptores de HFE/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Gonadotropina Coriónica/química , AMP Cíclico/biosíntesis , Diseño de Fármacos , Hormona Folículo Estimulante/análogos & derivados , Hormona Folículo Estimulante/antagonistas & inhibidores , Hormona Folículo Estimulante/metabolismo , Glutarredoxinas , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Conformación Proteica , Proteínas/química , Ratas , Receptores de HFE/metabolismo , Células de Sertoli/metabolismo , Relación Estructura-Actividad , Tiorredoxinas/química

RESUMEN

In many biological processes, defined regions of proteins are involved in selective recognition. These regions can often be mimicked with peptides and are the main targets for vaccine and drug development. The authors review the use of peptides, to define and ultimately mimic defined protein regions of interest. Especially the role of the Pepscan method is emphasized. This method has been proven to be a useful and fast tool in defining protein regions of interest. It is based on the simultaneously synthesis of multiple peptides coupled to solid supports. Hundreds of peptides can be produced and tested in a relatively short period of time. With the construction of random peptide libraries in recombinant DNA systems, it is now even possible to screen for peptidic determinants without the requirement of preliminary knowledge of primary structure. Having this information, the affinity of peptides can be further enhanced with the Pepscan approach. The power of this approach will be illustrated with results from studies on the development of synthetic vaccines and hormone analogues.

Asunto(s)

Péptidos , Bioensayo/métodos , Diseño de Fármacos , Técnicas Genéticas , Conformación Proteica , Vacunas Sintéticas

RESUMEN

The biochemical activities involved in the maintenance of Leydig cell functions, and the effects of hypophysectomy and human chorionic gonadotrophin (hCG) on these functions are largely unknown. In the present study, adult hypophysectomized rats were used as a model to determine the effects of these treatments on a number of biochemical and morphological parameters. After 33 days of hypophysectomy, the morphology of the Leydig cells had been drastically altered. In addition, alpha-naphthol and beta-naphthol esterase activity as well as the steroidogenic capacity of the Leydig cells were greatly reduced at this time. In contrast, the level of sterol carrier protein 2 (SCP2), a Leydig cell-specific protein, was affected by hypophysectomy much less than the other parameters measured. Two daily injections of hCG to rats hypophysectomized for 31 days resulted in no change in the morphology of the Leydig cells, or in their proliferative activity. Non-specific esterase activities were also unaffected by 2 days of treatment with hCG. However, two injections of hCG to rats hypophysectomized for 31 days resulted in nearly complete restoration of steroidogenic capacity, and a 3.5-fold increase in the level of SCP2. These findings indicate that hypophysectomy results in significant morphological and biochemical changes in Leydig cells, and that hCG is capable of restoring some of these capacities within a short time.

Asunto(s)

Gonadotropina Coriónica/farmacología , Hipofisectomía/efectos adversos , Células Intersticiales del Testículo/metabolismo , Proteínas de Plantas , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Esterasas/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/patología , Masculino , Ratas , Ratas Endogámicas , Esteroles/metabolismo

RESUMEN

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting. This distribution is very different in each of these two tissues. In liver, 66% of the transfer protein is present in the membrane-free cytosol as compared to 19% in the adrenal gland. In the latter tissue, the transfer protein is mainly found in the lysosomal/peroxisomal and the microsomal fraction at a level of 1093 and 582 ng per mg total protein, respectively (i.e., 17% and 35% of the total), and to a lesser extent in the mitochondrial fraction (11% of the total). Of all the membrane fractions isolated, the microsomal fraction from the liver and the mitochondrial fraction from the adrenal gland have the lowest levels of the transfer protein (i.e., 168 ng and 126 ng per mg total protein, respectively). These low levels correlate poorly with the active role proposed for this transfer protein in the conversion of cholesterol into bile acids and steroid hormones in these fractions. Using immunoblotting, it was demonstrated that in addition to the transfer protein (14 kDa) a cross-reactive 58 kD protein was present in the supernatant and the membrane fractions of both tissues. Cytochemical visualization in adrenal tissue with specific antibodies against the nonspecific lipid transfer protein showed that immunoreactive protein(s) were present mainly in the peroxisome-like structures.

Asunto(s)

Glándulas Suprarrenales/análisis , Proteínas Portadoras/análisis , Hígado/análisis , Proteínas de Plantas , Animales , Inmunohistoquímica , Ratas , Ratas Endogámicas , Valores de Referencia , Fracciones Subcelulares/análisis

RESUMEN

Sterol carrier protein2 (SCP2) also designated non specific lipid transfer protein (nsL-TP), added to tumour Leydig cell mitochondria as a pure compound or in cytosolic preparations, stimulates pregnenolone production two- to three-fold. This stimulation can be abolished by addition of anti rat SCP2 but not by preimmune IgG-antibodies. SCP2- levels in the cytosol are increased in less than two minutes after addition of lutropin (LH). This increased SCP2 level may contribute to stimulation of steroid production in intact cells. After hormonal stimulation the subcellular distribution of SCP2 changes. A two-fold increase of SCP2- levels in the supernatant fraction and four-fold decrease in extracts of the particulate fraction was observed 30 min after stimulation of tumour Leydig cells with LH and subsequent fractionation. This apparent shift of SCP2 can be explained by an altered association with membranes or a true relocation of the protein from the particulate to the supernatant fractions under the influence of the hormone.

Asunto(s)

Proteínas Portadoras/metabolismo , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/farmacología , Proteínas de Plantas , Pregnenolona/biosíntesis , Esteroles/metabolismo , Animales , Línea Celular , Tumor de Células de Leydig/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Mitocondrias/metabolismo , Fracciones Subcelulares/metabolismo , Neoplasias Testiculares/metabolismo

RESUMEN

The rate-determining step in steroidogenesis is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme. The transport of substrate for this reaction may be facilitated by sterol carrier protein2 (SCP2). In rat testis tissue SCP2 is specifically localized in the Leydig cells and tissue levels of SCP2 are regulated by luteinizing hormone (LH). The present study concerns short-term regulation of SCP2 in isolated rat Leydig cells. Levels of SCP2 in the membrane-free supernatant are increased 2-fold already after 2 min incubation with LH and remain elevated for 24 h. The same response occurs with cells preincubated in the presence of cycloheximide for 4 h. SCP2 levels are also 2-fold increased after incubation with dibutyryl cAMP or 4 beta-phorbol 12-myristate 13-acetate (PMA) whereas these compounds stimulate steroid production 5.5- and 2-fold respectively. Luteinizing hormone releasing hormone (LHRH), which can stimulate steroid production more than 3-fold, does not influence SCP2 levels, neither are SCP2 levels altered when LH is added in the presence of the Ca2+-channel blocker diltiazem or in the absence of extracellular Ca2+. A restoration of the LH effect on SCP2 levels was already obtained in the presence of 1 microM extracellular Ca2+. These results suggest that Ca2+ influx through the plasma membrane may play an important role in the control of SCP2 levels. In most of the experiments no correlation between steroid production and SCP2 levels could be observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Calcio/farmacología , Proteínas Portadoras/metabolismo , Células Intersticiales del Testículo/metabolismo , Proteínas de Plantas , Esteroles/metabolismo , Animales , Cinética , Tumor de Células de Leydig/metabolismo , Hormona Luteinizante/farmacología , Masculino , Ratas

RESUMEN

One single injection of ethylene dimethane sulfonate (EDS) to mature rats causes specific degeneration of testicular Leydig cells which is complete after 3 days. At this time no steroidogenic activities can be detected, indicating that Leydig cells are the source of steroids. The mechanism of this cytotoxic effect of EDS has been investigated with isolated cells. Extensive protein alkylation has been shown to occur in Leydig cells, Sertoli cells and hepatocytes. Steroid production by Leydig cells is always inhibited by EDS, but cytotoxic effects of EDS could only be demonstrated in Leydig cells from mature rats or tumour tissue and not in Leydig cells from immature rats. A new population of Leydig cells develops during the next 2-5 weeks after EDS treatment. In hypophysectomized rats this repopulation only occurs when hCG is given daily. FSH has no effects. The proliferative activity in the interstitial tissue increases within 2 days after administration of hCG or EDS and there are indications that LH and locally produced factors are involved in the proliferation of Leydig cells or Leydig cell precursor cells. Inhibition of cAMP production with inhibitors of adenylate cyclase results in an enhancement of the LH-stimulated steroid production similar to that observed with an LHRH agonist and phospholipase C (PLC). Since the effects of LHRH and PLC on protein phosphorylation and steroid production are similar and different from LH or active phorbol esters, it is proposed that LHRH and PLC may stimulate steroid production via liberation of calcium from a specific intracellular pool. Sterol carrier protein2 (SCP2) which is specifically localized in Leydig cells and regulated by LH probably plays a role in the delivery of cholesterol to the mitochondria although the mechanism of this carrier function is not clear. The results indicate that regulation of Leydig cell development and the steroidogenic activities by gonadotrophins and locally produced factors occur via different transducing systems and regulatory pathways.

Asunto(s)

Células Intersticiales del Testículo/metabolismo , Esteroides/biosíntesis , Animales , Calcio/farmacología , Diferenciación Celular , Gonadotropina Coriónica/farmacología , AMP Cíclico/metabolismo , Hormona Liberadora de Gonadotropina/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Hormona Luteinizante/farmacología , Masculino , Mesilatos/farmacología , Ratones , Mitocondrias/metabolismo , Modelos Biológicos , Ratas , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

In testis tissue from mature rats the non-specific lipid transfer protein (nsLTP), also called sterol carrier protein2 (SCP2), is concentrated in the Leydig cells and cannot be detected in Sertoli cells or germinal cells. Conclusions were reached after cell fractionation studies with normal testis tissue and after selective destruction of Leydig cells or germinal cells in vivo. The amount of nsLTP (SCP2) in testis tissue increased twofold 48 h after two daily injections of human chorionic gonadotrophin (100 i.u., s.c.) and decreased twofold after plasma luteinizing hormone levels were suppressed to almost undetectable levels with silicone elastomer implants containing testosterone. The specific localization in the Leydig cells and the luteinizing hormone-dependent cellular concentration of nsLTP/SCP2 support the possibility that this protein could play a role in the regulation of steroidogenesis by regulating the availability of cholesterol for the P450 side-chain cleavage enzyme in the mitochondria of Leydig cells.

Asunto(s)

Proteínas Portadoras/análisis , Células Intersticiales del Testículo/análisis , Proteínas de Plantas , Esteroles/análisis , Animales , Fraccionamiento Celular , Gonadotropina Coriónica/farmacología , Hormona Luteinizante/sangre , Masculino , Ratas , Ratas Endogámicas , Testosterona/farmacología

RESUMEN

In order to determine the localization of ribosomal protein genes on the chloroplast genome of Spirodela, we have followed two different approaches: First, antisera were prepared against purified 30S, 50S and 70S chloroplast ribosomal proteins from Spinacia. These antisera react with about two third of the chloroplast ribosomal proteins as shown by protein blot and immunoprecipitation experiments. Recombinant plasmids carrying the Spirodela BamHI-G or PstI-I cpDNA fragment both direct the synthesis of a 15 kD chloroplast ribosomal protein in a DNA dependent E. coli cell free system. This was confirmed by molecular weight determination, immunoprecipitation and competition immunoprecipitation experiments. Second, heterologous hybridization with the rps19 gene probe from Nicotiana revealed the localization of this gene on the chloroplast DNA of Spirodela within the BamHI-G fragment at the left junction of the large single copy region and the inverted repeat. Furthermore we show that the recombinant plasmid carrying Nicotiana rps19 also directs the synthesis of another chloroplast ribosomal protein in an E. coli cell free system. The identity of this protein is discussed.

RESUMEN

We have characterized the ribosomal proteins from Spinacia chloroplasts using two-dimensional gel electrophoresis. The 30S and 50S subunits contain 23-25 and 36 ribosomal proteins, respectively. In contrast to prokaryotic ribosomes, chloroplast ribosomes contain at least one (and possibly two) phosphorylated ribosomal proteins. Isolated chloroplasts synthesize in the presence of ((35)S) labeled methionine and cysteine at least seven 30S and thirteen 50S ribosomal proteins which are assembled into (pre)ribosomes. This suggests that about one third of the chloroplast ribosomal proteins is encoded by the chloroplast DNA itself. The identity of several labeled proteins in the two-dimensional gel electrophoretic patterns which did not comigrate with stained chloroplast ribosomal proteins is discussed.