RESUMEN

The integrity of the blood-brain barrier (BBB) plays an important role in maintaining a safe neural microenvironment in the brain. Loss of BBB integrity has been recognized as a major cause of profound brain alterations. Psychoactive drugs such as methamphetamine (METH) or cocaine are well-known drugs of abuse that can alter the permeability of the BBB via various mechanisms. In addition, the neurotoxicity of METH is well documented, and alterations in BBB function can contribute to this toxicity. A great deal of effort has been devoted to understanding the cellular and molecular mechanisms of the action of these drugs in the central nervous system. However, only a few investigations have focused on the effects of METH and cocaine on BBB function. The aim of this short review is to summarize our present knowledge of this subject.

Asunto(s)

Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiopatología , Cocaína/efectos adversos , Metanfetamina/efectos adversos , Animales , Barrera Hematoencefálica/virología , Moléculas de Adhesión Celular/metabolismo , Cocaína/química , VIH/efectos de los fármacos , VIH/fisiología , Humanos , Leucocitos/citología , Leucocitos/efectos de los fármacos , Metanfetamina/química

RESUMEN

ICAM-1 is a cell surface adhesion glycoprotein playing an essential role in inflammatory responses. We have investigated the effects of the thyroid hormone T3 on the expression of the ICAM-1 gene in C6 glioma cells. In these cells, T3 stimulated the ICAM-1 protein expression significantly after 24 h of treatment. The induction of ICAM-1 by cytokines such as interleukin 1beta or tumour necrosis factor TNF as well as by lipopolysaccharide or T3 can be suppressed by the two anti-inflammatory compounds dexamethasone and parthenolide. The C6 glioma cell line could then be a useful model for studying the effect of T3 hormone on the expression of specific genes in glial cells, especially genes involved in lymphocyte-glial cell interactions.

Asunto(s)

Barrera Hematoencefálica/efectos de los fármacos , Encefalitis/metabolismo , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Neuroglía/efectos de los fármacos , Triyodotironina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Actinas/metabolismo , Animales , Barrera Hematoencefálica/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Dexametasona/farmacología , Encefalitis/patología , Encefalitis/fisiopatología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Modelos Animales , Neuroglía/citología , Neuroglía/metabolismo , Ratas , Sesquiterpenos/farmacología , Triyodotironina/metabolismo , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Vimentina/metabolismo

RESUMEN

Transthyretin (TTR) is involved in the transport of thyroxine (T4) and retinol-binding protein (RBP) in cerebrospinal fluid (CSF) and serum. TTR is secreted in the CSF by the epithelial cells of choroid plexus. The binding of [(125)I]TTR to cultured ependymoma cells which form the brain cerebrospinal barrier, was studied to determine whether these cells carry receptor(s) for TTR. TTR was bound by ependymoma cells in a time-dependent manner reaching equilibrium within 2 h. Scatchard analysis was consistent with a single class of high-affinity binding sites with a K(d) of approximately 18 nM. Saturable high-affinity binding of human TTR has previously been described in rat primary hepatocytes and human renal adenocarcinoma, neuroblastoma, hepatoma and astrocytoma cells, and also transformed lung cells. Endocytosis of fluorescent or biotinylated TTR was observed in ependymoma cells in cytoplasmic vesicles but TTR did not colocalize with clathrin in endocytic coated vesicles. Endocytosis of TTR was inhibited by high sucrose concentration (0.45 M). Finally, ligand blotting and chemical-linking experiments revealed the presence of a approximately 100 kDa putative TTR receptor on the ependymoma cell membrane. Receptor binding of TTR provides a potential mechanism for the delivery of T4 within the central nervous system.

Asunto(s)

Neoplasias Encefálicas , Endocitosis/fisiología , Ependimoma , Prealbúmina/farmacocinética , Animales , Transporte Biológico/fisiología , Northern Blotting , Línea Celular Transformada/química , Línea Celular Transformada/metabolismo , Línea Celular Transformada/ultraestructura , Epéndimo/citología , Regulación Neoplásica de la Expresión Génica , Humanos , Radioisótopos de Yodo , Ratones , Ratones Transgénicos , Microscopía Electrónica , Prealbúmina/genética , ARN Mensajero/análisis , Ratas , Receptores de Albúmina/análisis , Receptores de Albúmina/metabolismo

RESUMEN

The passage of immunocompetent cells across the blood-brain barrier (BBB) is regulated at the level of the cerebral capillaries which have specific morphological and biochemical properties. We have developed and characterized an in vitro model of the BBB using immortalized human endothelial cells (ECV 304) induced by rat astrocytes. In this model, endothelial cells are attached together by continuous intercellular junctions with numerous tight junctions, develop a permeability barrier having a significant transcellular electrical resistance, possess high activities of gamma-glutamyl transpeptidase (gamma-GTP) and express the brain-type glucose transporter 1 (GLUT-1). These parameters are also characteristic of brain capillary endothelial cells. Under the culture conditions used, the ECV 304 cells express the intercellular adhesion molecule-1 (ICAM-1) on the external plasma membrane at concentrations which could permit lymphocyte adhesion to be studied.

Asunto(s)

Astrocitos/fisiología , Barrera Hematoencefálica/fisiología , Endotelio Vascular/fisiología , Uniones Intercelulares/fisiología , Animales , Astrocitos/citología , Astrocitos/ultraestructura , Permeabilidad de la Membrana Celular , Células Cultivadas , Electrofisiología , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Transportador de Glucosa de Tipo 1 , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Uniones Intercelulares/ultraestructura , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Proteínas de Transporte de Monosacáridos/análisis , Ratas , gamma-Glutamiltransferasa/análisis

RESUMEN

The effect of the thyroid hormone L-3,5,3'-triiodothyronine (T3) on the expression of ICAM-1, a cell surface glycoprotein playing a pivotal role in inflammatory responses, was investigated. In ECV 304 cells, T3 (30 nmol/L) markedly increased ICAM-1 protein expression, with a peak after 24 h of treatment. ECV 304 human cells express both alpha1 and beta1 T3 receptors. In an attempt to understand the molecular mechanisms leading to the induction of the ICAM-1 gene by T3, we have studied the effects of a synthetic glucocorticoid, dexamethasone, and of a sesquiterpene lactone, parthenolide, on this T3-stimulated expression of ICAM-1. Our results demonstrate a repressive effect of dexamethasone and parthenolide on the expression of the protein ICAM-1 stimulated by T3. Both anti-inflammatory compounds interfere with this T3-mediated pathway in a dose-dependent manner.

Asunto(s)

Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/genética , Triyodotironina/farmacología , Antiinflamatorios no Esteroideos/farmacología , Anticuerpos Monoclonales , Línea Celular , Humanos , Inflamación , Molécula 1 de Adhesión Intercelular/análisis , Cinética , Microscopía Inmunoelectrónica , Sesquiterpenos/farmacología , Triyodotironina/antagonistas & inhibidores

RESUMEN

The presence and synthesis of transthyretin, a major carrier protein of thyroxine in rat cerebrospinal fluid, was investigated in choroid plexus epithelial cells and ependymal cells by immunocytochemistry, in situ hybridization, and analysis by Northern and Western blot using a specific oligonucleotide probe and a specific polyclonal antibody to transthyretin. Choroid plexus epithelial cells expressed transthyretin at high levels in developing rat cerebral hemispheres and in cultured cells. These cells secreted transthyretin into the cerebrospinal fluid. In the developing rat brain transthyretin was present in the cytoplasm of ependymal cells, in vesicles in contact with the apical membrane and in cilia. In ependymal cell cultures this protein was particularly abundant in the cilia of these cells. In contrast, ependymal cells did not synthesize transthyretin. It is postulated that transthyretin is transported to ependymal cells from the cerebrospinal fluid by endocytosis.

Asunto(s)

Endocitosis/fisiología , Epéndimo/metabolismo , Prealbúmina/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/metabolismo , Western Blotting , Células Cultivadas , Plexo Coroideo/citología , Plexo Coroideo/metabolismo , Plexo Coroideo/fisiología , Epéndimo/citología , Epéndimo/fisiología , Inmunohistoquímica , Hibridación in Situ , Prealbúmina/biosíntesis , Prealbúmina/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar

RESUMEN

Apoptosis, or programmed cell death, is an active process of self-destruction, described a long time ago. However, the understanding of the molecular pathways which regulate programmed cell death is more recent and far from complete. Apoptosis occurs during embryonic and foetal development, and tissue remodeling, and its purpose is to assure homeostasis of cells and tissues. Apoptosis-defining morphological and biochemical changes are now well documented. Many physiological and non-physiological factors have been described as inducers of apoptosis. Several genes affecting various steps in programmed cell death must be expressed to trigger apoptosis. For example, ced-3 and ced-4 in the nematode C. elegans, and ICE, a gene found in mammals. In addition, the existence of genes suppressing apoptosis, like the human bcl-2 gene and a family of related bcl-2 genes was recently described. Several data dealing with these family of anti-apoptotic genes and some of their mechanisms of action are now currently available. It is clear that bcl-2 protects many cell lines from induced apoptosis. Other proteins, like bcl-xL, A1 or mcl-1 have the same anti-apoptotic function, but several molecules of the same family, like bcl-xS, bax-alpha or bak can trigger the opposite effect. It is known that bcl-2 can interact with other proteins. For example, bax, which can exist as a homodimer, is also able to form a heterodimer with bcl-2. A surexpression of bax in several cell lines allows to counteract the effect of bcl-2. R-ras p23 is another example, among others, of a protein interacting with bcl-2, and this results in an interruption of the apoptotic signal transduction pathway when bcl-2 is overexpressed. Some other explanations allowing a more detailed analysis of the molecular mechanisms of apoptosis and anti-apoptosis are discussed in this short review. Many interesting results suggest that bcl-2 is a death repressor molecule functioning in an anti-oxydant pathway, but other recent data seem to claim the contrary. Recently, the demonstration was made that apoptosis may require the activation of several classes of proteases. It seems now that bcl-2 has also a function of protease(s) inhibitor.

Asunto(s)

Apoptosis/genética , Genes bcl-2 , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Animales , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genética

RESUMEN

A study of the effect of the L-3,5,3'-triiodothyronine hormone on the expression of the mRNA of the adhesion molecule ICAM-1 led to the observation that the mRNA level is slightly up-regulated in human umbilical-cord endothelial cells. To analyze this induction at a molecular level, the search of T3 hormone receptors was undertaken. In this paper, we show that ECV 304 endothelial cells express the mRNAs encoding two thyroid hormone receptor isoforms alpha(alpha1 and alpha2) and one beta(beta1). This is, to our knowledge, a first important step towards the demonstration of the involvement of these receptors in the induction of the expression of ICAM-1 by the T3 hormone.

Asunto(s)

Endotelio Vascular/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Receptores de Hormona Tiroidea/biosíntesis , Triyodotironina/metabolismo , Línea Celular , Expresión Génica , Humanos , Molécula 1 de Adhesión Intercelular/genética , ARN Mensajero/biosíntesis , Triyodotironina/biosíntesis , Triyodotironina/genética , Triyodotironina/farmacología

RESUMEN

The AR4-2J cell line is derived from a transplantable tumour of the exocrine rat pancreas. Acinar in origin, this cell line contains significant amounts of amylase and can be grown in continuous culture. Many in vitro studies have been done using these cells; these studies were often complemented with in vivo experiments on animals. Particularly, many polypeptide hormones interacting with specific receptors located on the cell membrane have been analysed. The accurate knowledge of the hormone-receptor interactions has allowed to design interesting analogs of these hormones. In several cases, these compounds are powerful antagonists and are able to control cell proliferation induced by the corresponding polypeptide hormones. Other cell lines are useful to understand human pancreatic cancer. These human cell lines (Capan 1, Panc-1 for example) are of ductal origin and differ from AR4-2J cells, especially regarding the distribution of several polypeptide hormone and growth factor receptors. Both models are important for basic studies of neuropeptides, gastrointestinal peptides and their receptors, as well as for a better understanding of the underlying mechanisms of human pancreatic cancer.

Asunto(s)

Receptores de Superficie Celular/antagonistas & inhibidores , Animales , División Celular , Regulación de la Expresión Génica , Humanos , Estructura Molecular , Neoplasias Pancreáticas/patología , Péptidos , Ratas , Células Tumorales Cultivadas

RESUMEN

Using AR4-2J rat pancreatic carcinoma cells, the effects of a novel bombesin (BN) receptor antagonist [D-F5Phe6, D-Ala11]BN(6-13)OMe (BIM26226) on BN- or GRP-stimulated amylase release and binding of radio-labeled bombesin-like peptides to these cells were examined and compared to [D-Phe6,Leu13 psi(CH2NH)Leu14]BN(6-14) (Psi Bn(6-14)), one of the most potent BN receptor antagonists presently known. BN and GRP both stimulated amylase release with EC50 values in the nanomolar range. Both antagonists were devoid of agonist activity when tested alone. BIM26226 was most potent, antagonizing BN- or GRP-stimulated amylase release with IC50 values in the nanomolar range, whereas Psi Bn(6-14) was approximately ten times less potent. With 125I-[Tyr15]GRP bound to these cells, the binding affinities were BIM26226 > GRP > Psi Bn(6-14) >> neuromedin B. BIM 22626 was not able to inhibit binding of radio-labeled CCK-33, gastrin-17 or VIP. These results suggest that BIM26226 is one of the most potent and specific bombesin receptor antagonists in vitro and seems to be a useful tool to define the physiologic role of GRP in vivo.

Asunto(s)

Amilasas/metabolismo , Bombesina/análogos & derivados , Fragmentos de Péptidos/farmacología , Receptores de Bombesina/antagonistas & inhibidores , Animales , Unión Competitiva , Bombesina/metabolismo , Bombesina/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Péptido Liberador de Gastrina , Gastrinas/metabolismo , Humanos , Cinética , Neoplasias Pancreáticas , Péptidos/metabolismo , Péptidos/farmacología , Ratas , Células Tumorales Cultivadas

RESUMEN

A variety of studies has recently demonstrated that a group of peptides with relatively low molecular weights may play an important role in the rate of proliferation of tumor cells in vitro and in vivo. Among these peptides are bombesin and gastrin-releasing peptide (GRP) which both can function as autocrine growth factors in several neoplastic cells. These two neuropeptides also act as gut hormones and neuromediators. In this family of peptides, a shared C-terminal sequence is necessary for biological activity. The knowledge of this sequence provided a structural basis for the design of synthetic antagonists. Such agents are capable of interfering with the processing or the release of the autocrine growth factor or with its receptor, and might have a potential therapeutic utility. This short paper, focused on one class of neuropeptides, is not intended as an extensive review of this rapidly expanding field, but rather a survey of several topics presently under investigation.

Asunto(s)

Bombesina/farmacología , Bombesina/fisiología , División Celular/efectos de los fármacos , Neuropéptidos/farmacología , Neuropéptidos/fisiología , Secuencia de Aminoácidos , Anfibios , Animales , Bombesina/análogos & derivados , Bombesina/antagonistas & inhibidores , Humanos , Mamíferos , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias/patología , Neuropéptidos/antagonistas & inhibidores , Receptores de Bombesina/agonistas , Receptores de Bombesina/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal

RESUMEN

The immunoreactivity of the PEST region of mammalian tyrosine aminotransferase (TATase) was studied with a specific probe. An antipeptide serum was prepared using a synthetic peptide 385EFENDVEFTER395 (ER) corresponding to the main part of this important region of the liver enzyme. The antibodies were purified by affinity chromatography and analyzed by enzyme linked immunosorbent assay. Their use in immunoblotting experiments allowed the easy detection of the complete TATase molecule. The very high homology in rat and the human enzyme having the same PEST region, make these antibodies of interest for further studies of the human TATase.

Asunto(s)

Oligopéptidos/inmunología , Tirosina Transaminasa/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Reacciones Cruzadas , Humanos , Immunoblotting , Inmunoquímica , Datos de Secuencia Molecular , Oligopéptidos/química , Ratas , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Tirosina Transaminasa/química

RESUMEN

Mistletoe lectin (ML) I increases the production of cytokines by mononuclear cells and has been proposed as a useful biological response modifier in the treatment of cancer. Two other lectins, ML II and ML III, have been identified in mistletoe. We report that the N-terminal sequences of the three A chains of ML I, ML II and ML III are identical, and have interesting homology with the N-terminal sequences of the A chain of ricin-like toxins and of single-chain ribosome-inhibiting proteins. In addition, the three mistletoe lectins inhibit the growth of the human tumor cell line Molt 4, ML III being the most potent. followed by ML II and ML I. This inhibition is suppressed by addition of rabbit anti-ML I antibodies to the cultured cells. The data obtained suggest that the three lectins have amino acid sequences which show extensive homology and exert very similar biological effects. They may be derived from the same precursor.

Asunto(s)

Factores Inmunológicos/química , N-Glicosil Hidrolasas , Preparaciones de Plantas , Proteínas de Plantas/química , Proteínas Ribosómicas/antagonistas & inhibidores , Ricina/química , Toxinas Biológicas/química , Secuencia de Aminoácidos , División Celular/efectos de los fármacos , Preescolar , Humanos , Factores Inmunológicos/toxicidad , Leucemia de Células T/patología , Leucemia de Células T/terapia , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas de Plantas/toxicidad , Proteínas Ribosómicas/química , Proteínas Ribosómicas/toxicidad , Proteínas Inactivadoras de Ribosomas , Proteínas Inactivadoras de Ribosomas Tipo 1 , Proteínas Inactivadoras de Ribosomas Tipo 2 , Ricina/toxicidad , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Toxinas Biológicas/toxicidad , Tricosantina/química , Tricosantina/toxicidad , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

Tyrosine aminotransferase (L-tyrosine: 2 oxoglutarate aminotransferase; EC 2.6.1.5; TATase) is the first enzyme in the catabolic pathway of tyrosine. The gene of this transaminase is regulated by glucocorticoid hormones as well as via the cAMP pathway. This review gives a brief survey of the structural and physico-chemical properties of this well-known protein. A comparative study of the properties of TATase with other aminotransferases is also included to analyse this molecule for itself, and not only as a marker used in studies on enzymatic induction. Finally, the regulation of the gene expression is presented, in order to underline a few important features of this model.

Asunto(s)

Tirosina Transaminasa , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Regulación Enzimológica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Transaminasas/química , Transaminasas/metabolismo , Tirosina Transaminasa/genética , Tirosina Transaminasa/aislamiento & purificación , Tirosina Transaminasa/metabolismo

RESUMEN

Rat liver tyrosine aminotransferase has been expressed in Saccharomyces cerevisiae and Escherichia coli. In yeast, the extent of production is 20-fold higher than that in rat liver after induction by dexamethasone, and reaches 250-fold higher in an E. coli strain carrying the T7 RNA polymerase transcription system. About 250 mg pure and homogeneous enzyme was obtained from 50 g transformed E. coli cells. Determination of Mr and pI, as well as analysis of N- and C-terminal amino acids, suggest that the isolated protein is native. The catalytic properties, similar to those of the enzyme from rat liver, confirm that it is fully active and that post-translational modifications in the mammalian cells are not essential for activity. Pyridoxal 5'-phosphate strongly protects the enzyme against thermal inactivation. After denaturation, 10 thiol groups, out of 16 in the polypeptide chain, react with 5,5'-dithiobis(2-nitrobenzoic acid) whereas only five or six are accessible under native conditions. Two thiols are rapidly modified with concomitant inactivation of the apoenzyme, but pyridoxal 5'-phosphate partially protects them in the holoenzyme. The results are interpreted in the light of the structure/function relationship in this enzyme.

Asunto(s)

Tirosina Transaminasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Catálisis , Cromatografía en Gel , Clonación Molecular , Desoxirribonucleótidos , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Calor , Cinética , Mamíferos , Datos de Secuencia Molecular , Plásmidos , Conformación Proteica , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Tirosina Transaminasa/antagonistas & inhibidores , Tirosina Transaminasa/aislamiento & purificación , Tirosina Transaminasa/metabolismo

RESUMEN

Limited proteolysis was used to probe the structure of the apo- and holoenzyme of rat liver tyrosine aminotransferase. Both were subjected to trypsinolysis and the major fragments were isolated and characterized. Trypsin cleaves the apoenzyme after residues Arg57, Lys64, and Lys71 and the holoenzyme after Arg37 and Lys38. The difference in the accessibility of the enzyme deprived or associated with pyridoxal 5'-phosphate reflects two distinct conformations. The activity, the affinity for the ligands and the thermostability of the purified truncated enzyme forms are similar to those of the native apo- and holoenzyme. A model for the domain structure of mammalian tyrosine aminotransferase and a mechanism for its rapid turnover are proposed.

Asunto(s)

Apoenzimas/aislamiento & purificación , Hígado/enzimología , Tirosina Transaminasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Apoenzimas/química , Activación Enzimática , Hidrólisis , Cinética , Datos de Secuencia Molecular , Ratas , Relación Estructura-Actividad , Tripsina , Tirosina Transaminasa/química

RESUMEN

Antisera directed against synthetic peptides with sequences that correspond to selected regions of tyrosine aminotransferase may react with the protein without affecting its biological activity. The antiserum against the theoretical N-terminal dodecapeptide recognizes the enzyme and makes it possible to detect a blocked form of the enzyme. Another form shortened by seven aminoacids and starting with Thr 8 has been found. The isolation of tyrosine aminotransferase by one step affinity chromatography is now made possible; nevertheless the elution procedure remains a critical point. The strategy described should have further applications and allow the detailed exploration of the essential domains of aminotransferases, especially those involved in the function and the degradation of pyridoxal phosphate requiring enzymes.

Asunto(s)

Hígado/enzimología , Tirosina Transaminasa/aislamiento & purificación , Animales , Cromatografía de Afinidad/métodos , Terminación de la Cadena Péptídica Traduccional , Ratas , Tirosina Transaminasa/inmunología , Tirosina Transaminasa/metabolismo

RESUMEN

Our results show for the first time sequence data of the N-terminal part of tyrosine aminotransferase. This unblocked form of TATase, which has never been detected before, starts with a serine. This serine was found at position 29 of the primary structure of the enzyme deduced from the cDNA. We suggest that this free N-terminal amino acid is the extremity of a TATase form generated by proteolysis during the process of purification. Thus, proteolysis does not occur at the C-terminal as has been suggested before, but rather at the N-terminal region of the enzyme. To confirm this possibility, a peptide corresponding to the sequence of the seven carboxy-terminal amino acids of TATase was synthesized. It was coupled to ovalbumin or keyhole limpet hemocyanin, and the resulting conjugates were used to raise anti-peptide antibodies. The crude sera obtained were purified and their abilities to recognize TATase in ELISA and dot-blot experiments were proven. Our results demonstrate that the C-terminal part of the enzyme is present and well-recognized by the anti-peptide serum prepared. Furthermore, the anti-peptide serum reacts with TATase without inhibiting its enzymatic activity.

Asunto(s)

Hígado/enzimología , Tirosina Transaminasa , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática , Sueros Inmunes/inmunología , Sueros Inmunes/farmacología , Datos de Secuencia Molecular , Ratas , Tirosina Transaminasa/análisis , Tirosina Transaminasa/inmunología , Tirosina Transaminasa/metabolismo

RESUMEN

Transforming growth factor-beta (TGF-beta) is a multifunctional protein involved in the control of proliferation, differentiation and other functions in many cell types. The anchorage-independent growth of some established lines of untransformed fibroblasts in soft agar is induced by TGF-beta and requires in addition exogenous EGF for certain target cells, notably rat NRK-49 cells. The formation of colonies of NRK-49F cells is completely inhibited by the synthetic 11-beta substituted nor-steroid RU38486 added at a final concentration of 1.3 X 10(-5) M. We also explored the effect of TGF-beta on Daudi and Raji lymphoma cells by measuring the production of Epstein-Barr Virus (EBV) early antigens (EA). In Daudi cells an induction capacity giving rise to 10-16% positive EA-cells was observed; in Raji cells the induction only reached between 6 and 8%. The induction was partially inhibited by the anti-steroid RU38486 in both systems. Thus, RU38486 not only antagonizes the glucocorticoid hormone action but also interferes with the effects of TGF-beta in fibroblasts and in lymphoma cells. The molecular basis of the interactions observed was investigated by considering (1) the binding to specific receptors, (2) transfection experiments, in order to examine if the interference of the anti-steroid with TGF-beta activities occurs at the transcriptional level as in the case of glucocorticoid induction. The results suggest that the blocking by antiglucocorticoids of the effects of TGF-beta and glucocorticoids, in fibroblasts and lymphoma cells, occurs by different mechanisms.

Asunto(s)

División Celular/efectos de los fármacos , Dexametasona/farmacología , Estrenos/farmacología , Sustancias de Crecimiento/farmacología , Péptidos/farmacología , Animales , Línea Celular , Dexametasona/antagonistas & inhibidores , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Cinética , Linfoma , Mifepristona , Péptidos/antagonistas & inhibidores , Factores de Crecimiento Transformadores

RESUMEN

A synthetic peptide corresponding to the seven carboxy-terminal amino acids of tyrosine aminotransferase was coupled to ovalbumin or keyhole limpet haemocyanin, and the resulting conjugates were used to raise anti-peptide antibodies by immunization of rabbits. The crude sera were purified and tested for recognition of the whole enzyme by enzyme-linked immunosorbent assay and immunoprecipitation in extracts from [35S] methionine labeled hepatoma cells. Our results support the existence of an intact C-terminus. If processing takes place, it will rather occur at the N-terminus of this hepatic enzyme.

Asunto(s)

Tirosina Transaminasa , Complejo Antígeno-Anticuerpo , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Hemocianinas , Sueros Inmunes , Peso Molecular , Ovalbúmina , Tirosina Transaminasa/inmunología , Tirosina Transaminasa/metabolismo