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1.
Cell Mol Biol (Noisy-le-grand) ; 44(2): 333-42, 1998 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9593584

RESUMEN

Dramatic morphological, biochemical and cytological changes occur in parotid glands of rats and mice which have been treated with the beta-adrenergic receptor agonist isoproterenol (Ipr). Changes include glandular hypertrophy, induction of tissue-specific proline-rich proteins (PRPs), increases in cAMP, and occurrence of polyploidy. Similar changes also occur after feeding mice polyphenolic compounds commonly referred to as tannins. Data are presented which show that changes in cell cycle proteins are due to stimulation of the beta-adrenergic receptor by either isoproterenol or tannin treatment of mice. Both p34cdc2 mRNA and protein levels were elevated dramatically after mice were treated. Induction of p34cdc2 occurred within 24 hrs. and was transient during treatment. The beta1-adrenergic receptor antagonist atenolol blocked both tissue-specific expression of proline-rich proteins and induction of p34cdc2. Coincident with the increase in p34cdc2, cyclin-dependent kinase complexes containing cyclins A and B increased forty- and ten-fold, respectively. These results show that in mouse parotid glands activation of the beta1-adrenergic receptor by either the administration of isoproterenol or ingestion of dietary tannins induces synthesis of p34cdc2 and most likely contributes to the occurrence of polyploidy.


Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Proteína Quinasa CDC2/biosíntesis , Isoproterenol/farmacología , Glándula Parótida/efectos de los fármacos , Receptores Adrenérgicos beta 1/efectos de los fármacos , Proteínas y Péptidos Salivales/biosíntesis , Transducción de Señal/efectos de los fármacos , Antagonistas Adrenérgicos beta/farmacología , Animales , Areca/efectos adversos , Atenolol/farmacología , Proteína Quinasa CDC2/genética , Ciclo Celular/efectos de los fármacos , Cromatografía de Afinidad , Ciclina A/metabolismo , Ciclina E/metabolismo , Inducción Enzimática/efectos de los fármacos , Taninos Hidrolizables/farmacología , Ratones , Ratones Endogámicos A , Glándula Parótida/enzimología , Plantas Medicinales , Receptores Adrenérgicos beta 1/fisiología
2.
J Nat Prod ; 61(4): 529-33, 1998 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9584405

RESUMEN

Carmabins A and B have been isolated as linear lipotetrapeptides from the BuOH extract of the marine cyanobacterium Lyngbya majuscula. The planar structures were elucidated by extensive 2D NMR analysis, including 1H-15N HMBC and HMQC-TOCSY experiments, together with MS measurements.


Asunto(s)
Cianobacterias/química , Oligopéptidos/aislamiento & purificación , Cromatografía en Capa Delgada , Espectroscopía de Resonancia Magnética , Oligopéptidos/química , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta
3.
Proc Natl Acad Sci U S A ; 95(5): 2492-7, 1998 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-9482913

RESUMEN

Hereditary hemochromatosis (HH) is a common autosomal recessive disease characterized by increased iron absorption and progressive iron storage that results in damage to major organs in the body. Recently, a candidate gene for HH called HFE encoding a major histocompatibility complex class I-like protein was identified by positional cloning. Nearly 90% of Caucasian HH patients have been found to be homozygous for the same mutation (C282Y) in the HFE gene. To test the hypothesis that the HFE gene is involved in regulation of iron homeostasis, we studied the effects of a targeted disruption of the murine homologue of the HFE gene. The HFE-deficient mice showed profound differences in parameters of iron homeostasis. Even on a standard diet, by 10 weeks of age, fasting transferrin saturation was significantly elevated compared with normal littermates (96 +/- 5% vs. 77 +/- 3%, P < 0.007), and hepatic iron concentration was 8-fold higher than that of wild-type littermates (2,071 +/- 450 vs. 255 +/- 23 microg/g dry wt, P < 0.002). Stainable hepatic iron in the HFE mutant mice was predominantly in hepatocytes in a periportal distribution. Iron concentrations in spleen, heart, and kidney were not significantly different. Erythroid parameters were normal, indicating that the anemia did not contribute to the increased iron storage. This study shows that the HFE protein is involved in the regulation of iron homeostasis and that mutations in this gene are responsible for HH. The knockout mouse model of HH will facilitate investigation into the pathogenesis of increased iron accumulation in HH and provide opportunities to evaluate therapeutic strategies for prevention or correction of iron overload.


Asunto(s)
Antígenos HLA/genética , Antígenos HLA/fisiología , Hemocromatosis/genética , Hemocromatosis/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/fisiología , Complejo Mayor de Histocompatibilidad , Proteínas de la Membrana , Animales , Genes Recesivos , Hemocromatosis/sangre , Proteína de la Hemocromatosis , Humanos , Hierro/metabolismo , Riñón/inmunología , Riñón/metabolismo , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos , Ratones Noqueados , Bazo/inmunología , Bazo/metabolismo , Transferrina/metabolismo
4.
Proc Natl Acad Sci U S A ; 95(4): 1472-7, 1998 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-9465039

RESUMEN

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called HFE. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 --> Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with beta2-microglobulin (beta2m) and prevents cell-surface expression. A second mutation that has no effect on beta2m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37 degrees C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/beta2m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4 degrees C, the added soluble complex of HFE/beta2m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis.


Asunto(s)
Antígenos HLA/genética , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana , Receptores de Transferrina/metabolismo , Secuencia de Aminoácidos , Dimerización , Proteína de la Hemocromatosis , Humanos , Ligandos , Mutación Puntual , Pruebas de Precipitina , Unión Proteica , Relación Estructura-Actividad , Transfección , Transferrina/metabolismo , Microglobulina beta-2/metabolismo
5.
Proc Natl Acad Sci U S A ; 94(23): 12384-9, 1997 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-9356458

RESUMEN

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282-->Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with beta2-microglobulin; a second mutation, His-63-->Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with beta2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.


Asunto(s)
Antígenos HLA/genética , Antígenos HLA/metabolismo , Hemocromatosis/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de la Membrana , Mutación , Microglobulina beta-2/metabolismo , Animales , Transporte Biológico/genética , Células COS , Regulación de la Expresión Génica , Hemocromatosis/genética , Proteína de la Hemocromatosis
6.
Genome Res ; 7(5): 441-56, 1997 May.
Artículo en Inglés | MEDLINE | ID: mdl-9149941

RESUMEN

In the process of positionally cloning a candidate gene responsible for hereditary hemochromatosis (HH), we constructed a 1.1-Mb transcript map of the region of human chromosome 6p that lies 4.5 Mb telomeric to HLA-A. A combination of three gene-finding techniques, direct cDNA selection, exon trapping, and sample sequencing, were used initially for a saturation screening of the 1.1-Mb region for expressed sequence fragments. As genetic analysis further narrowed the HH candidate locus, we sequenced completely 0.25 Mb of genomic DNA as a final measure to identify all genes. Besides the novel MHC class 1-like HH candidate gene HLA-H, we identified a family of five butyrophilin-related sequences, two genes with structural similarity to a type 1 sodium phosphate transporter, 12 novel histone genes, and a gene we named RoRet based on its strong similarity to the 52-kD Ro/SSA lupus and Sjogren's syndrome auto-antigen and the RET finger protein. Several members of the butyrophilin family and the RoRet gene share an exon of common evolutionary origin called B30-2. The B30-2 exon was originally isolated from the HLA class 1 region, yet has apparently "shuffled" into several genes along the chromosome telomeric to the MHC. The conservation of the B30-2 exon in several novel genes and the previously described amino acid homology of HLA-H to MHC class 1 molecules provide further support that this gene-rich region of 6p21.3 is related to the MHC. Finally, we performed an analysis of the four approaches for gene finding and conclude that direct selection provides the most effective probes for cDNA screening, and that as much as 30% of ESTs in this 1.1-Mb region may be derived from noncoding genomic DNA.


Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Humanos Par 6 , Hemocromatosis/genética , Proteínas de la Membrana , ARN Citoplasmático Pequeño , Simportadores , Secuencia de Aminoácidos , Autoantígenos/genética , Bacterias/genética , Sitios de Unión , Northern Blotting , Butirofilinas , Proteínas Portadoras/genética , Clonación Molecular , Secuencia Conservada , ADN Complementario , Antígenos HLA/genética , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Histonas/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares , Proteínas/genética , Proteínas/metabolismo , Ribonucleoproteínas/genética , Análisis de Secuencia de ADN/métodos , Homología de Secuencia de Aminoácido , Lugares Marcados de Secuencia , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo I , Distribución Tisular , Factores de Transcripción , Transcripción Genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas
7.
J Biol Chem ; 272(22): 14025-8, 1997 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-9162021

RESUMEN

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), called HLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 --> tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 --> aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds beta2-microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease.


Asunto(s)
Antígenos HLA/genética , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana , Microglobulina beta-2/metabolismo , Secuencia de Bases , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Hemocromatosis/metabolismo , Proteína de la Hemocromatosis , Humanos , Datos de Secuencia Molecular , Mutación , Microglobulina beta-2/genética
8.
Proc Natl Acad Sci U S A ; 94(6): 2534-9, 1997 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-9122230

RESUMEN

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism that leads to excessive iron storage in the liver and other organs. Recently, between 83 and 100% of HH patients have been found to be homozygous for the same mutation in a novel major histocompatibility complex class I-like gene, called the HLA-H gene. The Cys-282 --> Tyr mutation in HH patients would be expected to disrupt the function of the HLA-H gene product by altering a critical disulfide bridge. As a first step in understanding the function of the HLA-H gene product, we generated an antibody to a C-terminal peptide and used it for immunolocalization of the HLA-H protein in the gastrointestinal tract of Finnish and American subjects presumed not to have HH. Although staining for the HLA-H protein was seen in some epithelial cells in every segment of the alimentary canal, its cellular and subcellular expression in the small intestine were quite distinct from those seen in other segments. In contrast to the stomach and colon, where staining was polarized and restricted to the basolateral surfaces, and in contrast to the epithelial cells of the esophagus and submucosal leukocytes, which showed nonpolarized staining around the entire plasma membrane, the staining in small intestine was mainly intracellular and perinuclear, limited to cells in deep crypts. Prior genetic evidence suggested that a defective HLA-H protein is the molecular basis of HH. Here we show that the HLA-H protein not only varies in its pattern of expression along the cranial/caudal axis of the gastrointestinal tract but that it has a unique subcellular localization in the crypts of the small intestine in proximity to the presumed sites of iron absorption.


Asunto(s)
Esófago/inmunología , Mucosa Gástrica/inmunología , Antígenos HLA/análisis , Hemocromatosis/inmunología , Antígenos de Histocompatibilidad Clase I/análisis , Mucosa Intestinal/inmunología , Proteínas de la Membrana , Corteza Cerebral/inmunología , Corteza Cerebral/patología , Colon , Duodeno , Epitelio/inmunología , Epitelio/patología , Esófago/patología , Finlandia , Vesícula Biliar/inmunología , Vesícula Biliar/patología , Mucosa Gástrica/patología , Antígenos HLA/biosíntesis , Antígenos HLA/genética , Hemocromatosis/genética , Hemocromatosis/patología , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Intestino Delgado , Hígado/inmunología , Hígado/patología , Complejo Mayor de Histocompatibilidad , Mutación Puntual , Estados Unidos
9.
Nat Genet ; 13(4): 399-408, 1996 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8696333

RESUMEN

Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.


Asunto(s)
Antígenos HLA/genética , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Evolución Biológica , Cromosomas Artificiales de Levadura , Cromosomas Humanos Par 6 , Clonación Molecular/métodos , Cisteína , Cartilla de ADN/química , Expresión Génica , Genes MHC Clase I , Marcadores Genéticos , Haplotipos , Proteína de la Hemocromatosis , Humanos , Desequilibrio de Ligamiento , Complejo Mayor de Histocompatibilidad , Datos de Secuencia Molecular , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido
10.
J Pharmacol Exp Ther ; 275(3): 1204-8, 1995 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8531082

RESUMEN

Injury of the rat carotid artery by gentle perfusion of air causes vascular thickening, similar to that seen in the clinic setting in humans after percutaneous angioplasty or bypass surgery to repair injured or diseased blood vessels. In the animal model as well as in patients, this stenosis appears to be the result of smooth muscle cell migration and proliferation. Various cell types in the lesion area may contribute by producing inflammatory cytokines, adhesion molecules and growth factors. In the present study, mycophenolate mofetil (MMF), an inosine monophosphate dehydrogenase inhibitor with antiproliferative and immunosuppressive properties, was tested for its ability to inhibit this process. With a daily oral dose of 30 mg MMF/kg started 6 days before injury to one carotid artery by air perfusion, MMF reduced cross-sectional areas of total vessel wall (intima-media) thickness by 17% to 25% and of neointimal thickness by 48% to 60% at 14 days after injury in four tests (P < .001 when MMF- and vehicle-treated groups were compared for these thickened areas, n = 29 or 30). In addition, intima/media ratios ranged from 0.26 +/- 0.03 to 0.46 +/- 0.04 for MMF-treated vs. 0.55 +/- 0.05 to 0.93 +/- 0.08 for vehicle-treated animals in the four different tests (P < .001). Starting MMF treatment at either 14 or 0 days before arterial injury made no difference in the degree of reduction, suggesting that any biological process that might be altered by MMF is not one that requires much time to become established. Intima/media ratios were 0.31 +/- 0.04 or 0.34 +/- 0.04 for MMF-treated vs. 0.55 +/- 0.05 or 0.65 +/- 0.07 for vehicle-treated animals (P < .001 for day 14 or 0, respectively, n = 30). Thus, MMF reduced the vascular thickening after carotid artery injury in rats, suggesting that this class of compound may be able to control the pathological processes that lead to restenosis.


Asunto(s)
Arterias Carótidas/efectos de los fármacos , Estenosis Carotídea/tratamiento farmacológico , Inmunosupresores/farmacología , Ácido Micofenólico/análogos & derivados , Animales , Arterias Carótidas/fisiopatología , Traumatismos de las Arterias Carótidas , Masculino , Ácido Micofenólico/farmacología , Ratas , Ratas Sprague-Dawley
11.
J Nat Prod ; 58(9): 1384-91, 1995 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-7494145

RESUMEN

Extracts and pure compounds isolated from four samples of Dysidea sp. sponges collected from two geographically distinct regions of the Indo-Pacific (Chuuk Atoll and Fiji) were assayed against five different enzyme assays, four of which are relevant to anticancer drug discovery and one of which (15-lipoxygenase) may detect compounds significant in modulating the development of atherosclerotic plaque. The pure compounds that inhibited various enzymes were polybrominated phenols and polybrominated phenoxyphenols. Fourteen of these phenols were isolated, six of which were new compounds. A variety of the phenols inhibited inosine monophosphate dehydrogenase (IMPDH), guanosine monophosphate synthetase, and 15-lipoxygenase. No activity was observed with protein tyrosine kinase pp60v-src or matrix metalloprotease.


Asunto(s)
Ligasas de Carbono-Nitrógeno , Inhibidores Enzimáticos/aislamiento & purificación , Fenoles/aislamiento & purificación , Éteres Fenílicos/aislamiento & purificación , Poríferos/química , Animales , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Ligasas/antagonistas & inhibidores , Inhibidores de la Lipooxigenasa , Estructura Molecular , Fenoles/química , Fenoles/farmacología , Éteres Fenílicos/química , Éteres Fenílicos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores
12.
Proc Natl Acad Sci U S A ; 92(15): 6981-5, 1995 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-7542783

RESUMEN

c-Src is a nontransforming tyrosine kinase that participates in signaling events mediated by a variety of polypeptide growth factor receptors, including the epidermal growth factor receptor (EGFR). Overexpression and continual ligand stimulation of the EGFR results in morphological transformation of cells in vitro and tumor development in vivo. Elevated levels of c-Src and the EGFR are found in a variety of human malignancies, raising the question of whether c-Src can functionally cooperate with the EGFR during tumorigenesis. To address this issue, we generated c-Src/EGFR double overexpressors and compared their proliferative and biochemical characteristics to those of single overexpressors and control cells. We found that in cells expressing high levels of receptor, c-Src potentiated DNA synthesis, growth in soft agar, and tumor formation in nude mice. Growth potentiation was associated with the formation of a heterocomplex between c-Src and activated EGFR, the appearance of a distinct tyrosyl phosphorylation on the receptor, and an enhancement of receptor substrate phosphorylation. These findings indicate that c-Src is capable of potentiating receptor-mediated tumorigenesis and suggest that synergism between c-Src and the EGFR may contribute to a more aggressive phenotype in multiple human tumors.


Asunto(s)
Transformación Celular Neoplásica , Receptores ErbB/metabolismo , Neoplasias/etiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Animales , Biomarcadores de Tumor , Neoplasias de la Mama/etiología , Carcinoma/etiología , Células Cultivadas , Neoplasias del Colon/etiología , Sinergismo Farmacológico , Ratones , Mitosis , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/farmacología
13.
Antiviral Res ; 21(4): 301-15, 1993 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8215302

RESUMEN

In cultured MRC-5 cells, ganciclovir (GCV) alone had good activity against both the established AD169 strain (IC50 8 and 9 microM) and a clinical isolate (IC50 14 microM) of human cytomegalovirus (CMV), while 3'-azido-3'-deoxythymidine (AZT) was relatively inactive [IC50 508 and > 800 (AD169 strain); > 800 microM (clinical isolate)]. When reductions in plaques were compared against reductions in the cellular metabolism of MTT at all GCV and AZT combination concentrations using an improved 3-dimensional linear regression analysis, AZT had an additive effect on the antiviral activity of GCV against the AD169 strain and potentiated the antiviral activity of GCV against the clinical isolate. Calculations showed that, in the presence of 50 microM AZT, the anti-CMV activity of GCV was unchanged for the AD169 strain, whereas the activity of GCV was increased approximately 5-10-fold for the clinical isolate. An increase in GCV efficacy for the AD169 strain first became apparent at 100 microM AZT with an approximately 3-fold increase in activity. In Swiss-Webster mice, the anti-CMV activity of GCV against murine CMV was unaffected when administered in combination with AZT. GCV given alone subcutaneously had an ED50 of 6 mg/kg which was unaffected by daily intraperitoneal doses of 320 mg/kg AZT. These results suggest that AZT will not adversely affect the efficacy of GCV against CMV in HIV-positive, non-neutropenic patients.


Asunto(s)
Citomegalovirus/efectos de los fármacos , Ganciclovir/farmacología , Infecciones por Herpesviridae/tratamiento farmacológico , Zidovudina/farmacología , Animales , Infecciones por Citomegalovirus/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Femenino , Infecciones por Herpesviridae/microbiología , Humanos , Ratones , Muromegalovirus/efectos de los fármacos , Análisis de Regresión
14.
J Immunol ; 148(10): 3021-7, 1992 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-1578127

RESUMEN

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.


Asunto(s)
Linfocitos B/enzimología , Inmunoglobulina M/fisiología , Receptores de Antígenos de Linfocitos B/fisiología , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , Animales , Anticuerpos Antiidiotipos/inmunología , Proteínas de Unión al GTP/fisiología , Inmunoglobulina M/inmunología , Isoenzimas/análisis , Ratones , Ratones Endogámicos , Fosforilación , Fosfolipasas de Tipo C/análisis
15.
Mol Cell Biol ; 11(9): 4760-70, 1991 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-1678856

RESUMEN

The retroviral oncogene v-erbB encodes a truncated form of the receptor for epidermal growth factor, an integral membrane protein-tyrosine kinase. By contrast, the oncogene v-src encodes a protein-tyrosine kinase that is a peripheral membrane protein. The morphologies and spectra of cells transformed by these two oncogenes differ. In an effort to identify the functional determinant(s) of these differences, we constructed and tested first deletion mutants of v-erbB and then chimeras between v-src and v-erbB. As reported previously, the absence of any membrane anchorage eliminated transformation by v-erbB. Anchorage of the cytoplasmic kinase domain of v-erbB to membranes with amino-terminal portions of the v-src protein permitted transformation. The phenotype and spectrum of transformation were those expected for v-erbB rather than for v-src. The transforming chimeras lost their biological activity if the signal for myristylation at the amino terminus of v-src was compromised by mutation. Biochemical fractionations revealed a correlation between transforming activity and the association of chimeric gene products with the membrane fraction of the cell. For reasons not yet apparent, the combined presence of membrane anchorage domains of v-src, and the transmembrane domain of v-erbB in the same chimera typically (but not inevitably) impeded transformation. Our results suggest that the specificity of transformation by v-erbB resides in the selection of substrates by the cytoplasmic domain of the gene product. The protein retains access to those substrates even when anchored to the membrane in the manner of a peripheral rather than a transmembrane protein.


Asunto(s)
Proteína Oncogénica pp60(v-src)/química , Proteínas Oncogénicas de Retroviridae/química , Alelos , Animales , Western Blotting , Fraccionamiento Celular , Línea Celular , Transformación Celular Neoplásica/genética , Células Cultivadas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteína Oncogénica pp60(v-src)/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Proteínas Oncogénicas v-erbB , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/metabolismo
16.
Proc Natl Acad Sci U S A ; 88(13): 5484-8, 1991 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-1712101

RESUMEN

Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein-tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nucleotide binding protein but does not induce the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Western blot analysis reveals that PLC-gamma 1 is tyrosine-phosphorylated in response to TCR stimulation. Nearly all of the PLC activity recovered in eluates from anti-phosphotyrosine immunoprecipitates was depleted by anti-PLC-gamma 1 antibodies. Stimulation of the TCR on mutants derived from Jurkat that are defective in TCR-induced PLC activation results in markedly reduced, if any, PLC activity recovered in phosphotyrosine immunoprecipitates and in no detectable PLC-gamma 1 tyrosine phosphorylation. Thus, the TCR functions like PTK growth factor receptors, but through an indirect interaction, to induce tyrosine phosphorylation of PLC-gamma 1. Since other studies have implicated two members of the src family of PTKs in TCR-mediated signal transduction, our findings suggest that the induction of tyrosine phosphorylation of PLC-gamma 1 by a mechanism involving a src-like kinase may be the means by which the TCR regulates PLC activity in T cells.


Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Fosfolipasas de Tipo C/metabolismo , Tirosina/análogos & derivados , Anticuerpos Monoclonales , Humanos , Técnicas In Vitro , Fosfatos de Inositol/fisiología , Fosforilación , Fosfotirosina , Transducción de Señal , Linfocitos T/fisiología , Células Tumorales Cultivadas , Tirosina/metabolismo
17.
Oncogene ; 5(1): 15-24, 1990 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-1969616

RESUMEN

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds and transforms the corresponding cells in culture. Neoplastic transformation by AEV is mediated principally by an oncogene known as v-erb-B. We have explored the means by which this gene is expressed from the genome of AEV and uncovered an important structural determinant for the potency of the oncogene. In order to define the boundaries of v-erb-B and the supplementary oncogene, v-erb-A, we sequenced all but a small portion of the genome of the ES4 strain of AEV. We then demonstrated that, during expression in infected cells, splicing fuses the first six amino acids of the retroviral gene gag to the body of the v-erb-B protein. In order to explore the impact of this fusion on the function of v-erb-B, we constructed vectors with Murine Leukemia Virus that express the oncogene either with or without the fusion to gag. Viruses generated from these two vectors differed greatly in their abilities to transform cells: fusion of v-erb-B with gag enhanced its transforming ability 50 to 100-fold as determined by focus transformation assays and growth in soft agar. Our data suggest that the difference in transforming ability is not due to alterations in transcription or translation but, rather, may result from changes in post-translational modification.


Asunto(s)
Transformación Celular Neoplásica , Genes gag , Oncogenes , Proteínas Oncogénicas de Retroviridae/genética , Retroviridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas v-erbB , Proteínas Oncogénicas Virales , Ratas , Proteínas Virales de Fusión/fisiología
18.
Mol Cell Biol ; 6(4): 1329-33, 1986 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-3023882

RESUMEN

The retroviral oncogene v-erb-B encodes a truncated version of the receptor for epidermal growth factor. To define the disposition of the v-erb-B protein within cells and across the plasma membrane, we raised antibodies against defined epitopes in the protein and used these in immunofluorescence to analyze cells transformed by v-erb-B. A small fraction of the v-erb-B protein was found on the plasma membrane in a clustered configuration. The bulk of the protein was located in the endoplasmic reticulum and Golgi apparatus. Epitopes near the amino terminus of the v-erb-B protein were displayed on the surface of the cell, whereas epitopes in the protein kinase domain were located exclusively within cells. We conclude that the v-erb-B protein spans the plasma membrane in a manner similar or identical to that of the epidermal growth factor receptor, even though the viral transforming protein does not possess the signal peptide that is thought to direct insertion of the receptor into the membrane.


Asunto(s)
Alpharetrovirus/genética , Virus de la Leucosis Aviar/genética , Receptores ErbB/genética , Eritroblastos/metabolismo , Membrana Eritrocítica/metabolismo , Genes Virales , Genes , Oncogenes , Animales , Anticuerpos , Médula Ósea , Receptores ErbB/análisis , Membrana Eritrocítica/ultraestructura , Técnica del Anticuerpo Fluorescente
20.
Biochem Pharmacol ; 33(1): 125-30, 1984 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-6584106

RESUMEN

R-24571 (calmidazolium), a derivative of the antimycotic agent miconazole, inhibited phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK), with an IC50 (the concentration causing 50% inhibition) of 5.3 microM. It also inhibited the calmodulin/Ca2+-stimulated enzymes, with IC50 values of 1.6 and 0.1 microM for myosin light chain kinase (MLCK) and phosphodiesterase respectively. Analysis of inhibition by R-24571 of PL-Ca-PK and MLCK revealed complex kinetics, suggesting that the agent interacted with the cofactors, the enzyme, and/or the cofactor-enzyme complexes. At saturating concentrations of the cofactors, R-24571 inhibited PL-Ca-PK and MLCK noncompetitively with their respective cofactors. Inhibition of MLCK by R-24571 was completely overcome by phosphatidylserine, indicating a strong hydrophobic interaction between R-24571 and the phospholipid in the presence of calmodulin. R-24571 also inhibited phosphorylation of various endogenous proteins in brain stimulated specifically by phosphatidylserine/Ca2+ or calmodulin/Ca2+. The present findings inducated that R-24571 has little specificity in inhibiting two types of Ca2+-dependent protein kinases sensitive to phospholipid or calmodulin.


Asunto(s)
Calcio/metabolismo , Calmodulina/antagonistas & inhibidores , Imidazoles/farmacología , Fosfolípidos/metabolismo , Inhibidores de Proteínas Quinasas , Animales , Bovinos , Humanos , Cinética , Leucemia Monocítica Aguda/enzimología , Quinasa de Cadena Ligera de Miosina , Porcinos
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