RESUMEN
Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.
Asunto(s)
Aorta/patología , Arteriosclerosis/metabolismo , Proteínas Portadoras/inmunología , Glicoproteínas , Vacunas Sintéticas/inmunología , Animales , Anticuerpos/sangre , Arteriosclerosis/inmunología , Arteriosclerosis/patología , Arteriosclerosis/terapia , Western Blotting , Células CHO , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Colesterol en la Dieta/farmacología , Cricetinae , Modelos Animales de Enfermedad , Humanos , Pruebas de Función Renal , Lipoproteínas/análisis , Conejos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Vacunas Sintéticas/administración & dosificaciónRESUMEN
The acceleration of atherosclerosis by polygenic (essential) hypertension is well-characterized in humans; however, the lack of an animal model that simulates human disease hinders the elucidation of pathogenic mechanisms. We report here a transgenic atherosclerosis-polygenic hypertension model in Dahl salt-sensitive hypertensive rats that overexpress the human cholesteryl ester transfer protein (Tg[hCETP]DS). Male Tg[hCETP]DS rats fed regular rat chow showed age-dependent severe combined hyperlipidemia, atherosclerotic lesions, myocardial infarctions and decreased survival. These findings differ from various mouse atherosclerosis models, demonstrating the necessity of complex disease modeling in different species. The data demonstrate that cholesteryl ester transfer protein can be proatherogenic. The interaction of polygenic hypertension and hyperlipidemia in the pathogenesis of atherosclerosis in Tg[hCETP]DS rats substantiates epidemiological observations in humans.
Asunto(s)
Proteínas Portadoras/genética , Enfermedad Coronaria/genética , Glicoproteínas , Hiperlipidemias/genética , Hipertensión/genética , Animales , Animales Modificados Genéticamente , Arteriosclerosis/etiología , Arteriosclerosis/genética , Arteriosclerosis/patología , Proteínas de Transferencia de Ésteres de Colesterol , Enfermedad Coronaria/etiología , Modelos Animales de Enfermedad , Humanos , Hiperlipidemias/complicaciones , Hipertensión/complicaciones , Longevidad , Masculino , Ratones , Fenotipo , Ratas , Ratas Endogámicas Dahl , Cloruro de Sodio , Especificidad de la EspecieRESUMEN
Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Glicoproteínas/farmacología , Selectinas/metabolismo , Animales , Western Blotting , Células CHO , Adhesión Celular , Cricetinae , Electroforesis/métodos , Citometría de Flujo , Glicoproteínas/química , Humanos , Espectrometría de Masas , Monosacáridos/análisis , Oligosacáridos/análisis , Unión Proteica , Ensayo de Unión Radioligante , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Células U937 , Regulación hacia ArribaRESUMEN
A series of vectors has been constructed to express the human T cell receptor V beta 5.3 under the control of the hybrid trc promoter in Escherichia coli. Transcriptional induction of the trc promoter was achieved chemically by using isopropyl beta-D-thiogalactopyranoside (IPTG) in a bacterial strain that harbors the lacIq gene, or thermally by using the mutant lacIts gene that encodes a temperature-sensitive lac repressor [Bukrinsky et al. (1988) Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli. Gene 70, 415-417]. Several of the plasmids tested also contain the E. coli heat-stable enterotoxin II (STII) signal sequence for protein secretion. In addition, the gene 10 leader sequence from bacteriophage T7 and a minicistron localized upstream of the V beta 5.3 coding sequence were tested for their potential effect on protein production. These elements increased protein yield two-fold when transcription was induced by IPTG, but had no detectable effect on protein yield when transcription was induced thermally. The highest protein yield was obtained when V beta 5.3 was expressed either from plasmid pKB containing the STII signal in strain LJ24, or from plasmid pKBi that lacks the signal sequence, in the protease deficient strain SG21173 (lon, htpR. clp). Both plasmids contain the lacIts gene, the trc promoter, the two transcription terminators of the rrnB operon, and a tetracycline selection marker. Production of V beta 5.3 using pKBi-V beta 5.3 in strain SG21173 in a 5-liter fermenter under controlled growth conditions yielded over 25 mg V beta 5.3/liter culture. Conversion of the lacIts to the lacIqts gene yielded vector pKBiq-V beta 5.3 which exhibits complete repression of the trc promoter at 30 degrees C. This stringent regulation of the thermally inducible trc promoter, the elimination of IPTG, the inclusion of the tetracycline resistance gene, and the high level of protein yield should render this expression system broadly useful for the high level production of heterologous proteins in E. coli, for both basic research and human therapeutic applications.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Regulación de la Expresión Génica/genética , Receptores de Antígenos de Linfocitos T/química , Proteínas Represoras/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Cartilla de ADN/química , Electroforesis en Gel de Poliacrilamida , Genes Bacterianos/genética , Genes Virales/genética , Marcadores Genéticos/genética , Vectores Genéticos/genética , Humanos , Isopropil Tiogalactósido/farmacología , Represoras Lac , Datos de Secuencia Molecular , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Receptores de Antígenos de Linfocitos T/aislamiento & purificaciónRESUMEN
A new approach has been used to extend the T(1/2) of human soluble complement receptor type 1 (sCR1) in rats. The albumin-binding domains B2A3 (BA) and B1A2B2A3 (BABA) from Streptococcal protein G were fused to the carboxyl terminus of sCR1, and the recombinant genes were expressed and amplified in Chinese hamster ovary cells. Western blot analysis and surface plasmon resonance measurements demonstrated the binding of rat serum albumin to both sCR1-BA and sCR1-BABA but not to sCR1. The in vitro complement inhibitory activity of the fusion proteins was shown to be similar to that of sCR1, indicating that neither the albumin-binding domains nor the presence of bovine serum albumin interfere with sCR1 function. Pharmacokinetic analysis showed that the T(1/2) of the distribution phase (T(1/2alpha)) was 3.3, 20.0 and 6.0 min for sCR1, sCR1-BA and sCR1-BABA, respectively. The T(1/2) of the elimination phase (T(1/2beta)) was 103, 297 and 170 min for sCR1, sCR1-BA and sCR1-BABA, respectively. The plasma elimination of sCR1-BA and sCR1-BABA was significantly (P < .05) prolonged as compared to sCR1. The proteins showed similar tissue distribution; at 4-hr postdosing, the highest levels of 125I-radioactivity per gram of tissue were localized in the urine, blood, liver, stomach, and small intestine.
Asunto(s)
Receptores de Complemento 3b/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Albúmina Sérica/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Células Cultivadas , Cricetinae , Cricetulus , Semivida , Humanos , Masculino , Datos de Secuencia Molecular , Conejos , Ratas , Ratas Sprague-Dawley , Ovinos , Distribución TisularRESUMEN
We have constructed a vector, pKBi, for the high-level expression of the variable beta chain 5.3 (V beta 5.3) of the human T-cell receptor in Escherichia coli. This vector incorporates the trc promoter, a polylinker, two transcription terminators, and the tetracycline resistance gene. Furthermore, the vector contains the lacIts gene that encodes a temperature-sensitive (ts) lac repressor, thus obviating both the need to use IPTG as a transcriptional inducer, and bacterial strains that harbor either the lacI or lacIq genes. The sequence of the lacIts gene shows an open reading frame of 1,080 nucleotides encoding 360 amino acids, and differs from the lacI gene at nucleotide 559 (with reference to the first nucleotide of the start codon). This nucleotide changes from G to A, causing amino acid residue 187 to change from glycine (GGC) to serine (AGC). This mutation imparts thermal sensitivity to the lac repressor protein. This is the first time that a TCR V beta region has been expressed at high levels (up to 28 mg/liter of culture) without fusion partners. The availability of the lacIts gene for thermal induction of the trc promoter, and the presence of the tetracycline resistance gene should make the expression vector pKBi particularly attractive for the efficient production of human therapeutic proteins in bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Vectores Genéticos/genética , Calor , Regiones Promotoras Genéticas , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Proteínas Represoras/genética , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Humanos , Represoras Lac , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta/genética , Mutación Puntual , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Resistencia a la Tetraciclina/genéticaRESUMEN
We have overproduced and purified wild type regA protein, a translational repressor encoded by bacteriophage T4. The repressor activity of the cloned regA protein has been tested on four known regA target genes (T4 genes: 44, 45, rpbA and regA) using in vitro coupled transcription-translation reactions. We have demonstrated the sensitivity of two additional T4 genes coding for alpha- and beta-glucosyltransferases to regA protein in vitro. The regA target site on the gene 44 messenger RNA has been identified through deletion analysis and RNase protection assays, using plasmids containing gene 44-lacZ fusions. The effect of regA protein on expression of 44P-beta-galactosidase fusion proteins was assayed in vitro, in coupled transcription-translation reactions. Analysis of deletion mutants of gene 44-lacZ localized the regA recognition region to between nucleotides -11 and +9 of the mRNA. RNase protection assays of g44-lacZ transcripts further defined the site of regA protein interaction to between nucleotides -10 and +2 of the mRNA. This region overlaps the gene 44 Shine-Dalgarno region and the A and U of the initiation codon.
Asunto(s)
ADN Viral/genética , Escherichia coli/genética , Genes Virales , Mutación , Regiones Promotoras Genéticas , ARN Mensajero/genética , Fagos T/genética , Proteínas Virales/metabolismo , Proteínas Estructurales Virales/genética , Secuencia de Bases , Clonación Molecular/métodos , ADN Viral/metabolismo , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Plásmidos , Unión Proteica , Biosíntesis de Proteínas , Mapeo Restrictivo , Ribonucleasas , Fagos T/metabolismo , Transcripción Genética , Proteínas Virales/genéticaRESUMEN
A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Genes ras , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Análisis Mutacional de ADN , Activación Enzimática , Proteínas Activadoras de GTPasa , Técnicas Inmunológicas , Técnicas In Vitro , Relación Estructura-Actividad , Proteínas Activadoras de ras GTPasaAsunto(s)
Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pirofosfatasas , Transducción de Señal , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transformación Celular Neoplásica , Clonación Molecular , ADN/genética , Femenino , Proteínas Activadoras de GTPasa , Humanos , Cinética , Datos de Secuencia Molecular , Oocitos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Placenta/metabolismo , Embarazo , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas Proto-Oncogénicas p21(ras) , Xenopus , Proteínas Activadoras de ras GTPasaRESUMEN
Bacteriophage T4 gene 32 encodes a single-stranded DNA binding protein required for T4 DNA replication, recombination, and repair. Previous attempts at cloning gene 32 have failed due to a presumably deleterious effect on host cell viability. In addition, overexpression of gene 32 would be expected to be limited by the autoregulatory ability of the gene 32 product g32P. A repetitive A + T-rich sequence flanking the ribosome binding site of gene 32 has been implicated in this translational regulation. To circumvent these problems, the wild-type gene for g32P has been reconstructed in M13 using restriction fragments from T4 g32am453 and synthetic oligodeoxynucleotides so that it no longer includes its native promoter and putative autoregulatory region. The g32am453 codon TAG was changed back to TGG as in wild-type gene 32 using site-directed oligodeoxynucleotide mutagenesis. In vectors containing the lambda leftward promoter PL, gene 32 is overexpressed with the resulting transcripts being depressed at g32P concentrations that repress the wild-type gene 32 transcripts.
Asunto(s)
Proteínas de Unión al ADN/genética , Fagos T/genética , Proteínas Virales/genética , Bacteriófago lambda/genética , Clonación Molecular , Replicación del ADN , ADN de Cadena Simple/metabolismo , Escherichia coli/genética , Regulación de la Expresión Génica , Mutación , Regiones Promotoras Genéticas , Relación Estructura-ActividadRESUMEN
The bacteriophage T4 translational repressor regA protein has been purified from an overproducing strain, and its activity has been studied in simple in vitro protein synthesis reactions. RegA protein was found to inhibit the translation of T4 genes 44, 45, and ORF45-1 in a concentration-dependent fashion. Expression of two other T4 genes which are insensitive to regA protein in vivo, genes 32 and 43, was unaffected by the presence of regA protein. Specific inhibition of synthesis of genes 44, 45, and ORF 45-1 proteins was achieved with 5-20 microM concentrations of regA protein, without the addition of any other T4 encoded proteins or cofactors. When in vitro protein synthesis was performed in two steps, uncoupling translation from transcription, regA protein had an inhibitory effect regardless of whether it was added at the initiation of transcription or only at the translation step. This indicates that regA protein functions during the translation step of protein synthesis in vitro in agreement with previous in vivo studies of regA protein.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Represoras/genética , Fagos T/genética , Factores de Transcripción/genética , Proteínas Virales/genética , Regulación de la Expresión Génica , Genes Virales , PlásmidosRESUMEN
The bacteriophage T4 regA gene codes for a regulatory protein that controls the expression of a number of T4 early genes, apparently at the level of translation. Restriction fragments containing the regA structural gene have been cloned into phage M13, and the nucleotide sequence has been determined. Translation of the DNA sequence predicted that regA protein contains 122 amino acids, with a Mr of 14,620. A DNA fragment carrying 85% of the coding sequence of regA has been cloned into the phage lambda leftward promoter PL expression vector pAS1, and a high level of truncated regA protein was produced by nalidixic acid induction. Protein chemical studies of the truncated regA protein gave results consistent with the nucleotide sequence of the regA gene. Subsequently, an intact regA gene was cloned into plasmid pAS1 and overexpressed. The regA protein produced in this way regulates the level of T4 45 and 44 proteins when their corresponding genes are carried on the same plasmid as the regA gene.