RESUMEN
Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor lines having either wild-type or oncogenic Ki-ras, treatment with an FTI/GGTI combination or with a DPI blocks Ki-Ras prenylation and induces markedly higher levels of apoptosis relative to FTI or GGTI alone. We demonstrate that these compounds can inhibit their enzyme targets in mice by monitoring pancreatic and tumor tissues from treated animals for inhibition of prenylation of Ki-Ras, HDJ2, a substrate specific for FPTase, and Rap1A, a substrate specific for GGPTase-I. Continuous infusion (72 h) of varying doses of GGTI in conjunction with a high, fixed dose of FTI causes a dose-dependent inhibition of Ki-Ras prenylation. However, a 72-h infusion of a GGTI, at a dose sufficient to inhibit Ki-Ras prenylation in the presence of an FTI, causes death within 2 weeks of the infusion when administered either as monotherapy or in combination with an FTI. DPIs are also lethal after a 72-h infusion at doses that inhibit Ki-Ras prenylation. Because 24 h infusion of a high dose of DPI is tolerated and inhibits Ki-Ras prenylation, we compared the antitumor efficacy from a 24-h FTI infusion to that of a DPI in a nude mouse/PSN-1 tumor cell xenograft model and in Ki-ras transgenic mice with mammary tumors. The FTI and DPI were dosed at a level that provided comparable inhibition of FPTase. The FTI and the DPI displayed comparable efficacy, causing a decrease in growth rate of the PSN-1 xenograft tumors and tumor regression in the transgenic model, but neither treatment regimen induced a statistically significant increase in tumor cell apoptosis. Although FTI/GGTI combinations elicit a greater apoptotic response than either agent alone in vitro, the toxicity associated with GGTI treatment in vivo limits the duration of treatment and, thus, may limit the therapeutic benefit that might be gained by inhibiting oncogenic Ki-Ras through dual prenyltransferase inhibitor therapy.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores Enzimáticos/farmacología , Transferasas Alquil y Aril/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/toxicidad , Farnesiltransferasa , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Prenilación de Proteína/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/metabolismoRESUMEN
A series of amino acid-based linkers was used to investigate the effects of various substituents upon the potency, pharmacokinetic properties, and conformation of macrocyclic farnesyl-protein transferase inhibitors (FTIs). As a result of the studies described herein, highly potent FTIs with improved pharmacokinetic profiles have been identified.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Aminoácidos/química , Animales , Células Cultivadas , Perros , Inhibidores Enzimáticos/síntesis química , Semivida , Concentración 50 Inhibidora , Tasa de Depuración Metabólica/fisiología , Conformación Molecular , Unión Proteica/efectos de los fármacos , RatasRESUMEN
Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The SNAr displacement reaction used to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of commercially available phenols quickly identified compounds which proved active in cells.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Éteres Fenílicos/farmacología , Transferasas Alquil y Aril/metabolismo , Animales , Unión Competitiva , Línea Celular , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Imidazoles/química , Concentración 50 Inhibidora , Biblioteca de Péptidos , Éteres Fenílicos/síntesis química , Ratas , Relación Estructura-ActividadRESUMEN
A series of aryloxy substituted piperazinones with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to have potent inhibitory activity in vitro and are promising agents for the inhibition of Ki-Ras signaling.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Piperazinas/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Farnesiltransferasa , Genes ras/efectos de los fármacos , Piperazinas/química , Polímeros/química , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The evaluation of SAR associated with the insertion of carbonyl groups at various positions of N-arylpiperazinone farnesyltransferase inhibitors is described herein. 1-Aryl-2,3-diketopiperazine derivatives exhibited the best balance of potency and pharmacokinetic profile relative to the parent 1-aryl-2-piperazinones.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Piperazinas/farmacología , Animales , Perros , Inhibidores Enzimáticos/farmacocinética , Farnesiltransferasa , Relación Estructura-ActividadRESUMEN
For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Genes ras , Inhibidores de Crecimiento/uso terapéutico , Neoplasias Mamarias Animales/tratamiento farmacológico , Virus del Tumor Mamario del Ratón , Metionina/análogos & derivados , Animales , Modelos Animales de Enfermedad , Farnesiltransferasa , Femenino , Humanos , Metionina/uso terapéutico , Ratones , Ratones Transgénicos , Fenotipo , TransgenesRESUMEN
Antibodies to hepatitis A virus (anti-HAV) were measured in children from two separate vaccine trials (n = 70) 4 weeks after a dose of inactivated hepatitis A vaccine (VAQTA). The geometric mean titers (GMTs) of anti-HAV were 49.3 and 45.2 mIU/mL by immunoassay, while reciprocal GMTs of neutralizing anti-HAV were 6.5 and 15.0 by an 80% radioimmunofocus inhibition test (RIFIT) and 55.6 and 92.0 by antigen reduction assay (HAVARNA). The GMT of antibody detected by radioimmunoprecipitation (RIPA) was > or =401. These data establish serologic correlates of protection against disease and show that RIPA is most sensitive for detection of early vaccine-induced antibody. Sera collected from adults (n = 20) 7 days after administration of immune globulin contained similar antibody levels by immunoassay (45.1 mIU/mL) and slightly higher GMTs of neutralizing antibody (27.5 by RIFIT and 146 by HAVARNA) but negligible precipitating antibody (GMT, 5.6). These results are best explained by differences in the affinity of antibodies for virus following active versus passive immunization.
Asunto(s)
Anticuerpos Antivirales/sangre , Hepatovirus/inmunología , Inmunización Pasiva , Vacunas contra Hepatitis Viral/inmunología , Adulto , Niño , Preescolar , Vacunas contra la Hepatitis A , Humanos , Pruebas de Precipitina , ARN Viral/análisis , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Pharmacological inhibitors of the housekeeping enzyme farnesyl transferase (FT) inhibit the growth of ras-transformed cells in vitro and in vivo without antiproliferative effects on normal cells. In one direction to analyze the basis for this selectivity and to study modes of drug resistance that arise in animals, we characterized a variant ras-transformed cell line, 749r-1, which was resistant to phenotypic reversion with FT inhibitors. The transformed phenotype, growth potential, and actin cytoskeleton of 749r-1 cells were unaffected by treatment with the FT inhibitor 1-739,749 at concentrations up to 30-fold higher than those sufficient to revert ras-transformed cells. Resistance correlated with a reduced ability of L-739,749 to inhibit the farnesylation of Ras and lamin B and with a reduction in the susceptibility of endogenous FT to drug inhibition. These effects were not due to mutation of the FT subunits, changes in intracellular drug accumulation, or amplification of the multiple drug resistance gene (MDR). However, a similar reduction in the ability of L-739,749 to inhibit Ras farnesylation was also seen in ras-transformed cells rendered resistant by ectopic expression of farnesyl-independent RhoB, suggesting some mechanistic overlap. We concluded that 749r-1 cells sustained a stable alteration that conferred drug resistance by a novel mechanism.
Asunto(s)
Transferasas Alquil y Aril , Transformación Celular Neoplásica/metabolismo , Genes ras , Transferasas/antagonistas & inhibidores , Actinas/metabolismo , Animales , Citoesqueleto/ultraestructura , Resistencia a Medicamentos , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Miristatos , Oligopéptidos/farmacología , Fenotipo , Procesamiento Proteico-Postraduccional , Ratas , Células Tumorales Cultivadas , Proteína de Unión al GTP rhoBAsunto(s)
Transferasas Alquil y Aril , Antineoplásicos/síntesis química , Inhibidores Enzimáticos/síntesis química , Piperazinas/síntesis química , Piperazinas/farmacología , Transferasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Transformada , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa , Ratones , Ratones Desnudos , Conformación Molecular , Estructura Molecular , Trasplante de Neoplasias , Fosfatos de Poliisoprenilo/metabolismo , Prenilación de Proteína , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Sarcoma Experimental/tratamiento farmacológico , Sarcoma Experimental/patología , Sesquiterpenos , Proteínas ras/metabolismoRESUMEN
Small-molecule inhibitors of the housekeeping enzyme farnesyltransferase (FT) suppress the malignant growth of Ras-transformed cells. Previous work suggested that the activity of these compounds reflected effects on actin stress fiber regulation rather than Ras inhibition. Rho proteins regulate stress fiber formation, and one member of this family, RhoB, is farnesylated in vivo. Therefore, we tested the hypothesis that interference with RhoB was the principal basis by which the peptidomimetic FT inhibitor L-739,749 suppressed Ras transformation. The half-life of RhoB was found to be approximately 2 h, supporting the possibility that it could be functionally depleted within the 18-h period required by L-739,749 to induce reversion. Cell treatment with L-739,749 disrupted the vesicular localization of RhoB but did not effect the localization of the closely related RhoA protein. Ras-transformed Rat1 cells ectopically expressing N-myristylated forms of RhoB (Myr-rhoB), whose vesicular localization was unaffected by L-739,749, were resistant to drug treatment. The protective effect of Myr-rhoB required the integrity of the RhoB effector domain and was not due to a gain-of-function effect of myristylation on cell growth. In contrast, Rat1 cells transformed by a myristylated Ras construct remained susceptible to growth inhibition by L-739,749. We concluded that Rho is necessary for Ras transformation and that FT inhibitors suppress the transformed phenotype at least in part by direct or indirect interference with Rho, possibly with RhoB itself.
Asunto(s)
Transferasas Alquil y Aril , Transformación Celular Neoplásica/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Genes ras , Proteínas de la Membrana/metabolismo , Oligopéptidos/farmacología , Transferasas/antagonistas & inhibidores , Animales , Secuencia de Bases , Chlorocebus aethiops , Cartilla de ADN , Farnesiltransferasa , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/genética , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Reacción en Cadena de la Polimerasa , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección , Proteína de Unión al GTP rhoA , Proteína de Unión al GTP rhoBRESUMEN
For Ras oncoproteins to transform mammalian cells, they must be post-translationally modified with a farnesyl group in a reaction catalysed by the enzyme farnesyl-protein transferase (FPTase). Inhibitors of FPTase have therefore been proposed as anti-cancer agents. We show that L-744,832, which mimics the CaaX motif to which the farnesyl group is added, is a potent and selective inhibitor of FPTase. In MMTV-v-Ha-ras mice bearing palpable tumours, daily administration of L-744,832 caused tumour regression. Following cessation of treatment, tumours reappeared, the majority of which regressed upon retreatment. No systemic toxicity was found upon necropsy of L-744,832-treated mice. This first demonstration of anti-FPTase-mediated tumour regression suggests that FPTase inhibitors may be safe and effective anti-tumour agents in some cancers.
Asunto(s)
Transferasas Alquil y Aril , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Metionina/análogos & derivados , Neoplasias de las Glándulas Salivales/tratamiento farmacológico , Transferasas/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/toxicidad , Femenino , Genes ras , Neoplasias Mamarias Experimentales/patología , Metionina/administración & dosificación , Metionina/uso terapéutico , Metionina/toxicidad , Ratones , Ratones TransgénicosRESUMEN
A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.
Asunto(s)
Actinas/metabolismo , Transferasas Alquil y Aril , Transformación Celular Neoplásica , Citoesqueleto/fisiología , Genes ras , Oligopéptidos/farmacología , Transferasas/antagonistas & inhibidores , Animales , Western Blotting , División Celular/efectos de los fármacos , Línea Celular , Citoesqueleto/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Farnesiltransferasa , Cinética , Proteínas Oncogénicas v-raf , Oncogenes , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/genética , Ratas , Proteínas Oncogénicas de Retroviridae/biosíntesis , Proteínas Oncogénicas de Retroviridae/genética , Transferasas/análisis , Transferasas/aislamiento & purificaciónRESUMEN
The Ras oncoprotein must be modified by farnesyl transferase (FTase) for biological activity. Therefore, inhibition of FTase may offer a means to block ras induced cell transformation. To address this hypothesis, we have introduced antisense and dominant inhibitory FTase expression plasmids into a panel of normal, mutant ras-, and mos- transformed rodent fibroblasts in an effort to genetically suppress FTase activity. Antisense FTase constructs reduced colony formation efficiency approximately 29% in normal and approximately 41% in ras-transformed cells relative to control plasmids. In contrast, antisense FTase plasmids did not exhibit a statistically significant effect on colony formation efficiency in mos-transformed transfectants. FTase alpha N199K is a mutant form of the alpha subunit of FTase that exhibits dominant inhibitory activity versus native FTase. Only mos-transformed transfectants exhibited expression of alpha N199K RNA in 15 of 16 fibroblast lines that were randomly selected and characterized. Our data suggest that genetic inhibition of FTase may result in a selection against animal cell growth.
Asunto(s)
Transferasas Alquil y Aril , División Celular/genética , Selección Genética , Transferasas/genética , Animales , Línea Celular Transformada , Farnesiltransferasa , Fibroblastos/citología , Vectores Genéticos , Humanos , Mutación , ARN sin Sentido , Ratas , Saccharomyces cerevisiae/genética , Supresión Genética , Transfección , Transferasas/antagonistas & inhibidoresRESUMEN
Pre- and postexposure prophylaxis against hepatitis A virus (HAV) infection with immune serum globulin (Ig) is only effective for 4-6 months. We compared the safety, tolerability and immunogenicity of a single i.m. injection of Ig with a single and booster dose of an inactivated hepatitis A virus vaccine (iHAV) in adults. Healthy volunteers (18-50 years) received a single Ig i.m. injection (n = 30), or iHAV i.m. (n = 15) at 0 and 24 weeks, or placebo (n = 4) at the same intervals. Anti-HAV seroconversion was measured by radioimmunoassay (RIA) and neutralizing antibodies by an antigen reduction assay. After Ig injection (0.06 ml/kg), anti-HAV seroconversion occurred in 100% of recipients at week 1, declining to 10% at week 12 and 0% by week 20. In contrast, after a single 25 ng dose, RIA seropositivity in iHAV vaccinees was 80% by week 2, reaching 100% by week 5 and persisted up to week 24, at which time anti-HAV geometric mean titres (GMT) were two fold higher than those seen at week 1 after Ig. Postbooster anti-HAV titres in iHAV recipients rose within 4 weeks to 73-fold greater than the peak GMT seen one week after Ig, and 400-fold higher than GMT at 12 weeks after Ig. Neutralizing antibody titres after iHAV followed a similar pattern, as observed for anti-HAV. iHAV was well tolerated; placebo and vaccine tolerability were indistinguishable, with no serious adverse experiences observed. In conclusion, active vaccination with a single iHAV dose may eventually replace Ig for pre-exposure prophylaxis.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hepatitis A/prevención & control , Vacunas contra Hepatitis Viral/administración & dosificación , Adolescente , Adulto , Femenino , Anticuerpos de Hepatitis A , Vacunas contra la Hepatitis A , Anticuerpos Antihepatitis/sangre , Hepatovirus/inmunología , Humanos , Inmunización Secundaria , Inmunoglobulinas/administración & dosificación , Masculino , Persona de Mediana Edad , Seguridad , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/efectos adversos , Vacunas contra Hepatitis Viral/efectos adversosRESUMEN
The development of the formalin-inactivated hepatitis A vaccine, VAQTA, culminates nearly two decades of the basic science studies of VAQTA in hepatitis A virology at the MRL. The master seed virus for production of VAQTA is derived from the F'(P18) variant of the strain CR326F which has been studied in human clinical trials and shown to the highly attenuated. The antigen is highly purified to make possible the consistency and thoroughness of its inactivation by formalin. Phase I clinical studies of VAQTA were initiated in 1989 and have progressed since that time to the recent Phase III clinical trials which demonstrated efficacy of a single dose of the vaccine in preventing clinical hepatitis A disease in pediatric populations in Monroe, NY.
Asunto(s)
Hepatovirus/inmunología , Vacunas contra Hepatitis Viral/biosíntesis , Animales , Formaldehído , Vacunas contra la Hepatitis A , Humanos , Vacunas de Productos Inactivados/biosíntesisRESUMEN
The Chinese hamster cell line, DC-3F, is heterozygous at the DHFR locus, and each allele can be distinguished on the basis of a unique DNA restriction pattern, protein isoelectric profile and in the abundancy of the DHFR mRNAs it expresses. Although each allele produces four transcripts, 1000, 1650 and 2150 nucleotides [corrected] in length, the relative distribution of these RNAs differs for each; the 2150 nt mRNA represents the major (60%) species generated from one allele, while the 1000 nt mRNA is the major species generated from the other. The allele that predominantly expresses the 2150 nt transcript is preferentially overexpressed when DC-3F cells are subjected to selection in methotrexate. We have analyzed the 3' ends of both DHFR alleles and have found that the three major mRNAs arise by readthrough of multiple polyadenylation signals. A four base deletion in one allele changes the consensus polyadenylation signal AAUAAA to AAUAAU, resulting in the utilization of a cryptic polyadenylation signal lying 21 bp upstream. Surprisingly, this mutation in the third polyadenylation signal appears to affect not only the utilization of this signal, but also the efficiency with which the first signal, located 1171 bp upstream from the third site, is utilized.
Asunto(s)
Poli A/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Alelos , Animales , Secuencia de Bases , Línea Celular , Cricetinae , Cricetulus , Pulmón , Datos de Secuencia Molecular , Mutagénesis , Oligodesoxirribonucleótidos , Plásmidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Mapeo Restrictivo , Eliminación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
To determine the safety and immunogenicity of an inactivated hepatitis A vaccine, 56 healthy adult volunteers were randomly assigned to receive an intramuscular injection of 6.3, 12.5 or 25 ng of inactivated hepatitis A vaccine or placebo at 0, 2 or 4, and 24 weeks. Adverse reactions occurred with similar frequency in vaccine and placebo recipients and consisted primarily of pain or tenderness at the injection site. By 4 weeks after a single 6.3, 12.5 or 25 ng injection, seven, nine and ten out of ten vaccinees, respectively, had antibody detectable by a HAV AB assay modified to increase its sensitivity tenfold. All vaccinees had antibodies detectable by this assay within 2 weeks of their second inoculation. Geometric mean antibody levels increased with higher doses of vaccine (p = 0.05). Neutralizing antibody was detected within 4 weeks of a single inoculation in all vaccinees. Neutralizing antibody was detected after the third inoculation at dilutions of greater than or equal to 1:2048 in all 12.5 and 25 ng vaccinees. All 19 vaccinees tested at 24 months still had HAV antibodies detectable by a modified HAV AB assay. This inactivated hepatitis A vaccine appears to be well tolerated and immunogenic at doses of 6.3-25 ng. The choice of dose and vaccination schedule may depend on the rapidity with which seroconversion is desired.
Asunto(s)
Hepatitis A/inmunología , Vacunas contra Hepatitis Viral/efectos adversos , Vacunas contra Hepatitis Viral/inmunología , Adolescente , Adulto , Antígenos Virales/aislamiento & purificación , Evaluación de Medicamentos , Femenino , Formaldehído , Vacunas contra la Hepatitis A , Anticuerpos Antihepatitis/sangre , Humanos , Masculino , Persona de Mediana Edad , Vacunas de Productos Inactivados/efectos adversos , Vacunas de Productos Inactivados/inmunologíaRESUMEN
In an effort to better understand the preferential resistance to actinomycin D displayed by the multidrug-resistant Chinese hamster lung cell line DC-3F/ADX, we have cloned from those cells a number of cDNAs representing p-glycoprotein gene transcripts. Of the 12 clones isolated, all represent pgp1 transcripts and one, pADX165, contains a 4304-base pair insert with an open reading frame encoding a 1276-amino acid protein that is the homolog of the mouse mdr3/mdr1a gene product. A domain by domain comparison of this protein with p-glycoproteins capable of supporting multidrug resistance, i.e. human mdr1, mouse mdr1/mdr1b, and mouse mdr3/mdr1a, shows that, in addition to the ATP binding sites, the second, fourth, and eleventh transmembrane domains and the four small intracellular loops, IC-1, IC-2, IC-4, and IC-5, are highly conserved and are therefore likely to be important for the maintenance of p-glycoprotein function. Of the remaining 11 cDNA clones, 9 were found to be truncated versions of pADX165. Two others, however, pADX185 and pADX124, contained internal deletions resulting in open reading frames capable of encoding lnovel forms of p-glycoprotein. S1 nuclease and RNase protection analysis demonstrated that these cDNAs represent transcripts present in a number of different multidrug-resistant Chinese hamster lung cell lines. Hence, both are considered to be splicing variants of the hamster pgp1 gene primary transcript.
Asunto(s)
Resistencia a Medicamentos , Glicoproteínas de Membrana/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , Cricetulus , ADN/genética , Expresión Génica , Humanos , Pulmón , Ratones , Datos de Secuencia Molecular , Empalme del ARN , ARN Mensajero/genética , Mapeo Restrictivo , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
The principal neutralizing determinant (PND) of human immunodeficiency virus HIV-1 is part of a disulfide bridged loop in the third variable region of the external envelope protein, gp120. Analysis of the amino acid sequences of this domain from 245 different HIV-1 isolates revealed that the PND is less variable than thought originally. Conservation to better than 80 percent of the amino acids in 9 out of 14 positions in the central portion of the PND and the occurrence of particular oligopeptide sequences in a majority of the isolates suggest that there are constraints on PND variability. One constraining influence may be the structural motif (beta strand--type II beta turn--beta strand--alpha helix) predicted for the consensus PND sequence by a neural network approach. Isolates with a PND similar to the commonly investigated human T cell lymphoma virus IIIB (HTLV-IIIB) and LAV-1 (BRU) strains were rare, and only 14 percent of sera from 86 randomly selected HIV-1 seropositive donors contained antibodies that recognized the PND of these virus isolates. In contrast, over 65 percent of these sera reacted with peptides containing more common PND sequences. These results suggest that HIV vaccine immunogens chosen because of their similarity to the consensus PND sequence and structure are likely to induce antibodies that neutralize a majority of HIV-1 isolates.