RESUMEN
A nef-deleted SHIV-NM-3rN (SHIV-NI) was previously shown to be nonpathogenic and to induce protective immunity. In the present study, a SHIV-NI expressing human interferon-gamma (SHIV-IFN-gamma) was constructed and the effect of co-expression of IFN-gamma on virus replication and immunopotentiation was investigated in macaques that were vaccinated with both viruses, by comparing cytokine responses during the first 4 weeks after vaccination. Peripheral blood mononuclear cells (PBMC) isolated from vaccinated macaques were stimulated with inactivated viral particles for 24 h, and the production of IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha and IFN-gamma was determined by ELISA and flow cytometry. All of the vaccinated macaques showed increases in cytokine production. However, the production of IFN-gamma (Th1-type cytokine) was more rapidly induced by SHIV-IFN-gamma vaccination, and IFN-gamma-producing cells appeared to be still increasing at 4 weeks after vaccination, although the difference of virus replication during the time was not significant in contrast to in vitro replication in cultured PBMC. These results suggest that co-expression of IFN-gamma with SHIV can modulate the antiviral immune responses into the Th1 type response, which would probably provide more protective immunity.
Asunto(s)
Citocinas/biosíntesis , VIH-1/inmunología , Interferón gamma/biosíntesis , Virus Reordenados/inmunología , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunación , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Eliminación de Gen , Genes nef/genética , VIH-1/genética , Interferón gamma/administración & dosificación , Interferón gamma/genética , Leucocitos Mononucleares/inmunología , Macaca mulatta , Virus Reordenados/genética , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/genética , Factores de Tiempo , Vacunas Atenuadas/inmunologíaRESUMEN
Stevia mixture, sweeteners extracted from the leaves of Stevia rebaudiana Bertoni, consists mainly of stevioside and rebaudioside A (glycosides of the diterpene derivative steviol). The aim of this study was to investigate human intestinal metabolism of stevia mixture and its alpha-glucose derivative (known in Japan as enzymatically modified stevia) by LC/MS/ESI analysis. Degradation was examined by incubating stevia mixture, enzymatically modified stevia, stevioside, rebaudioside A, alpha-monoglucosylstevioside, alpha-monoglucosylrebaudioside A and the aglycone, steviol with pooled human faecal homogenates (obtained from five healthy volunteers) for 0, 8 and 24 h under anaerobic conditions. Stevia mixture, enzymatically modified stevia, stevioside and rebaudioside A (0.2 mg/ml) were completely eliminated within 24 h, whereas no degradation of steviol (0.08 and 0.2 mg/ml) appeared to be found during the incubation period. Stevia mixture, stevioside and rebaudioside A appeared to be hydrolyzed to steviol by human intestinal microflora: this observation is consistent with previous rat metabolism studies. Similarly, enzymatically modified stevia appeared to be metabolized via stevia components and, finally, to steviol. This study suggests that there are apparently no species differences in intestinal metabolism of stevia mixture between rats and humans.
Asunto(s)
Sistema Digestivo/microbiología , Diterpenos de Tipo Kaurano , Heces/química , Glucósidos/metabolismo , Extractos Vegetales/metabolismo , Stevia/química , Edulcorantes/metabolismo , Adulto , Anaerobiosis , Animales , Bacterias Anaerobias/metabolismo , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Diterpenos/química , Heces/microbiología , Cromatografía de Gases y Espectrometría de Masas , Glucósidos/química , Humanos , Hidrólisis , Cinética , Masculino , Peso Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacocinética , Ratas , Especificidad de la Especie , Stevia/metabolismo , Relación Estructura-Actividad , Edulcorantes/química , Edulcorantes/farmacocinética , Terpenos/químicaRESUMEN
Monkeys that have been vaccinated with nef-deleted SHIVs were either fully or partially protected against challenge with acute pathogenic SHIV-89.6 P. Viruses isolated from these vaccinated monkeys were all found to be the 89.6 P challenge virus using PCR amplification and restriction enzyme analysis of the env region of the viruses. Analysis of the 3'-end of the env region and 5'-half of the nef region using a heteroduplex mobility assay revealed that the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys had quite an abundant and similar heterogeneous quasispecies population. In contrast, the viruses isolated from the vaccinated monkeys had different and fewer quasispecies indicating a selective immune pressure in the vaccinated monkeys. The in vitro replication of the viruses isolated from the vaccinated monkeys in human and macaque peripheral blood mononucular cells (PBMCs) as well as in established cell lines such as M8166 and HSC-F cells, were slow and delayed when compared to the parental 89.6 P and re-isolated viruses from unvaccinated 89.6 P-infected monkeys. Further comparison revealed that in HSC-F cells the viruses from vaccinated monkeys again showed delayed and weak CD4(+) cell down-modulation as well as having little or no effect on cell growth or cell viability on HSC-F cells and monkey PBMC. Thus we noticed that these re-isolated 89.6 P viruses from the vaccinated monkeys had changed or had been selected for low pathogenic viruses in the monkeys. This suggests that though the vaccination did not completely prevent the replication of the challenge virus in the monkeys it did contain the challenge virus by suppressing the pathogenic variants. This further enhances the prospects of this nef-deleted SHIV as the bases for effective anti-HIV vaccine candidates.
Asunto(s)
Vacunas contra el SIDA/inmunología , VIH-1/genética , VIH-1/patogenicidad , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Vacunas contra el SIDA/administración & dosificación , Animales , Línea Celular , Quimera , Efecto Citopatogénico Viral , Productos del Gen nef/genética , Células Gigantes , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/aislamiento & purificación , Análisis Heterodúplex , Humanos , Macaca mulatta , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
We investigated apoptosis induced by in vitro infection with the chimeric virus of simian immunodeficiency virus and human immunodeficiency virus (SHIV). Macaque and human peripheral blood mononuclear cells (PBMCs) were infected with pathogenic SHIV-89.6p (89.6p) or nonpathogenic SHIV-NM-3rN (NM-3rN). In macaque PBMCs, the extent of virus production and apoptosis induction in CD4(+) cells was much greater in 89.6p infection than in NM-3rN infection. The result was consistent with our previous study of in vivo SHIV infection. In human PBMCs, 89.6p replicated and induced apoptosis more extensively than did NM-3rN, when the cells were infected with the same infectious doses of the viruses. However, in cells infected with a high dose of NM-3rN, the levels of virus production and apoptosis induction were comparable to those in 89.6p infection. There was no significant difference in the extent of apoptosis induction between 89.6p and NM-3rN infection when growth curves of the two viruses matched. Thus, apoptosis induction by SHIV might depend quantitatively on the amount of virus production rather than on the strains of the virus. Moreover, the correlation between the extent of apoptosis induction and virus pathogenicity in macaque PBMCs has also been found in SHIV-infected macaques. This suggests that the profiles of SHIV infection in vitro reflect the in vivo phenomena. Therefore, the in vitro evaluation of apoptosis induction by SHIV could be useful as a safety test for the development of live-attenuated vaccines.
Asunto(s)
Apoptosis , VIH/fisiología , Leucocitos Mononucleares/citología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Células Cultivadas , VIH/genética , Humanos , Leucocitos Mononucleares/virología , Macaca , Recombinación Genética , Virus de la Inmunodeficiencia de los Simios/genéticaRESUMEN
The present study examined how changes would occur in sex-role attitudes of female high school students. Six hundred (600) randomly sampled female high school students from a metropolitan area completed a questionnaire. Partial Order Scalogram Analysis (POSA) was conducted for three variables: complaint against sex discrimination, interest in women's independence, and attitude toward women's achievement in the public arena. POSA found two separate change routes for sex-role attitudes. In the first, the complaint process, increasing complaint against sex discrimination led to positive attitude toward women's achievement in the public arena, which in turn to heightened interest in women's independence. For the second, the interest process, heightened interest in women's independence was followed by positive attitude toward women's achievement, and then by more complaint against sex discrimination. Results also suggested not only that the complaint process was facilitated by external factors like discrimination, and that the interest process by increasing interest in self. But students in the last stage of both processes experience sex-role conflict.
Asunto(s)
Actitud , Identidad de Género , Psicología del Adolescente , Instituciones Académicas , Estudiantes/psicología , Adolescente , Conflicto Psicológico , Femenino , Feminismo , Humanos , Prejuicio , Encuestas y CuestionariosRESUMEN
In accordance with our recent results obtained with cultured rat hepatocytes [Fujioka, T. & Ui, M. (2001) Eur. J. Biochem. 268, 25-34], epidermal growth factor (EGF) gave rise to transient tyrosine phosphorylation of insulin receptor substrates (IRS-1 and IRS-2), thereby activating the bound phosphatidylinositol 3-kinase in human epidermoid carcinoma A431 cells normally abundant in EGF receptors (EGFR) and Chinese hamster ovary (CHO) cells transfected with full-length EGFR. These actions of EGF, although much smaller in magnitude than those of insulin or IGF-I in the same cells, were accompanied by tyrosine phosphorylation of EGFR rather than insulin or IGF-I receptors, never observed in wild-type CHO cells expressing no EGFR, and totally inhibited by an inhibitor of EGFR kinase, AG1478, that was without effect on insulin or IGF-I actions. Recombinant IRS-1 was phosphorylated on tyrosines upon incubation with purified EGFR from A431 cells and 32P-labeled ATP. When CHO cells were transfected with C-terminal truncated EGFR lacking three NPXY motifs responsible for direct binding to phosphotyrosine-binding domains of IRSs, no effect of EGF could be observed. We suggest that tyrosine phosphorylation of IRS-1 or IRS-2 could mediate EGFR-induced activation of phosphatidylinositol 3-kinase in mammalian cells.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal , Androstadienos/farmacología , Animales , Células CHO , Sistema Libre de Células , Cricetinae , ADN Complementario/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Immunoblotting , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas/metabolismo , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Estructura Terciaria de Proteína , Quinazolinas , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Tirosina/metabolismo , Tirfostinos/farmacología , WortmaninaRESUMEN
Sphingosine 1-phosphate (S1P), a novel lipid mediator, is concentrated in the fraction of lipoproteins that include high density lipoprotein (HDL) and low density lipoprotein (LDL) in human plasma. Here, we show that oxidation of LDL resulted in a marked reduction in the S1P level in association with a marked accumulation of lysophosphatidylcholine (LPC). We therefore investigated the role of the lipoprotein-associated lipids especially S1P in the lipoprotein-induced cytoprotective or cytotoxic actions in human umbilical vein endothelial cells. The viability of the cells gradually decreased in the absence of serum or growth factors in the culture medium. The addition of oxidized LDL (ox-LDL) accelerated the decrease in the cell viability. LPC and 7-ketocholesterol mimicked ox-LDL actions. On the other hand, HDL and LDL almost completely reversed the serum deprivation- or ox-LDL-induced cytotoxicity. Exogenous S1P mimicked cytoprotective actions. Moreover, the S1P-rich fraction and chromatographically purified S1P from HDL exerted cytoprotective actions, but the rest of the fractions did not. The cytoprotective actions of HDL and S1P were associated with extracellular signal-regulated kinase (ERK) activation and were almost completely inhibited by pertussis toxin and PD98059, an ERK kinase inhibitor. The HDL-induced action was specifically desensitized in the S1P-pretreated cells. Taken together, these results indicate that the lipoprotein-associated S1P and the lipid receptor-mediated signal pathways may be responsible for the lipoprotein-induced cytoprotective actions. Furthermore, the decrease in the S1P content, in addition to the accumulation of cytotoxic substances such as LPC, may be important for the acquisition of the cytotoxic property to ox-LDL.
Asunto(s)
Endotelio Vascular/metabolismo , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Lisofosfolípidos , Esfingosina/metabolismo , Venas Umbilicales/metabolismo , Células Cultivadas , Carbón Orgánico/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Fase G1 , Humanos , Lipoproteínas HDL/química , Lipoproteínas LDL/química , Oxidación-Reducción , Fase de Descanso del Ciclo Celular , Esfingosina/análogos & derivados , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacosRESUMEN
Fos family proteins form stable heterodimers with Jun family proteins, and each heterodimer shows distinctive transactivating potential for regulating cellular growth, differentiation, and development via AP-1 binding sites. However, the molecular mechanism underlying dimer specificity and the molecules that facilitate transactivation remain undefined. Here, we show that BAF60a, a subunit of the SWI.SNF chromatin remodeling complex, is a determinant of the transactivation potential of Fos/Jun dimers. BAF60a binds to a specific subset of Fos/Jun heterodimers using two different interfaces for c-Fos and c-Jun, respectively. Only when the functional SWI.SNF complex is present, can c-Fos/c-Jun (high affinity to BAF60a) but not Fra-2/JunD (no affinity to BAF60a) induce the endogenous AP-1-regulated genes such as collagenase and c-met. These results indicate that a specific subset of Fos/Jun dimers recruits SWI.SNF complex via BAF60a to initiate transcription.
Asunto(s)
Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Dimerización , Mutación , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , Eliminación de SecuenciaRESUMEN
Exogenous sphingosine 1-phosphate (S1P) increased cytosolic Ca(2+) concentration, stimulated thymidine incorporation (DNA synthesis) and inhibited cell migration in rat aortic smooth-muscle cells (AoSMCs). Although exogenous sphingosine, a substrate of sphingosine kinase or a precursor of S1P, markedly induced the intracellular accumulation of S1P, the lipid failed to mimic the S1P-induced actions. In contrast, dihydrosphingosine 1-phosphate (DHS1P), an S1P receptor agonist, duplicated these S1P actions even though DHS1P was approx. 20-50-fold less potent than S1P. The pharmacological properties of DHS1P for the S1P receptor subtypes Edg-1, Edg-3, Edg-5 and Edg-6 were compared in Chinese hamster ovary (CHO) cells that were overexpressing the respective receptor. In these S1P-receptor-overexpressing cells, DHS1P was approx. 20-30-fold less potent than S1P for the displacement of [(3)H]S1P binding and inositol phosphate response in Edg-5-expressing CHO cells, as was the case for AoSMCs. However, it was slightly (not more than 3-fold) less potent than S1P in cells expressing Edg-1, Edg-3 or Edg-6. Of the above-mentioned four types of S1P receptor, Edg-5 was abundantly expressed in AoSMCs, as demonstrated by Northern blotting. These results suggest that the intracellular accumulation of S1P is not necessary for the S1P-induced Ca(2+) response, for the stimulation of DNA synthesis or for the inhibition of cell migration. Thus these S1P-induced actions might be mediated through extracellular (or cell-surface) S1P receptors in AoSMCs: Edg-5 might be a most important receptor subtype.
Asunto(s)
Aorta , Movimiento Celular/efectos de los fármacos , ADN/biosíntesis , Lisofosfolípidos , Músculo Liso Vascular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Esfingosina/farmacología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Células CHO , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Cricetinae , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptores de Superficie Celular/genética , Receptores Lisofosfolípidos , Esfingosina/metabolismo , Especificidad por Sustrato , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Short-term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 when the cells had been submitted to primary culture from 1-18 h. Tyrosine-phosphorylated IRS-1 and IRS-2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3-kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3-kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15-30 s and with similar EC(50)s in the nM range in both cases. A possible involvement of insulin receptors in these insulin-like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor beta-subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin-like growth factor-I (IGF-I) for a short period responded to the subsequent addition of EGF, whereas EGF-treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF-I. Thus, the EGF receptor-initiated signalling was mediated by PtdIns 3-kinase associated with tyrosine-phosphorylated IRSs in short-term cultured rat hepatocytes.
Asunto(s)
Factor de Crecimiento Epidérmico/fisiología , Hepatocitos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/fisiología , Receptor de Insulina/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática , Hepatocitos/metabolismo , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas/metabolismo , Fosforilación , Ratas , Ratas Wistar , Factores de Tiempo , Tirosina/metabolismoRESUMEN
The concentration of sphingosine 1-phosphate (S1P) in plasma or serum is much higher than the half-maximal concentration of the sphingolipid needed to stimulate its receptors. Nevertheless, the inositol phosphate response to plasma or serum mediated by Edg-3, one of the S1P receptors, which was overexpressed in Chinese hamster ovary cells, was much smaller than the response expected from the total amount of S1P in these samples. The inositol phosphate response to exogenous S1P was markedly attenuated in the presence of charcoal-treated low-S1P serum. The inhibitory effect was lost by boiling but not by dialysis of the serum. The inhibitory action of the serum was specific to S1P and was associated with the trapping of exogenous S1P; the inositol phosphate response to P(2)-purinergic agonists was somewhat enhanced by the charcoal-treated serum. Among the components of plasma or serum, lipoproteins such as low-density and high-density lipoproteins showed a stronger activity for trapping S1P than lipoprotein-deficient serum. Consistent with this observation, we detected a 15-100-fold higher amount of S1P per unit amount of protein in lipoproteins than in the lipoprotein-deficient serum. Thus even though the protein content of the lipoprotein fraction contributes to only 4% of the total protein content of plasma or serum, more than 60% of S1P is distributed in this fraction. These results suggest that the tight binding of S1P to the components of serum or plasma, including lipoproteins, may interfere with the S1P binding to its receptors and thereby attenuate the lipid-receptor-mediated actions in the cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Lipoproteínas/metabolismo , Lisofosfolípidos , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Animales , Proteínas Sanguíneas/farmacología , Células CHO , Carbón Orgánico/farmacología , Cricetinae , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Diálisis , Fosfatos de Inositol/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Inhibidor NF-kappaB alfa , Unión Proteica/efectos de los fármacos , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores Lisofosfolípidos , Esfingosina/antagonistas & inhibidores , Esfingosina/sangre , Esfingosina/farmacología , TransfecciónRESUMEN
The baseline activity of cyclic nucleotide phosphodiesterase 4 was markedly lowered by primary culture of rat hepatocytes with herbimycin A for 4 h [Eur. J. Biochem. 260 (1999) 398-408.]. We now report that insulin added to this preparation of hepatocytes, which had been completely freed of herbimycin, increased the thus lowered phosphodiesterase activity, consequently antagonizing glucagon-induced production of cAMP and activation of glycogen phosphorylase. The insulin receptor beta-subunits and alpha-tubulin were tyrosine-phosphorylated upon the addition of insulin. The phosphorylation of alpha-tubulin afforded conditions unfavorable for microtubule assembly that is responsible for phosphodiesterase inhibition. These effects of insulin observed in herbimycin-pretreated hepatocytes were not inhibited by wortmannin that actually abolished insulin-induced activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) under the same conditions. The physiological significance of the insulin action not mediated by PtdIns 3-kinase in herbimycin-pretreated hepatocytes is discussed.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/efectos de los fármacos , Glucagón/farmacología , Hepatocitos/efectos de los fármacos , Insulina/farmacología , Quinonas/farmacología , 1-Metil-3-Isobutilxantina/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Benzoquinonas , Células Cultivadas , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/enzimología , Lactamas Macrocíclicas , Masculino , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilasas/efectos de los fármacos , Fosforilasas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Rifabutina/análogos & derivados , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Tirosina/metabolismoRESUMEN
The authors argue that complementary hostile and benevolent components of sexism exist across cultures. Male dominance creates hostile sexism (HS), but men's dependence on women fosters benevolent sexism (BS)--subjectively positive attitudes that put women on a pedestal but reinforce their subordination. Research with 15,000 men and women in 19 nations showed that (a) HS and BS are coherent constructs that correlate positively across nations, but (b) HS predicts the ascription of negative and BS the ascription of positive traits to women, (c) relative to men, women are more likely to reject HS than BS, especially when overall levels of sexism in a culture are high, and (d) national averages on BS and HS predict gender inequality across nations. These results challenge prevailing notions of prejudice as an antipathy in that BS (an affectionate, patronizing ideology) reflects inequality and is a cross-culturally pervasive complement to HS.
Asunto(s)
Características Culturales , Prejuicio , Estereotipo , Mujeres , Adulto , África , Américas , Asia , Australia , Comparación Transcultural , Europa (Continente) , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Psicológicos , Muestreo , Encuestas y CuestionariosRESUMEN
We investigated the role of endogenous AP-1 in human tumor cell lines by introducing SupJunD-1, a dominant-negative mutant of AP-1, using vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors. Single inoculation of six human tumor cell lines, originating from osteosarcomas, non-small cell lung carcinomas or cervical carcinomas, with recombinant SupJunD-1 virus at a high multiplicity of infection readily inhibited colony formation in soft agar. We detected no significant changes in expression levels of AP-1 components c-Jun or Fra-1, adhesion molecules CD44 or E-cadherin, or cell cycle regulator p53, which are encoded by genes previously reported to be under the control of AP-1 in some mouse or human cell lines. By varying the dosage of VSV-G-pseudotyped retrovirus, we were able to change the proviral copy number of supjunD-1 from 1 to approximately 10 and monitor suppression of endogenous AP-1 function as assessed by growth characteristics of the tumor cell lines, we found a SupJunD-1 dosage which significantly suppressed anchorage-independent growth without affecting the cellular growth in monolayer cultures at all. We conclude that endogenous AP-1 levels necessary for oncogenic activity are much higher than those sufficient to support normal growth.
Asunto(s)
Glicoproteínas de Membrana , Neoplasias/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Células 3T3 , Animales , Western Blotting , Cadherinas/biosíntesis , División Celular , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Genes Dominantes , Humanos , Receptores de Hialuranos/biosíntesis , Ratones , Plásmidos/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Retroviridae/genética , Factores de Tiempo , Factor de Transcripción AP-1/fisiología , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas del Envoltorio Viral/metabolismoRESUMEN
In this study, we tried a DNA vaccination regime in rhesus macaques using a full genome HIV-1 plasmid. The HIV-1 genome is under the control of its original LTR promoter, but has a mutated zinc finger motif gene in the nucleocapsid region. Due to the lack of genomic RNA packaging, the plasmid produces only noninfectious viral particles. We repeatedly injected four macaque monkeys intramuscularly with the naked DNA over a period of 40 weeks. To evaluate the humoral and cell-mediated immunity provided by this DNA vaccination, no other booster or other recombinant viral vectors were used. Immunological responses against HIV-1 were elicited in all of the vaccinated monkeys: stable anti-HIV-1 Env antibodies were raised in two monkeys and CTL activities were induced in the other monkeys. The macaques were intravenously challenged at 54 weeks with 100 TCID(50) of SHIV-NM-3rN, which possesses an envelope gene homologous to the one in the vaccinated plasmid. In all of the vaccinated macaques, the peak plasma viral loads induced by the challenge virus were two to three orders of magnitude lower than those of the naive controls. These results suggest that a DNA vaccination regime with a full genome plasmid alone is potentially efficacious and provides a new possibility for the development of an AIDS vaccine.
Asunto(s)
Vacunas contra el SIDA/inmunología , Genoma Viral , VIH-1/inmunología , Macaca mulatta/inmunología , Macaca mulatta/virología , Plásmidos/genética , Vacunas de ADN/inmunología , Vacunas contra el SIDA/química , Vacunas contra el SIDA/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , ADN Viral/análisis , ADN Viral/genética , Productos del Gen env/genética , Productos del Gen env/inmunología , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/genética , Antígenos VIH/inmunología , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , VIH-1/fisiología , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Macaca mulatta/sangre , Masculino , Datos de Secuencia Molecular , Pruebas de Neutralización , Plásmidos/inmunología , Provirus/genética , Provirus/fisiología , ARN Viral/sangre , ARN Viral/genética , Alineación de Secuencia , Síndrome de Inmunodeficiencia Adquirida del Simio/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/virología , Vacunación , Vacunas de ADN/química , Vacunas de ADN/genética , Carga Viral , Dedos de ZincRESUMEN
The mechanism of cell growth was investigated in GIT medium-supplemented in vitro assay using high and low metastatic mouse hepatoma cell sublines, G-5 and G-1, respectively. G-5 cells exhibited high growth rate compared to G-1 cells. The PI3-kinase inhibitor LY294002 and P70 S6 kinase inhibitor rapamycin partially blocked both G-1 and G-5 cell growth, suggesting that these two kinases are involved in hepatoma cell growth. In contrast, the MEK1 inhibitor PD98059 partially blocked G-5 cell growth but not G-1 cell growth. MAP kinases (MAPK) in both G-1 and G-5 cells were indistinguishably phosphorylated, yet MEK-dependent MAPK activation was observed only in G-5 cells. In G-1 cells, MAPK was phosphorylated in a manner not connected to MEK activation. Thus, the low degree of cell growth in G-1 cells was attributable to disruption of the MEK-dependent MAPK cascade. However, the molecular mechanism whereby MAPK phosphorylation does not parallel MAPK activation in G-1 cells remains unknown. Here, we suggest that there may be an as yet unidentified MAPK phosphorylation pathway in malignantly transformed cells, which may affect in vivo cell growth and metastatic capacities of cancers.
Asunto(s)
División Celular/fisiología , Neoplasias Hepáticas Experimentales/patología , Quinasa 1 de Quinasa de Quinasa MAP , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , División Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Densitometría , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Sustancias de Crecimiento/farmacología , Immunoblotting , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/enzimología , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/inmunología , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/metabolismo , Células Tumorales CultivadasRESUMEN
Sphingosine 1-phosphate (S1P) stimulates thymidine incorporation (DNA synthesis), cell growth and cell migration in human aortic endothelial cells (HAECs). The extent of the S1P-induced responses are comparable to those stimulated by vascular endothelial growth factor, one of the most potent stimulators of angiogenesis. These responses to S1P were mimicked by dihydrosphingosine 1-phosphate, an S1P receptor agonist, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S1P also induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAP kinase). The activation of these enzymes was inhibited again by PTX and also by suramin, a non-selective receptor antagonist. S1P-induced DNA synthesis and ERK activation were inhibited by PD98059, an ERK kinase inhibitor, but not by SB203580, a p38 MAP kinase inhibitor. In contrast, cell migration and p38 MAP kinase activation, in response to S1P, were inhibited by SB203580 but not by PD98059. In HAECs, high-affinity S1P binding activity and expression of Edg-1 and Edg-3 mRNA were detected. These results suggest that S1P might be a novel angiogenesis factor and that the lipid-induced proliferation and migration of endothelial cells are possibly mediated through cell-surface S1P receptors, Edg-1 and Edg-3, which are linked to signalling pathways.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/efectos de los fármacos , Proteínas I-kappa B , Proteínas Inmediatas-Precoces/metabolismo , Lisofosfolípidos , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Antineoplásicos/farmacología , Unión Competitiva , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , ADN/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Endotelio Vascular/citología , Proteínas de Unión al GTP/metabolismo , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidor NF-kappaB alfa , Toxina del Pertussis , ARN Mensajero/biosíntesis , Receptores Lisofosfolípidos , Transducción de Señal , Esfingosina/farmacología , Suramina/farmacología , Factores de Virulencia de Bordetella/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
To investigate the role of apoptosis in the progressive loss of CD4+ lymphocytes in HIV infection, we have used macaques infected with SHIV, a hybrid virus of HIV and simian immunodeficiency virus (SIV). In the present study, we sequentially analyzed apoptosis induction in the acute phase of SHIV infection. Four macaques infected with a pathogenic SHIV, SHIV89.6P, and four macaques infected with a nonpathogenic SHIV, NM-3rN, were analyzed during the first 2 or 4 weeks postinfection. In the 89.6P-infected macaques the number of peripheral CD4+ cells sharply decreased at 2 weeks postinfection and was maintained below 50/microl until 4 weeks postinfection, while in the NM-3rN-infected macques the number of the CD4+ cells did not change significantly. Plasma viral loads peaked at 2 and 2-3 weeks postinfection, and the peak values were about 1 x 10(9) and 10(6)-10(7) copies/ml in the 89.6P- and the NM-3rN-infected macaques, respectively. In the 89.6P-infected macaques, Fas antigen expression and the extent of apoptosis in PBMCs and peripheral lymph nodes increased at 1-2 weeks postinfection. A high number of apoptotic cells was also observed in thymus sections 2 and 4 weeks postinfection. On the other hand, apoptosis was scarcely induced in the NM-3rN-infected macaques. These results suggest that the extent of apoptosis induction is closely correlated with the pathogenicity of SHIV and that the apoptosis induction in peripheral lymphoid tissues and thymus, where T cell maturation occurs, may play an important role in the depletion of CD4+ lymphocytes in 89.6P infection.
Asunto(s)
Apoptosis , Linfocitos T CD4-Positivos/patología , VIH/inmunología , Ganglios Linfáticos/patología , Virus de la Inmunodeficiencia de los Simios/inmunología , Timo/patología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Animales , Anticuerpos Antivirales/inmunología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Femenino , Humanos , Estudios Longitudinales , Macaca , Masculino , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Carga Viral , Receptor fas/biosíntesisRESUMEN
Several cDNA encoding G-protein-coupled receptors, i.e. Edg-1,-3,-5,-6 and -8, have recently been identified as sphingosine 1-phosphate (S1P) receptors. However, the role of the respective receptor subtype has not been well defined. In C6 glioma cells, exogenous S1P induced expression of fibroblast growth factor-2 (FGF-2), a potent neurotrophic factor, which was associated with the stimulation of extracellular signal-regulated kinase (ERK) and the expression of early growth response-1 (Egr-1). S1P also stimulated phospholipase C (PLC)/Ca(2+) system and phospholipase D (PLD). In this study, we sought to identify S1P receptors responsible for these S1P-induced actions. Of five S1P receptor subtypes, Edg-1 and Edg-5 are expressed in the glioma cells, as evidenced by Northern blotting. We therefore prepared the cells overexpressing these S1P receptor subtypes and compared the intrinsic activities to stimulate these signaling pathways and their sensitivity to pertussis toxin (PTX). The potency of S1P and dihydrosphingosine 1-phosphate (DHS1P), another S1P receptor agonist, to stimulate the Edg-1 and Edg-5 receptors was also examined. We found that the intrinsic activity that stimulated ERK/Egr-1/FGF-2 system was much higher in Edg-1 than in Edg-5. Furthermore, DHS1P was as potent as S1P in activating ERK in control C6 cells, a pattern also observed in cells overexpressing Edg-1. On the other hand, the stimulation of the PLC/Ca(2+) system and PLD induced by S1P was PTX-insensitive, and the potency of S1P in activating PLD was roughly one order higher than that of DHS1P in control C6 cells; similar responsiveness to such pharmacological tools were observed in Edg-5-overexpressing cells. Taken together, these results suggest that Edg-1 may be the main receptor mediating the stimulation of ERK/Egr-1/FGF-2 system but that Edg-5 may be responsible for the stimulation of PLC-Ca(2+) system and PLD in native C6 glioma cells.
Asunto(s)
Proteínas Inmediatas-Precoces/metabolismo , Lisofosfolípidos , Sistema de Señalización de MAP Quinasas/fisiología , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Animales , Calcio/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Proteínas de Unión al GTP/metabolismo , Expresión Génica/fisiología , Glioma , Proteínas Inmediatas-Precoces/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neuronas/enzimología , Toxina del Pertussis , ARN Mensajero/análisis , Ratas , Receptores de Superficie Celular/genética , Receptores Lisofosfolípidos , Esfingosina/farmacología , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We constructed three simian-human immunodeficiency viruses (SHIVs) lacking regulatory gene(s) and analyzed their induction of protective immunity against challenge infection with gene-intact SHIV in rhesus macaques. Inoculation of SHIV-dn lacking nef and SHIV-drn lacking nef and vpr induced transient viremia, while that of SHIV-dxrn lacking nef, vpr, and vpx induced no viremia. The SHIVs with fewer deletions were more effective in inducing neutralizing antibodies and cytotoxic T lymphocyte responses. When these macaques were challenged with parental gene-intact SHIV-NM-3rN, all the SHIV-dn-vaccinated macaques and two of the four SHIV-drn-vaccinated macaques showed complete resistance. The other two SHIV-drn-vaccinated macaques and all SHIV-dxrn-vaccinated macaques did not show complete resistance, but they did show suppression of replication of the challenge virus. These results suggested that as more genes were deleted, protective immunity was decreased.