RESUMEN

Viral oncogenes, mutated cellular oncogenes, or other adventitious agents that might contaminate vaccine preparations on inoculation of the host will encounter a T cell-mediated immune response which will play a determining role in the progression of neoplastic events or replication of contaminating viral agents. Using SV40 T antigen tumour systems as a model we discuss the regions of the oncoprotein that have an impact on tumourigenicity and the role of CD8 T lymphocyte immune responses in eliminating potential tumour cells. In addition, we discuss measures that counteract T cell immune responses to abrogate T cell-mediated immunosurveillance.

Asunto(s)

Genes Virales , Oncogenes , Linfocitos T Citotóxicos/inmunología , Vacunas Virales , Animales , Línea Celular , Humanos , Inmunocompetencia , Huésped Inmunocomprometido

RESUMEN

The cytotoxic T-lymphocyte response to wild-type simian virus 40 large tumor antigen (Tag) in C57BL/6 (H2(b)) mice is directed against three H2-D(b)-restricted epitopes, I, II/III, and V, and one H2-K(b)-restricted epitope, IV. Epitopes I, II/III, and IV are immunodominant, while epitope V is immunorecessive. We investigated whether this hierarchical response was established in vivo or was due to differential expansion in vitro by using direct enumeration of CD8(+) T lymphocytes with Tag epitope/major histocompatibility complex class I tetramers and intracellular gamma interferon staining. The results demonstrate that epitope IV-specific CD8(+) T cells dominated the Tag-specific response in vivo following immunization with full-length Tag while CD8(+) T cells specific for epitopes I and II/III were detected at less than one-third of this level. The immunorecessive nature of epitope V was apparent in vivo, since epitope V-specific CD8(+) T cells were undetectable following immunization with full-length Tag. In contrast, high levels of epitope V-specific CD8(+) T lymphocytes were recruited in vivo following immunization and boosting with a Tag variant in which epitopes I, II/III, and IV had been inactivated. In addition, analysis of the T-cell receptor beta (TCRbeta) repertoire of Tag epitope-specific CD8(+) cells revealed that multiple TCRbeta variable regions were utilized for each epitope except Tag epitope II/III, which was limited to TCRbeta10 usage. These results indicate that the hierarchy of Tag epitope-specific CD8(+) T-cell responses is established in vivo.

Asunto(s)

Antígenos Transformadores de Poliomavirus/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Virus 40 de los Simios/inmunología , Animales , Anticuerpos/inmunología , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada/inmunología , Transformación Celular Viral , Epítopos de Linfocito T/genética , Femenino , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunización , Interferón gamma/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus Vaccinia/metabolismo

RESUMEN

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.

Asunto(s)

Antígenos Transformadores de Poliomavirus/inmunología , Neoplasias del Plexo Coroideo/inmunología , Epítopos de Linfocito T/inmunología , Epítopos Inmunodominantes/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada , Neoplasias del Plexo Coroideo/prevención & control , Progresión de la Enfermedad , Rayos gamma , Genes Virales , Antígenos H-2/biosíntesis , Antígenos H-2/inmunología , Antígeno de Histocompatibilidad H-2D , Tolerancia Inmunológica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos

RESUMEN

SV40 large tumor Ag (Tag) contains four H-2b-restricted (I, II/III, IV, and V) CTL epitopes. A hierarchy exists among these CTL epitopes. CTL directed against epitopes I, II/III, and IV are readily detected following immunization of H-2b mice with SV40, Tag-transformed syngeneic cells, or a vaccinia recombinant that expresses full-length Tag, while epitope V-specific CTL are not. The mechanisms that define this hierarchy remain unknown. Initial studies have shown that the locations of epitopes I and V within SV40 Tag do not determine the immunological potencies of these epitopes. Like the wild-type Tag, derivatives in which the locations of epitopes I and V were precisely reversed within Tag failed to induce epitope V-specific CTL, but did induce epitope I-specific CTL. The use of an S206G-substituted epitope I variant (GAINNYAQKL) revealed that the S206G variant sequence induced CTL when located within the native epitope I context, but failed to do so when located within the epitope V context of Tag. Mutagenesis of residues adjacent to the S206G-substituted epitope I variant revealed that the identity of the residue flanking the amino terminus of the S206G variant was critical when it resided within the epitope V location, but not within the epitope I location. These results demonstrate that effects imposed by both regional context and adjacent residues can modulate immunogenicity, but that the relative importance of such effects varies in an epitope-dependent manner.

Asunto(s)

Sustitución de Aminoácidos/genética , Antígenos Virales de Tumores/genética , Epítopos de Linfocito T/genética , Glicina/genética , Antígenos H-2/genética , Serina/genética , Virus 40 de los Simios/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/inmunología , Animales , Presentación de Antígeno/genética , Antígenos Virales de Tumores/inmunología , Antígenos Virales de Tumores/metabolismo , Células Clonales , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Variación Genética/inmunología , Glicina/inmunología , Antígenos H-2/inmunología , Antígeno de Histocompatibilidad H-2D , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Serina/inmunología , Virus 40 de los Simios/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo

RESUMEN

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.

Asunto(s)

Antígenos Virales , Herpesvirus Humano 2/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/virología , Epítopos/genética , Antígenos H-2 , Herpes Genital/inmunología , Herpes Genital/prevención & control , Herpes Genital/virología , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/aislamiento & purificación , Antígenos de Histocompatibilidad Clase I , Inmunidad Mucosa , Inmunización , Memoria Inmunológica , Masculino , Ratones , Ratones Endogámicos C57BL , Recombinación Genética , Virus Vaccinia/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología

RESUMEN

Simian virus 40 (SV40) has been shown to be associated with a number of human tumours. Two other human papova viruses, BKV and JCV, infect humans at a relatively high frequency and are activated upon immune suppression. The T antigens of both of these viruses share considerable homologies with the transforming protein T antigen of SV40. We have used SV40 T antigen specific cytotoxic T lymphocyte (CTL) clones to discriminate among the T antigens of SV40, BKV and JCV. These CTL clones directed to four distinct CTL epitopes serve as specific probes and can differentiate subtle alterations or deletions in the CTL epitopes relative to SV40 T antigen. Using this strategy, we have been able to authenticate three SV40 viruses isolated from humans as all four distinct CTL epitopes in the T antigens encoded by these three SV40 human isolates (SVCPC, SVMEN, and SVPML-1) were found to be identical to prototype SV40. We have further identified a 198 amino acid deletion T antigen variant of SVCPC. The finding of a deletion mutant in the SVCPC virus population suggests that the cellular immune response may play a role in the selection of antigenic loss variants.

Asunto(s)

Antígenos Transformadores de Poliomavirus/inmunología , Biomarcadores de Tumor/inmunología , Poliomavirus/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Virus BK/inmunología , Células Cultivadas , Células Clonales/inmunología , Mapeo Epitopo , Epítopos de Linfocito T/química , Antígenos H-2/química , Humanos , Virus JC/inmunología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Virus 40 de los Simios/inmunología

RESUMEN

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.

Asunto(s)

Antígenos/inmunología , Epítopos/inmunología , Antígenos H-2/inmunología , Modelos Inmunológicos , Fragmentos de Péptidos/inmunología , Conformación Proteica , Receptores de Antígenos de Linfocitos T/química , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Antígenos/química , Antígenos/metabolismo , Epítopos/química , Epítopos/metabolismo , Antígenos H-2/química , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Relación Estructura-Actividad

RESUMEN

SV40-transformed mKSA cells (H-2d) readily induce progressively growing tumors in adult syngeneic BALB/c mice while expressing the full complement of H-2d MHC class I antigens. BALB/c mice previously immunized with SV40, soluble SV40 T antigen, or irradiated SV40-transformed syngeneic, allogeneic, or xenogeneic cells reject an mKSA tumor challenge even though these mice have been considered low- or nonresponders to T antigen due to difficulty in demonstrating SV40 T antigen-specific CTL. We have investigated the role of H-2d-restricted CTL in the rejection of SV40 tumors in BALB/c mice. Immunization of BALB/c mice with SV40 induced T antigen-specific CTL which were largely. H-2Ld-restricted. However, following repeated in vitro restimulation with mKSA cells, CTL emerged which recognized a subdominant H-2Kd-restricted epitope corresponding to T antigen residues 499-507. Immunization of BALB/c mice with a recombinant vaccinia virus expressing the T499-507 epitope provided partial protection against a challenge of syngeneic mKSA tumor cells and induced the generation of T499-507-specific CTL. These results indicate that a subdominant H-2Kd-restricted CTL epitope can participate in the rejection of SV40 tumors in BALB/c mice.

Asunto(s)

Antígenos Transformadores de Poliomavirus/inmunología , Antígenos H-2 , Infecciones por Papillomavirus/inmunología , Virus 40 de los Simios/inmunología , Linfocitos T Citotóxicos/inmunología , Infecciones Tumorales por Virus/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Transformadores de Poliomavirus/genética , Línea Celular , Mapeo Epitopo , Epítopos/genética , Rechazo de Injerto/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Virus 40 de los Simios/genética , Virus Vaccinia/genética

RESUMEN

An immunological hierarchy among three H-2Db-restricted cytotoxic T lymphocyte (CTL) determinants in simian virus 40 (SV40) large T antigen (Tag) was described previously: determinants I and II/III are immunodominant, whereas determinant V is immunorecessive. To assess the immunogenicity of each determinant individually and define mechanisms that contribute to the immunorecessive nature of determinant V, we constructed a panel of recombinant vaccinia viruses (rVVs) expressing minigenes encoding these determinants in various polypeptide contexts. We found the following. (i) Immunization of mice with an rVV encoding full-length SV40 Tag resulted in priming for CTL responses to determinants I and II/III but not determinant V. (ii) rVVs encoding peptide I or II/III in the cytosol or targeted to the endoplasmic reticulum (ER) were highly antigenic and immunogenic. (iii) rVVs encoding peptide V minigenes were antigenic and immunogenic if the peptide was targeted to the ER, expressed in the cytosol with short flanking sequences, or expressed from within a self-protein, murine dihydrofolate reductase. (iv) Presentation of the nonflanked peptide V (preceded by a Met codon only) could be enhanced by using a potent inhibitor of the proteasome. (v) H-2Db-epitope V peptide complexes decayed more rapidly than complexes containing epitope I or II/III peptides. In brefeldin A blocking experiments, functional epitope V complexes were detected longer on targets expressing ER-targeted epitope V than on targets expressing forms of epitope V dependent on the transporter associated with antigen processing. Therefore, limited formation of relatively unstable cell surface H-2Db complexes most likely contributes to the immunorecessive nature of epitope V within SV40 Tag. Increasing the delivery of epitope V peptide to the major histocompatibility complex class I presentation pathway by ER targeting dramatically enhanced the immunogenicity of epitope V.

Asunto(s)

Antígenos Virales de Tumores/inmunología , Epítopos de Linfocito T/inmunología , Virus 40 de los Simios/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Presentación de Antígeno , Retículo Endoplásmico/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/inmunología

RESUMEN

The simian virus 40 large T antigen induces tumors in a wide variety of tissues in transgenic mice, the precise tissues depending on the tissue specificity of the upstream region controlling T-antigen expression. Expression of mutant T antigens that contain a subset of the protein's activities restricts the spectrum of tumors induced. Others showed previously that expression of a mutant large T antigen containing the N-terminal 121 amino acids (T1-121) under control of the lymphotropic papovavirus promoter resulted in slow-growing choroid plexus tumors, whereas full-length T antigen under the same promoter induced rapidly growing CPR tumors, T-cell lymphomas, and B-cell lymphomas. In those instances, the alteration in tumor induction or progression correlated with inability of the mutant large T antigen to bind the tumor suppressor p53. In the study reported here, we investigated the capacity of an N-terminal T antigen segment (T1-127) expressed in conjunction with small t antigen under control of the rat elastase-1 (E1) promoter to induce pancreatic tumors. The results show that pancreases of transgenic mice expressing T1-127 and small t antigen display acinar cell dysplasia at birth that progresses to neoplasia. The average age to death in these mice is within the range reported for transgenic mice expressing full-length T antigen under control of the E1 promoter. These results indicate that sequestering p53 by binding is not required for the development of rapidly growing acinar cell carcinomas. In addition, we provide evidence that small t antigen is unlikely to be required. Finally, we show that the p53 protein in acinar cell carcinomas is wild type in conformation.

Asunto(s)

Antígenos Transformadores de Poliomavirus/fisiología , Carcinoma de Células Acinares/virología , Neoplasias Pancreáticas/virología , Animales , Carcinoma de Células Acinares/patología , Genes p53 , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Elastasa Pancreática/genética , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas

Asunto(s)

Clonación Molecular/métodos , Regulación Fúngica de la Expresión Génica , Proteínas Recombinantes/biosíntesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Proteínas de Unión al ADN , Galactosa , Vectores Genéticos , Operón , Regiones Promotoras Genéticas/genética

RESUMEN

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.

Asunto(s)

Antígenos Transformadores de Poliomavirus/inmunología , Antígenos H-2/inmunología , Virus 40 de los Simios/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Transformadores de Poliomavirus/genética , Sitios de Unión , Línea Celular , Citotoxicidad Inmunológica , Epítopos/inmunología , Variación Genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/inmunología , Eliminación de Secuencia

RESUMEN

Simian virus 40 tumor (T) antigen contains three H-2Db-and one H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes (sites). Two of the H-2Db-restricted CTL epitopes, I and II/III, are separated by 7 amino acids in the amino-terminal one third of T antigen. In this study, we determine if the amino acids separating these two H-2Db-restricted CTL epitopes are dispensable for efficient processing and presentation. In addition, the importance of amino acid residues lying within and flanking the H-2Db-restricted epitopes I and II/III for efficient processing, presentation, and recognition by site-specific CTL clones was determined by using T-antigen mutants containing single-amino-acid substitutions between residues 200 and 239. Using synthetic peptides in CTL lysis and major histocompatibility complex class I stabilization assays, CTL recognition site I has been redefined to include residues 206 to 215. Substitutions in amino acids flanking either site I or site II/III did not affect recognition by any of the T-antigen-specific CTL clones. Additionally, the removal of the 7 residues separating site I and site II/III did not affect CTL recognition, thus demonstrating that these two epitopes when arranged in tandem in the native T antigen can be efficiently processed and presented to CTL clones. Differences in fine specificities of two CTL clones which recognize the same epitope (Y-1 and K-11 for site I and Y-2 and Y-3 for site II/III) have been used in conjunction with synthetic peptide variants to assign roles for residues within epitopes I and II/III with respect to TCR recognition and/or peptide-major histocompatibility complex association.

Asunto(s)

Antígenos Transformadores de Poliomavirus/genética , Antígenos H-2/genética , Virus 40 de los Simios/genética , Virus 40 de los Simios/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Antígenos Transformadores de Poliomavirus/metabolismo , Secuencia de Bases , Línea Celular , Epítopos/genética , Epítopos/metabolismo , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidad H-2D , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual

RESUMEN

Immunization of C57BL/6 mice with syngeneic cells transformed by simian virus 40 large T antigen (SV40 T ag) induces the generation of T antigen-specific cytotoxic T lymphocytes (CTL) which are restricted by the major histocompatibility class I antigens H-2Db and H-2Kb. Previous studies have shown that the H-2Db-restricted CTL response is directed to at least three distinct epitopes (I, II/III, and V) in the SV40 T antigen which have been precisely mapped using deletion mutagenesis and overlapping synthetic peptides. Although in vivo the CTL response to SV40 T antigen is dominated by the H-2Kb class I antigen, the precise location of the H-2Kb-restricted epitope(s) was not known, and whether there was multiplicity of H-2Kb-restricted epitopes remained unclear. In this study, we have defined the minimal recognition epitope for the SV40-specific H-2Kb-restricted CTL clone Y-4 as T antigen residues 404-411 by using T antigen deletion and point mutants and synthetic peptides. DNA sequence analysis of the region encoding residues 404-411 from the T antigens expressed in three independently isolated CTL clone Y-4 escape variants identified inactivating mutations capable of abrogating CTL recognition. Estimation of CTL precursor (CTLp) frequencies by limiting dilution analysis revealed that CTLp specific for epitope IV represent a large percentage of the total CTL response elicited by the intact T antigen in H-2b mice. Immunization of B6 mice with cells expressing a T antigen derivative deleted of residues 404-411 revealed that site IV represents the only immunodominant H-2Kb-restricted epitope within T antigen.

Asunto(s)

Antígenos Transformadores de Poliomavirus/genética , Antígenos H-2/genética , Infecciones por Polyomavirus/inmunología , Virus 40 de los Simios/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , Infecciones Tumorales por Virus/inmunología , Animales , Presentación de Antígeno/genética , Antígenos Transformadores de Poliomavirus/inmunología , Línea Celular , Citotoxicidad Inmunológica/genética , Antígenos H-2/inmunología , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Ratones , Ratones Endogámicos C57BL , Mutación , Infecciones por Polyomavirus/genética , Virus 40 de los Simios/genética , Infecciones Tumorales por Virus/genética

RESUMEN

The relationship between event-related potentials (ERPs) and cognitive functioning was studied in patients with Parkinson's Disease (PD) but without dementia. Auditory and visual stimuli were used; 30 subjects participated in the auditory study and 20 in the visual study. Patient groups did not differ with respect to gender, age, education, illness duration, and level of cognitive functioning. Visual stimuli were 2.3 cpd sinusoidal grating patterns randomly presented in an oddball paradigm (oblique vs. vertical spatial orientation). Auditory stimuli were tones presented at 70 dB SPL at a rate of 1.1/second, also using the oddball paradigm (1.5K vs. 1K tones). All patients were given neuropsychological tests to measure verbal fluency, memory, visual spatial perception, and abstract reasoning. P300 and N200 abnormalities correlated with a number of these measures, such that longer ERP latencies were related to lower scores on tests of cognitive functioning. Patterns of results suggest that auditory and visual ERPs correlate with different subsets of neuropsychological functions in nondemented PD patients and that N200 may provide a new metric for clinical use.

Asunto(s)

Cognición/fisiología , Potenciales Relacionados con Evento P300/fisiología , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/psicología , Trastornos del Conocimiento/fisiopatología , Trastornos del Conocimiento/psicología , Demencia/psicología , Potenciales Evocados Auditivos/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Estimulación Luminosa , Conducta Verbal/fisiología , Percepción Visual/fisiología , Escalas de Wechsler

RESUMEN

The yeast Snf1p kinase is required for normal expression of many genes involved in utilization of non-glucose carbon. Snf1p is known to associate with several proteins. One is Sip1p, a protein that becomes phosphorylated in the presence of Snf1p and thus is a candidate Snf1p kinase substrate. We have isolated the SIP1 gene as a multicopy suppressor of the gal83-associated defect in glucose repression of GAL gene expression. Multicopy SIP1 also suppressed the gal82-associated defect in glucose repression, suggesting that SIP1, GAL83 and GAL82 function interdependently. Multicopy SIP1 gene reduces GAL1, GAL2, GAL7 and GAL10 gene expression three- to fourfold in cells grown in the presence of glucose but has no effect in cells grown on nonrepressing carbon. Sip1-deletion cells exhibited a two- to threefold increase in GAL gene expression compared to wild-type cells when grown on glucose. These studies show that SIP1 is a catabolite repression-specific negative regulator of GAL gene expression. Northern analysis revealed two SIP1 transcripts whose relative abundance changed with carbon source. Western blots revealed that Sip1p abundance is not markedly affected by carbon source, suggesting that Sip1p may be regulated post-translationally.

Asunto(s)

Proteínas Portadoras , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Genes Supresores , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Proteínas Quinasas Activadas por AMP , Secuencia de Bases , Represión Enzimática/genética , Proteínas Fúngicas/metabolismo , Galactoquinasa/biosíntesis , Galactosa/metabolismo , Eliminación de Gen , Glucosa/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/genética , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Transducción de Señal/genética , Especificidad por Sustrato , Factores de Transcripción/metabolismo

RESUMEN

We studied acute and chronic effects of levo-acetyl-carnitine (LAC) on event-related potentials (ERPs) in 3 monkeys trained in a "go"/"no-go" visual "oddball" discrimination task. The stimuli were 2.5 cpd sinusoidal gratings differing in their respective orientation only (0 degrees or 45 degrees). Each monkey was trained to release a lever during a prespecified time window. Target stimulus presentation probabilities were between 0.25 and 0.5. ERPs had comparable mean latencies and amplitudes in all monkeys. Primary evoked potentials recorded to either the target or non-target stimulus did not change significantly as a result of LAC treatment. On the other hand, P300 latency decreased following LAC administration, with a maximum occurring in 15-20 min. The major effects of LAC were consistent within each animal and for all three of them.

Asunto(s)

Acetilcarnitina/farmacología , Encéfalo/fisiología , Cognición/efectos de los fármacos , Potenciales Evocados Visuales/efectos de los fármacos , Percepción Visual/efectos de los fármacos , Animales , Conducta Animal , Encéfalo/efectos de los fármacos , Aprendizaje Discriminativo/efectos de los fármacos , Electroencefalografía , Macaca fascicularis , Masculino , Desempeño Psicomotor/efectos de los fármacos , Percepción Visual/fisiología

RESUMEN

Transient evoked potentials were recorded simultaneously over 5 electrodes placed in a horizontal row across the occiput. A range of spatial frequencies were presented as either full-field or hemifield stimuli. Subjects were 11 normal observers and 5 patients with lesions causing a homonymous hemianopic field defect. The shortest latency peak response was at approximately 70 msec, a negative potential (N70). For all spatial frequencies, full-field stimuli evoked a lower amplitude N70 at the midline than the sum of N70 amplitudes to two hemifield stimuli, suggesting partial cancellation. The latency and amplitude of N70 increased as spatial frequency increased. N70 and P100 differed in respect to their response to spatial frequency and field size, further suggesting that they may not be subsets of a unitary response. For hemifield stimulation, N70 had an ipsilateral maximum and attenuated or completely reversed in polarity across the midline. Consistent with the data of normals using hemifield stimuli, in 5 patients a full-field stimulus elicited an N70 lateralized contralaterally to the homonymous hemianopia, i.e., the ipsilateral N70 was absent. The absolute amplitude difference between the left and right electrodes was significant for hemifield stimulation in normals and full-field stimulation in the patients, but not for full-field stimulation in normals. Our results imply that the evaluation of N70 hemispheric distribution is useful for the evaluation of paramacular visual field defects.

Asunto(s)

Encéfalo/fisiología , Potenciales Evocados Visuales/fisiología , Fóvea Central/fisiología , Adulto , Anciano , Análisis de Varianza , Electroencefalografía , Femenino , Hemianopsia/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Estimulación Luminosa , Tiempo de Reacción/fisiología

RESUMEN

Efficient transcription of many Saccharomyces cerevisiae genes requires the GAL11 Protein. GAL11 belongs to a class of transcription activator that lacks a DNA-binding domain. Such proteins are thought to activate specific genes by complexing with DNA-bound proteins. To begin to understand the domain structure-function relationships of GAL11 we cloned and sequenced a homologue from the yeast Kluyveromyces lactis, Kl-GAL11. The two predicted GAL11 proteins show high overall amino acid conservation and an unusual amino acid composition including 18% glutamine, 10% asparagine (S. cerevisiae) or 7% (K. lactis), and 8% proline (K. lactis) or 5% (S. cerevisiae) residues. Both proteins have runs of pure glutamines. Sc-GAL11 has glutamine-alanine runs but in Kl-GAL11 the alanines in such runs are replaced by proline and other residues. The primary sequence similarity is reflected in functional similarity since a gal11 mutation in K. lactis creates phenotypes similar to those seen previously in gal11-defective S. cerevisiae. In addition, Kl-GAL11 complements a gal11-defect in S. cerevisiae by partially restoring induction of GAL1 expression, growth on nonfermentable carbon sources, and phosphorylation of GAL4.

Asunto(s)

Proteínas Fúngicas/genética , Kluyveromyces/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido Nucleico , Transactivadores , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Galactosa/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Prueba de Complementación Genética , Lactosa/farmacología , Complejo Mediador , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Factores de Transcripción/química , Factores de Transcripción/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

The GAL4 protein of Saccharomyces cerevisiae is a DNA-binding transcriptional activator that is highly specific for the GAL genes. In vivo levels of GAL gene transcription are closely correlated with the phosphorylation state of GAL4. In vivo levels of GAL gene transcription are also affected by the activity of the GAL11 (SPT13) protein, a protein that has been implicated as a global auxiliary transcriptional factor. Here we examine the influence of GAL11 (SPT13) on the phosphorylation state of GAL4. Cells bearing a gal11 deletion mutation are defective in the production or maintenance of GAL4III, a phosphorylated form of GAL4 that is associated with higher levels of GAL gene transcription. In addition, the gal11 deletion cells are reduced in total GAL4 protein. However, the fivefold-reduced expression of the GAL1 gene observed in gal11 deletion cells cannot be due solely to reduced levels of total GAL4 protein, since gal11 deletion cells amplified for GAL4 production are still markedly reduced in GAL4 protein-dependent transcription. Thus, these data demonstrate that the GAL11 protein augments GAL4 protein-dependent transcription in a manner that is tightly coupled to the formation or maintenance of a phosphorylated form of GAL4.

Asunto(s)

Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transactivadores , Factores de Transcripción/metabolismo , Western Blotting , Proteínas de Unión al ADN , Genes Fúngicos , Complejo Mediador , Mutación , Fosforilación , Saccharomyces cerevisiae/metabolismo