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1.
J Exp Med ; 180(6): 2389-94, 1994 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-7964513

RESUMEN

Major histocompatibility complex (MHC) class I molecules bind peptides of 8-10 residues in the endoplasmic reticulum (ER) and convey them to the cell surface for inspection by CD8-expressing T cells (TCD8+). Antigenic peptides are predominantly derived from a cytosolic pool of polypeptides. The proteolytic generation of peptides from polypeptides clearly begins in the cytosol, but it is uncertain whether the final proteolytic steps occur before or after peptides are transported into the ER by the MHC-encoded peptide transporter (TAP). To study the trimming of antigenic peptides in the secretory pathway in the absence of cytosolic processing, we used an NH2-terminal signal sequence to target to the ER of TAP-deficient cells, "tandem" peptides consisting of two defined TCD8+ determinants arranged from head to tail. We find that in contrast to cytosolic proteases in TAP-expressing cells, which are able to liberate antigenic peptides from either end of a tandem peptide, proteases (probably aminopeptidases) present in an early secretory compartment preferentially liberate the COOH-terminal determinant. These findings demonstrate that proteolytic activities associated with antigen processing are not limited to the cytosol, but that they also exist in an early secretory compartment. Such secretory aminopeptidases may function to trim TAP-transported peptides to the optimal size for binding to class I molecules.


Asunto(s)
Antígenos Virales/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T Citotóxicos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Endopeptidasas/metabolismo , Retículo Endoplásmico/inmunología , Humanos , Virus de la Influenza A/inmunología , Células L , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Células Tumorales Cultivadas , Virus de la Estomatitis Vesicular Indiana/inmunología
2.
J Exp Med ; 186(7): 1087-98, 1997 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-9314557

RESUMEN

Jaw1 is an endoplasmic reticulum (ER) resident protein representative of a class of proteins post translationally inserted into membranes via a type II membrane anchor (cytosolic NH2 domain, lumenal COOH domain) in a translocon-independent manner. We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein. Peptide delivery mediated by Jaw1 to class I molecules was equal or better than that mediated by the adenovirus E3/19K glycoprotein signal sequence, and was sufficient to enable cytofluorographic detection of newly recruited thermostabile class I molecules at the surface of TAP-deficient cells. Deletion of the transmembrane region retargeted Jaw1 from the ER to the cytosol, and severely, although incompletely, abrogated its TAP-independent peptide carrier activity. Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1. These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.


Asunto(s)
Presentación de Antígeno/inmunología , Proteínas Portadoras/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas de la Membrana/metabolismo , Western Blotting , Linfocitos T CD8-positivos/inmunología , Línea Celular , Citosol/metabolismo , Endopeptidasas/metabolismo , Retículo Endoplásmico/enzimología , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Microscopía Inmunoelectrónica , Péptidos/metabolismo , Inhibidores de Proteasas/farmacología , Proteínas Recombinantes de Fusión , Transformación Genética , Virus Vaccinia/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo
3.
J Exp Med ; 186(4): 479-87, 1997 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-9254646

RESUMEN

We found that the presentation of a H-2Kd-restricted determinant from influenza virus nucleoprotein (NP) to T cells is strictly dependent on expression of the transporter associated with antigen presentation (TAP), regardless of whether NP is expressed as a cytosolic or secreted NP (SNP). Introducing an N-linked glycosylation site into the determinant selectively reduced presentation of SNP. This indicates that glycosylation does not interfere with TAP-transported peptides, and therefore that cytosolic peptides derived from SNP must have been exposed to the glycosylation machinery of the endoplasmic reticulum (ER) before their existence in the cytosol. Based on these findings, we propose that TAP-dependent processing of at least some ER-targeted proteins entails the reimportation of protein from the secretory pathway to the cytosol, where the protein is processed via the classical pathway.


Asunto(s)
Presentación de Antígeno , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase I/fisiología , Nucleoproteínas/metabolismo , Proteínas de Unión al ARN , Proteínas del Núcleo Viral/metabolismo , Animales , Transporte Biológico , Citosol/metabolismo , Glicosilación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Proteínas de la Nucleocápside , Fragmentos de Péptidos/metabolismo
4.
Aviat Space Environ Med ; 64(8): 717-25, 1993 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8368985

RESUMEN

The majority of antihistamines have sedative effects; however, it is claimed that Hismanal (astemizole) does not possess central nervous system side effects. A three-factor, repeated measures, double-blind design was used to compare the effects of singl oral doses of 1) Benadryl (diphenhydramine), 50 mg; 2) Hismanal, 10 mg; and 3) placebo, on two mood scales, physiological measures, sleepiness, the ability to detect ingestion of an antihistamine versus placebo, and symptoms in 28 healthy men. Higher tension, greater fatigue, and lower activity levels were reported post-Benadryl (p < 0.05). Lower vigor-activity and higher confusion-bewilderment post-Hismanal and Benadryl were noted 1 h postingestion (p < 0.05), with confusion being lower and activity higher for Hismanal than for Benadryl (p < 0.05). Low vigor-activity, high confusion, and increased sleepiness post-Benadryl persisted for 3 h, while fatigue-inertia persisted for 7 h (p < 0.05). Results suggest that Hismanal is superior to Benadryl for avoidance of subjective effects; however, neither antihistamine was entirely devoid of subjective effects.


Asunto(s)
Astemizol/efectos adversos , Difenhidramina/efectos adversos , Adulto , Afecto/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Método Doble Ciego , Humanos , Masculino , Percepción , Pulso Arterial/efectos de los fármacos , Fases del Sueño/efectos de los fármacos
5.
Aviat Space Environ Med ; 64(8): 726-34, 1993 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8368986

RESUMEN

Classic antihistamines (H1 antagonists) have sedative effects and can impair psychomotor performance. It is claimed that Hismanal (astemizole) does not possess central nervous system side effects. A three-factor, repeated measures, double-blind design was used to compare the effects of single oral doses of (1) Benadryl (diphenhydramine)-50 mg, (2) Hismanal-10 mg, and (3) placebo on a battery of 11 cognitive information-processing tasks and performance ratings in 28 healthy men. Performance decrements were seen at 1 h post-Benadryl ingestion on Following Directions (p < 0.05), at 1.5 h on Unstable Tracking (p < 0.05), and at 3 h on serial addition/subtraction (p < 0.05). No decrements in performance were found post-Hismanal. Subjects perceived their performance as poorer following Benadryl ingestion versus placebo and Hismanal for 3 h postingestion (p < 0.05). Results demonstrate performance effects post-Benadryl ingestion for 1 h longer than previously reported. Tasks which demonstrated sensitivity to antihistamines were those which required an element of sustained attention and those which required a visual-motor response.


Asunto(s)
Astemizol/farmacología , Difenhidramina/farmacología , Percepción/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Adulto , Cognición/efectos de los fármacos , Humanos , Masculino , Pruebas Psicológicas , Factores de Tiempo
6.
Appl Ergon ; 20(1): 26-32, 1989 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15676712

RESUMEN

This study determined the perceived safety and comfort of an alternating tread stair and a conventional ship's ladder. The alternating tread stair and the conventional ship's ladder were also compared with respect to travel time and missteps. Subjects in military uniform ascended and descended both devices under load and no-load conditions. In the load condition, subjects performed trials while carrying a 9-kg tool-box; in the no-load condition, trials were performed without the tool-box. Results indicate that the alternating tread stair is perceived to be safer and more comfortable to use. Moreover, the alternating tread stair had significantly fewer missteps.

9.
Hum Factors ; 33(4): 459-69, 1991 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-1955204

RESUMEN

The effects of physical image quality on reading and on perceived image quality from CRT and hard copy displays (photographs) were studied. The results showed that as the image quality of a display increased, indicated by an increase in the value of the modulation transfer function area (MTFA), the reading speed and subjective image quality ratings increased. This change in reading speed and perceived image quality occurred similarly for both hard-copy and soft-copy conditions. If the image qualities of the displayed text are similar, hard-copy and soft-copy displays will yield equivalent reading speeds.


Asunto(s)
Presentación de Datos , Lectura , Percepción Visual , Adulto , Femenino , Humanos , Masculino , Tiempo de Reacción
10.
J Immunol ; 156(9): 3374-81, 1996 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-8617963

RESUMEN

T lymphocytes are believed to play a major role in protection against malaria. Previous experiments using in vivo depletion of CD8+ T cells, reconstitution with CD8+ T splenic cells, and adoptive transfer of CD8+ CTL clones demonstrated that protection against the exoerythrocytic stage of the murine strain, Plasmodium berghei malaria, was CD8+ T cell-dependent. Despite evidence for the critical role of CD8+ CTL, neither the cellular nor the molecular requirements for CD8+ T cell induction or for recognition of malaria Ags are known. In this study, we wished to define the role of CD8+ T cells and MHC class I molecules by using the P. berghei malaria attenuated sporozoites (SPZ) protection model in beta 2-microglobulin (beta 2m) knockout (-/-) mice. In contrast to observations that beta 2m-/- mice are resistant to many infectious diseases by compensatory mechanisms involving non-class I-restricted T cells, we found that beta 2m-/- mice failed to be protected against P. berghei SPZ, although immunization with attenuated SPZ induced production of IL-2, INF-gamma, anti-circumsporozoite protein IgG, and proliferative T cells. The lack of compensatory mechanisms involving non-CD8+ T cells was particularly evident in the failure to adoptively transfer protective immunity with wild-type SPZ-immune splenic T cells. From our data it can be concluded that CD8+ T cells induced during immunization with attenuated SPZ must recognize liver-expressed Ags presented by class I molecules to engage effectively in a response leading to destruction of the malaria parasites.


Asunto(s)
Presentación de Antígeno , Antígenos de Protozoos/inmunología , Linfocitos T CD8-positivos/inmunología , Eritrocitos/parasitología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Malaria/inmunología , Malaria/prevención & control , Plasmodium berghei/inmunología , Animales , Antígenos de Protozoos/sangre , Femenino , Inmunoterapia Adoptiva , Malaria/sangre , Vacunas contra la Malaria/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium berghei/crecimiento & desarrollo , Bazo/parasitología , Bazo/trasplante , Vacunas Atenuadas/inmunología , Microglobulina beta-2/genética
11.
Eur J Immunol ; 28(4): 1339-46, 1998 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9565374

RESUMEN

TAP can efficiently transport peptides up to twice as long as those bound to MHC class I molecules, suggesting a role for endoplasmic reticulum (ER) proteases in the trimming of TAP-transported peptides. To better define ER processing of antigenic peptides, we examined the capacity of TAP-deficient cells to present determinants derived from ER-targeted proteins encoded by recombinant vaccinia viruses. TAP-deficient cells failed to present antigenic peptides from internal locations in secreted proteins to MHC class I-restricted T lymphocytes. The same peptides were liberated from the C termini of a secreted protein and the lumenal domains of two membrane proteins delivered to the ER via different routes. These findings suggest that proteases in the secretory compartment can liberate C-terminal antigenic peptides from virtually any context. We propose that this activity often participates in the removal of N-terminal extensions from TAP-transported peptides, thereby creating optimally sized products for MHC class I binding. We further demonstrate that ER trimming of C termini can occur if we express an appropriate carboxypeptidase in the secretory pathway. The absence of such trimming under normal circumstances suggests that carboxypeptidase activity is generally deficient in the ER, consistent with the concordance between the specificity of TAP and MHC class I molecules for the same types of C-terminal residues.


Asunto(s)
Presentación de Antígeno , Proteínas Portadoras/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Transporte Biológico/inmunología , Endopeptidasas/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/inmunología
12.
J Immunol ; 159(2): 554-64, 1997 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-9218569

RESUMEN

The proteasome is believed to participate in the generation of a large percentage of peptide ligands for MHC class I molecules. This conclusion is based largely on the activities of peptidyl aldehydes that block proteasome activity. We tested the ability of a panel of proteasome inhibitors to affect the generation of MHC class I binding peptides in mouse L929 cells. Included in the panel are peptidyl aldehydes and a microbial product, lactacystin, that blocks proteasome activity in a distinct and more specific manner. Contrary to expectations, proteasome inhibitors failed to block the generation of a large portion of high affinity peptides as inferred by measuring cell surface expression of newly synthesized MHC class I molecules. These findings were confirmed by examining the effects of the inhibitors on the presentation of individual antigenic determinants from endogenously synthesized or exogenously delivered influenza virus proteins. Presentation of peptides derived from exogenous basic polymerase 1, endogenous basic polymerase 1, and nonstructural-1 proteins was decreased by inhibitors in a manner consistent with proteasomal involvement. Presentation of peptides derived from endogenous nucleoprotein was not significantly affected by the proteasome inhibitors, while presentation of exogenous hemagglutinin and nucleoprotein was enhanced by the proteasome inhibitors. These data are consistent with the involvement of both proteasomes and nonproteasomal cytosolic proteases in the generation of a significant portion of MHC class I binding peptides.


Asunto(s)
Presentación de Antígeno , Cisteína Endopeptidasas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Complejos Multienzimáticos/inmunología , Péptidos/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno/efectos de los fármacos , Antígenos Virales/inmunología , Línea Celular , Inhibidores Enzimáticos/farmacología , Ligandos , Ratones , Orthomyxoviridae/inmunología , Complejo de la Endopetidasa Proteasomal
13.
J Immunol ; 160(10): 4859-68, 1998 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-9590233

RESUMEN

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.


Asunto(s)
Presentación de Antígeno , Cisteína Endopeptidasas/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Complejos Multienzimáticos/fisiología , Fragmentos de Péptidos/metabolismo , Proteínas Virales/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Complejo de la Endopetidasa Proteasomal , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas , Proteínas Virales/inmunología
14.
Immunol Rev ; 172: 97-108, 1999 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-10631940

RESUMEN

MHC class I molecules function to present peptides comprised of eight to 11 residues to CD8+ T lymphocytes. Here we review the efforts of our laboratory to understand how cells generate such peptides from viral gene products. We particularly focus on the nature of substrates acted on by cytosolic proteases, the contribution of proteasomes and non-proteasomal proteases to peptide generation, the involvement of ubiquitination in peptide generation, the intracellular localization of proteasome generation of antigenic peptides, and the trimming of peptides in the endoplasmic reticulum.


Asunto(s)
Antígenos Virales/genética , Antígenos Virales/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Animales , Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Cisteína Endopeptidasas/metabolismo , Citosol/inmunología , Citosol/metabolismo , Endopeptidasas/metabolismo , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Humanos , Ligandos , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Procesamiento Proteico-Postraduccional , Ubiquitinas/metabolismo
15.
J Immunother ; 21(2): 127-31, 1998 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9551364

RESUMEN

We have taken several approaches to investigate the capacity of the secretory pathway to liberate major histocompatibility complex (MHC) class I-restricted antigenic peptides from precursor polypeptides. Cells lacking the peptide transporter (TAP) are unable to deliver peptides from cytosolic antigens to class I molecules. TAP can be bypassed by targeting peptides directly to the endoplasmic reticulum (ER) using NH2-terminal signal sequences. This results in the generation of enormous numbers of MHC class I complexes (50,000 peptides/cell), and recombinant vaccinia viruses expressing such peptides are highly immunogenic. In contrast to signal sequence-targeted peptides, peptides are liberated very inefficiently from internal locations in ER-targeted full-length proteins, indicating that the secretory pathway has a limited capacity for generating antigenic peptides from most polypeptide contexts. We have, however, identified a location in proteins from which peptides can be liberated in numerous contexts in the secretory pathway. Placing a number of different peptides at the COOH termini of a secreted protein and two proteins with type II membrane anchors resulted in their TAP-independent presentation. These findings demonstrate that the secretory compartment possesses proteases able to liberate COOH-terminal antigenic peptides from virtually any context, entirely consistent with a role for these proteases in the processing of TAP-transported antigenic peptide precursors.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Presentación de Antígeno/inmunología , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Animales , Humanos
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