RESUMO
BACKGROUND: Psychological safety-speaking up about ideas and concerns, free from interpersonal risk-are essential to the high-risk environment, such as healthcare settings. Psychologically safe working is particularly important in mental health where recovery-oriented approaches rely on collaborative efforts of interprofessional teams to make complex decisions. Much research focuses on antecedents and outcomes associated with psychological safety, but little focus on the practical steps for how to increase psychological safety across and at different levels of a healthcare organisation. AIMS: We explore how a mental health organisation creates an organisation-wide plan for building the foundations of mental health and how to enhance psychological safety. METHODS: This review encompasses strategies across psychological safety and organisational culture change to increase psychological safety at an individual, team and organisational level. We set out a comprehensive overview of the types of strategies and interventions for increasing the ethos of psychological safety and setting the foundations for delivering an organisation-wide programme on this topic. We also provide a list of key targeted areas in mental health that would maximally benefit from increasing psychological safety-both in clinical and non-clinical settings. CONCLUSIONS: Psychological safety is a crucial determinant of safe and effective patient care in mental health services. This paper provides the key steps and considerations, creating a large-scale programme in psychological safety with a focus on mental health and drawing from the current literature, providing concrete steps for how our current understanding of psychological safety into practice.
RESUMO
Posttranslational modification of peptide antigens has been shown to alter the ability of T cells to recognize major histocompatibility complex (MHC) class I-restricted peptides. However, the existence and origin of naturally processed phosphorylated peptides presented by MHC class I molecules have not been explored. By using mass spectrometry, significant numbers of naturally processed phosphorylated peptides were detected in association with several human MHC class I molecules. In addition, CD8(+) T cells could be generated that specifically recognized a phosphorylated epitope. Thus, phosphorylated peptides are part of the repertoire of antigens available for recognition by T cells in vivo.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/imunologia , Fosfopeptídeos/imunologia , Fosfopeptídeos/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Antígenos/química , Antígenos/imunologia , Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Citocinas/metabolismo , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Antígenos HLA/imunologia , Humanos , Interferon gama/biossíntese , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Fosfopeptídeos/químicaRESUMO
Minor histocompatibility antigens (mHAgs) present a significant impediment to organ and bone marrow transplantation between HLA-identical donor and recipient pairs. Here we report the identification of a new HLA-A*0201-restricted mHAg, HA-8. Designation of this mHAg as HA-8 is based on the nomenclature of Goulmy (Goulmy, E. 1996. Curr. Opin. Immunol. 8:75-81). This peptide, RTLDKVLEV, is derived from KIAA0020, a gene of unknown function located on chromosome 9. Polymorphic alleles of KIAA0020 encode the alternative sequences PTLDKVLEV and PTLDKVLEL. Genotypic analysis demonstrated that the HA-8-specific cytotoxic T lymphocyte (CTL) clone SKH-13 recognized only cells that expressed the allele encoding R at P1. However, when PTLDKVLEV was pulsed onto cells, or when a minigene encoding this sequence was used to artificially translocate this peptide into the endoplasmic reticulum, it was recognized by CTLs nearly as well as RTLDKVLEV. This indicates that the failure of CTLs to recognize cells expressing the PTLDKVLEV-encoding allele of KIAA0020 is due to a failure of this peptide to be appropriately proteolyzed or transported. Consistent with the latter possibility, PTLDKVLEV and its longer precursors were transported poorly compared with RTLDKVLEV by transporter associated with antigen processing (TAP). These studies identify a new human mHAg and provide the first evidence that minor histocompatibility differences can result from the altered processing of potential antigens rather than differences in interaction with the relevant major histocompatibility complex molecule or T cell receptor.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Menor/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Células Clonais , Primers do DNA/genética , Epitopos/química , Epitopos/genética , Feminino , Humanos , Masculino , Espectrometria de Massas , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/genética , Dados de Sequência Molecular , Linhagem , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.
Assuntos
Antígenos/imunologia , Epitopos Imunodominantes/imunologia , Mutação Puntual , Proteínas Quinases , RNA Helicases , RNA Nucleotidiltransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linfócitos T CD8-Positivos/imunologia , RNA Helicases DEAD-box , DNA Complementar , Feminino , Antígenos H-2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neoplasias Experimentais/imunologiaRESUMO
Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant beta2-microglobulin (beta2m) and a pool of randomly synthesised nonamer peptides. This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography. The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool. Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells. This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus. Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies. We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.
Assuntos
Antígenos HLA/metabolismo , Biblioteca de Peptídeos , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia em Gel , Clonagem Molecular , Escherichia coli/genética , Antígenos HLA/química , Antígenos HLA/genética , Humanos , Dados de Sequência MolecularRESUMO
We eluted peptides from class I molecules of HLA-A2.1(+) breast adenocarcinoma and loaded reverse phase high-performance liquid chromatography (HPLC) fractions onto dendritic cells to prime naive CD8(+) T cells. Fractions that supported growth of tumor-specific cytotoxic T lymphocytes were analyzed by nano-HPLC micro-ESI tandem mass spectrometry. Six HLA-A2.1-binding peptides, four 9-mers (P1-P4) differing in the COOH-terminal residue, and two 10-mers (P5 and P6) with an additional COOH-terminal alanine, were identified in one fraction. Peptide sequences were homologous to cyclin B1. We primed CD8(+) T cells from another HLA-A2.1(+) healthy donor with synthetic peptides and generated P4-specific responses. We also detected memory T cells specific for one or more of these peptides in patients with breast cancer and squamous cell carcinomas of the head and neck (SCCHN). T cells from one patient, restimulated once in vitro, could kill the tumor cell line from which the peptides were derived. Immunohistochemical analysis of tumor lines and tissue sections showed cyclin B1 overexpression and aberrant localization in the cytoplasm instead of the nucleus. Sequencing genomic DNA and cDNA corresponding to P1-P6 region showed that differences in COOH-terminal residues were not due to either DNA mutations or errors in transcription, suggesting a high error rate in translation of cyclin B1 protein in tumors.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/imunologia , Ciclina B/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Antígenos de Neoplasias/genética , Sequência de Bases , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/citologia , Carcinoma de Células Escamosas/patologia , Células Cultivadas , Ciclina B/biossíntese , Ciclina B/genética , Ciclina B1 , DNA , DNA Complementar , Feminino , Expressão Gênica , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Neoplasias de Cabeça e Pescoço/patologia , Nível de Saúde , Humanos , Memória Imunológica , Dados de Sequência Molecular , Mutagênese , Peptídeos/genética , Peptídeos/imunologia , RNA , Doadores de Tecidos , Células Tumorais CultivadasRESUMO
T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.
Assuntos
Antígeno HLA-A2 , Melanoma/imunologia , Proteínas de Membrana/metabolismo , Monofenol Mono-Oxigenase/imunologia , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Antígenos de Neoplasias/imunologia , Asparagina/metabolismo , Ácido Aspártico/biossíntese , Células Clonais , Epitopos , Humanos , Melanoma/enzimologia , Modelos Biológicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
A subtractive analysis of peptides eluted from major histocompatibility complex (MHC) class I human histocompatibility leukocyte antigen (HLA)-A2.1 molecules purified from either human immunodeficiency virus type-1 (HIV-1)-infected or uninfected cells was performed using micro high-performance liquid chromatography and mass spectrometry. Three peptides unique to infected cells were identified and found to derive from a single protein, human vinculin, a structural protein not known to be involved in viral pathogenesis. Molecular and cytofluorometric analyses revealed vinculin mRNA and vinculin protein overexpression in B and T lymphocytes from HIV-1-infected individuals. Vinculin peptide-specific CTL activity was readily elicited from peripheral blood lymphocytes of the majority of HLA-A2.1+, HIV+ patients tested. Our observations suggest that atypical vinculin expression and MHC class I-mediated presentation of vinculin-derived peptides accompany HIV infection of lymphoid cells in vivo, with a resultant induction of antivinculin CTL in a significant portion of HIV+ (HLA-A2.1+) individuals.
Assuntos
Linfócitos B/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Antígeno HLA-A2/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T/imunologia , Vinculina/biossíntese , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Citotoxicidade Imunológica , Citometria de Fluxo , Antígeno HLA-A2/isolamento & purificação , Humanos , Complexo Principal de Histocompatibilidade , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Valores de Referência , Vinculina/imunologia , Vinculina/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/imunologiaRESUMO
CD4(+) T cells play a critical role in generating and maintaining immune responses against pathogens and alloantigens, and evidence suggests an important role for them in antitumor immunity as well. Although major histocompatibility complex class II-restricted human CD4(+) T cells with specific antitumor reactivities have been described, no standard method exists for cloning the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spectrometry sequencing techniques and molecular cloning to isolate a unique melanoma Ag recognized by a CD4(+) tumor-infiltrating lymphocyte (TIL) line. The HLA-DRbeta1*0101-restricted Ag was determined to be a mutated glycolytic enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detected in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity was enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutated amino acid residue was a T cell receptor contact. Defining human tumor Ag recognized by T helper cells may provide important clues to designing more effective immunotherapies for cancer.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígeno HLA-DR1/imunologia , Melanoma/imunologia , Triose-Fosfato Isomerase/imunologia , Sequência de Aminoácidos , Antígenos de Neoplasias/genética , Linhagem Celular , Epitopos/genética , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma/enzimologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Triose-Fosfato Isomerase/genéticaRESUMO
Formation of major histocompatibility complex class I-associated peptides from membrane proteins has not been thoroughly investigated. We examined the processing of an HLA-A*0201-associated epitope, YMDGTMSQV, that is derived from the membrane protein tyrosinase by posttranslational conversion of the sequence YMNGTMSQV. Only YMDGTMSQV and not YMNGTMSQV was presented by HLA-A*0201 on cells expressing full-length tyrosinase, although both peptides have similar affinities for HLA-A*0201 and are transported by TAP. In contrast, translation of YMNGTMSQV in the cytosol, as a minigene or a larger fragment of tyrosinase, led to the presentation of the unconverted YMNGTMSQV. This was not due to overexpression leading to saturation of the processing/conversion machinery, since presentation of the converted peptide, YMDGTMSQV, was low or undetectable. Thus, presentation of unconverted peptide was associated with translation in the cytosol, suggesting that processing of the full-length tyrosinase occurs after translation in the endoplasmic reticulum. Nevertheless, presentation of YMDGTMSQV in cells expressing full-length tyrosinase was TAP (transporter associated with antigen processing) and proteasome dependent. After inhibition of proteasome activity, tyrosinase species could be detected in the cytosol. We propose that processing of tyrosinase involves translation in the endoplasmic reticulum, export of full-length tyrosinase to the cytosol, and retransport of converted peptides by TAP for association with HLA-A*0201.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Sequência de Aminoácidos , Apresentação de Antígeno/genética , Apresentação de Antígeno/fisiologia , Sequência de Bases , Transporte Biológico Ativo , Linhagem Celular , Citosol/imunologia , Citosol/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Epitopos/genética , Expressão Gênica , Genes MHC Classe I , Antígenos HLA-A/química , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/imunologia , Oligodesoxirribonucleotídeos/genética , Biossíntese de Proteínas , Linfócitos T Citotóxicos/imunologiaRESUMO
The first step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular , Genes Reporter , Histonas/química , Humanos , Immunoblotting , Substâncias Macromoleculares , Microscopia de Fluorescência , Sinais de Localização Nuclear , Proteínas Nucleares/genética , Proteína 1 de Modelagem do Nucleossomo , Ligação Proteica , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Leveduras/genética , Leveduras/metabolismo , beta CarioferinasRESUMO
The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.
Assuntos
Actinina/metabolismo , Biomarcadores , Contração Muscular , Proteínas Musculares/metabolismo , Músculo Liso Vascular/citologia , Citoesqueleto de Actina/ultraestrutura , Actinina/química , Actinina/imunologia , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Diferenciação Celular , Células Cultivadas , Galinhas , Coturnix , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Peso Molecular , Proteínas Musculares/química , Proteínas Musculares/imunologia , Músculo Liso Vascular/química , Peptídeos/químicaRESUMO
A number of bacterial protein toxins, including adenylate cyclase (AC) toxin from Bordetella pertussis, require the product of an accessory gene in order to express their biological activities. In this study, mass spectrometry was used to demonstrate that activated, wild-type AC toxin was modified by amide-linked palmitoylation on the epsilon-amino group of lysine 983. This modification was absent from a mutant in which the accessory gene had been disrupted. A synthetic palmitoylated peptide corresponding to the tryptic fragment (glutamine 972 to arginine 984) that contained the acylation blocked AC toxin-induced accumulation of adenosine 3',5'-monophosphate, whereas the non-acylated peptide had no effect.
Assuntos
Toxina Adenilato Ciclase , Lisina/metabolismo , Palmitatos/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Hemólise , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Ovinos , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/química , Fatores de Virulência de Bordetella/toxicidadeRESUMO
Two autoantibiotics produced by the fungus Candida albicans have been identified, with the aid of mass spectrometry, as 2-phenylethanol and 3-beta-hydroxyethylindole. The cyclodipeptides, cyclo(proline-leucine) and cyclo(proline-phenylalanine) were also isolated from cultures of Candida albicans, but did not inhibit the growth of the fungus.
Assuntos
Antibacterianos/biossíntese , Candida/metabolismo , Etanol , Indóis/biossíntese , Cromatografia , Dipeptídeos/isolamento & purificação , Raios Infravermelhos , Análise EspectralRESUMO
Peptides extracted from HLA-A2.1 class I major histocompatibility complex (MHC) molecules expressed on the antigen processing mutant CEMx721.174.T2 were characterized by electrospray ionization-tandem mass spectrometry. Only seven dominant peptides were found, in contrast to over 200 associated with HLA-A2.1 on normal cells. These peptides were derived from the signal peptide domains of normal cellular proteins, were usually larger than nine residues, and were also associated with HLA-A2.1 in normal cells. These results suggest that proteolysis of signal peptide domains in the endoplasmic reticulum is a second mechanism for processing and presentation of peptides for association with class I molecules.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos/metabolismo , Antígeno HLA-A2/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Antígenos/química , Antígenos/imunologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Antígeno HLA-A2/química , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Peptídeos/química , Peptídeos/imunologia , Sinais Direcionadores de Proteínas/química , Linfócitos T/imunologiaRESUMO
Class II major histocompatibility complexes bind peptides in an endosome-like compartment. When the class II null cell line 721.174 was transfected with class II DR3 genes, DR molecules were produced in normal amounts. However, the DR molecules were abnormally conformed and unstable because deletion of an antigen-processing gene had impaired intracellular formation of most class II-peptide complexes. Yet, 70 percent of the DR molecules still bore peptides, 80 percent of which were 21- to 24-amino acid fragments of the class II-associated invariant chain. These peptides were rare on DR3 from control cells. Thus, a defect in the main antigen-processing pathway revealed a process in which DR molecules bind long peptides derived from proteins present in the same compartment.
Assuntos
Genes MHC da Classe II , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Antígeno HLA-DR3/genética , Peptídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Deleção de Genes , Antígeno HLA-DR3/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , TransfecçãoRESUMO
Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.
Assuntos
Antígenos/metabolismo , Antígeno HLA-A2/metabolismo , Espectrometria de Massas , Peptídeos/metabolismo , Sequência de Aminoácidos , Antígenos/química , Antígenos/imunologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Técnicas de Imunoadsorção , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Linfócitos T/imunologiaRESUMO
A nuclear isoform of myosin I beta that contains a unique 16-amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.
Assuntos
Núcleo Celular/metabolismo , Proteínas Motores Moleculares , Miosinas/metabolismo , RNA Polimerase II/metabolismo , RNA/biossíntese , Transcrição Gênica , Células 3T3 , Actinas/metabolismo , Amanitinas/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Dactinomicina/farmacologia , Éxons , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Dados de Sequência Molecular , Miosinas/química , Miosinas/genética , Miosinas/imunologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Testes de Precipitina , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismoRESUMO
Between 650 and 2000 different peptides are associated with the major histocompatibility complex class II molecule I-Ad. Sequences for nine of these were obtained by a combination of automated Edman degradation and tandem mass spectrometry. All of the peptides are derived from secretory or integral membrane proteins that are synthesized by the antigen-presenting cell itself. Peptides were 16 to 18 residues long, had ragged NH2-and COOH-termini, and contained a six-residue binding motif that was variably placed within the peptide chain. Binding data on truncated peptides suggest that the peptide binding groove on class II molecules can be open at both ends.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
The minor histocompatibility antigen (mHag) HA-1 is the only known mHag for which mismatching is correlated with the development of severe graft versus host disease (GvHD) after human leukocyte antigen-identical bone marrow transplantation. HA-1 was found to be a nonapeptide derived from an allele of the KIAA0223 gene. The HA-1-negative allelic counterpart encoded by KIAA0223 had one amino acid difference from HA-1. Family analysis with HA-1 allele-specific polymerase chain reaction showed an exact correlation between this allelic polymorphism and the HA-1 phenotype. HA-1 allele typing of donor and recipient should improve donor selection and allow the determination of bone marrow transplantation recipients with high risk for HA-1-induced GvHD development.