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K Sakaguchi

Publications and source records attributed to K Sakaguchi.

At least 19 recordsLinked to original sources
PubMed1996

Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.

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.

Amino Acid Sequence↗
PubMed1996

Characterization of DNA ligase from the fungus Coprinus cinereus.

DNA ligase was highly purified from the fungus Coprinus cinereus at the miotic recombination stage, pachytene. The pachytene DNA ligase showed three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimated by the [32P]AMP-labeling assay. These three polypeptides were susceptible to reaction with an mAb against a 16-amino-acid sequence in human DNA ligase I, which is conserved in C-terminal regions of mammalian, vaccinia virus and yeast DNA ligases. Since rapidly purified preparations from fresh pachytene cells exhibited a single polypeptide of DNA ligase with a molecular mass of 88 kDa, the smaller polypeptides seemed to be limited-degradation products of the 88-kDa polypeptide during the isolation and purification procedures. K(m) values for ATP and (dT)20 hybridized with (dA)n were 1.5 microM and 90 nM, respectively. This enzyme was capable of joining (dT)20.(rA)n and (rA)12-18 (dT)n as well as (dT)20.(dA)n and able to ligate blunt-ended DNA in the presence of poly(ethylene glycol) 6000. DNA ligases were also partially purified from zygotene cells at the meiotic pairing stage and mitotic mycelium cells. In their molecular mass, immuno-reactivity, K(m) value and substrate specificity, they were indistinguishable from pachytene DNA ligase. These results suggest that the fungus C. cinereus at the pachytene stage contains DNA ligase with a molecular mass of 88 kDa as a main or a single species, which is quite similar to DNA ligases from the zygotene and mycelium cells in molecular and catalytic properties.

Amino Acid Sequence↗
PubMed1996

Interaction of Shc with adaptor protein adaptins.

The role of Shc as a substrate of receptors for growth factors and cytokines is well established. To gain further insight into the function of Shc in signal transduction, we used an affinity method to identify potential Shc-binding proteins. Incubation of bovine brain lysates with a glutathione S-transferase (GST)-Shc fusion protein immobilized on glutathione-Sepharose beads resulted in the binding of cellular proteins of approximately 115, 110, and 100 kDa as well as those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides revealed that the 100-kDa protein was almost identical to beta-adaptin and that the 110- and 115-kDa proteins were almost identical to alphaA-adaptin. Using immunoblot analysis, anti-alpha-adaptin antibody recognized several proteins of 100 approximately 115 kDa, and anti-beta-adaptin antibody recognized a 100-kDa protein, suggesting that alphaA-, alphaC-, and beta-adaptins are bound to the GST-Shc fusion protein. Immunoblot analysis with anti-alpha-adaptin antibody revealed that alpha-adaptin was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates, suggesting a specific interaction of Shc and adaptins in intact cells. A binding study using mutant GST-Shc fusion proteins revealed that the collagen homologous region (amino acids 233-377) of Shc was required for adaptin binding. Conversely, the collagen homologous region of Shc inhibited the binding of adaptins to GST-Shc. In addition, adaptin was able to bind mutant fusion proteins containing amino acids 233-369, 233-355, 346-369, and 346-355 of Shc, but failed to bind a mutant containing amino acids 233-345, suggesting that amino acids 346-355 (RDLFDMKPFE) in the collagen homologous region of Shc are required for adaptin binding. Thus, this study indicates the specific interaction of Shc with alpha- and beta-adaptin components of plasma membrane adaptor proteins that are thought to be involved in receptor endocytosis.

Adaptor Protein Complex 2↗
PubMed1996

Growth hormone-independent expression of insulin-like growth factor I messenger ribonucleic acid in extrahepatic tissues of the chicken.

The sex-linked dwarf (SLD) chicken, which lacks GH receptor (GHR), and its normal littermates provide a useful experimental system to investigate GH-dependent cellular responses. The GH dependence of insulin-like growth factor I (IGF-I) expression in tissues was examined in SLD and normal chickens of the Gifu 20 strain. Four weeks after hatching, the most abundant expression of IGF-I messenger RNA (mRNA) was observed in liver of normal chickens, whereas no IGF-mRNA expression was detected in that organ of dwarf chickens. On the contrary, in extrahepatic tissues such as spleen, lung, brain, kidney, heart, intestine, thymus, and muscle, IGF-I mRNA expression was equally observed in normal and GHR-lacking dwarf chickens. In the testis, expression of IGF-I mRNA was enhanced by about 5-fold in dwarf chickens showing an expression level comparable to that in normal liver. On day 16 in the embryonic stage, IGF-I mRNA was expressed in muscle, brain, eye, heart, and lung in both normal and SLD chick embryos. However, no IGF-I mRNA expression was observed in liver or kidney of normal and dwarf chick embryos. These results suggest that in chicken, IGF-I mRNA is expressed in liver in a GH-dependent manner after hatching, whereas in other tissues, mRNA expression is independent of GH and GHR before and after hatching, except for testis, in which GH seems to inhibit IGF-I mRNA expression.

Animals↗
PubMed1996

Pup contact induces the expression of long form prolactin receptor mRNA in the brain of female rats: effects of ovariectomy and hypophysectomy on receptor gene expression.

Prolactin receptor (PRL-R) mRNA expression levels in the female rat brain (cerebrum) during pup contact stimulation were determined by the reverse transcription-PCR method. The high expression levels of long form PRL-R mRNA found in the brain of lactating rats were markedly reduced by removal of pups, and long form PRL-R mRNA levels were recovered by resumption of pup contact. Interestingly, pup contact stimuli of nulliparous virgin rats also markedly induced long form but not short form PRL-R mRNA expression in the brain in 1.3 days, together with the expression of maternal behaviour. In ovariectomized (OVX) or hypophysectomized (HYPOX) virgin rats, or in OVX plus HYPOX virgin rats, however, brain long form PRL-R mRNA was not significantly induced by pup contact stimuli for as long as 7 days, while maternal behaviour was fully expressed in these rats after 7 days of pup contact. The in situ hybridization experiments revealed that the long form PRL-R mRNA induced in virgin rats in contact with pups or in lactating rats was localized in the epithelial cells of the choroid plexus. No significant increase in mRNA was detected in other regions of the brain, such as the hypothalamus or cortex, in these maternal female rats. These results suggest that pup contact induces the expression of long form PRL-R mRNA in the choroid plexus of the brain in the presence of female sex steroid and pituitary hormones for the rapid expression of maternal behaviour. Our studies also suggested that maternal behaviour can be expressed in OVX or HYPOX rats after exposure to pups for 7 days without any significant increase in brain PRL-R mRNA expression.

Animals↗
PubMed1995

Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes.

A number of Ags recognized by class I-restricted, melanoma-specific T cells have recently been identified. In this report we demonstrated that tumor-infiltrating lymphocytes (TIL) from melanoma patient 1413 recognize a tumor Ag, tyrosinase, in the context of HLA-A24. This Ag had previously been shown to be recognized by an HLA-A24-restricted TIL, TIL 888, as well as HLA-A2-restricted, melanoma-specific T cells isolated from two additional patients. The peptide epitope recognized by TIL 1413 was then identified through the use of sequential deletions of the tyrosinase cDNA, as well as through prediction of HLA-A24 binding peptides based on a previously identified motif. Two peptides, a 9-amino acid peptide (AFLPWHRLF) and an overlapping 10-amino acid peptide (AFLPWHRLFL) containing an additional leucine at the carboxyl terminus, were both recognized by TIL 1413. Anti-peptide-specific CTL could be induced by repeated stimulation of peripheral blood lymphocytes from melanoma patient 1413, and this CTL line specifically recognized both HLA-A24+ B cell lines pulsed with the peptide and HLA-A24+ tyrosinase+ melanoma cells. This peptide thus represents a reagent that may be used to generate melanoma-specific T cells for adoptive immunotherapy, as well as in peptide vaccines for HLA-A24+ melanoma patients.

Amino Acid Sequence↗
PubMed1995

Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.

Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.

Adenosine Triphosphate↗
PubMed1995

Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes.

The role of tumor-specific T cells in mediating the regression of metastatic melanoma has been suggested by the clinical response of patients to treatment with tumor-infiltrating lymphocytes (TIL). A number of Ags recognized by class I-restricted melanoma-specific T cells have recently been isolated, raising the hope that this will lead to the development of improved therapies. In this study, we report the cloning of a tumor Ag recognized by T cells from melanoma patient 888. Previously, we reported that TIL 888, grown from the tumor of this patient, recognized tyrosinase in an HLA-A24-restricted fashion. This line, when infused into the autologous patient, resulted in complete regression of multiple metastases. Three years later, a second TIL line, TIL 1290, was isolated from a recurrent pelvic tumor. Infusion of a mixture of TIL 888 and TIL 1290 cell lines into the patient resulted in complete regression of a residual abdominal mass and the patient remains disease-free 2 yr later. The TIL 1290 cell line, which recognized melanoma in an HLA-A24-restricted manner, failed to recognize tyrosinase. TIL 1290 was then used to screen an 888 melanoma cDNA library, and an Ag was isolated that did not correspond to any found in sequence databases. This gene, termed p15, was found to be expressed in a variety of normal tissues, and a peptide epitope recognized by TIL 1290 was found to represent the product of an nonmutated gene. Screening of additional cDNA pools resulted in the isolation of a second clone which stimulated TIL 1290. This clone also appeared to represent a transcript of the p15 gene, indicating that this gene may encode the predominant Ag recognized by TIL 1290.

Amino Acid Sequence↗
PubMed1995

Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.

Four of ten HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-gamma release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gp100, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the development of immunotherapies for patients with melanoma.

Amino Acid Sequence↗
PubMed1995

EGF-induced activation of 70-kDa S6 kinase in CHO cells expressing human EGF receptors.

We investigated epidermal growth factor (EGF)-induced activation of 85-kDa/110-kDa phosphatidylinositol (PI)-3-kinase and 70-kDa S6 kinase in Chinese hamster ovary cells expressing the human EGF receptor. EGF-induced activation of p70 S6 kinase was comparable to that induced by insulin, whereas that of PI-3-kinase in anti-phosphotyrosine immunoprecipitates was very small compared with insulin. Wortmannin, a p85/p110 PI-3-kinase inhibitor, inhibited EGF-induced activation of p70 S6 kinase in a dose-dependent manner. Given that several proteins homologous to catalytic subunit of p85/p110 PI-3-kinase have been identified and that wortmannin inhibits distinct form of PI-3-kinase, the present results suggest that wortmannin-sensitive kinases that resemble catalytic subunit of p85/p110 PI-3-kinase may participate in the signaling pathway from EGF receptors to p70 S6 kinase.

Androstadienes↗
PubMed1995

Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.

MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2+ melanoma patients, as well as from 2 of 4 PBL from HLA-A2+ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2+ MART-1+ melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2+ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.

Amino Acid Sequence↗
PubMed1995

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells.

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.

Animals↗
PubMed1995

Backbone dynamics of the oligomerization domain of p53 determined from 15N NMR relaxation measurements.

The backbone dynamics of the tetrameric p53 oligomerization domain (residues 319-360) have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and heteronuclear NOEs were measured for 39 of 40 non-proline backbone NH vectors at both field strengths. The overall correlation time for the tetramer, calculated from the T1/T2 ratios, was found to be 14.8 ns at 35 degrees C. The correlation times and amplitudes of the internal motions were extracted from the relaxation data using the model-free formalism (Lipari G, Szabo A, 1982, J Am Chem Soc 104:4546-4559). The internal dynamics of the structural core of the p53 oligomerization domain are uniform and fairly rigid, with residues 327-354 exhibiting an average generalized order parameter (S2) of 0.88 +/- 0.08. The N- and C-termini exhibit substantial mobility and are unstructured in the solution structure of p53. Residues located at the N- and C-termini, in the beta-sheet, in the turn between the alpha-helix and beta-sheet, and at the C-terminal end of the alpha-helix display two distinct internal motions that are faster than the overall correlation time. Fast internal motions (< or = 20 ps) are within the extreme narrowing limit and are of uniform amplitude. The slower motions (0.6-2.2 ns) are outside the extreme narrowing limit and vary in amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer Graphics↗
PubMed1995

Analysis of MHC-specific peptide motifs. Applications in immunotherapy.

The structural features which underlie peptide binding to MHC molecules permit the binding of a diverse array of peptides. Polymorphic residues of class I, and to a lesser extent, class II molecules, determine the peptide selectivities associated with various allomorphs. The motifs which are described here and elsewhere in the literature mainly reflect peptide features which contribute to high affinity binding. While high affinity MHC binding is not an absolute prerequisite for the immunologic relevance of a peptide, motifs provide general guidelines for eliciting and characterizing cellular responses to epitopes presented by a given MHC allomorph or group of related allomorphs. The utility of motifs is underscored by emerging developments in the clinical application of peptides to elicit specific and effective cellular responses.

HLA-A2 Antigen↗
PubMed1995

Refined solution structure of the oligomerization domain of the tumour suppressor p53.

The NMR solution structure of the oligomerization domain of the tumour suppressor p53 (residues 319-360) has been refined. The structure comprises a dimer of dimers, oriented in an approximately orthogonal manner. The present structure determination is based on 4,472 experimental NMR restraints which represents a three and half fold increase over our previous work in the number of NOE restraints at the tetramerization interface. A comparison with the recently solved 1.7 A resolution X-ray structure shows that the structures are very similar and that the average angular root-mean-square difference in the interhelical angles is about 1 degree. The results of recent extensive mutagenesis data and the possible effects of mutations which have been identified in human cancers are discussed in the light of the present structure.

Animals↗