Biomedical subjects

K Sakaguchi

Publications and source records attributed to K Sakaguchi.

At least 217 records · Page 12Linked to original sources

PubMed1994

Removal of zinc is required for processing of the mature nucleocapsid protein of human immunodeficiency virus, type 1, by the viral protease.

In human immunodeficiency virus, RNA selection and packaging during assembly involve the two retroviral-type fingers of the nucleocapsid protein that are held in a constrained configuration by coordinated zinc ions. In this report, we demonstrate that the nucleocapsid protein in a metal bound state is resistant to cleavage by the viral protease, but upon removal of zinc ions by chelating agents, it is hydrolyzed within the first zinc finger between Phe-16 and Asn-17. However, 3-nitrosobenzamide and cupric ions, which release zinc through oxidation of the cysteine residues of the finger, render the nucleocapsid protein resistant to cleavage. Since protease inhibitors and 3-nitrosobenzamide restrict processes relating to steps early in infection, the cleavage of the nucleocapsid protein may represent an essential event that can be exploited for the design of novel antiviral agents.

Amino Acid Sequence↗

PubMed1994

High-resolution structure of the oligomerization domain of p53 by multidimensional NMR.

The three-dimensional structure of the oligomerization domain (residues 319 to 360) of the tumor suppressor p53 has been solved by multidimensional heteronuclear magnetic resonance (NMR) spectroscopy. The domain forms a 20-kilodalton symmetric tetramer with a topology made up from a dimer of dimers. The two primary dimers each comprise two antiparallel helices linked by an antiparallel beta sheet. One beta strand and one helix are contributed from each monomer. The interface between the two dimers forming the tetramer is mediated solely by helix-helix contacts. The overall result is a symmetric, four-helix bundle with adjacent helices oriented antiparallel to each other and with the two antiparallel beta sheets located on opposing faces of the molecule. The tetramer is stabilized not only by hydrophobic interactions within the protein core but also by a number of electrostatic interactions. The implications of the structure of the tetramer for the biological function of p53 are discussed.

Base Sequence↗

PubMed1994

Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are binding sites of Shc in intact cells.

Autophosphorylation of receptor tyrosine kinases provides binding sites for signaling proteins containing Src homology 2 (SH2) domains. We determined the binding sites of Shc, SH2-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing EGF receptor mutants in which autophosphorylation sites, either alone or in combination, were replaced by phenylalanine. Binding of Shc to EGF receptor mutants lacking single tyrosine residues at 1148 or 1173 decreased by approximately 60 or approximately 15%, respectively, whereas other single point mutants bound the wild-type level of Shc. Binding of Shc markedly decreased in mutants lacking both tyrosine residues at 1148 and 1173. In peptide inhibition assay, phosphorylated nonameric peptide representing tyrosine 1148, DNPDpYQQDF, but not pentameric peptide, pYQQDF, inhibited the binding of glutathione S-transferase-Shc SH2 domain fusion protein to in vitro autophosphorylated EGF receptors, suggesting that N-terminal sequences adjacent to phosphotyrosine are necessary for the association of Shc. Based on results of peptide inhibition assays in which phosphorylated peptides representing tyrosines 992, 1148, and 1173 inhibited Shc binding to the receptor, we constructed another EGF receptor mutant in which one of these tyrosine residues was retained. The amount of Shc bound to mutant receptors retaining tyrosines 1148, 1173, or 992 was approximately 80, approximately 40, or approximately 10% of wild-type level, respectively. These results indicate that tyrosine 1148 of activated human EGF receptors is a major binding site of Shc and tyrosine 1173 is a secondary binding site in intact cells.

Amino Acid Sequence↗

PubMed1994

Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.

The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.

Adult↗

PubMed1994

Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes.

Four melanoma proteins, MART-1, gp100, tyrosinase, and tyrosinase-related protein-1 (gp75) were evaluated for recognition by HLA-A2-restricted melanoma-specific cytotoxic T lymphocytes (CTLs) derived from the tumor-infiltrating lymphocytes (TIL) of 10 different patients. 9 of 10 TIL recognized MART-1, 4 recognized gp100 (including 3 that also recognized MART-1), but none of the TIL recognized tyrosinase or gp75. Based on the known HLA-A2.1 peptide binding motifs, 23 peptides from MART-1 were synthesized in an attempt to identify the epitopes recognized by TIL. Three peptides were recognized by TIL when pulsed on T2 target cells. One of the 9-mer peptides, AAGIGILTV, was most effective in sensitizing the T2 cells for TIL lysis. This peptide was recognized by 9 of 10 HLA-A2-restricted melanoma-specific CTLs. Therefore, this peptide appears to be a very common immunogenic epitope for HLA-A2-restricted melanoma-specific TIL and may be useful for the development of immunotherapeutic strategies.

Amino Acid Sequence↗

PubMed1994

Expression on outer membranes of mannose residues, which are involved in osteoclast formation via cellular fusion events.

Osteoclast, the bone-resorbing cell, is formed from hematopoietic precursors via cell-cell fusion. To evaluate the possibility that under certain specific conditions mannose residues may be expressed on the mammalian cell surface, we examined the action of pradimicin derivatives, which bind specific sugars such as the mannose residue, on the formation of osteoclast induced in the coculture of mouse spleen cells with mouse stromal cells, a process in which cell-cell fusion is involved. Osteoclast formation was inhibited by treatment of this coculture system with pradimicin at the later stage (day 4-7), and this inhibition was specifically abrogated by mannose-rich yeast mannan. During the 8-day cocultivation, osteoclast formation was blocked by the pradimicin on days 6 and 7, when mononuclear preosteoclasts fused into multinucleated osteoclasts. With an interactive laser cytometer ACAS570, fluorescein isothiocyanate-labeled pradimicin was observed to bind osteoclast progenitors at the fusion stage and to have no binding affinity for osteoclast progenitors at the early stage (day 0-3) or for osteoclasts, which were formed after performing fusion between mononuclear preosteoclasts. These results suggest that mannose residues were expressed on outer membranes of monocytes under pathophysiological conditions and that they were involved in the osteoclast formation via cellular membrane fusion events.

Animals↗

PubMed1994

Molecular interactions between interferon consensus sequence binding protein and members of the interferon regulatory factor family.

Interferon (IFN) consensus sequence binding protein (ICSBP) is a transcription factor expressed mostly in the cells of the immune system. ICSBP belongs to the IFN regulatory factor (IRF) family, which also includes IRF-1, IRF-2, and the IFN-alpha-stimulated gene factor 3 gamma (ISGF3 gamma). We show here that ICSBP forms a complex with IRF-1 or IRF-2 both in vivo and in vitro and, in the presence or absence of the target DNA, with the IFN-stimulated response element (ISRE). Further, electrophoretic mobility shift assays show that this interaction greatly enhances the otherwise very low binding affinity of ICSBP to the ISRE. We show, on the other hand, that ICSBP inhibits binding of the IFN-alpha-stimulated gene factor 3 gamma to the ISRE. Through these interactions ICSBP is likely to exert complex modulatory functions in the regulation of IFN-stimulated genes.

Base Sequence↗

PubMed1994

CD45 regulation of tyrosine phosphorylation and enzyme activity of src family kinases.

Previous analyses have suggested that the CD45 tyrosine phosphatase activates src family tyrosine kinases p56lck and p59fyn by dephosphorylating regulatory COOH-terminal residues. We have examined the status of p56lck and p59fyn in murine and human CD45- T cell lines. Surprisingly, despite the fact that p56lck and p59fyn were spontaneously hyperphosphorylated, the tyrosine kinase activity of both enzymes was increased in CD45- versus CD45+ cells. In vitro exposure of hyperphosphorylated p56lck to CD45 decreased enzyme activity to near-basal levels. Lck from CD45- cells was hyperphosphorylated on the cyanogen bromide digestion fragment that contains the negative regulatory residue Tyr-505, and the identity of this site of phosphorylation was confirmed by trypsin digestion followed by high performance liquid chromatography. Loss of CD45 results, therefore, in a paradoxical hyperphosphorylation of the COOH-terminal tyrosine and increased src family kinase enzymatic activity.

Amino Acid Sequence↗

PubMed1994

Definition of a DQ3.1-specific binding motif.

A quantitative peptide binding assay using purified DQA1*0301/DQB1*0301 (DQ3.1) molecules was developed and validated by examining the correlation between the data obtained in the binding assay with those obtained in inhibition of Ag presentation assays. By the combined use of large libraries of synthetic peptides and of substitution and truncation analogues, a putative DQ3.1 motif was defined. Its most prominent feature is the requirement for two small and/or hydrophobic residues spaced at positions i + 2 and i + 4. This motif is quite different from the motif recognized by DR molecules, but similar to the motif previously defined for certain IA alleles (the putative mouse homologue of DQ). These data suggest that various class II isotypes have evolved to present different peptide structures to each other, thus maximizing the repertoire of different epitopes available to T cell scrutiny.

Amino Acid Sequence↗

PubMed1994

A meiotic DNA polymerase from a mushroom, Agaricus bisporus.

A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.

Agaricus↗

PubMed1994

Definition of specific peptide motifs for four major HLA-A alleles.

Allele-specific motifs for the human MHC class I molecules, HLA-A1, A3, A11, and A24 were characterized by three complementary approaches. First, amino acid sequence analysis of acid eluted peptide pools from affinity purified class I molecules defined putative motifs 9 or 10 amino acids in length and bearing critical anchor residues at position 2 and at the COOH-terminal. These motifs were distinct, with the exception of the HLA-A3 and A11 motifs that were very similar to each other. Second, the correctness of these putative motifs was verified by analyzing the binding capacity of polyalanine peptide analogues to purified HLA-A molecules. Several alternative anchor residues that were not obvious from the pooled peptide sequencing analysis were identified. Third, sequences of individual peptides eluted from HLA-A1, A11, and A24 were determined by tandem mass spectrometry. Nonamers were the predominant species, although peptides of 8, 10, 11, and 12 amino acids in length were also identified. These peptides displayed anchor residues predicted by the specific motifs at position 2 and at the COOH-terminal, regardless of peptide length. Synthetic versions of the naturally processed peptides were shown to bind to the appropriate HLA-A alleles with IC50 values in the 0.3- to 200-nM range. A rational approach to search Ags with known amino acid sequences for epitopes restricted by some of the most common HLA-A types and of potential clinical importance is now feasible.

Alleles↗

PubMed1994

Naturally processed peptides longer than nine amino acid residues bind to the class I MHC molecule HLA-A2.1 with high affinity and in different conformations.

An equilibrium binding assay was used to directly measure the relative affinities of naturally processed 9-mer, 10-mer, and 12-mer peptides for the human class I MHC molecule HLA-A2.1. The peptides exhibited a range of affinities with IC50 values of 11 to 214 nM. The mode of interaction between these peptides and HLA-A2.1 was examined using peptides in which Asp had been substituted for suspected anchor residues. Regardless of length, the previously identified Leu at position 2 relative to the amino terminus was critical for peptide binding. While the carboxyl terminal residue was also critical for the binding of a 9-mer peptide, it was much less important in the binding of longer peptides. Additional residues close to the carboxyl terminus that contained aliphatic hydrocarbon side chains were of similar or greater importance in peptide binding. In addition, residue at position 3 also appeared to be important for the binding of longer peptides. The data suggest that different naturally occurring longer peptides can bind in different conformations to class I MHC molecules. While one of these is similar to the kinked conformation described by others, another conformation would involve an extension of the carboxyl terminus out of the class I binding site. The ability of MHC molecules to accommodate the same peptide in different conformations would appear to have distinct advantages to the immune system.

Amino Acid Sequence↗

PubMed1994

Drosophila DNA polymerase delta. Purification and characterization.

A DNA polymerase with properties similar to mammalian polymerase delta has been isolated to near homogeneity from early embryos of Drosophila melanogaster. A combination of exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that this enzyme has a total molecular mass of 185 kDa and is composed of 138- and 47-kDa polypeptides. Its isoelectric point is 6.8. This polymerase activity is strongly inhibited by N-ethylmaleimide, aphidicolin, and high KCl concentration but is relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate. There was no reaction in an immunological test using monoclonal antibody against Drosophila DNA polymerase alpha. In a final purification step, this polymerase activity was accompanied by 3'-->5'-exonuclease activity as expected proof-reading activity. This polymerase activity is remarkably stimulated by mouse proliferating cell nuclear antigen, which is structurally and immunologically very similar to a Drosophila counterpart. These properties clearly indicate this enzyme belongs to the category of DNA polymerase delta.

Animals↗

PubMed1994

A new meiotic protein factor which enhances activity of meiotic DNA polymerase from Coprinus cinereus.

Meiotic cells of Coprinus contain a protein that can be identified by its ability to enhance activity of the meiotic DNA polymerase reported previously (8). Its activity is found only during the prophase stages in meiotic cells, and is accompanied by the meiotic polymerase. The protein, which was purified to near homogeneity, is a single polypeptide with a molecular mass of 30k, and shows no activities of nuclease, ATPase, or DNA-binding protein. The protein could enhance the meiotic polymerase activity by at least 5 fold. The other Coprinus polymerases were not influenced by the protein. The protein could increase Vmax value of the meiotic polymerase, but not the Km. The significance of this protein to meiotic DNA synthesis is considered in relation to other biochemical properties of meiotic cells.

Chromatography, Gel↗

PubMed1994

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component.

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.

Amino Acid Sequence↗

PubMed1994

Peripheral T cell response to A-gliadin in celiac disease: differential processing and presentation capacities of Epstein-Barr-transformed B cells and fibroblasts.

Celiac disease (CD) is a small intestinal disorder characterized by the malabsorption of most nutrients. Disease pathogenesis appears to be associated with immune-mediated pathology. Susceptibility is associated with genes coding for DQw2 class II molecules. In the present report we investigated T cell responses to A-gliadin (AGL), a major alpha-gliadin component known to activate disease. Gliadin-specific lines were generated from a CD patient and a normal donor. Three major points were revealed by the analysis of these T cells: (1) On the basis of mapping experiments using Epstein-Barr virus (EBV) lines and DR-transfected fibroblasts and DR-, DP-, and DQ-specific monoclonal antibodies (mAb), all responses appeared to be DR-restricted. Thus, in contrast to the strong association of disease susceptibility with DQ molecules, no DQ-restricted, gliadin-specific response was detectable. (2) Fine specificity analysis, using a panel of synthetic peptides spanning the entire alpha-gliadin component molecule, revealed that the clones derived from the normal donor were DR53-restricted and AGL 21-40-specific, while clones derived from the CD patient were DR7-restricted and peptide 1-20-specific. (3) Both whole AGL and AGL 1-20 were presented to the patient-derived clones with much higher efficiency by DDR-transfected fibroblasts than by EBV lines. These data suggested that fibroblasts processed this determinant efficiently, while EBV lines were unable to do so. Indeed, analysis of a panel of truncated AGL 1-20 analogs revealed that peptide AGL 1-8, which contained the minimal T cell epitope, was presented with equal efficiency by fixed or irradiated EBV and irradiated DR7-transfected fibroblasts.

Adult↗

PubMed1994

Eicosapentaenoic acid inhibits bone loss due to ovariectomy in rats.

Eicosapentaenoic acid (EPA), one of the polyunsaturated fatty acids, is well-known to have a wide variety of beneficial biological functions. In the present work we demonstrate another new beneficial effect of EPA on bone metabolism in vivo. Ovariectomized rats were divided into 4 groups under the same calorie intake condition; (1) normal diet, (2) low calcium diet (1.5 mg/day), (3) EPA-enriched diet (160 mg/day/kg), (4) EPA-enriched and low calcium diet. These diets were continued for 35 consecutive days. The bone weight of the femora and tibiae decreased significantly in the low calcium group, but the decrease was inhibited in the EPA-low calcium group. Moreover, in the rupture test, which indicates bone strength, the femora in the low calcium group were easier to break than in the normal calcium diet groups. In the EPA-low calcium group the strength of the bone was equivalent to that in the normal diet group. These results suggest that an EPA-enriched diet prevents the loss of bone weight and strength caused by oestrogen deficiency or inadequate nutrition. There is a possibility that EPA could be developed to be a novel anti-osteoporosis drug.

Animals↗

PubMed1994

Differential regulation of prolactin receptor mRNA expression in rat liver and kidney by testosterone and oestradiol.

Prolactin (PRL) exerts a wide variety of physiological effects on mammalian tissues through its receptor (PRL-R) on the target cells. PRL-R in rat tissue consists of two isoforms, the long and the short form, and the regulatory mechanisms of their mRNA expression in tissues are complex and diverse. The present study reports the differential regulation of PRL-R mRNA expression in rat liver and kidney by testosterone and oestradiol. Using Northern blot analysis, short form PRL-R mRNA was clearly detected in female rat liver and male rat kidney, and long form PRL-R mRNA was faintly observed only in female rat liver. However, the reverse transcription-polymerase chain reaction method enabled efficient analysis of mRNA levels in short and long forms of PRL-R in the liver and kidney of both male and female rats. The mRNA levels for the long and short forms of PRL-R were depressed in the liver of male rats but not in that from female rats during sexual maturation. Castration of male rats resulted in the induction of the mRNAs for these two forms of PRL-R in the liver. Testosterone, but not oestradiol, completely blocked the induction by castration of liver PRL-R gene expression. In kidney, in contrast, mRNA levels for both forms of PRL-R were depressed in female rats but not in male rats after sexual maturation. Administration of oestradiol, but not of testosterone, caused marked repression of short form PRL-R mRNA, particularly in the kidney of male rats. The levels of long form PRL-R mRNA in the kidney was less affected by the administration of oestradiol. These results have suggested that the expression of PRL-R mRNAs in rat liver and kidney is differentially regulated by testosterone and oestrogen.

Animals↗