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M Takiguchi

Publications and source records attributed to M Takiguchi.

At least 145 records · Page 8Linked to original sources
PubMed1996

Regulation of CCAAT/enhancer-binding protein family members by stimulation of glutamate receptors in cultured rat cortical astrocytes.

Regulation of mRNA levels, DNA binding activities, and phosphorylation of CCAAT/enhancer-binding protein (C/EBP) family members by stimulation of glutamate receptors were studied in cultured rat cortical astrocytes. Indirect immunofluorescence and immunoblot analyses with specific antibodies to C/EBP family members revealed that both C/EBPbeta and C/EBPdelta but not C/EBPalpha are expressed in the nuclei of astrocytes. After exposure to glutamate, C/EBPbeta mRNA levels increased within 10 min, reached the maximal level at about 1 h, and returned to the basal level within 6 h. In contrast, C/EBPdelta mRNA levels decreased by 6 h and were recovered within 12 h. These changes in mRNA levels were accompanied by an increase and a decrease in proteins for C/EBPbeta and C/EBPdelta, respectively. Elevation of C/EBPbeta mRNA levels by glutamate treatment required an increase in intracellular Ca2+ concentration and depended on activations of protein kinase C and calmodulin-dependent protein kinases. Gel mobility shift analysis using nuclear extracts from the glutamate-treated cells showed increases in C/EBP site binding activities 2 h after the exposure to glutamate. Moreover, glutamate stimulated phosphorylation of C/EBPbeta in 32P-labeled astrocytes in a Ca2+-dependent manner. These results suggest that glutamate regulates functions of C/EBP family members in brain astrocytes through changes in mRNA levels of C/EBPbeta and C/EBPdelta as well as through phosphorylation of C/EBPbeta.

Animals↗
PubMed1996

Regulation of CTL recognition by receptors for HLA-DR molecules.

NK cells and some T cells have receptors for MHC class I molecules that negatively regulate the recognition of these cells. In the present study, we show alpha beta CTL clones carrying the receptors for HLA-DR molecules that inhibit this cytotoxicity. The cytotoxicity of two CTL clones possessing NK activity was inhibited by the surface expression of HLA-DR and HLA class I molecules on the target cells. The inhibitory effect of DR molecules on the cytotoxicity of the CTL clones was confirmed by using T2 transfectants expressing HLA-DR3. These results suggest that receptors for both HLA-DR and HLA class I molecules negatively regulate the recognition of T cells.

Antigen Presentation↗
PubMed1996

Dual recognition of a human cytotoxic T-cell clone for melanoma antigens.

It is well known that tumor-specific CTLs have a crucial role in the elimination of tumors and that different CTL populations recognize tumor antigens in MHC-restricted and MHC-unrestricted manners. We have established two alpha beta CTL clones that recognize melanoma antigens in both human lymphocyte antigen (HLA)-A2-restricted and HLA-unrestricted manners. Flow cytometry analysis showed that these CTL clones carry CD3, CD8, and alpha beta T-cell receptor (TCR) and express low levels of CD56. In contrast, these CTL clones do not express CD16, indicating that they do not contain natural killer cells. TCR analysis of these CTL clones using an anchored PCR method revealed that each clone carries a single alpha beta TCR. Both CTL clones contained the same Valpha and Vbeta gene segments although they carried different Jalpha and Jbeta gene segments. Taken together, these results confirm that CTL clones that carry a single alpha beta TCR recognize melanoma antigens in both HLA-A2-restricted and HLA-unrestricted manners. It is strongly suggested that the dual recognition of these CTL clones for the melanoma antigens is mediated by TCRs. The novel mechanism for antitumor immunity by these CTLs may be important in the effective elimination of tumors in vivo.

Amino Acid Sequence↗
PubMed1996

CCAAT/enhancer-binding protein beta (C/EBP beta) binds and activates while hepatocyte nuclear factor-4 (HNF-4) does not bind but represses the liver-type arginase promoter.

In an attempt to elucidate the mechanism governing liver-specific transcription of the arginase gene, we previously detected two protein-binding sites designated footprint areas A and B at positions around--90 and --55 bp, respectively, relative to the transcription start site of the rat arginase gene. Based on the finding that area A was bound by a liver-selective factor(s) related to CCAAT/enhancer-binding protein (C/EBP), we performed cotransfection assay and showed that C/EBP family members and a related factor, albumin D-element-binding protein (DBP) stimulate transcription from the arginase promoter. In addition to area A, a recombinant C/EBP beta protein bound to area B, which appeared to be primarily responsible for activation by C/EBPs. We unexpectedly found that the arginase promoter activity stimulated by C/EBPs and DBP was repressed by another liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4). Analysis of chimeras formed between the arginase promoter and the herpes simplex virus thymidine kinase promoter allowed us to delimit the negative HNF-4-responsive element into the region overlapping with footprint area B. However, no apparent binding of HNF-4 was observed in this negative element. We speculate that HNF-4 is involved in fine regulation of the arginase gene in the liver or shutdown of the gene in nonhepatic tissues without direct binding to the promoter region.

Animals↗
PubMed1996

Coinduction of nitric oxide synthase, argininosuccinate synthetase, and argininosuccinate lyase in lipopolysaccharide-treated rats. RNA blot, immunoblot, and immunohistochemical analyses.

Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of NOS (iNOS), AS, and AL was analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS, and AL were coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.

Animals↗
PubMed1996

Class I-restricted presentation of an HIV-1 gp41 epitope containing an N-linked glycosylation site. Implications for the mechanism of processing of viral envelope proteins.

Uncertainty exists over the site of processing of viral envelope (env) proteins for recognition by CTL. The extracellular domains of env proteins are not present in the cytosol, the site where the class I Ag processing pathway begins. Rather, the ecto-domains of env proteins are cotranslationally translocated into the endoplasmic reticulum during biosynthesis. To clarify the site of processing of viral env proteins, we examined the processing of an HLA B*3501-restricted epitope in the extracellular domain of the HIV-1 env protein. Although this epitope contains an N-linked glycosylation signal sequence, CTL specific for this epitope recognize a nonameric peptide that has not been previously modified by attachment of oligosaccharide. This was demonstrated in two ways. First, an env-specific B*3501-restricted CTL clone recognized a nonglycosylated, synthetic nonamer representing the minimal B*3501-restricted epitope, but not the glycosylated or deglycosylated forms. Second, the naturally processed, B*3501-restricted, env peptide is identical with a nonglycosylated, synthetic nonamer. Thus, the naturally processed form of an env epitope containing an N-linked glycosylation site is derived from env protein that is not glycosylated at the relevant asparagine during biosynthesis. Since the addition of N-linked oligosaccharides occurs only after the glycosylation signal sequence (N-X-S/T) is translocated into the endoplasmic reticulum, the initial processing reaction for this epitope may take place in the cytosol. Low-frequency failure of signal sequence containing polypeptides to engage the translocation apparatus, resulting in synthesis and degradation in the cytosol, may represent an important mechanism for the generation of class I-restricted CTL responses.

Amino Acid Sequence↗
PubMed1996

An HLA-B35-restricted epitope modified at an anchor residue results in an antagonist peptide.

Peptides associated with HLA-B35 commonly have a proline or occasionally a serine residue in the P2 anchor position of the peptide, with a tyrosine at the C terminus. Based on this motif, we identified an octamer epitope from influenza A matrix protein which is presented by HLA-B35. The requirements for MHC binding and T cell receptor contact have been analyzed using analogs of this peptide with substitutions at positions 1, 2, 4, 7 and 8. The natural epitope contains a serine residue at P2 of the peptide. Substitution of this residue with proline (the favored amino acid in this position in B35-associated peptides) considerably enhances binding to HLA-B35 in the T2-B35 cell line, but the peptide is not recognized by the majority of CTL clones and can antagonize recognition of the index peptide. This suggests that a conservative substitution at the P2 anchor position results in a conformational change in the peptide-MHC surface exposed to the T cell receptor.

Amino Acid Sequence↗
PubMed1996

Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket.

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cells expressing human beta2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B*5101 binding self-peptides, 27 peptides bound to HLA-B*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B*5102 and B*5103 molecules showed that a single amino acid substitution of Tyr for His at residue 171(B*5102) and that of Gly for Trp at residue 167 (B*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B*5102 or HLA-B*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.

Amino Acid Sequence↗
PubMed1996

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules.

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76. 2%) of these peptides bound to HLA-A*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules.

Binding Sites↗
PubMed1996

Refined peptide HLA-B*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding.

HLA-B*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B*3501-restricted antigens is the characterization of peptide-HLA-B*3501 interactions. In this study, peptide-HLA-B*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 - 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B*3501 binding cleft.

Amino Acid Sequence↗
PubMed1996

Identification and DNA typing of two Cw7 alleles (Cw*0702 and Cw*0704) in Japanese, with the corrected sequence of Cw*0702.

Two alleles encoding HLA-Cw7 antigens, tentatively called C7J1 and C7J2, have been identified in Japanese using a PCR-SSCP method. The nucleotide sequence of full-length C7J1 cDNAs showed a high degree of homology to the reported Cw*0702 sequence except in exon 1. We then resequenced the allele carried by the cell line JY in which Cw*0702 was first identified, according to a request from the WHO Nomenclature Committee. The results revealed complete identity between the corrected Cw*0702 sequence and the C7J1 sequence. On the other hand, the C7J2 sequence was completely identical to the reported Cw*0704 sequence. Sequences specific for Cw*0702 and Cw*0704 were confirmed using PCR-RFLP and PCR-SSO methods. Moreover, association analysis with other HLA locus alleles showed positive associations of Cw*0702 with HLA-B7, -B39, and -B67 and of Cw*0704 with HLA-B70 in Japanese.

Alleles↗
PubMed1996

Role of HLA-B*5101 binding nonamer peptides in formation of the HLA-Bw4 public epitope.

HLA-Bw4 is one of two HLA-B public epitopes which are discriminated by specific alloantisera and mAb. It is believed that the 77-83 of HLA-B molecules form the Bw4 epitope recognized by specific antibodies. This epitope is also recognized by NKB1 receptors on NK cells. We investigated the role of a peptide bound to HLA-B molecules on the formation of the Bw4 epitope recognized by two HLA-Bw4-specific mAb, TU109 and TU48, which recognized the difference of the Bw4 epitope among HLA-B52, -B52 and -B53. Recognition of the HLA-B*5101-peptide complex by these mAb was examined using a panel of HLA-B*5101 binding nonamer peptides. The sequence of HLA-B*5101 binding peptides has a minimum influence on the binding of TU48 mAb to HLA-B*5101 molecules. In contrast, the binding of TU109 mAb to HLA-B*5101 molecules was critically influenced by the sequence of a peptide bound to HLA-B*5101 molecules. TU109 mAb did not recognize HLA-B*5101 binding peptides carrying small or negatively charged residues at P8. The results were confirmed by a panel of mutant peptides at P8. Taken together, these results indicate that a positively charged or neutralized side chain of P8 is critical for the epitope formation of Bw4 recognized by this mAb.

Animals↗
PubMed1996

Induction of the C/EBP beta gene by dexamethasone and glucagon in primary-cultured rat hepatocytes.

The synthetic glucocorticoid, dexamethasone, and glucagon cooperatively elevated the level of mRNA for the transcription factor CCAAT/enhancer binding protein beta (C/EBP beta) in primary-cultured rat hepatocytes. In response to dexamethasone and/or glucagon, C/EBP beta mRNA started to increase as early as 30 min, reached a maximum within 2 h, and then gradually decreased. The administration of cycloheximide, a protein synthesis inhibitor, led rather to an increase in C/EBP beta mRNA, which suggested that a labile negative protein factor(s) is involved in regulation of the C/EBP beta mRNA level. Cycloheximide further augmented the increases in C/EBP beta mRNA by dexamethasone and/or glucagon. Therefore, C/EBP beta mRNA accumulation in response to these hormones is apparently independent of ongoing protein synthesis. The elevation of the C/EBP beta mRNA level by these hormones was accounted for by increases in the rate of transcription of the C/EBP beta gene, as deduced on nuclear run-on analysis. Gel mobility shift analysis revealed that the DNA-binding activity of C/EBP beta was increased cooperatively by dexamethasone and glucagon. These results suggest that the C/EBP beta gene is primarily induced by glucocorticoids and/or glucagon and that the accumulated C/EBP beta protein is then involved in secondary activation of target genes in response to these hormones in the liver.

Animals↗
PubMed1996

[A case of saccular aneurysm of the coronary artery].

A 42-year-old man with chest oppression was diagnosed as having a saccular aneurysm of the coronary artery by cardiac catheterization. At the time of operation on April 26, 1993, the aneurysm, measured 15 mm in diamer, and arose from the proximal segment of the first septal branch of the left anterior descending coronary artery. Several coronary arteriovenous fistulae from the aneurysm to the pulmonary artery were also noted. The stem of the aneurysm and the coronary arteriovenous fistulae were ligated and the aneurysm was resected. We considered the etiology to be mainly arteriosclerosis on the basis of the aneurysm pathology. And this aneurysm was also considered to have congenital factors because of the existence of arteriovenous fistulae. There were no symptoms after the operation and he was discharged at 24th postoperative day. Now he continues to do well.

Adult↗
PubMed1996

Identification of multiple HIV-1 cytotoxic T-cell epitopes presented by human leukocyte antigen B35 molecules.

OBJECTIVE: To identify HIV-1 cytotoxic T lymphocyte (CTL) epitopes presented by human leukocyte antigen (HLA)-B35 molecules that are associated with the accelerated progression of AIDS using a reverse immunogenetic approach. METHODS: 8-mer to 11-mer sequences carrying two anchor residues at position 2 and the carboxy-terminus were selected from HIV-1SF2 strain. Sixty-four peptides matched to these sequences were synthesized and tested by a peptide binding assay using RMA-S-B*3501 cells. Peripheral blood lymphocytes (PBL) from two HIV-1-infected donors carrying HLA-B35 were stimulated once-weekly with each HLA-B*3501 binding peptide. The CTL activity of the cultured cells for the HLA-B35-positive target cells loaded with the corresponding peptides was examined after the second and fourth stimulation. Furthermore, the CTL activity of the cultured cells possessing HLA-B*3501-restricted HIV-1 peptide-specific CTL activity were examined for the HLA-B*3501-positive target cells infected with the recombinant vaccinia virus containing corresponding HIV-1 gene. RESULTS: HIV-1 peptide-specific HLA-B*3501-restricted CTL was induced in PBL of HIV-1 infected donors by in vitro stimulation with 11 out of 27 HLA-B*3501-binding HIV-1 peptides. The specific CTL induced with 10 peptides killed the cells infected with recombinant vaccinia virus expressing the corresponding HIV-1 proteins. Out of these HIV-1 peptide epitopes, two epitopes were also presented by HLA-B51 molecules. CONCLUSION: In addition to the four HLA-B35-restricted HIV-1 CTL epitopes that have been previously reported, nine HLA-B35-restricted HIV-1 CTL epitopes were identified in the present study. These multiple epitopes will be useful in studies for immunopathogenesis of AIDS.

Antigen Presentation↗