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S Hirosawa

Publications and source records attributed to S Hirosawa.

At least 73 records · Page 4Linked to original sources
PubMed1994

Recognition DNA sequence of a novel putative transcription factor, BCL6.

The BCL6 gene involved in the 3q27 translocation associated with B-cell lymphomas encodes a novel Cys2-His2 zinc finger protein. We generated a fusion protein of glutathione S-transferase and zinc finger domain of BCL6 to determine recognition sequences of BCL6 with polymerase chain reaction using random oligonucleotides of 26 bases as a ligand. A consensus of 14 nucleotides consisting of (T/A)NCTTTCNAGG(A/G)AT was identified in the recognition sequences. In a gel mobility shift assay, the probe containing the 14-nucleotide recognition sequence formed a complex with the fusion protein and nuclear proteins from Burkitt's cell lines overexpressing the BCL6 transcripts. The consensus sequence was protected from the digestion by nuclease in a DNase I footprinting assay. In conclusion, BCL6 may be involved in tumorigenesis by binding to the consensus sequences of the other genes.

Base Sequence↗
PubMed1994

Presence of functional cyclic AMP responsive element in the 3'-untranslated region of the human thrombomodulin gene.

We characterized the transcriptional regulatory function of the 3'-untranslated region of the thrombomodulin gene by transient transfection assays in human umbilical endothelial cells. Deletion analyses of the 3'-untranslated region indicated that the region containing the consensus sequence of the cyclic AMP (cAMP) responsive element (position 2092) showed an increased transcriptional activity in response to cAMP. Gel-shift analysis showed that a band representing the fragment containing position 2092 was retarded when incubated with nuclear extracts from the cells treated with cAMP. In addition, the region downstream of the cAMP responsive element was found to function negatively in the gene expression. These results indicate that the 3'-untranslated region has a functional cAMP responsive element and plays an important role in the regulation of the thrombomodulin gene expression.

Base Sequence↗
PubMed1994

Molecular cloning of the breakpoint for 3q27 translocation in B-cell lymphomas and leukemias.

Reciprocal exchanges between chromosomal region 3q27 and three loci of the Ig genes have been reported in cases of B-cell type non-Hodgkin's lymphoma. We have cloned a region containing a breakpoint junction of 3q27 from a cell line established from a patient with Burkitt's lymphoma carrying t(3;22)(q27;q11). The region cloned was shown to contain an Ig lambda light chain gene fused to a gene on chromosome 3q27. This finding was subsequently confirmed by fluorescence in situ hybridization. Extra nucleotides were present at the joining site. The heptamer-like and nonamer-like sequences separated by an intervening 24 bp were present in the region corresponding to the breakpoint of 3q27, suggesting that a misrecombination in Ig gene rearrangement may be involved in the translocation. Southern blot analysis with a 3q27-specific probe showed rearrangements in three additional patients with B-cell malignancies with the t(3;14)(q27;q32). The breakpoints of all four cases clustered within a limited 3-kb region on chromosome 3q27. The region of 3q27 involved in the translocation was designated as the BCL5 locus. The transcripts from the BCL5 locus were detected in normal tissues and hematopoietic cell lines, and the increased expression of transcript of aberrant size was detected in the established cell line carrying t(3;22). These observations suggest that a gene located at 3q27 is involved in the translocation and that its deregulation plays a role in the malignant transformation of B cells.

Base Sequence↗
PubMed1994

Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Krüppel-like zinc-finger protein.

Chromosomal translocations involving band 3q27 are the recently described nonrandom cytogenetic abnormalities in B-cell malignancies. We have previously cloned the breakpoint region of 3q27, designated as the BCL5 locus, from the B-cell line carrying the t(3;22). The cDNA for the BCL5 gene was cloned from the human liver cDNA library. The nucleotide sequencing analysis showed that the BCL5 gene encodes a potential transcription factor containing six repeats of the Cys2-His2 zinc-finger motif resembling the Drosophila segmentation gene Krüppel. The calculated molecular weight was 78.8 kD, which was supported by an in vitro transcription and translation experiment. A part of the sequence was essentially identical to that of a genomic fragment, ZNF51, previously reported to be located at 3qter. The translocation occurred in the 5' region of the BCL5 gene, and the protein-coding exons were fused to the Ig-lambda gene in a head-to-head configuration in the cell line carrying t(3;22). The BCL5 cDNA probe detected a major transcript of 3.8 kb in Burkitt's lymphoma cell lines and an aberrant transcript in the t(3;22) cell line, whereas no transcript was detected in myeloid, monocytoid, erythroid, T-lymphoid, and Epstein-Barr virus-immortalized B-lymphoblastoid cell lines.

Amino Acid Sequence↗
PubMed1994

Secretion of alpha 2-plasmin inhibitor is impaired by amino acid deletion in a small region of the molecule.

Alpha 2-plasmin inhibitor (alpha 2PI) deficiency Okinawa results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of the deletion of Glu137 in the amino acid sequence of alpha 2PI. To examine the effects of replacing the amino acid occupying position 137 or deleting its neighboring amino acid on alpha 2PI secretion, we used oligonucleotide-directed mutagenesis of alpha 2PI cDNA to change the codon specifying Glu137 or delete a codon specifying its neighboring amino acid. The effects were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of media from transiently transfected COS-7 cells. Replacement of Glu137 with an amino acid other than Cys had little effect on alpha 2PI secretion. In contrast, deletion of an amino acid in a region spanning a sequence of less than 30 amino acids including positions 127 and 137 severely impaired the secretion. The results suggest that structural integrity of the region, rather than its component amino acids, is important for the intracellular transport and secretion of alpha 2PI.

Amino Acid Sequence↗
PubMed1994

Variant translocation of the BCL6 gene to immunoglobulin kappa light chain gene in B-cell lymphoma.

A lymphoma cell line with a variant type of translocation, t(2;3)(p11;q27), was established from a patient who had received liver transplantation. To elucidate the molecular mechanism of the t(2;3)(p11;q27) chromosomal translocation, we compared the structures of both derivative (der) chromosomal breakpoints with those of their germline predecessors. We noted that the BCL6 gene on chromosome 3 was juxtaposed with the immunoglobulin kappa light chain (Ig kappa) gene on chromosome 2 in a head-to-head configuration. The breakpoint of the BCL6 gene was within a previously reported breakpoint cluster region. The breakpoint on chromosome 2 was within the intron between the leader (L) and variable (V) sequences of one of the V kappa genes, which was fused to the J kappa 3 (J = joining) segment. At chromosomal junctures, a direct repeat duplication of chromosome 3 sequences and a deletion of chromosome 2 sequences were discovered. These results are consistent with a translocation model with illegitimate pairing of staggered double-stranded DNA breaks at 3q27 and 2p11, repair, and ligation to generate der(3) and der(2) chromosomes.

Base Sequence↗
PubMed1994

Determination of plasma tissue factor antigen and its clinical significance.

To investigate the clinical significance of determination of plasma tissue factor (TF) antigen, we have developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for plasma TF, using two different monoclonal antibodies against TF apoprotein, 6B4 (catching antibody) and 5G9 (detecting antibody), and tetramethyl benzidine/H2O2 as substrates. Titration curves of recombinant human TF in buffer containing Triton X-100 were linear within the range from 50 to 2000 pg/ml. The total assay time was 3 h. Ultracentrifugation and immunoblot analysis indicated that human plasma and urine contained 50,000 g sedimentable and non-sedimentable forms of TF, both of which were detected by our ELISA method. Plasma and urine concentrations of TF in healthy subjects and patients with various diseases were measured by the ELISA method. In healthy subjects, plasma and urinary TF levels were found to be 149 +/- 72 pg/ml (n = 30) and 175 +/- 60 pg TF/urine creatinine mg (n = 95), respectively. TF was increased in plasma of patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, vasculitis associated with collagen diseases, diabetic microangiopathy and chronic renal failure receiving haemodialysis, but not in the plasma of endotoxaemic patients without DIC. The plasma TF/serum creatinine ratio did not show a positive correlation. Measurement of TF antigen in plasma may be useful for evaluating the endothelial damage and cell destruction in TF-containing tissues.

Adult↗
PubMed1994

Compound heterozygous protein C deficiency caused by two mutations, Arg-178 to Gln and Cys-331 to Arg, leading to impaired secretion of mutant protein C.

The protein C gene in a patient apparently homozygous for protein C deficiency was analyzed. Two different point mutations, each located in a different allele, were detected to reveal that the patient is a compound heterozygote. Mutation of Arg-178 (CGG) to Gln (CAG) [mutation I] was detected in exon VII, in the vicinity of activation peptide cleavage site by thrombin. Mutation of Cys-331 (TGC) to Arg (CGC) [mutation II] was found in exon IX, at one of the sites involved in disulfide bond formation in the catalytic domain of the heavy chain. The alteration of Cys-331 to Arg disables the formation of the disulfide bond and would alter the protein conformation. Transient expression assays using COS-7 cells transfected with protein C expression vectors containing each one of these two mutations suggested that each of the two mutations would lead to the protein C deficiency by an impairment of secretion of the respective mutant proteins.

Alleles↗
PubMed1994

[Molecular study of hematological diseases].

With the recent advances in molecular technology, diagnostic procedures of the diseases at a DNA level have been introduced in hematological fields. The diagnostic methods used are Southern blotting to detect gene rearrangements, Northern blotting to find gene expressions, RT-PCR (reverse transcriptase-polymerase chain reaction) to identify transcribed fusion messages, and PCR-SSCR (single strand conformation polymorphism) to detect mutated genes. Rearrangements within major Bcr (breakpoint cluster region) were observed in almost all cases in chronic myelogenous leukemia, and breakpoint were found within minor Bcr in Philadelphia-positive leukemia. The rearrangements within the second intron of the retinoic acid receptor-alpha and sixth intron (bcr 1), third intron (bcr 3) and sixth exon (bcr 2) of the PML gene were detected in all cases with acute promyelocytic leukemia. In malignant lymphoma, the rearrangements of immunoglobulin and T-cell receptor genes, and new genes such as Bcl-1, Bcl-2, Bcl-5, Tal-1, and Tal-2 were also reported and rearrangements of the Bcl-5 gene were found in this study using Bcl-5 specific probe which we have cloned. Point mutations and deletions of the genes involved in the coagulation and fibrinolysis system have been reported. One base insertion resulting in elongation of carboxy terminal region and one amino acid deletion in alpha 2-plasmin inhibitor gene were found in two cases of its deficiency. Further study revealed that mutated proteins were retained in the endoplasmic reticulum in the cells. With the development of the PCR method, identification of gene mutation is gradually carried out as a routine work.

DNA↗
PubMed1994

The organization of the BCL6 gene.

The chromosomal translocation involving 3q27 has been recently described in B-cell malignancies, especially in diffuse large cell lymphomas. We have previously cloned the breakpoint cluster region of 3q27 designated as the BCL6 locus, previously known as BCL5, and subsequently cloned the cDNA for the BCL6 (we previously reported it as BCL5) gene encoding a novel Cys2-His2 zinc-finger protein, which locates adjacent to the breakpoints and is activated through the translocation. To elucidate whether rearrangements occur within the BCL6 gene, we characterized the genomic structure of the gene. The BCL6 gene encompasses about 26 kilobases (kb) and consists of nine exons. Translation start site is located in exon 3 and zinc-finger motif is distributed in the exons 6 to 9. We have identified at least two types of mRNA alternatively spliced, which contain or do not contain exon 2 of 134 bp coding for the 5' untranslated region. A large intron 1 of 9 kb is not efficiently spliced out, which might result in the creation of minor 10-12-kb transcripts observed in the Northern blot analysis in addition to major 3.8-kb transcripts. The breakpoints are clustered around the first exon, and the putative regulatory region of the BCL6 gene is removed through the translocation, leading to the over-expression of the gene.

Amino Acid Sequence↗
PubMed1993

Functional characterization of the 5'-regulatory region of the human thrombomodulin gene.

Thrombomodulin, a glycoprotein expressed in endothelial cells, has an important role in the blood coagulation system as a modulator. Functional characterization of the 5'-regulatory region of the human thrombomodulin gene was carried out to identify elements necessary for its expression. We used a series of dissected gene constructs containing the bacterial chloramphenicol acetyltransferase gene in transient transfection assays on human umbilical vein endothelial cells. The region extending from -290 to -33 of the 5' end flanking sequence is required for the full expression of this gene. Within this region, four potential Sp1 sites were found, and the sequences of Sp1 sites were mutated to identify their role in the promoter activity of the gene, showing that the two Sp1 sites at -207 and -141 are important for the full activity of the thrombomodulin promoter. Site-directed mutation analysis identified sequence elements GCAATC at -110 as a functioning CAAT box. Another three regions, -290 to -223, -99 to -68, and -67 to -33 have unidentified positively and negatively acting elements. A silencer element was located in the region spanning from -947 to -772 bases of the 5' end flanking region. These data indicate that the expression of the thrombomodulin gene is regulated by various elements which act positively or negatively.

Base Sequence↗
PubMed1993

Consistent involvement of the 3' half part of the first BCR intron in adult Philadelphia-positive leukaemia without M-bcr rearrangement.

About one half of Philadelphia (Ph)-positive acute leukaemia (AL) patients and rare cases of Ph-positive chronic myelogenous leukaemia (CML) show rearrangements within the first intron of the BCR gene on chromosome 22. We studied breakpoints within the first BCR intron in 22 adult patients with Ph-positive leukaemia; 21 with AL and one with CML, which lacked rearrangements within the major bcr (M-bcr). With a series of genomic probes from this intronic region, we detected chromosomal breaks in all 22 patients within the 35 kb area, corresponding almost to the 3' half portion of the intron. The breakpoints were distributed throughout this region but we could not identify any special cluster of breakpoints in this area. Our data support consistent involvement of the 3' half part of the first BCR intron in Ph-positive leukaemias without M-bcr rearrangement, and indicate relatively wide scattering of breakpoints in this portion of the intron.

Adult↗
PubMed1993

Localization of the human alpha 2-plasmin inhibitor gene (PLI) to 17p13.

We previously assigned the human alpha 2-plasmin inhibitor gene (PLI) to 18p11.1-->q11.2 by isotopic in situ hybridization. However, results obtained subsequently via Southern blot analysis of somatic cell hybrids and fluorescence in situ hybridization studies indicate that the gene is located at 17p13. Accordingly, we reassign PLI to 17p13.

Blotting, Southern↗
PubMed1993

[Thrombomodulin as a clinical marker in thrombotic thrombocytopenic purpura. Japan TTP Study Group].

Endothelial cell injury may be common and a fundamental mechanism in thrombotic thrombocytopenic purpura (TTP). Thrombomodulin (TM) is an endothelium-associated cofactor for thrombin-induced protein C activation. TM also abrogates virtually all of thrombin's procoagulant activities. Soluble TM exists in circulating plasma as heterogeneous fragments and is regarded as a molecular marker, reflecting injury of endothelial cells. The plasma TM level is elevated in patients with TTP as compared with healthy subjects. It may reflect damage to vascular endothelial cells or organ failure in patients with TTP. Although, at present, there are no alternative means superior to TM for specifically evaluating the endothelial damage, further studies are needed before soluble TM can be recommended as a standard molecular marker for TTP.

Animals↗
PubMed1993

[Changes and progress in the treatment of TTP--protocols of therapeutic plasma infusion and plasma exchange--therapeutic results by the Japanese and Canadian Study Groups].

Plasma exchange (PE) and plasma infusion (PI) are effective in the treatment of TTP (thrombotic thrombocytopenic purpura). Comparison of PE and PI in the treatment of TTP was carried out by the Canadian Apheresis Study Group, which reported that PE was superior to PI. The Japanese TTP Study Group made a therapeutic protocol in which a smaller volume of Fresh frozen plasma (FFP) was used in the treatment with PI. The efficacy of PE and PI was similar. The results suggest that the volume of FFP necessary for effective treatment is not very large.

Blood Component Transfusion↗
PubMed1992

[Computed tomographic evaluation of advanced gastric cancer].

Pre-operative CT of 75 gastric cancer lesions in 74 patients were evaluated for CT diagnosis of N-factor, n-factor and pancreatic invasion. 1. Sensitivity of CT diagnosis of N-factor was depended upon the size of the lymph node; Group A, where all nodular density within the fat plane of anatomical lymph node location were called abnormal, revealed 73.8% sensitivity. Sensitivity of group B was 69.8%, group C was 60.4% and group D was 28.6%. Pre-education of operators did not significantly affect the sensitivity. 2. CT detected No. 3, 7, 8, 9, 12, 14, and 16 in higher sensitivity, and No. 2, 4, 5 and 15 in lower sensitivity as N-factor. 3. Overall CT sensitivity of n-factor diagnosis was 48.5% and specificity was 95.6% and accuracy was 88.5%. No. 3, 7 and 8 showed higher sensitivity and specificity, but low sensitivity and specificity were noted at No. 1 and 4. 4. Pancreatic invasion was evaluated objectively from ROC, and sensitivity was 80.0% and 73.3% with and without operative findings, respectively.

Adult↗
PubMed1992

Frequent rearrangements of retinoic acid receptor alpha gene and myl gene, and rare mutations of RAS and FMS genes in acute promyelocytic leukemia.

To investigate leukemogenesis of acute promyelocytic leukemia (APL), we studied the involvements of retinoic acid receptor alpha (RAR alpha) and myl genes, and also the frequency of N-RAS, K-RAS, H-RAS, and FMS point mutations in sixteen patients with APL. By Southern blot analysis, the rearrangements of RAR alpha gene were detected in 13 patients (81.2%), and myl gene in 14 (87.5%). Either RAR alpha or myl gene rearrangements were found in all patients including one with normal karyotype. Breakpoints of both genes were clustered. By direct sequencing, no point mutations were found at codons 12, 13, and 61 of N-, K-, and H-RAS genes, and at codons 301 and 969 of FMS gene. These data indicate that myl-RAR alpha translocation occurs frequently in APL, whereas RAS and FMS mutations are rare in APL. It may be suggested that leukemogenesis of APL is different from other subtypes of acute myelogenous leukemia, and multistep leukemogenesis may not be a prevalent feature in APL.

Adolescent↗