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Biomedical subjects

S Misawa

Publications and source records attributed to S Misawa.

At least 91 records · Page 5Linked to original sources
PubMed1996

Tumor-specific rearrangements of the immunoglobulin heavy-chain gene in B-cell non-Hodgkin's lymphoma detected by in situ hybridization.

We have recently described the potential use of fluorescence in situ hybridization (FISH) to detect tumor-specific rearrangements of the immunoglobulin heavy-chain (IgH) gene in interphase nuclei. Using yeast artificial chromosome (YAC) clone Y6 containing variable region (VH) gene and bacteriophage clones Ig gamma, we analyzed 70 patients with B-cell non-Hodgkin's lymphoma (NHL) and compared the results with those obtained by the conventional G-banding method. Tumor-specific rearrangements of the IgH gene equivalent to 14q32 translocations were defined as separate signals of VH and Ig gamma genes or those of Ig gamma genes and referred to split signals. Twenty-nine patients (41.4%) showed split signals. Among these, 13 did not show 14q32 translocations by G-banding: three with other chromosomal abnormalities, one with normal karyotype, and nine with no analyzable metaphases. The partner sites of 14q32 translocations were identified in 17 patients by FISH: t(3;14)(q27;q32) including a complex variant was observed in nine patients, t(14;18)(q32;q21) in four, t(8;14)(q24;q32) in three, t(14;19)(q32;q13) in one, and t(11;14)(q13;q32) in one. Six of nine patients with t(3;14) or its variant and one of three with t(8;14) were diagnosed as having respective translocations only by FISH. Translocation t(3;14) was found most commonly, and was correlated histologically with diffuse lymphoma with large-cell components. These results indicate that interphase FISH with IgH gene probes promises to be a rapid and reliable method for use in the diagnosis of B-cell NHL.

Adult↗
PubMed1996

T-cell lymphoma presenting with pericardial and pleural effusion as the initial and primary lesion: cytogenetic and molecular evidence.

A 90-year-old woman was admitted with progressive dyspnea. Chest roentgenogram and computed tomography revealed a massive pericardial effusion and bilateral pleural effusion, but no lymphomatous lesion was seen. A diagnosis of malignant lymphoma was made by cytological and immunological studies of the cells obtained from the pericardial effusion. Chromosome analysis showed a clonal abnormality and T-lineage clonality was determined by the rearrangement of the T-cell receptor gamma gene. The patient achieved remission with chemotherapy, but she later relapsed, with right pleural effusion, and died. She exhibited no lymphomatous features throughout the clinical course, indicating the possibility of malignant lymphoma originating from the pericardium and/or pleura.

Aged↗
PubMed1996

TP53 mutations in myelodysplastic syndrome.

Mutations of the TP53 tumor suppressor gene, contributing to the development and progression of a wide variety of human malignancies, are found in some of the patients with myelodysplastic syndromes (MDS). Previous reports revealed that TP53 mutations were found in 0-25% of patients with MDS and are closely associated with a complex abnormal karyotype including such chromosomal losses as -5/5q-, -7/7q- and/or 17p-, which are known to be frequent in therapy-related leukemias. We have also detected TP53 mutation in 10 (14%) of 70 patients with MDS. All of the mutations were detected at the time of diagnosis, which suggest the TP53 mutation may play a role in the development of MDS. Those patients with a TP53 mutation had a poor prognosis regardless of leukemic transformation or not. The reported mutational spectra of TP53 in MDS and ANLL differ from those of colon and lung cancers. Compared with other hematological disorders, the spectrum of TP53 mutations in MDS and ANLL is assumed to be associated with pathogenic exposure to known or unknown carcinogens, as suggested by the chromosomal findings. Further studies are required to clarify the pathogenesis of this heterogenous disease entity.

Genes, p53↗
PubMed1996

Alterations of CDKN2 gene structure in childhood acute lymphoblastic leukemia: mutations of CDKN2 are observed preferentially in T lineage.

We analyzed homozygous deletions and mutations of the CDKN2(p16(INK4A)/MTS1) gene, using polymerase chain reaction and Southern blot analysis, in 120 children with acute lymphoblastic leukemia (ALL). Homozygous deletion was found in 17 of 89 (19%) precursor B-ALL patients, in 11 of 24 (46%) T-ALL patients, and in 0 of 7 other phenotype ALL patients. After excluding 28 (23%) patients who showed a homozygous deletion of CDKN2, we found that three patients (3%) had mutation at exon 2 of CDKN2 using PCR-SSCP and sequencing strategy. One had a CGA to TGA nonsense mutation (Arg to stop) at codon 72, one had a 1-bp deletion at codon 117, and the third had a 2-bp deletion at codon 70, resulting in frameshifts in the two latter patients. All three of these patients were T phenotype ALL, and the incidence of mutation in the 24 T-ALL patients examined was 13%. In contrast, no mutation was detected in the remaining patients with precursor-B or other type ALL (0/96). Our results suggest that mutational inactivation of the CDKN2 gene may contribute to the leukemogenic growth, especially in some patients with T-ALL.

Adolescent↗
PubMed1996

Primary acute myeloid leukemia cells negative for c-kit receptor protein expression do not proliferate in vitro in response to colony-stimulating factors.

This study examined the effects of recombinant human stem cell factor (SCF) alone or in combination with colony-stimulated factors (CSFs) on colony formation of leukemic progenitors obtained from 29 acute myeloid leukemia (AML) patients. C-kit receptor (c-kitR) protein expression was examined using an immunofluorescence method and was detected on more than 10% of leukemic cells in 20 of 29 cases (c-kitR+). SCF alone could stimulate formation of colonies in 14 of 20 c-kitR+ cases. Granulocyte(G)-CSF, granulocyte-macrophage (GM)-CSF, and interleukin (IL)-3 alone stimulated colony growth in 18, 17 and 16 of c-kitR+ cases, respectively. In contrast, colony and cluster formations from eight of nine c-kitR- cases was not stimulated at all by SCF alone nor SCF in combination with CSF nor with any CSF alone. However, the in vitro culture behavior of fluorescence-activated cell sorter-sorted c-kitR- cells from c-kitR+ cases did not significantly differ from c-kitR+ cells. These results suggest that responses of leukemic progenitors to CSFs were variable but may be predicted by their c-kitR expressions, and that c-kitR(-) cases may have a different disease entity from c-kitR(+) cases.

Adult↗
PubMed1996

Detection and quantification of TEL/AML1 fusion transcripts by polymerase chain reaction in childhood acute lymphoblastic leukemia.

We investigated TEL/AML1 fusion mRNA in 108 children with acute lymphoblastic leukemia (ALL) (86 B-lineage ALL, 15 T-ALL, two mixed lineage ALL, and five other phenotypes) using reverse transcriptase-polymerase chain reaction (RT-PCR). TEL/AML1 transcripts were found in 14 patients (13%) including three relapsed patients, and were unexceptionally limited to B-lineage ALL patients. The incidence of TEL/AML1 transcripts among B-lineage ALL was 16% (14/86). The reciprocal AML1/TEL transcripts were detected in 12 (86%) of the 14 cases expressing a TEL/AML1 transcript. In three cases, the TEL gene was fused to exon 3 of the AML1 gene, and to exon 2 in the remaining cases. To evaluate the amount of TEL/AML1 molecules for the quantification of a minimal residual disease (MRD), a plasmid vector which contained either a long TEL/AML1 PCR product (464 bp) or a short one (425 bp) was used as a competitor. We amplified RNAs obtained from bone marrow (BM) at complete remission or from peripheral blood stem cell (PBSC) harvests in two representative cases. For one PBSC harvest showing a positive result, a competitive PCR was carried out to quantify the amount of MRD. A 1:4 dilution series of competitor vectors was constructed, and each vector was added to a PCR reaction which contain a constant amount of cDNA obtained from the PBSC harvest. An equivalent point was compared to that of corresponding samples at diagnosis. Using this method, MRD in the PBSC harvest was 3.9:10(3). Our results elucidated the incidence, lineage-specificity, and variant forms of TEL/AML1 fusion transcripts in childhood ALL. Since the percentage of other chromosomal translocations in childhood ALL is not more than 5%, TEL/AML1 transcript would be the most feasible clone-specific marker for these patients. In addition, our method could be a powerful tool for quantification of the TEL/AML1 transcript and for the detection of MRD.

Adolescent↗
PubMed1996

Genetic polymorphism of the 3' VNTR region of the human dopaminergic function gene DAT1 (human dopamine transporter gene) in the Mongolian population.

The hypervariable region of the dopamine transporter gene (DAT1) was amplified from samples in the Mongolian population. This region includes a variable number of tandem repeats of a 40-bp core unit in the 3' untranslated region of DAT1. Vandenbergh et al. (1992) reported variability in the number of repeats of this 3' flanking region ranging from 3 to 11 times in white and black populations. We examined polymorphism at the DAT1 locus in 78 native Mongolian subjects. We found alleles with 7 to 13 repeats, which is different from the findings of Vandenbergh et al. (1992). The allele distribution of the Mongolian population is similar to that in the Japanese population, reported previously by Nakatome et al. (1995). Chi-square analysis showed a significant lack of homogeneity between our findings in Mongolian subjects and those reported previously in white and black populations. The DAT1 locus was estimated to have a heterozygosity index of 14.1%, and the polymorphic information content was calculated to be 0.16.

Base Sequence↗
PubMed1996

Microsatellite instability is an early genetic event in myelodysplastic syndrome but is infrequent and not associated with TGF-beta receptor type II gene mutation.

We examined microsatellite instability (MSI) at 10 loci of dinucleotide repeats using the polymerase chain reaction (PCR) in patients with myelodysplastic syndrome (MDS). Bone marrow DNA was obtained from 45 patients repeatedly during the disease course and fibroblast DNA was also collected from 19 of them as a normal control. Three of the 19 patients showed an alteration at more than three loci, when the allele length was compared between their fibroblast DNA and the initial marrow DNA. On the other hand, none of the 45 patients showed an alteration when the initial sample was compared with the latest one. One of the three patients with MSI had refractory anemia and two refractory anemia with ring sideroblasts and none of them showed disease progression, complex chromosome abnormality, karyotypic evolution, or mutation of N-RAS or TP53. Moreover, a frameshift mutation within 10 repeating adenines of transforming growth factor beta type II receptor gene, which was recently recognized as a critical target of MSI, was not found in any of the patients including the three with MSI. These findings suggest that MSI is an early but infrequent genetic event and is independent of other critical genetic aberrations in the development of MDS.

Bone Marrow↗
PubMed1996

Internal tandem duplication of the flt3 gene found in acute myeloid leukemia.

We analyzed mRNA expression of the flt3 gene in 30 patients with acute myeloid leukemia (AML) and 50 with acute lymphoblastic leukemia (ALL). Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of flt3 was observed in 61 patients; 22 (73%) with AML and 39 (78%) with ALL. Among these, five patients with AML (one M2, two M4, and two M5) showed unexpected longer transcripts with a primer combination which could amplify the transmembrane (TM) domain through the juxtamembrane (JM) domain. For those patients who expressed flt3 mRNA, the extracellular domain of the flt3 gene was also examined by RT-PCR, but no length abnormality was seen in this region. We further analyzed the TM domain through the second tyrosine kinase domain by genomic amplifications. The five patients who showed aberrant flt3 transcripts exhibited abnormal longer PCR products in addition to the germline products at a region corresponding to the JM through the first TK (TK1) domains. Sequence analyses of the abnormal RT-PCR products demonstrated that partial sequences were tandemly duplicated. Because all these altered transcripts were in-frame, deduced protein products could be expected. Sequence analyses of the genomic DNA revealed that three of the five patients showed a simple internal duplication within exon 11; one had an internal duplication (26 bp) with a 4-bp insertion; and in the fifth patient, a 136-bp sequence from the 3' part of exon 11 to intron 11 and the first 16-bp sequence of exon 12 were each duplicated with 1-bp insertion. In order to confirm the tumor specificity of these alterations, DNA samples obtained at complete remission were also analyzed in the three patients harboring an flt3 duplication, but no abnormal PCR product other than germline was detected in any of the samples. Our results suggest that an internal tandem duplication at the JM/TK1 domains of the flt3 gene is a somatic change detected preferentially in AML, possibly containing a monocytic component.

Acute Disease↗
PubMed1995

Cloning of cDNA for rat eosinophil major basic protein.

We have determined the complete nucleotide sequence for the cDNA encoding rat eosinophil major basic protein (MBP) using the rapid amplification of cDNA ends (RACE) procedure. The deduced amino acid sequence revealed that the rat prepro-MBP has three functional domains, namely the signal peptide, the acidic peptide that contains numerous acidic amino acids, and the mature MBP, as in human and guinea pig MBP.

Amino Acid Sequence↗
PubMed1995

Simultaneous determination of plasma phenytoin and its primary hydroxylated metabolites in carbon tetrachloride-intoxicated rats by high-performance liquid chromatography.

We have developed a simple and sensitive method for the simultaneous determination of phenytoin (PHT), 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in rat plasma by high-performance liquid chromatography. The three substances were separated on a reversed-phase column (5 microns TSK gel ODS-80TM, 250 mm x 4.6 mm I.D.) using acetonitrile-0.008 M NaH2PO4 (pH 6) (35:65, v/v) as a mobile phase at a flow-rate of 0.8 ml/min. Absorbance was monitored at 215 nm. The quantification limit was 50 ng/ml for each of PHT, m-HPPH and p-HPPH. The mean recoveries for DPH, m-HPPH and p-HPPH from plasma were 95.6 +/- 3.6, 94.5 +/- 4.2 and 98.6 +/- 2.9%, respectively.

Animals↗
PubMed1995

Translocations and amplification of the BCL2 gene are detected in interphase nuclei of non-Hodgkin's lymphoma by in situ hybridization with yeast artificial chromosome clones.

Translocation of the BCL2 gene in B-cell malignancies carrying t(14;18) and amplification of the BCL2 gene in a cell line (HBL-2) derived from a non-Hodgkin's lymphoma (NHL) were detected specifically in both metaphase spreads and interphase nuclei by fluorescence in situ hybridization (FISH) using yeast artificial chromosomes (YACs). A YAC clone containing the BCL2 gene yA153A6, a 360-kb clone spanning from approximately 60 kb upstream of BCL2 exon 1 to approximately 60 kb 3' of the minor breakpoint cluster region, was used for single-color FISH analysis. Seven patients with NHL and one patient with acute lymphoblastic leukemia were analyzed for BCL2 translocations. Interphase nuclei of NHL patients showed three signals when hybridized with the yA153A6 probe. This was expected because the YAC clone spans the BCL2 breakpoint regions on 18q21.3. In a patient with acute lymphoblastic leukemia, a positive signal for BCL2 was detected on der(14) at band 14q32.33 by single-color FISH with the yA153A6 probe, whereas no signals were detected on der(18). The amplification of BCL2 in the HBL-2 cell line was observed on a characteristic abnormal chromosome 18, add(18)(q23); the periodic pattern of the fluorescent signal of this region was suggestive of an amplicon. Using double-color FISH with YAC clones containing the more centromeric 18q21.3 gene gastrin-releasing peptide (y302F10) and the 14q32.33 gene (IgH; Y6), we detected t(14;18) by showing the juxtaposition of the 18q21.3 and 14q32.33 bands on the derivative chromosome 18. Interphase FISH with these YAC clones provided a rapid procedure for the diagnosis of B-cell malignancies carrying t(14;18). In addition, we showed that translocations and amplification of the BCL2 gene can be detected at the single-cell level.

Adult↗
PubMed1995

Microbial secretion of biologically active human transforming growth factor alpha fused to the Streptomyces protease inhibitor.

A secretory production system for the active form of transforming growth factor alpha (TGF alpha) was established in Streptomyces lividans using a gene encoding the secretory protease inhibitor, Streptomyces subtilisin inhibitor (SSI). It was demonstrated that deletion of one of the putative dual ssi terminators is effective to extracellularly produce a heterologous polypeptide in a fused form. The recombinant fusion protein, SSI::TGF alpha, was purified to homogeneity by a combination of hydrophobic chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). It was noteworthy that the SSI::TGF alpha hybrid protein exhibited bifunctional activity: the TGF alpha activity for cell growth promotion and the inhibitory activity of SSI. Taken together with the results of analytical gel filtration, these findings strongly indicate that each moiety in the fusion protein correctly folds and the whole hybrid molecule exists in a dimeric form, which results in its bifunctional activity.

Bacterial Proteins↗
PubMed1995

Interphase and metaphase detection of the breakpoint of 14q32 translocations in B-cell malignancies by double-color fluorescence in situ hybridization.

The breakpoint of 14q32 translocations found in B-cell malignancies was delineated specifically in both metaphase spreads and interphase nuclei by double-color fluorescence in situ hybridization (FISH) using bacteriophage clones containing the human immunoglobulin gamma chain gene locus (Ig gamma) and a cosmid clone, CY24-68, containing VH segments. CY24-68 is more telomeric than Ig gamma, separated by approximately 1 megabase (Mb). FISH studies were performed on four patients with non-Hodgkin's lymphoma (NHL), one with acute lymphoblastic leukemia (ALL), one with plasma cell leukemia (PCL), and three cell lines. In each patient with t(8;14), t(14;18), and t(3;14), the signal of Ig gamma gene was observed on der(14) and that of CY24-68 at respective partner sites of these translocations, 8q24.1, 18q21.3, and 3q27. Interphase nuclei with a signal of Ig gamma clearly separated from that of CY24-68 were more frequently encountered in all of the patients (45% to 74%) than those in normal controls (4% to 5%). Even in cases where only interphase nuclei were available for FISH studies, 14q32 translocations are detected as shown in two patients each with NHL and t(11;14)-carrying PCL. In two cell lines, HS-1 derived from ALL carrying t(8;14) and FR4 derived from a plasmacytoma carrying a complex form of t(8;14), the signal of Ig gamma was observed at the breakpoint region 8q24.1 of the der(8) in addition to the der(14), indicating that translocation event occurred within the Ig gamma locus. Intense Ig gamma signal was found at the breakpoint region on the der(14)t(11;14) in HBL-2 derived from NHL, indicating amplification of the Ig gamma gene, and presumably the resultant chimeric DNA between Ig gamma and DNA sequences at 11q13. The present approach allowed us to unequivocally detect tumor-specific breakpoints of 14q32 translocations. Furthermore, interphase FISH provides a rapid diagnostic procedure to detect 14q32 translocations in B-cell malignancies.

Burkitt Lymphoma↗
PubMed1995

TP53 mutations emerge at early phase of myelodysplastic syndrome and are associated with complex chromosomal abnormalities.

We examined TP53 mutation in 57 patients with myelodysplastic syndrome (MDS) at either the MDS phase or at the terminal leukemic phase using polymerase chain reaction-mediated single-strand conformation polymorphism (PCR-SSCP) analysis. TP53 mutations within exons 5 through 8 were found in seven patients. All these mutations were detected at the presentation of MDS whether these patients showed leukemic transformation or not. TP53 mutations were frequently found in patients with loss of the short arm of chromosome 17 (17p-) (three of seven patients with 17p-, 43%) and complex karyotypic abnormalities (five of 14, 38%). Among the seven patients with the TP53 mutation, four patients progressed to acute leukemia within 7 months from the diagnosis of MDS, and the remaining three died within 7 months without leukemic transformation. These findings suggest that mutations of the TP53 can be implicated in leukemic transformation and a poor prognosis in MDS.

Acute Disease↗
PubMed1995

Copper metabolism leading to and following acute hepatitis in LEC rats.

The accumulation process of copper (Cu) in the liver and the following metabolic disorder of Cu were examined in LEC rats, a mutant strain which accumulates Cu with age and shows spontaneous acute hepatitis and/or hepatoma. Cu concentration in the liver of female rats was approximately 220 micrograms/g liver at 2 weeks of age, decreased to 100 micrograms/g liver at 4-6 weeks, and then started to increase with age linearly to the highest concentration of 250 micrograms/g liver at 16 weeks. Although the Cu level expressed by concentration (microgram/g liver) decreased during weaning, it increased linearly with age when it was expressed by content (mg/liver), indicating a constant and preferential accumulation of Cu in the liver. Cu concentration stopped increasing at 16 weeks in the liver, followed by a sudden decrease to 1/2 the highest level. Biological markers (serum lactate dehydrogenase and glutamic-oxaloacetic transaminase activities) for liver damage started to increase, together with the appearance of signs of jaundice, when Cu attained the highest concentration. Distributions of Cu and zinc (Zn) in the supernatant fraction of the liver indicated that both metals were mostly distributed to metallothionein (MT) and, to a small extent, to superoxide dismutase on a gel filtration column throughout the course of the experiments. Serum Cu concentration started to increase in a form of ceruloplasmin, together with serum marker enzyme activities for liver damage. Cu concentration in the kidneys also started to increase after the increase of serum Cu. The results indicate that Cu accumulates in the form of MT in the liver of LEC rats to a maximum level of approximately 250 micrograms/g liver, and then decreases suddenly with the onset of acute hepatitis. The maximum level seems to be related to the capacity of MT synthesis, and acute hepatitis is assumed to occur when Cu accumulates beyond the capacity. Serum Cu started to increase, from the abnormally low level, when the metal accumulated beyond the capacity of MT synthesis in the liver, and it was partly reabsorbed by the kidneys and the rest was excreted into urine. Changes in iron and zinc levels were determined and discussed in relation to those of Cu.

Acute Disease↗