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Biomedical subjects

S Misawa

Publications and source records attributed to S Misawa.

At least 127 records · Page 7Linked to original sources
PubMed1994

Nonrandom chromosomal rearrangements of 14q32.3 and 19p13.3 and preferential deletion of 1p in 21 patients with multiple myeloma and plasma cell leukemia.

Structural chromosomal abnormalities and their break-points were characterized in 17 patients with multiple myeloma (MM) and 4 with plasma cell leukemia by banding. Chromosome 14q32 translocations with a variety of partners were detected in 13 patients, and a variant translocation t(8;22)(q24.1;q11) was detected in 1. Three recurrent 14q32 translocations have been identified: t(6;14)(p21.1;q32.3) occurring in 3 cases, and t(11;14)(q13;q32.3) and t(14;18) (q32.3;q21.3) each occurring in 2 cases. Translocations t(1;14)(q21;q32.3), t(3;14)(p11;q32),t(7;14)(q11.2;q32.3), and t(11;14)(q23;q32.3) were found in each patient, whereas in the remaining 2 patients, partner chromosomes could not be determined. The band 19p13.3 was newly delineated as a recurrent breakpoint involved in translocations in MM. Chromosomes 1 and 6 were also commonly involved in structural abnormalities (14 and 10 patients, respectively), although no particular bands were noted. However, the short arm of chromosome 1 was preferentially involved in deletion, suggesting a certain antioncogene on 1p associated with the development of myeloma. In addition; fluorescence in situ hybridization was successfully applied to determine the nature of the structural abnormalities in a patient with t(8;22) translocation. The present findings suggest that there may be subsets of 14q32 translocations specific to MM.

Adult↗
PubMed1994

High-level expression of Mycoplasma arginine deiminase in Escherichia coli and its efficient renaturation as an anti-tumor enzyme.

The arginine deiminase (AD) gene was cloned from Mycoplasma arginini and expressed in the cytosol of Escherichia coli as inclusion bodies with an expression level of at least 20% of the total bacterial proteins. The inclusion bodies were solubilized with 6 M guanidine hydrochloride (Gdn-HCl) under reducing conditions, in order to avoid incorrect disulfide-bond formation of the recombinant (r-) AD molecules, and renaturation was performed under various refolding conditions. The optimum renaturation conditions were found to be incubation for 90 h at pH 7.5 and 15 degrees C. The resulting completely refolded r-AD was purified to homogeneity by anion-exchange and arginine-affinity chromatography and its activity yield was 72.5%. The specific activity of the purified r-AD was comparable to and its amino acid composition was identical to those of Mycoplasma AD, and NH2-terminal sequence analysis revealed that its methionine residue corresponding to the translation initiation codon had been removed completely. Anti-tumor activity analyses showed that r-AD inhibited the growth of two mouse cell lines, hepatoma MH134 and fibrosarcoma Meth A, strongly in vitro at concentrations in excess of 10 ng ml-1. Moreover, when MH134-implanted mice were given single intravenous injections of r-AD at doses of 50 mg kg-1 and higher, their survival times were prolonged significantly. These results, taken together, indicate that the enzymatic properties and biological actions of r-AD were highly consistent with those of Mycoplasma AD.

Amino Acid Sequence↗
PubMed1994

Enzymic O-sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A-44.

The enzymic O-sulfation of Tyr residues in a recombinant hirudin variant-1 (rHV-1) and its analog in which Glu61 and Glu62 were replaced by Tyr, [E61Y, E62Y]rHV-1, was carried out by use of sulfotransferase isolated from an anaerobic bacterium from the human intestine, Eubacterium A-44. Although rHV-1 was not sulfated by this enzyme, the sulfation of [E61Y, E62Y]rHV-1 was observed, and three kinds of sulfated analog, whose C-terminal six amino acid residues were -PYY(SO3H)YLQ, -PYYY(SO3H)LQ, and -PYY(SO3H)Y(SO3H)LQ, were obtained. Among the sulfated hirudin analogs tested here, the Tyr62 and Tyr63 bisulfated [E61Y, E62Y]rHV-1 showed the strongest thrombin inhibition with the inhibition constant (Ki) of 0.0430 pM, followed by the Tyr63 monosulfated analog (Ki = 0.0593 pM) and the Tyr62 monosulfated one (Ki = 0.158 pM). The Tyr63 monosulfated analog and Tyr62 and Tyr63 bisulfated one were more potent inhibitors of thrombin than unsulfated rHV-1. The increase in affinity caused by sulfation was predominantly due to an increase in the association-rate constant.

Amino Acid Sequence↗
PubMed1994

Detection of 14q32 translocations in B-cell malignancies by in situ hybridization with yeast artificial chromosome clones containing the human IgH gene locus.

Partner sites of 14q32 translocations found in B-cell malignancies were detected by fluorescence in situ hybridization (FISH) using yeast artificial chromosome (YAC) clones, Y20 and Y6, containing the human Ig heavy chain (IgH) gene locus. Y20 spans a 160-kb upstream and 40-kb downstream region of the JH segments on chromosome band 14q32.33. Y6 is 300-kb upstream of Y20, and spans a further 320-kb telomeric region. The human DNA sequences amplified by Alu polymerase chain reaction of the YAC clones were used as probes for FISH to study six patients with non-Hodgkin's lymphoma (NHL), one patient with acute lymphoblastic leukemia, and one cell line FR4 established from a plasmacytoma. Three telomeric YAC clones each specific for 3q, 8q, and 18q were also used to further characterize 14q32 translocations. The IgH YACs were successfully applied to detect cytogenetically invisible subtelomeric translocation of the IgH gene locus to each partner site in t(14;18), t(8;14), and t(14;19), and to identify t(3;14) (q27;q32.33) in three patients with 14q32 translocation of unknown origin. Furthermore, complex translocations involving more than three chromosomes were detected in an NHL patient with t(8;14), and t(3;12), and in the FR4 with der(14)t(8;14), der(8)dic(1;8), and del(1)(q21). The technique would be a useful tool in elucidating the mechanisms of a 14q32 translocation in B-cell malignancies.

Aged↗
PubMed1994

High-level expression of porcine muscle adenylate kinase in Escherichia coli: effects of the copy number of the gene and the translational initiation signals.

Porcine muscle adenylate kinase (ADK) was overproduced in Escherichia coli using the expression plasmid with double A-T-G codon at the translational starting site and the Shine-Dalgarno (SD) sequence 10 bp apart from the first A-T-G. We used the expression vectors pKK223-3 and pMK2. pMK2 is about 10-20 times larger in copy number than pK223-3. For both vectors, duplication of A-T-G was effective and the quantity of the expressed ADK from the double A-T-G plasmid was 2 approximately 4-fold more than that achieved when only one A-T-G was present. The amount of the produced ADK was maximum in the case of using pMK2 with double A-T-G. The overproduced ADK formed inclusion bodies in E. coli. It was solubilized in 6 M guanidine hydrochloride and refolded. Through two steps of column chromatography, ADK was purified. It has the same amino acid composition and grossly the same activity as that reported by Schirmer et al. (1970). Its amino acid sequence of the NH2-terminal region was identical with that deduced from the cDNA sequence including the NH2-terminal methionine.

Adenylate Kinase↗
PubMed1994

Trimethadione metabolism, a useful indicator for assessing hepatic drug-oxidizing capacity.

The metabolism of trimethadione (TMO), a useful indicator of hepatic drug-oxidizing capacity in rats and humans, was studied using 14 different forms of rat cytochrome P450 (CYP1A1, 1A2, 2A1, 2A2, 2B1, 2B2, 2C6, 2C7, 2C11, 2C12, 2C13, 2E1, 3A2 and 4A2) and three forms of human cytochrome P450 (CYP1A2, 2C and 3A4). TMO N-demethylation was increased by treating rats with phenobarbital. CYP2C11 and 2B1 had high TMO N-demethylase activity, but 1A1 and 1A2 had low activity. Antibodies raised to CYP2C11 and 2B1/2 inhibited TMO N-demethylation in hepatic microsomes of untreated and phenobarbital-treated rats, respectively. In a reconstituted system, human CYP3A4 and 2C produced efficiently dimethadione (DMO), but CYP1A2 did not catalyse TMO N-demethylation. Antibodies raised to CYP3A2 and 2C11 inhibited TMO N-demethylation in human hepatic microsomes. These results indicated that the N-demethylation of TMO is catalysed mainly by CYP2C11 and 2B1 in rat hepatic microsomes, and that human CYP3A4 and an unspecified isoform of the 2C subfamilies contribute to TMO N-demethylation in human liver.

Animals↗
PubMed1994

Myelomonocytic crisis with t(5;17) and a p53 mutation in a patient with chronic myelogenous leukemia.

We report a 64-year-old Japanese man with chronic myelogenous leukemia (CML) who expired with myelomonocytic crisis. Cytogenetic analyses of chronic phase (CP) and accelerated phase (AP) cells revealed a Philadelphia chromosome and an isochromosome for the long arm of chromosome 17, i(17q). This karyotype was replaced by another karyotype in blast crisis (BC), resulting in near triploidy with t(5;17) (p15;p11) and loss of chromosome 17 pter-->p11. Interphase fluorescent in situ hybridization studies with a chromosome 17 specific alpha satellite DNA probe confirmed the presence of a clonal change in BC. In addition, single-strand conformation polymorphism analysis and PCR-direct sequencing of BC cells revealed a point mutation at codon 203 of the p53 gene, GTG to GAG (Val to Glu), and loss of the normal allele. In contrast, no alterations of the p53 gene were found in CP and AP cells. Therefore, progression of CML in this patient appeared to be related to loss of 17p, as well as a mutation in the p53 gene.

Alleles↗
PubMed1994

Polymorphism of pentanucleotide repeats in the 5' flanking region of glutathione S-transferase (GST) pi gene.

The upstream sequence of the glutathione S-transferase pi gene contains pentanucleotide (ATAAA) repeats. Analysis of the region using polymerase chain reaction indicated that the repeat sequence was polymorphic and segregation of the polymorphic alleles was codominant heredity. Heterozygosity of the new VNTR was 0.818 in healthy Japanese and 0.794 in American whites. Allelic frequencies among healthy controls and alcoholics as well as other diseases were not significantly different.

Alleles↗
PubMed1994

Prognostic significance of loss of a chromosome 17p and p53 gene mutations in blast crisis of chronic myelogenous leukaemia.

In 31 cases of chronic myelogenous leukaemia (CML) we examined the prognostic significance of chromosomal loss of a 17p and p53 mutations at the onset of blast crisis (BC). p53 mutations were closely related to a shortened survival in CML-BC (P < 0.005 by the logrank test), whereas loss of a 17p by itself was not a poor prognostic indicator. The prognostic significance of loss of a 17p, however, emerged when combined with its predominance in the metaphases analysed. This predominance might easily and rapidly be screened by polymerase chain reaction-based analysis in about half of the cases.

Adolescent↗
PubMed1994

Multiple aberrant splicing of the p53 transcript without genomic mutations around exon-intron junctions in a case of chronic myelogenous leukaemia in blast crisis: a possible novel mechanism of p53 inactivation.

We found three truncated p53 transcripts in a patient with chronic myelogenous leukaemia in blast crisis carrying chromosome 17 abnormalities. Sequencing of these transcripts revealed complete absence of the entire exons 7, 8 and 9 in one, exons 8 and 9 in another, and exon 10 in the other. Sequencing analysis of genomic DNA, however, revealed no mutation in exons 6-10 and their flanking introns. These results suggest that the aberrant p53 transcripts in this case might not result from splicing mutations but from an unknown affected splicing process.

Base Sequence↗
PubMed1994

[A case of Corynebacterium jeikeium septicemia].

We report a case of Corynebacterium jeikeium septicemia associated with malignant lymphoma. The patient is a 58-year-old male who was diagnosed as malignant lymphoma on August 1992. May 15, 1993, he was admitted to our hospital because of oliguria, abdominal flatulence and vomiting which developed a few days before admission. Anticancer regimen were started. In the middle of July, white blood cell (WBC) count dropped to 100/mm3 and body temperature rose to 39 degrees C. He was been treated with Ceftazidime and Piperacillin. C. jeikeium was recovered from blood culture. Antibiotics were switched to minocycline and vancomycin. He died of septic shock and pneumonia. Autopsy revealed the presence of the colonies of Rods. Which were morphologically compatible with C. jeikeium were observed in lung tissue and in the small pulmonary vessels.

Bacteremia↗
PubMed1994

[Distribution of pulmonary neuroendocrine cells--an immunohistochemical study of the lung in autopsied infants including sudden infant death syndrome].

The distribution of pulmonary neuroendocrine cells (PNEC) was analyzed immunohistochemically in 14 victims of sudden infant death syndrome (SIDS), and 10 cases of infant death unrelated to SIDS, excluding congenital heart disease. Lung tissue sections were immunostained with antibodies against chromogranin A (CGA), calcitonin (CT) and gastrin-releasing peptide (GRP). CT/GRP immunoreactivity decreased in older infants of each group, while CGA immunoreactivity showed almost no decrease. Serial section analysis showed some PNEC produced CGA, CT and GRP. However, CGA-immunoreactive PNEC sometimes lacked of CT/GRP immunoreactivity. The difference of PNEC distribution between SIDS and the control cases could not be verified. To date, there have been no studies reported of PNEC distribution in infants by using CGA expression. CGA is considered to be the most useful marker for detecting PNEC in infant lung. Our findings suggest that substances produced by PNEC changed with postnatal development both in SIDS and the control group. This result may be one clue to clarifying the development and function of small airways in infants, allowing further progress in SIDS research.

Calcitonin↗
PubMed1994

Sex identification of old skeletal remains by DNA analysis: the report of three cases.

We analyzed the DNA in the remains of three skeletons affected by severe post-mortem modification to identify the sex of the cases. We analyzed DNA extracted from bone, bone marrow and a tooth by Southern blot hybridization with Y chromosome specific probe and PCR to amplify the sex chromosome specific sequence. The first case had been preserved underground in a deep grave for two months, the second case was found in a forest after more than five years, and the last case had been kept in a styrofoam box for few years. The detective sensitivity of Southern hybridization improved to nearly the same level as PCR with the aid of computed image processing. Although PCR was of obvious benefit when analyzing small amounts of biological evidence, amplification was impossible in case the target sequence became lost or changed. As a result, we identify the sexes of the three cases by Southern blot hybridization or PCR using bone, bone marrow and tooth DNA. The old skeletal remains were suitable for sex identification in spite of severe post-mortem change, due to preservation in a condition protected from external influences.

Adult↗
PubMed1994

[Application of DNA polymorphism to forensic medicine].

Recent recombinant DNA technology has provided some novel and powerful methods for forensic science application. Human genomic DNA can be analyzed directly for individual identification and paternity testing on the basis of variations in its sequence. The analysis of DNA isolated from forensic biological evidence provides valuable information relating to the identification of the source of the sample. Restriction fragment length polymorphism (RFLP) testing, using a combination of single locus probe (SLP) that vary highly among individuals, produces a DNA fingerprint or profile. The PCR method using STR (microsatellite) and mitochondrial DNA analysis (mtDNA) is suitable for examination of the forensic biological samples (bloodstains, hairs, seminal stains, bones, tooth). For sex identification of bloodstains, bleached skeletons and teeth, Southern blot hybridization with Y-chromosome specific probe (pHY10; 3.4 kb) and PCR amplifying with sex chromosome specific fragments, can be used. Mitochondrial DNA RFLPs and 9-bp deletion type of mtDNA may suggest the characteristics of the human races. For the purpose of resolving the genetic basis of human uniqueness, we reported the isolation of human-specific sequences using the technique of genome subtraction (HS5, 2282 bp).

Animals↗