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Biomedical subjects

S Mizuno

Publications and source records attributed to S Mizuno.

At least 145 records · Page 8Linked to original sources

The location of Z- and W-linked marker genes and sequence on the homomorphic sex chromosomes of the ostrich and the emu.

Perhaps the most striking fact about early Cenozoic avian history some 70 million years ago was the rapid radiation of large, flightless, ground-living birds. It has been suggested that, for a time, there was active competition between these large terrestrial birds and the early mammals. Probably reflecting the above noted early start of Ratitae of the infraclass Eoaves, the presumptive sex chromosomes of their present day survivors, such as the emu and the ostrich, largely remained homomorphic. The signs of genetic differentiation between their still-homomorphic Z and W chromosomes were tested by using two marker genes (Z-linked ZOV3 and the gene for the iron-responsive element-binding protein) and one marker sequence of a part of a presumptive pseudogene (W-linked EE0.6 of the chicken). Their homologues, maintaining 71-92% identities to the chicken counterparts, were found in both the emu (Dromaius novaehollandiae) and the ostrich (Struthio camelus). Their locations were visualized on chromosome preparations by fluorescence in situ hybridization. In the case of the emu, these three marker sequences were localized on both members of the fifth pair of a female, thus revealing no sign yet of genetic differentiation between the Z and the W. The finding was the same with regard to both members of the fourth pair of male ostriches. In the female ostrich, however, the sequence of the gene for the iron-responsive element-binding protein was missing from one of the pairs, thus revealing the differentiation by a small deletion of the W from the Z.

Animals↗

Molecular cloning, structural characterization, and chromosomal mapping of the human LECT2 gene.

We originally isolated LECT2 (leukocyte cell-derived chemotaxin 2) as a 16-kDa secreted protein having a human neutrophil chemotactic activity, then cloned human and bovine LECT2 cDNAs and demonstrated the liver-specific expression of the protein. LECT2 is thought to be a multifunctional protein, because it was recently found to be identical to chondromodulin-II a growth stimulator of chondrocyte cells. We report here the cloning and the structural analysis of the human LECT2 gene. The gene spans approximately 8 kb and consists of four exons and three introns. Primer extension analysis revealed that several transcription initiation sites occur within 70-230 nucleotides upstream of the translation initiation codon. Several transcriptional control sequences relevant to the liver-specific expression have been identified at the 5' untranslated region of the human LECT2 gene. The human LECT2 gene was mapped to chromosome 5q31.1-q32 by fluorescence in situ hybridization. This region contains a cluster of cytokine genes including IL-4, IL-5, and IL-9.

Base Sequence↗

Molecular cloning of human and bovine LECT2 having a neutrophil chemotactic activity and its specific expression in the liver.

We previously reported the purification and amino acid sequence of a novel neutrophil chemotactic protein termed LECT2 (leukocyte cell-derived chemotaxin 2). In this paper we report molecular cloning of human and bovine LECT2 cDNAs based on the amino acid sequence of the purified protein. The deduced amino acid sequence of human LECT2 (hLECT2) shows an 86% identity to bovine LECT2 (bLECT2). The deduced primary structures of LECT2 were highly homologous to the repeated units of Mim-1 protein (myb induced myeloid protein-1). The mim-1 gene is one of the known myb target genes and is specifically expressed in normal and transformed immature granulocytes in the chicken. Northern blot analysis of normal human tissues demonstrated that the hLECT2 gene is specifically expressed in the adult and fetal livers. In addition, several human hepatoma cell lines also expressed LECT2 mRNA, suggesting that hepatic cells in the liver produce LECT2 protein.

Amino Acid Sequence↗

Granulocyte colony-stimulating factor and its receptor in acute promyelocytic leukemia.

Expression of granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) and in vitro proliferative response to G-CSF were investigated by quantitative immunofluorescence and [3H] thymidine uptake, respectively, in a series of acute myeloid leukemias (AML). The results indicated that G-CSFR was detected at high levels in acute promyelocytic leukemia (APL) cells, in comparison with other types of AML. Moreover, APL cells were also seen to predominantly proliferate in response to G-CSF. Based on these observations, we administered recombinant human G-CSF to a patient with APL in the third relapse that was resistant to both cytotoxic agents and all trans retinoic acid, in an attempt to sensitize the leukemic cells to cell-cycle-dependent agents. Complete remission was achieved. The finding that APL cells are exquisitely responsive to G-CSF supports the view that G-CSF is useful for augmentation of their vulnerability to cell-cycle specific agents.

Adult↗

Unordered arrangement of chromosomes in the nuclei of chicken spermatozoa.

The arrangement of chromosomes in the elongated sperm nuclei of chicken was studied using fluorescence in situ hybridization with probes specific for telomeres of all chromosomes, a microchromosome, the long arm of chromosome 6, the large heterochromatic block on the Z-chromosome, and the same heterochromatic block plus subtelomeric sites on macrochromosomes 1-4. The positions of all probes vary from one sperm to another. No order in chromosome arrangement is apparent. It is suggested that large chromosome size and small chromosome number correlate with constant positions of chromosomes and vice versa. Based on the known quantity of repetitive units of the repeat on the Z-chromosome, the degree of compaction of chromatin in the chicken sperm nucleus is estimated as ca 0.7 Mb/ microm. As judged from the length of the heterochromatic region of the Z-chromosome at the lampbrush stage, the total length of the Z-chromosome in mature sperm is 2.5-4 times that of the sperm nucleus.

Animals↗

A new method to prevent wound infection: a controlled clinical trial in patients with combined liver and bile duct resection.

INTRODUCTION: Despite advances in antibiotic prophylaxis, postoperative wound infection remains a major source of morbidity after digestive surgery. Its prevention is a challenging problem, especially in high-risk patients. The authors introduced a new method to prevent surgical wound infections and evaluated its efficacy in a prospective, randomized trial in markedly high-risk patients. METHODS: Patients with biliary tract carcinoma who were scheduled to undergo combined liver and extrahepatic bile duct resection with biliary reconstruction were randomly assigned to one of two groups, well matched in terms of clinical characteristics at baseline. In one group the new treatment was employed (sealed group, n=31), and in the other the wound was treated in the usual fashion (open group, n=28). In the sealed group, povidone-iodine gel was administered to the subcutaneous tissue, and the skin and peritoneum were approximated with a continuous suture. Wound infection was registered up to 30 days after surgery. RESULTS: Wound infection occurred in 18 patients: 5 (16%) patients in the sealed group and 13 (46%) in the open group (P<0.05). All 18 underwent preoperative percutaneous transhepatic biliary drainage and had positive bile culture findings. In 13 of these 18 patients (72%) the microorganisms isolated from the infected wound were identical to those in the bile. CONCLUSIONS: Our results confirm the close association between infected bile and wound infection in hepatobiliary surgery. Our new method, "direct wound sealing," is simple, easy to perform, virtually cost-free, and has the potential to prevent wound infections even in markedly high-risk patients.

Anti-Infective Agents, Local↗

Flow cytometric analysis of cell cycle fractions in oral leukoplakia.

Flow cytometric analysis of cell cycle fractions and DNA ploidy was performed on 39 biopsy specimens of oral leukoplakia. The aneuploidy rate of these leukoplakias was 9/39 (23.1%) and the mean DNA index of the aneuploid lesions was 1.34. The aneuploidy rate was significantly higher in severely dysplastic lesions (8/17) than in mildly dysplastic (1/15, P<0.02) and nondysplastic (0/7, P<0.05) lesions. No significant differences in the percentages of each cell cycle fraction were seen between the diploid and the aneuploid leukoplakias. However, the S-phase fraction of the severely dysplastic lesions (23.0%) among the diploid leukoplakias was higher than those of the mildly dysplastic (12.4%) and nondysplastic (15.5%) lesions, and the difference between the severely dysplastic and the mildly dysplastic lesions was statistically significant (P<0.001). These results suggest that flow cytometric analysis of cell cycle fractions and DNA ploidy might offer additional information for assessing the malignant potential of oral leukoplakias.

Aneuploidy↗

Determination by modified RT-PCR of transcript amounts from genes involved in sex-steroid synthesis in chicken organs including brain.

Sex steroid hormones in mammals have shown to be synthesized not only in gonads but also in non-steroidogenic organs such as the brain. Steroid hormones in the brain were indicated to be involved in sex behavior and brain differentiation. In avian species, an experimental injection of androgen into the brain suggested the existence of a steroidogenic pathway. However, no studies have demonstrated the expression of genes involved in such a steroidogenic pathway in the brain, or in other non-steroidogenic organs of birds such as the liver and kidney. In this study, we have modified the RT-PCR procedure to analyze the expression of the steroidogenic genes, P-450scc, 3beta-HSD, P-450c17, 17beta-HSD and P-450arom in non-steroidogenic organs of chicken including the brain. The RT-PCR has demonstrated the presence of mRNAs from genes in non-steroidogenic as well as steroidogenic organs of chicken. The amounts of mRNAs from these genes (except for P-450c17) among the non-steroidogenic organs were found to be greatest in the brain.

Animals↗

Specific chromomeres on the chicken W lampbrush chromosome contain specific repetitive DNA sequence families.

Chromomeres 1 and 3 of the chicken W lampbrush chromosome contain most of the EcoRI and XhoI repeat sequence families respectively. These chromomeres were stained with DAPI and their sizes relative to other W chromomeres were observed. Their relative contents of EcoRI and XhoI repeats were determined using fluorescence in situ hybridization with genomic probes for each of the two repeat families. There were two types of W chromosome in the chickens (White Leghorn and Rhode Island Red) used in this study with respect to the amount of EcoRI repeat. A high-copy-number type has about 4000 copies of the 1.2-kb repeat per genome and shows a large fluorescence signal on W chromomere 1. A low-copy-number type has about 700 copies per genome and does not have a detectable chromomere 1 on W chromosome, nor does it show FISH labelling in the region normally occupied by chromomere 1. The genome of Fayoumi chickens has about one-sixth the amount of the XhoI sequence family of White Leghorns. W lampbrush chromomere 3 is much smaller and its FISH labelling with the XhoI probe is much weaker in Fayoumis than in White Leghorns. These results demonstrate that in the chicken W chromosome, specific chomomeres are occupied by specific DNA repeat sequence families.

Animals↗

A panel set for epitope analysis of myeloperoxidase (MPO)-specific antineutrophil cytoplasmic antibody MPO-ANCA using recombinant hexamer histidine-tagged MPO deletion mutants.

A major target protein of antineutrophil cytoplasmic antibody with a perinuclear staining pattern (P-ANCA) has been identified as myeloperoxidase (MPO). Recombinant deletion mutants of MPO, eight fragments of the heavy-chain subunit, and two fragments of the light chain subunit were expressed in E. coli using a pQE expression vector. The recombinant hexamer histidine-tagged fragments were partially purified as the denatured proteins on a Ni2+-charged nitrirotriacetic acid column. The recombinant fragments were reacted with a rabbit polyclonal antibody to human MPO in Western blotting. In addition, the reactivities of the proteins with MPO-ANCA-positive sera of four patients with renal diseases were examined by Western blotting. The profile of the reactivity showed that different sera recognized different sets of fragments of the heavy chain, whereas no serum reacted with the fragments of the light chain. These results indicate that the sera of patients with MPO-ANCA-positive diseases showed varied reactivities with the different fragments. Furthermore, an ELISA system using a set of the fragments completely purified by Sephacryl S-200HR column chromatography was established. The panel set is useful for subclassification of MPO-ANCA-related diseases.

Animals↗

Genes for thrombopoietin and c-mpl are not responsible for familial thrombocythaemia: a case study.

The underlying molecular basis for familial thrombocythaemia (FT). an extremely rare form of primary thrombocythaemia which occurs in an autosomal dominant manner, is currently unknown. We have investigated a family with FT and clarified whether we could detect alteration(s) in the genes coding for c-mpl and its ligand, thrombopoietin (TPO). There was no difference in platelet c-mpl mRNA expression levels between the affected and non-affected individuals in the family. Nucleotide sequence analysis of the c-mpl cDNA for the proband revealed no abnormality. We identified an intragenic tetranucleotide short tandem repeat system in the TPO gene and found non-linkage between the TPO locus and the FT phenotype. We conclude that genes for c-mpl and TPO are not responsible for thrombocythaemia in our FT family.

Adult↗

Suppression of thermotolerance development through cycloheximide-induced negative control of stress protein gene expression.

Expression of a luciferase reporter gene by Chinese hamster ovary cells under the control of the human heat shock protein (hsp) 70 gene promoter was suppressed by incubation at 37 degrees C after treatment with cycloheximide (CHX) during 42 degrees C heat shock exposure. The CHX-induced suppression of hsp gene expression induced no development of thermotolerance. However, 42 degrees C heat shock treatment without CHX followed by CHX inhibition of protein synthesis during recovery incubation at 37 degrees C induced thermotolerance expression by inducing enhanced synthesis of hsps including hsp70 in subsequent heat challenge incubation at 43 degrees C. The results demonstrated a direct correlation between suppression (induction) of stress protein gene expression and non-expression (expression) of thermotolerance. Kinetic analysis showed that the CHX suppression of hsp gene induction was greater than the CHX inhibition of protein synthesis, and that it depended on the severity of heat stress: it decreased with increasing heat stress doses. Moreover, prior feeding of the proline analog L-azetidine 2-carboxylic acid abrogated the CHX-induced suppression of hsp gene expression. In addition, CHX treatment during heat cell-killing at 43 degrees C induced protection of cells. These results were well explained by the proposed model of negative or positive control of stress protein gene expression depending on the level of free hsp70, which may be modulated by both the rate of protein synthesis and the severity of heat stress.

Adaptation, Physiological↗

Induction of granulomas in interferon-gamma gene-disrupted mice by avirulent but not by virulent strains of Mycobacterium tuberculosis.

To gain a better understanding of the pathological role of interferon-gamma (IFN-gamma) in specific granuloma formation, IFN-gamma gene-deficient mice (BALB/c and C57BL/6) were produced. The IFN-gamma gene in embryonic stem (ES) cells was disrupted by inserting the beta-galactosidase gene (lacZ) and the neomycin resistance gene (neo) at the translation initiation site in exon 1 by homologous recombination. Six-week-old IFN-gamma-deficient and wild-type mice were inoculated with 10(3)-10(7) bacilli of various strains of Mycobacterium tuberculosis (Kurono, H37Rv, H37Ra and BCG Pasteur) through their tail veins. The mice were examined 7 weeks later for granuloma formation. The avirulent BCG Pasteur and H37Ra strains (10(3)-10(4) bacilli/ml) induced granulomas in the spleen, liver and lungs of IFN-gamma-deficient mice. The granulomas consisted of epithelioid macrophages and Langhans multinucleate giant cells, but lacked caseous necrosis. The virulent Kurono and H37Rv strains induced disseminated abscesses but not granulomas in various organs of IFN-gamma-deficient mice and Mac-3-positive macrophages were not detected in the abscess lesions. These results suggest that IFN-gamma may be primarily responsible for macrophage activation and that other factor(s) may be involved in the granuloma formation mechanism.

Animals↗

A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II-DNA complex.

Amrubicin is a novel, completely synthetic 9-aminoanthracycline derivative. Amrubicin and its C-13 alcohol metabolite, amrubicinol, inhibited purified human DNA topoisomerase II (topo II). Compared with doxorubicin (DXR), amrubicin and amrubicinol induced extensive DNA-protein complex formation and double-strand DNA breaks in CCRF-CEM cells and KU-2 cells. In this study, we found that ICRF-193, a topo II catalytic inhibitor, antagonized both DNA-protein complex formation and double-strand DNA breaks induced by amrubicin and amrubicinol. Coordinately, cell growth inhibition induced by amrubicin and amrubicinol, but not that induced by DXR, was antagonized by ICRF-193. Taken together, these findings indicate that the cell growth-inhibitory effects of amrubicin and amrubicinol are due to DNA-protein complex formation followed by double-strand DNA breaks, which are mediated by topo II.

Anthracyclines↗

Up-regulation of p27Kip1 correlates inversely with anchorage-independent growth of human cancer cell lines.

We examined the correlation between anchorage-independent growth and cell cycle-related molecules using 39 human cancer cell lines. They consisted of lung-, colon-, stomach-, breast-, ovarian-, brain-, renal- and melanoma-derived cell lines. Their anchorage-independent growth ability varied, but was not clearly related to the tissue of origin. There was a tendency for the levels of cyclin D1, cyclin E, cyclin A, p27, and p21 to show a tissue-dependent expression pattern. Statistical analysis revealed an inverse correlation of the p27 level with anchorage-independent growth (r=-0.456, P<0.01). Thus, the regulation of p27 is suggested to be linked to the anchorage independence of human cancer cells.

Cell Adhesion↗