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Biomedical subjects

Y Mukai

Publications and source records attributed to Y Mukai.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of genes for a pollen allergen, Cry j 2, of Cryptomeria japonica.

BACKGROUND: Cry j 2 is one of the major pollen allergens of Cryptomeria japonica. The polymorphism of Cry j 2 isoforms and the conservation of the structure of Cry j 2 in coniferous species remain to be analyzed. METHODS: A cDNA library derived from the pollen of C. japonica was screened using a fragment of Cry j 2 cDNA. Restriction fragment length polymorphism analysis was performed to examine the diversity of Cry j 2 genes. The promoters of Cry j 2 genes were isolated with a commercially available cloning kit. Clonal variations in the expression of Cry j 2 in pollen were examined by RNA gel blot analysis, and the conservation of the structure of the Cry j 2 gene in coniferous species was evaluated by DNA gel blot analysis. RESULTS: We isolated three cDNA clones encoding novel isoforms of Cry j 2. We also sequenced a total of 16 promoter regions from 10 specimens. The sequences of promoter regions of Cry j 2 genes were highly divergent. The amount of Cry j 2 mRNA also varied considerably. The Cry j 2 gene was found to be conserved among species belonging to Taxodiaceae and Cupressaceae but to vary between Taxodiaceae and Pinaceae. CONCLUSIONS: The coding and promoter regions of Cry j 2 genes contain large numbers of polymorphisms. Our analysis revealed large variations in the expression of Cry j 2 at the transcriptional level, and we suggest that conserved homologs of Cry j 2 confer cross-allergenicity among Taxodiaceae and Cupressaceae.

Allergens↗
PubMed2006

C3-6 laminoplasty takes over C3-7 laminoplasty with significantly lower incidence of axial neck pain.

Five-lamina (C3-7) procedure is the most popular cervical laminoplasty and there have been no studies on the most appropriate number of laminae to be opened. We prospectively reduced the range of laminoplasty from C3-7 to C3-6 in 2002 and compared the outcome of C3-6 laminoplasty (n=37) to that of C3-7 laminoplasty (n=28). In both groups, neurological gain was satisfactory, radiographic changes were minimal, and postoperative MRI indicated sufficient expansion of the dura and the spinal cord. Average operating period was significantly shorter, and length of the operative wound was significantly less in the C3-6 group than in the C3-7 group. Postoperative axial neck pain was significantly rarer after C3-6 laminoplasty than after C3-7 laminoplasty (5.4% vs. 29%, P=0.015). Due to its simplicity and various benefits, C3-6 laminoplasty is a promising alternative to conventional C3-7 laminoplasty for treatment of multisegmental compression myelopathy.

Adult↗
PubMed2006

Optimization of anti-tumor necrosis factor-alpha single chain Fv displayed on phages for creation of functional antibodies.

In this study, we converted the immunoglobulin-type anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (Mab) to a scFv-type antibody in order to assess its basic properties. The immunoglobulin VH and VL genes were isolated from the hybridoma that produced an anti-TNF-alpha neutralizing Mab, and they were then linked together to create scFvs of the VL-VH or VH-VL-form. The binding affinity to TNF-a was retained in both scFvs. Interestingly, the VL-VH-type scFv effectively inhibited the TNF-alpha-mediated cytotoxicity, while this neutralization activity was dramatically decreased in the VH-VL-type scFv. These results suggest that the VL-VH-type scFv is a suitable template to create improved versions of the anti-TNF-alpha antibody using a phage display system, and they also show that the structural format must be taken into account in manufacturing scFvs.

Antibodies, Blocking↗
PubMed2005

High-resolution physical mapping of the secalin-1 locus of rye on extended DNA fibers.

High-resolution mapping of secalin-1 (Sec-1) locus has been performed by fluorescence in situ hybridization to extended DNA fibers of rye (Secale cereale, 2n = 14), employing DNA probes of lambda phage clones containing the omega-secalin gene. The fluorescent signals to rye extended DNA fibers revealed continuous strings of 45 microm, corresponding to the size of 147 kb DNA. To determine the copy number of Sec-1 locus on DNA fibers, a 1.2-kb fragment including the entire coding region of the omega-secalin gene and a 1.0-kb fragment of the promoter region were amplified by PCR as probes for another fiber FISH. The physical position of these sequences was visualized as alternating fluorescent spots by multicolor in situ hybridization. Alternating signals of two DNA probes reflected the tandem repeated organization of the Sec-1 locus having 15 copies of the gene. The present findings based on fiber FISH analysis support the contention that the omega-secalin genes are arranged in a head-to-tail fashion separated by 8 kb of spacer sequences with a total length of 145 kb.

Base Sequence↗
PubMed2004

In situ chromosomal localization of rDNA sites in "Safed Musli" Chlorophytum ker-gawl and their physical measurement by fiber FISH.

Fluorescence In Situ Hybridization (FISH) technique has been applied on somatic chromosomes and extended DNA fibers in the medicinally important species of Chlorophytum to elucidate physical localization and measurement of the rDNA sites using two rRNA multigene families homologous to 45S and 5S rDNA. The two species of Chlorophytum, namely C. borivillianum and C. comosum, both with 2n = 28, reveal diversity for copy number and localization of rDNA sites. C. borivillianum is comprised of five 45S-rDNA sites:one each in the secondary constriction region of chromosomes 7, 8, 9; one in the subtelomeric region of the short arm of chromosome 2 and the telomeric region of the short arm of chromosome 12; and one 5S-rDNA site in the subtelomeric region of the long arm of chromosome 1. In C. comosum, there are three 45S-rDNA sites (one each in the short arm of chromosomes 12, 13, and 14) and two 5S-rDNA sites (in the secondary constriction regions of chromosomes 2 and 13). Fiber FISH analysis conducted on extended DNA fibers revealed variation in the size of continuous tandem strings for the two r-DNA families. Taking the standard value of native B DNA equivalent to 3.27 kb for 1 mum, it was estimated that the physical size of continuous DNA strings is of the order of approximately 90 kb, 180 kb, and 300 kb for 45S-rDNA and of the order of 60 kb, 150 kb for 5S-rDNA in C. comosum, grossly in correspondence to their respective physical sizes at metaphase.

Asparagaceae↗
PubMed2004

Allelic diversity of S-RNase at the self-incompatibility locus in natural flowering cherry populations (Prunus lannesiana var. speciosa).

In the Rosaceae family, which includes Prunus, gametophytic self-incompatibility (GSI) is controlled by a single multiallelic locus (S-locus), and the S-locus product expressed in the pistils is a glycoprotein with ribonuclease activity (S-RNase). Two populations of flowering cherry (Prunus lannesiana var. speciosa), located on Hachijo Island in Japan's Izu Islands, were sampled, and S-allele diversity was surveyed based on the sequence polymorphism of S-RNase. A total of seven S-alleles were cloned and sequenced. The S-RNases of flowering cherry showed high homology to those of Prunus cultivars (P. avium and P. dulcis). In the phylogenetic tree, the S-RNases of flowering cherry and other Prunus cultivars formed a distinct group, but they did not form species-specific subgroups. The nucleotide substitution pattern in S-RNases of flowering cherry showed no excess of nonsynonymous substitutions relative to synonymous substitutions. However, the S-RNases of flowering cherry had a higher Ka/Ks ratio than those of other Prunus cultivars, and a subtle heterogeneity in the nucleotide substitution rates was observed among the Prunus species. The S-genotype of each individual was determined by Southern blotting of restriction enzyme-digested genomic DNA, using cDNA for S-RNase as a probe. A total of 22 S-alleles were identified. All individuals examined were heterozygous, as expected under GSI. The allele frequencies were, contrary to the expectation under GSI, significantly unequal. The two populations studied showed a high degree of overlap, with 18 shared alleles. However, the allele frequencies differed considerably between the two populations.

Alleles↗
PubMed2003

The organization of genes tightly linked to the Ha locus in Aegilops tauschii, the D-genome donor to wheat.

The grain hardness locus, Ha, is located at the distal end of the short arm of chromosome 5D in wheat. Three polypeptides, puroindoline-a, puroindoline-b, and grain softness protein (GSP-1), have been identified as components of friabilin, a biochemical marker for grain softness, and the genes for these polypeptides are known to be tightly linked to the Ha locus. However, this region of the chromosome 5D has not been well characterized and the physical distance between the markers is not known. Separate lambda clones containing the puroindoline-a gene and the puroindoline-b gene have been isolated from an Aegilops tauschii (the donor of the D genome to wheat) genomic lambda library and investigated. Considerable variation appears to exist in the organization of the region upstream of the gene for puroindoline-b among species closely related to wheat. Using in situ hybridization the genes for puroindoline-a, -b, and GSP-1 were demonstrated to be physically located at the tip of the short arm of chromosome 5 of A. tauschii. Four overlapping clones were isolated from a large-insert BAC library constructed from A. tauschii and of these one contained genes for all of puroindoline-a, puroindoline-b, and GSP-1. The gene for puroindoline-a is located between the other two genes at a distance no greater than approximately 30 kb from either gene. The BAC clone containing all three known genes was used to screen a cDNA library constructed from hexaploid wheat and cDNAs that could encode novel polypeptides were isolated.

Chromosomes, Plant↗
PubMed2003

The sugary-type isoamylase gene from rice and Aegilops tauschii: characterization and comparison with maize and arabidopsis.

Genes for an isoamylase-like debranching enzyme have been isolated from rice and Aegilops tauschii, the donor of the D genome to wheat. The structures of the genes are very similar to each other and to the maize SU1 isoamylase gene and consist of 18 exons spread over approximately 7.5 kb. Southern analysis and fluorescent in situ hybridization showed the Ae. tauschii gene to be located in the proximal region of the short arm of chromosome 7D, thus showing synteny with the localization of the rice isoamylase gene on rice chromosome 8. Analysis of the expression pattern of wheat sugary isoamylase genes indicates that they are strongly expressed in the developing endosperm 6 days after flowering. Three distinct Sugary-type cDNA sequences were isolated from the wheat endosperm that are likely to correspond to the products of the three genomes. The deduced amino acid sequence of rice and wheat Sugary-type isoamylase is compared with other sequences available in the database and the results demonstrate that there are three types of isoamylase sequences in plants: those containing 18 exons (the Sugary-type isoamylase gene), those containing 21 exons, and those containing only 1 exon. It is possible that different combinations of isoamylase genes are expressed in different tissues.

Amino Acid Sequence↗
PubMed2003

Extended chromatin and DNA fibers from active plant nuclei for high-resolution FISH.

The conventional protocol for isolation of cell wall free nuclei for release of DNA fibers for plants involves mechanical removal of the cell wall and separation of debris by sieve filtration. The mechanical grinding pressure applied during the process leaves only the more tolerant G(1) nuclei intact, and all other states of active nuclei that may be present in the target tissues (e.g., leaf) are simply crushed/disrupted during the isolation process. Here we describe an alternative enzymatic protocol for isolation of nuclei from root tip tissue. Cell wall free nuclei at a given stage of cell cycle, free of any cell debris, could be realized in suspension that are fit for preparation of extended fibers suitable for fiber FISH applications. The protocol utilizes selective harvest of active nuclei from root tip tissue in liquid suspension under the influence of cell wall-degrading enzymes, and provides opportunities to target cell cycle-specific nuclei from interphase through division phase for the release of extended DNA fibers. Availability of cell cycle-specific fibers may have added value in transcriptional analysis, DNA:RNA hybridization, visualization of DNA replication and replication forks, and improved FISH efficiency.

Cell Cycle↗
PubMed2002

Hemophagocytic syndrome as the primary clinical symptom of Hodgkin's disease.

It is now well recognized that hemophagocytic syndrome (HPS) is occasionally associated with malignant lymphomas. However, its association with Hodgkin's disease has been only rarely reported. We present here a 72-year-old woman manifesting with HPS as the primary and solitary clinical symptom of Hodgkin's disease. She had been suffering from high-grade fever and anemia for more than a month. Based on the findings in bone marrow aspirates, she was diagnosed as having HPS. In spite of extensive surveys including various cultures, serological tests for collagen disease, abdominal and cardiac sonography, chest computed tomography (CT), and renal biopsy, the origin of the fever was not determined. She was treated with steroid pulse therapy and then referred. Radiological studies revealed only mild hepatosplenomegaly and small lymph node swellings around celiac and common hepatic arteries. Reevaluation of the bone marrow specimen revealed the infiltration of small numbers of CD30-, CD15-, and EBER-1-positive large-sized lymphocytes with bizarre nucleus. Under the diagnosis of Hodgkin's disease, she was treated with combination chemotherapy containing pirarubicin, cyclophosphamide, vincristine, and prednisolone. However, it was not effective and she died of rapidly progressive hepatic failure on the 5th day of the chemotherapy. Autopsy was performed, which showed proliferation of lymphoma cells in para-aortic lymph nodes. We believe that diagnostic survey to rule out the underlying lymphoma should be vigorously performed for patients with hemophagocytic syndrome of unknown origin.

Aged↗
PubMed2002

The structural organisation of the gene encoding class II starch synthase of wheat and barley and the evolution of the genes encoding starch synthases in plants.

Wheat and barley contain at least four classes of starch synthases in the endosperm, granule bound starch synthase I (GBSSI) and starch synthases I, II and III (SSI, SSII, SSIII). In this work, SSII in barley is shown to be associated with the starch granule by using antibodies. A cDNA from barley encoding SSII and the genes for SSII from barley and Aegilops tauschii ( A. tauschii, the D genome donor to wheat) are characterised. Fluorescent in situ hybridisation (FISH) and PCR were used to localise the wheat SSII gene to the short arm of chromosome 7, showing synteny with the location of the rice SSII gene to the short arm of chromosome 6. Comparison of the genes encoding SSII of A. tauschii, barley and Arabidopsis showed a conserved exon-intron structure although the size of the introns varied considerably. Extending such comparison between the genes encoding starch synthases (GBSSI, SSI, SSII and SSIII) from A. tauschii and Arabidopsis showed that the exon-intron structures are essentially conserved. Separate and distinct genes for the individual starch synthases therefore existed before the separation of monocotyledons and dicotyledons.

Base Sequence↗
PubMed2002

Fatal reactivation of hepatitis B virus following cytotoxic chemotherapy for acute myelogenous leukemia: fibrosing cholestatic hepatitis.

Hepatitis B virus (HBV) is a well known pathogen that sometimes causes fulminant hepatitis in patients undergoing cytotoxic chemotherapy. Fibrosing cholestatic hepatitis (FCH) is a recently recognized unique variant of viral hepatitis, which has been occasionally reported in HBV-infected recipients of liver, renal, or bone marrow transplantation. We present here a 48-yr-old male in whom HBV was reactivated during post-remission chemotherapy for acute myelogenous leukemia, which resulted in rapidly fatal outcome. He manifested with deterioration of liver function in association with enormous replication of HBV. Liver biopsy showed marked ballooning of hepatocytes, cholestasis, and periportal fibrosis with minimum infiltrates. Immunostaining revealed that hepatocytes were strongly positive for hepatitis B surface antigen. Under the diagnosis of FCH, he was treated with lamivudine and interferon beta, which was not effective. Autopsy showed severe atrophy of the liver and marked degeneration of hepatocytes. Hematologists should be aware that FCH is a fatal complication that can develop under post-chemotherapy immunosuppressed conditions. Although there is no convincing evidence, prophylactic administration of lamivudine seems to be a reasonable strategy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2001

Dual-specificity protein phosphatase Yvh1p, which is required for vegetative growth and sporulation, interacts with yeast pescadillo homolog in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae dual-specificity protein phosphatase Yvh1p, identified as vaccinia VH1 homolog, regulates cell growth, sporulation, and glycogen accumulation. Transcription of YVH1 is induced by lowering temperature and nitrogen starvation. Using the yeast two-hybrid system, we searched for Yvh1p-interacting proteins, including substrates and regulatory subunits of Yvh1p. Two clones were identified encoding a segment of YPH1 (yeast pescadillo homolog), which is essential for cell cycle progression in yeast. Deletion analysis revealed that the catalytic domain of Yvh1p and the BRCT domain of Yph1p are sufficient for this interaction. We found that the multicopy of YPH1 not only suppressed slow growth but also decreased IME2 expression in the yvh1 disruptant. These observations indicate that Yph1p plays a role in sporulation in addition to cell cycle progression, and is a candidate for a substrate or a regulatory subunit of Yvh1p.

Blotting, Northern↗
PubMed2001

Activation and localization of cartilage-derived morphogenetic protein-1 at the site of ossification of the ligamentum flavum.

Localization and expression of cartilage-derived morphogenetic protein (CDMP)-1 in tissues at the site of ossification of the ligamentum flavum (OLF) were examined by immunohistochemistry and in situ hybridization. The CDMP-1 protein and messenger ribonucleic acid (mRNA) were localized in spindle-shaped cells and chondrocytes in the OLF tissues. CDMP-1 was not detected in cells in non-ossified sites. These data indicate that CDMP-1 is locally activated and localized in spindle-shaped cells and chondrocytes at the site of OLE. Given the previously reported promoting action of CDMP-1 for chondrogenesis, the current results suggest that CDMP-1 may be involved in the progression of OLF, leading to the narrowing of spinal canal and thus causing severe clinical manifestations.

Aged↗
PubMed2001

Interaction of pleurocidin and its analogs with phospholipid membrane and their antibacterial activity.

A 25-mer cationic peptide pleurocidin, isolated from the winter flounder, has broad antibacterial activity. To clarify the structure-activity relationship, its properties and biological activity were examined. CD measurements showed that pleurocidin took an alpha-helical structure in the presence of DOPC/DOPG (3:1, anionic) vesicles. Very weak hemolytic activity of pleurocidin was observed and its antibacterial activity was moderate. Tryptophan fluorescence shift measurements showed that pleurocidin interacted weakly with a neutral phospholipid, but strongly with an acidic phospholipid. The peptide exhibited weak dye-leakage activity for DOPC (neutral) vesicles and moderate activity for acidic vesicles. From experiments on dye-leakage activity and membrane translocation of the peptide, it seemed likely that pleurocidin, like magainin 2, forms pores in the lipid membrane. A study of amino acid substitution in pleurocidin revealed that alpha-helicity, rather than hydrophobicity, affects the properties and activity of the peptide.

Animals↗
PubMed2001

Physical arrangement of retrotransposon-related repeats in centromeric regions of wheat.

Cereal centromeres commonly contain many repetitive sequences that are derived from Ty3/gypsy retrotransposon. FISH analysis using a large DNA insert library of wheat identified a 67-kb clone (R11H) that showed strong hybridization signals on the centromeres. The R11H clone contains Ty3/gypsy retrotransposon-related sequences; both integrase and CCS1 family sequences were identified. Subsequently, we isolated additional 23 large-insert clones which also contained the integrase and CCS1 sequences. Based on the number of the integrase repeats in the clones determined by DNA gel blot analysis, we concluded that the retrotransposon-like sequences are tandemly repeated in wheat centromeres in ca. 55-kb interval on average. This conclusion is consistent with the results of FISH analysis on the extended DNA fibers.

Base Sequence↗
PubMed2001

Ancestral MADS box genes in Sugi, Cryptomeria japonica D. Don (Taxodiaceae), homologous to the B function genes in angiosperms.

In flowering plants, flower organ identity is controlled by the ABC genes, including several MADS box genes. We present two MADS box genes of a conifer, Cryptomeria japonica D. Don. The genes, CjMADS1 and CjMADS2, were related to the angiosperm B function genes which determine the identities of petals and stamens. A phylogenetic analysis showed that these genes form a new clade outside the angiosperm B group, that is, PISTILLATA (PI) and APETALA3 (AP3) lineages. CjMADS1 had a PI-group specific motif and CjMADS2 had AP3-group specific motifs at the C terminal end, respectively. CjMADS1 was expressed in male strobili (or cones) throughout its development, while CjMADS2 was transiently expressed during male strobilus development. The specific expression in the male reproductive organ indicated that the B function is maintained in gymnosperms. Our cladistic analysis suggests that the gene duplication event which generated B function gene lineages predates the divergence of angiosperms and gymnosperms and that the gene duplication which produced the two genes of C. japonica occurred in an ancestral conifer species.

Amino Acid Sequence↗