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Biomedical subjects

K Hagiwara

Publications and source records attributed to K Hagiwara.

At least 91 records · Page 5Linked to original sources
PubMed1998

Expression of human alpha-lactalbumin in transgenic tobacco.

alphaLA-Lactalbumin (alphaLA), a major milk protein, is the regulatory subunit of lactose synthase. To assess the production of recombinant alphaLA in plants, the cDNAs for human alphaLA with or without its own signal sequence were introduced into tobacco plants under the control of the cauliflower mosaic virus 35S promoter. The gene integration and expression at the mRNA level were confirmed in several regenerated plants, while the expression at the protein level could be confirmed only in a transgenic tobacco transformed with the gene containing the signal sequence. The tobacco-expressed alphaLA migrated in SDS-PAGE with identical mobility to alphaLA prepared from human milk, indicating that the signal peptide of human alphaLA was correctly processed to yield a mature protein in tobacco plants. The expressed alphaLA (ca. 5 microg/g of fresh leaves) was found in the soluble fraction and eluted from a DEAE-Sepharose column in the same salt concentration range as the milk alphaLA. The partially purified tobacco-alphaLA was fully active in the synthesis of lactose when combined with galactosyltransferase. Thus, the transgenic tobacco produces a fully active mature alphaLA in a soluble form.

Cloning, Molecular↗
PubMed1998

A preliminary study aimed at the detection of Leishmania parasites in subjects with cutaneous leishmaniasis using polymerase chain reaction.

As a basic study for future diagnosis of cutaneous leishmaniasis, we tried to detect Leishmania parasites representing different species in the subgenera Leishmania and Viannia from subject patients with cutaneous leishmaniasis by using the polymerase chain reaction (PCR) with the subgenus Viannia specific primer. Four out of the 14 specimens revealed an amplified DNA of 70 bp specific for the subgenus Viannia (L. braziliensis complex). No bands were detected in the rest of the specimens belonging to the subgenus Leishmania and unclassified groups. The base sequences of the amplified DNA corresponded with those of the L. (V). braziliensis kinetoplast minicircle. We concluded that PCR using the present primer specific for the subgenus Viannia would be useful in detecting Leishmania parasites in lesions of cutaneous leishmaniasis caused by the L. braziliensis complex.

Animals↗
PubMed1998

Comparative studies of the detection rates of Leishmania parasites from formalin, ethanol-fixed, frozen human skin specimens by polymerase chain reaction and Southern blotting.

In this study, detection rates of Leishmania parasites from human skin were compared among three different types of specimens, formalin-fixed, ethanol-fixed, and frozen, by polymerase chain reaction (PCR) and Southern blotting. For this purpose, we used biopsy specimens collected from 19 leishmaniasis patients and performed PCR and Southern hybridization with the probe specific for Leishmania (Viannia) braziliensis complex. Among these 19, 16 specimens were from cutaneous leishmaniasis (CL), one, diffuse cutaneous leishmaniasis (DCL) and 2, mucocutaneous leishmaniasis (MCL) and were formalin-fixed and paraffin-embedded. The causative agents for one case of CL and one case of DCL were already identified as L. (Leishmania) complex. Six specimens of CL were preserved in 100% ethanol. Two specimens of MCL were frozen tissues. PCR using the formalin-fixed and paraffin-embedded specimens revealed positive bands at 70 bp in 9 (47.4%) out of 19 specimens of CL, MCL and DCL. Southern blotting detected the signals in 12 (63.2%) out of the 19. PCR using the 100% ethanol-fixed specimens revealed positive bands in 4 (66.7%) out of 6, and Southern blotting also detected the signals in 4 (66.7%) out of the 6. PCR and Southern blotting using 2 frozen specimens of MCL were always positive (100%). Although we failed to detect significant differences by Chi-square test between the results from the formalin-fixed, paraffin-embedded specimens and those from 100% ethanol-fixed ones, we concluded that ethanol-fixed specimens, convenient for transportation and storage, would be more useful for diagnosis of leishmaniasis by PCR in a developing country.

Adolescent↗
PubMed1998

Phacomatosis pigmentovascularis type IIb associated with Sturge-Weber syndrome and pyogenic granuloma.

A case of phacomatosis pigmentovascularis (PPV) in a 6-year-old girl with Sturge-Weber syndrome, pyogenic granuloma, and other complications is described. It is relatively rare that a complete form of Sturge-Weber syndrome was associated with PPV. A review of the literature on PPV, focusing on total number of reported cases and etiological speculations, is presented. To our knowledge, a total of 118 cases of PPV, including the present one, have been reported to date. Regardless of many speculations, the true etiology remains unknown. The average "density" of mast cells (MCs) per mm2 appearing in the central region of the pyogenic granuloma was calculated to be 86.3/mm2 and that in the adjacent nevus flammeus was 37.9/mm2. The "density" of mast cells in pyogenic granuloma separately calculated from ten other cases was 105.5 +/- 28.6/mm2 (mean +/- SD), compared with that in normal skin, 6.85 +/- 4.9/mm2 (n = 20). There was a significant difference between the two, indicating that MCs are closely associated with angiogenesis in pyogenic granuloma.

Biopsy, Needle↗
PubMed1998

Transforming growth factor beta type II receptor (TGF beta RII) mutation in gastric lymphoma without mutator phenotype.

A new mutation in the serine-threonine kinase domain of the transforming growth factor beta type II receptor (TGF beta RII) was found in a case of diffuse, B cell non-Hodgkin's lymphoma of the stomach. A missense mutation (ACA to GCA, Thr to Ala) was detected in exon 5, and a wild type allele was also present. This is the first naturally occurring mutation in the kinase domain of this gene identified in human primary lymphoma. The replication error at three loci was negative, and the poly A tract of exon 3, which is frequently a target of mismatch repair genes, was intact. Malignant lymphoma of B cell origin in the stomach is an addition to an expanding catalogue of tumors with TGF beta RII alterations, and the biological sequelae of the change in the functional domain and the clinical characteristics of the patient in this study are intriguing.

Aged↗
PubMed1998

Determination of the nucleotide sequence of Bombyx mori cytoplasmic polyhedrosis virus segment 9 and its expression in BmN4 cells.

Cloning and sequencing of segment 9 of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strains H and I were performed. The segment consisted of 1,186 bp harboring 5' and 3' noncoding regions and an open reading frame from positions 75 to 1037, encoding a protein with 320 amino acids, termed NS5. Comparison of the nucleotide sequences of NS5 for the two strains indicated 37 point differences resulting in only six amino acid replacements. Homology search showed that NS5 has localized similarities to human poliovirus RNA-dependent RNA polymerase and human rotavirus NS26. By Western blot analysis, NS5 was found in BmCPV-infected midgut cells, but not in polyhedra or virus virions, and was mainly detectable in the nucleus in BmCPV-infected BmN4 cells. Immunoblot analysis with anti-NS5 and antipolyhedrin antibodies displayed marked differences in the period of expression of NS5 and polyhedrin: the polyhedrin molecule was first detected 2 or 3 days after infection with BmCPV, whereas the expression of NS5 was initiated within a few hours. In addition, the level of polyhedrin increased as the infection developed, whereas the amount of NS5 remained essentially constant. When segment 9 was expressed with a baculovirus expression system, the resulting NS5 protein possessed the ability to bind to the double-stranded RNA genome. These results suggest that NS5 is expressed in early stages of infection and contributes to regulation of genomic RNA function.

Amino Acid Sequence↗
PubMed1998

Binding of lactoferrin to bacterial cells of the Clostridium species and their agglutination.

Cell agglutination in cell suspensions of 10 strains of Clostridium by lactoferrin (Lf) was observed by obtaining the ratio of increased absorbance (RIA) at 450 nm. The RIA values were very different among the species, being higher in the cell suspensions with bovine Lf (bLf) than in those with human Lf. The binding ability of bLf to the bacterial cells was also observed by an enzyme-linked ligand-binding assay, using the conjugate of iron-free or iron-saturated bLf with horseradish peroxidase (HRPO). The binding ability of bLf was very different among the 10 species, and showed a significant correlation with the cell agglutination of each strain. bLf formed a complex with the cells of C. perfringens, did not dissociate in 2 M NaCl or 4 M urea, but did dissociate in 1 M KSCN. These results suggest that the agglutination of cells of the Clostridium species by bLf is probably caused by the cooperative action of at least electrostatic and hydrophobic interactions between bLf and certain components of the cell surface.

Agglutination↗
PubMed1998

A method for analysis of cell to cell transmission of Theileria sergenti piroplasms.

We tried to develop an experimental system for quantifying piroplasm transmission in Theileria sergenti infection using a SCID mouse model, of which red blood cells (RBC) had been substituted with bovine (Bo). A mouse hybridoma cell producing antibody against Holstein RBC was established for distinguishing Holstein RBC from Japanese Black RBC by a immuno-staining method. T. sergenti piroplasms in RBC were stained by 4',6-diamidino-2-phenylindole. With the aid of these techniques the piroplasm transmission from one to another race of Bo-RBCs could successfully be quantified in SCID mice.

Animals↗
PubMed1997

Effects of vitamin E deficiency and glutathione depletion on stress protein heme oxygenase 1 mRNA expression in rat liver and kidney.

Heme oxygenase 1 (HO-1) is a stress protein and has been suggested to provide defense mechanisms against agents that may induce oxidative injury. Vitamin E (VE) is considered to function as an important cellular antioxidant. Rats were fed a VE-deficient (0E) or a VE-sufficient (10E) diet for 6 weeks and then were intraperitoneally administered buthionine sulfoximine (BSO), a glutathione (GSH)-depleting reagent. Whereas HO-1 mRNA levels were undetectable in untreated 0E and 10E rat livers, BSO administration induced HO-1 mRNA expression in both 0E and 10E rat livers. High levels of HO-1 mRNA expression were observed in particular in BSO-treated 0E rat livers. The time-course of changes in HO-1 mRNA expression in 0E rat liver after BSO administration showed that HO-1 mRNA expression was transiently induced at 2.5 hr after BSO treatment, the earliest time examined. In addition, to determine whether VE deficiency and GSH depletion affect the expression of HO-1 mRNA in other tissues, we also examined the time-course of HO-1 mRNA expression in BSO-treated 0E rat kidney. The expression pattern of HO-1 mRNA in the kidney was very similar to that in the liver, and the peak was also observed at about 2.5 hr after BSO administration. Interestingly, histologic assessment of liver and kidney showed that VE deficiency and GSH depletion induced injury in the kidney, but not in the liver.

Animals↗
PubMed1997

Regulation and immunohistochemical analysis of stress protein heme oxygenase-1 in rat kidney with myoglobinuric acute renal failure.

Intramuscular injection of hypertonic glycerol solution to rats results in acute renal injury. In this model, the proximal tubules are characteristically damaged. After glycerol injection renal glutathione (GSH) levels drastically decreased. On the other hand, stress protein heme oxygenase-1 (HO-1) was induced. When N-acetyl cysteine was administered to rats before 1 h glycerol injection, renal function was obviously improved. In this condition, the renal GSH content were sustained in the normal levels and HO-1 protein levels were decreased compared with those of glycerol-treated rats. Induction of HO-1 was accompanied by reduced renal GSH content. In addition, to investigate whether the location of HO-1 protein induced by glycerol injection is restricted to injured region or not in the kidney, we determined the localization of HO-1 protein using immunohistochemical staining. HO-1 protein was identified in the epithelia of the distal tubules, Henle's loop and collecting ducts, but not in the injured proximal tubules.

Acetylcysteine↗
PubMed1997

Mutation analysis of the transforming growth factor-beta type II receptor in human cell lines resistant to growth inhibition by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta) binds the type II TGF-beta growth factor receptor (RII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the RII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-beta resistance. To further investigate the type, frequency and tissue distribution of RII mutations, we selected 24 human cancer cell lines from various tissues which were previously reported to be resistant to the inhibitory effects of TGF-beta. We developed protocols for non-isotopic SSCP analysis of PCR products from genomic DNA samples, and we tested them for microsatellite instability. PCR-SSCP analysis followed by DNA sequencing identified deletion mutations in the exon 3 poly-adenine tract in three colon tumor cell lines: LS174T and SW48 had a single base deletion and LS411 had a two base deletion. Among the 24 previously unreported cell lines, only these three demonstrated microsatellite instability. These and other recent data indicate that RII mutations are essentially confined to colon and gastric cancers with microsatellite instability. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.

Cell Division↗
PubMed1997

The modulation effect of vitamin E on prostaglandin E2 level and ornithine decarboxylase activity at the promotion phase of lung tumorigenesis in mice.

The present study was undertaken to investigate a mechanism of the inhibitory effect of vitamin E in urethane-induced lung tumorigenesis in mice. We assayed ornithine decarboxylase (ODC) activity and the prostaglandin E2 (PGE2) level in lung at 8 weeks after urethane injection (promotion phase). Excessive vitamin E feeding or indomethacin treatment suppressed the urethane-induced increase in ODC activity, while exogenous PGE2 overcame the effect of vitamin E on ODC activity. Furthermore, the amount of PGE, and the level of ODC activity were well correlated. These results indicate that the vitamin E-induced decrease in PGE2 level probably contributes to the inhibition of ODC induction and the prevention of tumor development in the lung.

Animals↗
PubMed1997

Crystallization and preliminary X-ray diffraction studies of tulip aryl acylamidase: a key enzyme in plant herbicide detoxification.

Crystals of aryl acylamidase (E.C. 3.5.1.13) from tulip bulbs have been obtained by the hanging-drop vapor-diffusion method using polyethylene glycol (PEG) 8000 as a precipitant. The crystals belong to space group P2(1)2(1)2(1) with unit-cell dimensions a = 68.7, b = 80.1 and c = 112.9 A. Assuming two molecules of molecular weight of 34 kDa in the asymmetric unit, V(m) is 2.28 A(3) Da(-1), indicating a solvent content of approximately 46%. The intensity data have been collected to 2.5 A resolution with an R(merge) of 0.067.

Journal Article↗
PubMed1997

FHIT mutations in human primary gastric cancer.

Allelic deletion of multiple regions on the short arm of chromosome 3 (3p) implies the presence of multiple important tumor suppressor genes in human carcinogenesis. The FHIT gene, identified recently in chromosome 3p14.2, shows frequent allelic deletion and aberrant transcripts in gastrointestinal tumors. After determining the intron sequences flanking each of the coding exons of the FHIT gene and designing intron primers to facilitate mutation analysis of genomic DNA samples, we analyzed the complete coding sequences in matched cancer and normal tissues from 40 cases with primary gastric cancer using intron primers, PCR-single-strand conformation polymorphism analysis, and direct sequencing. A somatic missense mutation in exon 6, codon 61, ACG (threonine) --> ATG (methionine) was found in a signet ring cell adenocarcinoma. We also evaluated allelic deletion in these tumors by PCR-based microsatellite analysis; allelic deletion occurred in 42.1% (16 of 38) of evaluable cases. This is the first report of a somatic missense mutation of the FHIT gene in a primary tumor. Presence of a point mutation and frequent allelic deletions are consistent with the hypothesis that FHIT gene alterations are involved in the development of primary gastric cancers.

Acid Anhydride Hydrolases↗
PubMed1997

The inhibitory effect of vitamin E on pulmonary polyamine biosynthesis, cell proliferation and carcinogenesis in mice.

We investigated the modulating effect of vitamin E on pulmonary polyamine biosynthesis, cell proliferation and carcinogenesis in mice treated with urethane. Pulmonary ornithine decarboxylase induction and subsequent polyamine accumulation were observed during the initiation and promotion phases of the urethane-induced lung carcinogenesis in mice. The increases of ODC activity and polyamine level during both phases were almost inhibited when a high vitamin E diet was provided. The urethane-increased level of pulmonary proliferating cell nuclear antigen as a marker of cell proliferation during the carcinogenesis was inhibited by vitamin E treatment. Also, vitamin E suppressed the urethane-induced elevation of pulmonary cyclooxygenase activity as a marker of tumor promotion. In conjugation with these events, vitamin E reduced the development of lung tumors in mice treated with urethane. These results indicated that vitamin E could act as a useful chemopreventive agent against lung carcinogenesis in mice due to the regulation of cell proliferation.

Animals↗
PubMed1997

The inhibitory effect of vitamin E on K-ras mutation at an early stage of lung carcinogenesis in mice.

In this study, we investigated the effect of vitamin E on the activation of the K-ras oncogene with a 61st codon A-->T mutation at an early stage of urethane-induced lung carcinogenesis in mice. Thirty days after urethane injection, the K-ras mutation was detected in 64% of lung samples tested by mutant-allele-specific amplification. The consumption of a supplemented diet with about 20-times more vitamin E than the control diet, only during the promotion phase or during both the initiation and promotion phases of lung carcinogenesis, reduced the frequency of the mutation to 36 and 18%, respectively. Also, vitamin E suppressed the level of proliferating cell nuclear antigen as a marker of cell proliferation in the lungs of mice treated with urethane. These results support the notion that vitamin E is a useful chemopreventive agent against lung cancer.

Animals↗
PubMed1997

Mutation analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers.

Mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene have been detected in several human cancers exhibiting microsatellite instability. To extend analyses of this gene, we previously investigated the exon-intron organization of the TGFbeta RII gene and defined seven exons and flanking intron sequences. In this study, we further determined the nucleotide sequences surrounding these seven exons and designed eight sets of intron-based primers to examine the entire coding region of the TGFbeta RII gene. Using these primers, we screened genomic DNA sequences from 30 sporadic colorectal cancers for mutations of the TGFbeta RII gene. TGFbeta RII mutations were detected in two of 30 tumors and both displayed microsatellite instability. One had a deletion in a polyadenine tract in exon 3 and the other had a point mutation in the kinase domain located in exon 7. There were no mutations in exons 1, 2, 4, 5 and 6. These results further implicate the polyadenine tract and kinase domain as mutational hotspots in the TGFbeta RII gene in cells with genomic instability and suggest that TGFbeta RII gene mutations occur rarely in cells lacking genomic instability.

Base Sequence↗