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Biomedical subjects

K Harada

Publications and source records attributed to K Harada.

At least 163 records · Page 9Linked to original sources
PubMed2000

Evolution of specificity in an immune network.

A dynamic antigen response of the immune network is discussed, based on shape-space modelling. The present model extends the shape-space modelling by introducing the evolution of specificity of idiotypes. When the amount of external antigen increases, a measure of stability of the immune network is lost and thus the network can respond to the antigen. It is shown that specific and non-specific responses emerge as a function of antigen amounts. A specific response is observed with a fixed-point attractor, and a non-specific response is observed with a chaotic attractor for the lymphocyte population dynamics. The network topology also changes between fixed-point and chaotic attractors. For some antigen amounts, chaotic attractors will vanish or become long-lived super-transient states. A dynamic bell-shaped response function will thus emerge. The relevance of long-lived chaotic transient states embedded in fixed-point attractors is discussed with respect to immune functions.

Animals↗
PubMed2000

Recycling foam countercurrent chromatography

A new sample injection method for foam countercurrent chromatography (CCC), named the "recycle injection system", has been developed. In this system, the effluent from the liquid outlet is directly returned into the column through the sample feed line so that the sample solution is continuously recycled for repetitive foam fractionation. The utility of this system was demonstrated in the separations of microcystin extract and bacitracin complex from large volumes of sample solution. Microcystins were separated and enriched in decreasing order of hydrophobicity. Bacitracin A, a hydrophobic major component, in the bacitracin complex was highly enriched in the foam fraction and almost completely isolated from other components. This recycling foam CCC method may be effectively applied for separation and enrichment of various foam-active components from crude natural products.

Journal Article↗
PubMed2000

The functions of circulatory polymorphonuclear leukocytes in diabetic patients with and without diabetic triopathy.

We determined circulatory polymorphonuclear leukocytes (PMN) functions of superoxide anion production, adhesion and aggregation in 38 type 2 diabetic patients with and without diabetic triopathy. Tumor necrosis factor (TNF)-alpha-stimulated superoxide production and N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated aggregation in diabetic patients with triopathy were significantly greater than those in diabetics without triopathy. The more diabetic complications existed, the more TNF-alpha-stimulated superoxide was produced by PMN. These results suggest that the activated PMN contributes to a progression of diabetic triopathy in type 2 diabetic patients.

Cell Adhesion↗
PubMed2000

Lipopolysaccharide augments expression and secretion of vascular endothelial growth factor in rat ventricular myocytes.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is highly expressed in the myocardium under various stimuli including hypoxia and ischemia. On the other hand, lipopolysaccharide (LPS) causes systemic inflammatory response syndrome (SIRS), which consists of systemic pathophysiological changes related to vascular hyperpermeability. To test the hypothesis that VEGF is one of the important mediators of SIRS, we examined effects of LPS on the VEGF expression and secretion in cultured neonatal rat ventricular myocytes. LPS (10 microg/ml) rapidly (within 1 h) augmented the levels of VEGF mRNA in these cells. Pharmacological inhibition of nucleic factor-kappaB or tyrosine kinases did not affect the LPS-induced augmentation of VEGF mRNA expression, while these treatments markedly suppressed the up-regulation of inducible nitric oxide synthase (iNOS) expression by LPS. The VEGF concentrations in the conditioned media were also significantly increased by the LPS treatment of 6 h. In conclusion, LPS augments VEGF expression and secretion in rat ventricular myocytes, suggesting that VEGF may be involved in pathogenesis of SIRS. LPS may induce VEGF mRNA through the signaling pathways that are distinct from those responsible for the iNOS induction.

Animals↗
PubMed2000

Structure determination and binding kinetics of a DNA aptamer-argininamide complex.

The structure of a DNA aptamer, which was selected for specific binding to arginine, was determined using NMR spectroscopy. The sequence forms a hairpin loop, with residues important for binding occurring in the loop region. Binding of argininamide induces formation of one Watson-Crick and two non-Watson-Crick base pairs, which facilitate generation of a binding pocket. The specificity for arginine seems to arise from contacts between the guanidino end of the arginine and phosphates, with atoms positioned by the shape of the pocket. Complex binding kinetics are observed suggesting that there is a slow interconversion of two forms of the DNA, which have different binding affinities. These data provide information on the process of adaptive recognition of a ligand by an aptamer.

Arginine↗
PubMed2000

Immunohistochemical analysis of cell-matrix adhesion molecules and their ligands in the portal tracts of primary biliary cirrhosis.

Intraepithelial migration of lymphoid cells (epitheliotropism) through the basement membrane of the bile duct is a key event in the development of chronic non-suppurative destructive cholangitis (CNSDC) and eventual immune-mediated bile duct loss in primary biliary cirrhosis (PBC). Cell-to-cell and cell-to-extracellular matrix proteins may play an important role in the development of CNSDC. To address the events of epitheliotropism in CNSDC, the expression of cell-matrix adhesion molecules such as integrins alpha1, alpha3, alpha4, alpha5, and alpha6 and the distribution of fibronectin, laminin, and collagen type IV were studied immunohistochemically, with emphasis on both infiltrating lymphocytes and the bile ducts, in frozen sections from 15 PBC cases and 34 controls (chronic viral hepatitis, extrahepatic biliary obstruction, and normal liver). In PBC and chronic viral hepatitis, most of the infiltrating lymphoid cells expressed integrin alpha4, while such expression was less common in extrahepatic biliary obstruction and normal liver. A biliary basement membrane-like structure was delineated by immunostaining of collagen type IV and laminin in PBC and controls. On the basement membrane of CNSDC, fibronectin, a ligand of integrin alpha4, was strongly and frequently expressed in PBC, while such expression on the biliary basement membrane was rare in other controls. These results suggest that increased fibronectin expression on the biliary basement membrane and integrin alpha4-fibronectin interaction facilitate adhesion and the penetration of infiltrating alpha4-expressing lymphocytes into the biliary epithelial layer in PBC.

Antigens, CD↗
PubMed2000

A case of monoclonal gammopathy associated with acute myelomonocytic leukemia with eosinophilia suggested to be the result of lineage infidelity.

Acute myelomonocytic leukemia (AMMoL) accompanied by monoclonal gammopathy is a rare condition, and its pathogenesis and the cytogenetic mechanism of such leukemogenesis have not been determined in detail. A case of AMMoL with eosinophilia accompanied by immunoglobulin G kappa monoclonal gammopathy is described. Immunophenotypic studies of the peripheral blood and bone marrow mononuclear cells revealed no evidence of abnormally proliferating cells of B-lineage. DNA analyses of bone marrow mononuclear cells containing leukemic cells revealed rearrangement of the kappa-light chain (Igkappa) gene and c-myc and c-jun proto-oncogenes. The intensities of the rearranged bands for these genes on Southern blot analysis suggested the existence of a major population of leukemic cells with rearranged Igkappa gene and minor population(s) of leukemic cells with rearranged c-myc and/or c-jun proto-oncogene(s) in the patient's bone marrow and indicated the occurrence of genetic evolutionary changes in leukemic cells in this patient before starting chemotherapy. These results suggest that these leukemic cells are the most likely candidate for immunoglobulin G kappa monoclonal protein production, and structural abnormalities of c-myc and c-jun proto-oncogenes may have contributed to the evolution of leukemic cells in this patient.

Blotting, Southern↗
PubMed2000

Suppression of tumor necrosis factor alpha-induced matrix metalloproteinase 9 production by the introduction of a super-repressor form of inhibitor of nuclear factor kappaBalpha complementary DNA into immortalized human salivary gland acinar cells. Prevention of the destruction of the acinar structure in Sjögren's syndrome salivary glands.

OBJECTIVE: We have previously shown that specific enhancement in acinar cells of proteolytic activity induced by tumor necrosis factor alpha (TNFalpha) may be responsible for the destruction of the acinar structure in the salivary glands of patients with Sjögren's syndrome. Because matrix metalloproteinase 9 (MMP-9) is regulated by nuclear factor kappaB (NF-kappaB), we investigated the effect of a super-repressor form of inhibitor of nuclear factor kappaBalpha (srIkappaBalpha) on the suppression of TNFalpha-induced MMP-9 production in acinar cells. METHODS: Two srIkappaBalpha complementary DNA (cDNA)-transfected acinar cell clones (ACMT-6 and ACMT-7) and 1 empty vector-transfected cell clone (ACpRc-1) were established. After treatment of cell clones with TNFalpha, the expression of MMP-9 was examined. In addition, the effect of TNFalpha on cell growth and the morphogenetic behavior of cell clones cultured on type IV collagen-coated dishes were examined. RESULTS: TNFalpha induced the production of MMP-9 in the ACpRc-1 cell clone, but greatly suppressed MMP-9 production in ACMT-6 and ACMT-7 clones. No apparent cytotoxic effect of TNFalpha treatment was observed in these cell clones. When ACpRc-1 cells were seeded on type IV collagen-coated dishes in the presence of both TNFalpha and plasmin, type IV collagen interaction with the cells was lost and the cells entered apoptosis. However, even when ACMT-6 and ACMT-7 cells were cultured under the same culture conditions as those for ACpRc-1, these cell clones attached to the substrate and grew consistently without showing apoptosis. Conclusion. These observations indicate that suppression of TNFalpha-induced MMP-9 production by the introduction of srIkappaBalpha cDNA corrected the aberrant in vitro morphogenesis of acinar cells grown on type IV collagen.

Blotting, Western↗
PubMed2000

Expression of genes encoding vascular endothelial growth factor and its Flk-1 receptor in the chick embryonic heart.

Vascular endothelial growth factor (VEGF) is known to play an essential role in embryonic vascular development. The heart is one of the main organs that produce VEGF, but it is still unknown how expression of VEGF gene is regulated in embryonic cardiac myocytes. Thus, we cloned cDNAs encoding VEGF and its receptor (a KDR/flk-1 or Quek1 homologue) from cultured 10-day-old chick embryonic ventricular myocytes (CEVM). Reverse transcription-polymerase chain reaction revealed that the chick VEGF mRNAs consisted of at least four different species corresponding to the isoforms of 190, 166, 146 and 122 amino acids. In the embryonic heart and CEVM, the isoforms of 166 and 122 amino acids were dominant. Northern blot analysis detected an abundance of VEGF mRNA in both the embryonic heart and CEVM, even at the basal state. The levels of VEGF mRNA in CEVM were significantly augmented by forskolin (100 microM), or phorbol 12-myristate, 13-acetate (200 nM) in a time-dependent manner in CEVM. In contrast, the basal levels of VEGF mRNA were attenuated by genistein (100 microM), but not by H89 (100 microM) or bisindolylmaleimide (75 microM). Northern blot analysis also detected the chick flk-1 mRNA in abundance in the embryonic heart, and to a much lesser extent in CEVM. The expression levels of VEGF and flk-1 mRNA species were continuously high in the 6, 8 and 10-day-old chick embryonic hearts. In the 10-day-old embryonic hearts, in situ hybridization confirmed that mRNA encoding VEGF was mainly expressed in ventricular myocytes. In contrast, the flk-1 mRNA was detected in the microvascular endothelial cells, and to a lesser extent in the ventricular myocytes. These data suggest that VEGF is produced in embryonic ventricular myocytes, even at the basal state, and that the levels of VEGF mRNA may be differently regulated by various protein kinases. VEGF produced by the chick embryonic heart may play important roles in embryonic cardiovascular development by acting on surrounding endothelial cells and, possibly, on ventricular myocytes themselves.

Amino Acid Sequence↗
PubMed2000

The high level of hCDC10 gene expression in neuroblastoma may be associated with favorable characteristics of the tumor.

BACKGROUND: The biological behavior of neuroblastomas detected through mass screening (MS, </=1 year of age) and that of mass screening-negative later-presenting (MSN, >1 year of age) neuroblastomas have been reported to differ in many studies. To investigate the biological differences between these two groups, we analyzed the differences in mRNA profiles. MATERIALS AND METHODS: We analyzed the mRNA profiles of MS and MSN neuroblastomas using differential display, and cloned and sequenced the bands differentially expressed between these two groups. Using the RNA analysis by polymerase chain reaction (RNA-PCR) method, the relative amount of mRNA in tumor tissue in each sample was measured. Associations between relative amount of mRNA and clinical and genetic variables related to patient prognosis and the effect of the level of mRNA expression on survival probability were investigated using statistical methods. RESULTS: Using differential display and RNA-PCR, we found that the mRNA for the human homologue of the yeast cdc10 gene (hCDC10) identified in Saccharomyces cerevisiae was expressed at a higher level in the MS group of patients than in the MSN group of patients (0.554 +/- 0.197 for MS neuroblastoma, n = 24 and 0.244 +/- 0.179 for MSN neuroblastoma, n = 10, P < 0.01), and this difference was suggested to be independent of the histologic subtype of tumor. A high level of hCDC10 mRNA expression in neuroblastomas (relative amount of hCDC10 mRNA > 0.35) was also suggested to be associated with younger age at diagnosis (</=1 year of age, P < 0.01), favorable clinical stage (I, II, and IVs, P < 0. 01), and favorable histology in the Shimada classification (P < 0. 01), whereas a low level of hCDC10 mRNA expression (relative amount of hCDC10 mRNA </=0.35) was suggested to be associated with the progression of clinical stage (P < 0.01) and N-myc gene amplification (>1 copy, P < 0.05). Patients with neuroblastomas with a high level of hCDC10 mRNA expression were suggested to have a better prognosis than those with a low level of hCDC10 mRNA expression (P < 0.01). CONCLUSIONS: A high level of hCDC10 mRNA expression in neuroblastomas may be associated with favorable clinical and biological characteristics, and the expression of hCDC10 mRNA in neuroblastomas may affect the clinical and biological characteristics of this type of tumor.

Cell Cycle Proteins↗
PubMed2000

Pediatric acoustic schwannoma showing rapid regrowth with high proliferative activity.

Acoustic schwannoma is a slow-growing tumor and usually occurs in adult patients. We report a rare pediatric case of acoustic schwannoma with high proliferative potential. A 10-year-old boy was diagnosed as having a right cerebellopontine angle tumor. The tumor was subtotally resected. Histological examination revealed a typical acoustic schwannoma with a few mitotic figures. Chromosomal analysis showed no abnormality on the long arm of chromosome 22 associated with neurofibromatosis type 2. The lesion re-grew rapidly as an acoustic schwannoma, necessitating subtotal resection on three occasions and CyberKnife radiosurgery. The immunohistochemical MIB-1 staining indices of the specimens obtained at the first, second, and third operations were 2.3%, 4.6% and 14.7%, respectively. The immunohistochemical proliferative potential of acoustic schwannoma is discussed.

Biomarkers, Tumor↗
PubMed2000

Developmental expression of myotonic dystrophy protein kinase in brain and its relevance to clinical phenotype.

To investigate the pathophysiologic role of myotonic dystrophy protein kinase (DMPK) in the brain in myotonic dystrophy (MD), the developmental characteristics of DMPK immunoreactivity in the central nervous system and its alteration with disease were studied. Eleven patients' brain with MD (5 congenital form, 6 adult form) were examined by immunohistochemistry using a specific antibody against synthetic DMPK peptides, antipeptide DM1, and compared with 30 control brains, including 16 age-matched controls. In controls, DM1-immunoreactive neurons appeared in the early fetal frontal cortex and cerebellar granule cell layer, persisting through 29 weeks of gestation and then disappearing. In contrast, immunoreactive neurons continued to persist in the cerebral cortex and cerebellar granule cell layer of MD patients. When we counted DM1-immunoreactive neurons, the increase over controls was greater in the congenital form of MD than in the adult form, and was greater in the cerebrum than in the cerebellum in both forms of MD. DM1 immunostaining was predominantly nuclear, mirroring Western blotting of subcellular fractions. Differences in DM1 expression related to development and to the two forms of MD may be closely related to the pathogenesis of mental retardation in this disease.

Adult↗
PubMed2000

Transfer of the mitochondrial rps10 gene to the nucleus in rice: acquisition of the 5' untranslated region followed by gene duplication.

Mitochondrial ribosomal protein S10 (rps10) is encoded by the mitochondrial genome in potato and pea. Here we show that the rps10 gene is absent from the mitochondrial genome of rice and has been transferred to the nucleus. Cloning and transcriptional analysis show that there are two rps10 genes in the rice nuclear genome and that their transcripts differ in abundance. Western analysis detected the RPS10 protein in the soluble fraction of rice mitochondria, although neither RPS10 has any obvious N-terminal presequence for targeting to mitochondria. This result suggests that targeting information is present in the internal region of rice RPS10. Genomic sequence analysis indicated that each rps10 gene has an intron in the 5' untranslated region (5' UTR) and that these intron sequences are homologous to each other. This result strongly suggests that a duplication event occurred after transfer of the rps10 gene to the nucleus. The duplicated rps10 genes have since been translocated to different chromosomes, because the two rps10 genes were mapped on chromosomes 6 and 12 by RFLP analysis. Interestingly, the 5' UTR and the intron of the rice rps10 genes are homologous to sequences found in several rice genes with various functions, such as osk4, EF-1beta2 and RAG1, suggesting a common origin and a functional role for the 5' UTR. Acquisition of the 5' flanking region might have accelerated the activation of the mitochondrial rps10 gene which was transferred to the nuclear genome.

5' Untranslated Regions↗
PubMed2000

The development of a cell array and its combination with laser-scanning cytometry allows a high-throughput analysis of nuclear DNA content.

We developed a cell array device for analyzing cellular characteristics such as DNA ploidy, numerical chromosomal aberrations, and antigen expression in multiple specimens in a single experiment. Fifty (10 x 5) spots, 2 mm in diameter, were arrayed in an area of 30 x 16 mm on a glass slide, and approximately 1,000 cells were placed on each spot. To demonstrate the usefulness of the cell array, we measured nuclear DNA content using laser-scanning cytometry for DNA ploidy analysis in nine human tumor cell lines and normal lymphocytes. Combining the cell array with laser-scanning cytometry allows not only measurement of nuclear DNA content for 50 samples but also easy comparison of DNA ploidy among the samples in a single experiment. In addition, we used the cell array for fluorescence in situ hybridization using a DNA probe specific for the pericentromeric region of chromosome 11.

Cell Count↗
PubMed2000

Separation of spiramycin components using high-speed counter-current chromatography.

High-speed counter-current chromatography was successfully applied to the separation of the main components of spiramycin. A 25-mg quantity of sample was separated using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:6:5:5), and the fractions were analyzed by high-performance liquid chromatography and fast atom bombardment mass spectrometry. The separation yielded 13.4, 0.7 and 1.7 mg of spiramycins I, II, and III with purities of 98.2, 92.3 and 97.4%, respectively.

Anti-Bacterial Agents↗
PubMed2000

First report on the distribution of orally administered microcystin-LR in mouse tissue using an immunostaining method.

The purpose of this study was to investigate the distribution of microcystin-LR (MCLR) orally administered to mice using an immunostaining method. MCLR was orally dosed at 500 microg/kg to aged Balb/C and ICR mice and their lethality was 23.9%. The former was more sensitive to MCLR than the latter, suggesting that oral toxicity by MCLR is related to the animal strains tested, although the pathological and immunostaining changes were essentially the same in both strains. According to this method the distribution of MCLR and related compounds were indicated as the red staining. Particularly, livers of dead aged mice were intensively stained. The main route of absorption was considered to be the small intestine because the villi contained a large amount of MCLR in both surface epithelial cells and lamina propria, resulting in erosion. The absorbed MCLR was contained in blood plasma and moved to the liver, lung, and heart, and finally to capillaries of the whole body. Excretion of MCLR was shown in the mucous from goblet cells in both the small intestine and large intestine.

Animals↗
PubMed2000

Immunoaffinity purification method for detection and quantification of microcystins in lake water.

We have developed a new clean-up method, which consisted of solid-phase extraction on a Sep-Pak PS-2 (styrene-divinylbenzene copolymer) or Excelpak SPE-GLF (polymethacrylate) cartridge instead of conventional ODS silica gel and silica gel together with following immunoaffinity purification using anti-microcystin-LR monoclonal antibodies. This newly developed method was demonstrated to eliminate co-existing substances and to concentrate microcystins in the lake water. The recoveries from lake water (1 liter) spiked with 100 ng each of microcystins-RR, -YR and -LR were 85.5, 89.2 and 92.2%, respectively, with coefficients of variation of 3.3-7.6%. Only 3 h were required to complete the total procedures starting from the microcystin extraction, the immunoaffinity purification, and the quantification using HPLC. The detection limits for all of the 3 microcystins in lake water were 0.005 microg/l. Applicability of this method has been demonstrated by measuring the concentrations of microcystins in water samples collected from lakes where water blooms occurred, which turned out to be 0.012-0.177 microg/l of total microcystins.

Antibodies, Monoclonal↗