Biomedical subjects

M Shibano

Publications and source records attributed to M Shibano.

At least 19 recordsLinked to original sources

PubMed2002

Airway epithelial cells promote transmigration of eosinophils in a new three-dimensional chemotaxis model.

BACKGROUND: Prominent infiltration of eosinophils in airway mucosa is the pathognomonic sign of asthma. The role of airway epithelial cells in eosinophil infiltration, however, has not been fully elucidated. OBJECTIVE: The aim of this study is to develop a new in vitro transmigration system composed of airway epithelial cells and extracellular matrix, and to investigate the role of airway epithelial cells in eosinophil infiltration. METHODS: A layer of type I collagen gel was formed in Netwell, and BEAS-2B bronchial epithelial cells were cultured on the gel. Then the wells covered with epithelial monolayer were filled with medium, inverted, and new upper chambers were constructed on the gel side by applying a ring cap. After further incubation with or without exogenous cytokines for 48 h, eosinophils or neutrophils were loaded in upper chambers (the gel side) and cells transmigrated to lower chambers (the epithelial cell side) were counted. Immunohistochemical analyses were also performed. RESULTS: While a simple collagen gel hardly promoted eosinophil migration even in the presence of eotaxin or RANTES, significant numbers of eosinophils migrated to lower chambers in the presence of the epithelial cells. Replacement of medium in the lower chamber (the epithelial cell side) with fresh medium, addition of exogenous eotaxin or RANTES in the upper chamber (the gel side), or pre-treatment of eosinophils with anti-CCR3 all inhibited transmigration. We found that the epithelial cells produced and deposited extracellular matrix proteins such as type IV collagen onto the type I collagen gel. Separately, we found that type IV collagen itself was capable of enhancing eotaxin-induced eosinophil migration in a standard chemotaxis assay. Neutrophils also efficiently migrated in the present transmigration system. Pre-treatment of epithelial cells with TNF-alpha and IL-4 enhanced eosinophil transmigration, while that of neutrophils was enhanced by TNF-alpha but suppressed by IL-4. CONCLUSION: By utilizing a new in vitro transmigration system mimicking the airway mucosa, we have demonstrated that airway epithelial cells play an essential role in transmigration of eosinophils and that multiple factors such as chemokines, extracellular matrix proteins and exogenous inflammatory cytokines are involved in efficient transmigration.

Bronchi↗

PubMed2001

Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan-class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A-deficient B-cell line (JY5), a PIG-A-deficient K562 cell line, an Epstein-Barr virus-transformed B-cell line (TK-14(-)) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A-deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34(+) cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.

3T3 Cells↗

PubMed2001

Differential response of mast cells separated from various organs and basophils of dogs to the fluoroquinolone antimicrobial levofloxacin.

Histamine releases induced by the fluoroquinolone antimicrobial levofloxacin (LVFX) were investigated using mast cells separated from various organs and peripheral basophils of dogs, being the most susceptible species to quinolone derivatives, in both in vivo and in vitro systems. An intravenous infusion of LVFX at 30 mg/kg over a 30-min period produced endogenous histamine release from 5 min, and a maximum at 30 min, in which the plasma LVFX concentration was approximately 50 microM. A close correlation (r = 0.87, n = 20) between histamine and LVFX concentrations in plasma during the infusion was observed. In the in vitro study, LVFX at 30 microM or more caused histamine release from mast cells separated from the liver and skin, but not from the gastric mucosa, lung, and peripheral basophils. More exactly, the liver mast cells were most susceptible to LVFX among the organs tested. On the other hand, compound 48/80, a prototype histamine liberator, elicited the histamine release from the liver or skin mast cells at 10 microg/ml, and the calcium ionophore A23187 at 1 microM exhibited the histamine release from the mast cells derived from all organs examined. Histochemical analysis revealed that the liver and skin mast cells had positive reaction for both alcian blue and safranin staining, but the gastric mucosa and lung mast cells were only positive for alcian blue staining, indicating that LVFX preferably activated the connective tissue-type mast cells rather than the mucosal-type mast cells. The degranulation of the liver and skin mast cells brought about by either LVFX or compound 48/80, unlike the calcium ionophore A23187, was blocked by pretreatment with pertussis toxin, suggesting the involvement of pertussis toxin-sensitive G proteins. The results obtained from the canine experiments strongly suggest that LVFX induces histamine release from the connective tissue-type mast cells distributed mainly in the liver, somewhat in the cutaneous tissue, through the activation of pertussis toxin-sensitive G proteins.

Animals↗

PubMed2001

Effects of fukinolic acid and cimicifugic acids from Cimicifuga species on collagenolytic activity.

The inhibitory collagenolytic activity (47-64% inhibition in 0.22-0.24 microM) of fukinolic acid and cimicifugic acids A, B, and C, which are esters of fukiic acid (3',4'-dihydroxybenzyl tartaric acid) was more potent than that (20-37% inhibition in 0.23-0.24 microM) of cimicifugic acids D, E, F, which are esters of pscidic acid (4'-hydroxybenzyl tartaric acid). Since fukiic acid showed weaker inhibition, and caffeic acid, ferulic acid, isoferulic acid, and p-coumaric acid showed far weaker activities, the entire structures of fukinolic acid and cimicifugic acids A, B, and C proved to be responsible for the inhibitory activities. Trypsin and pronase E hydrolyzed collagen nonselectively alone or in addition to collagenase. These collagenolytic activities were also inhibited by fukinolic acid. These results show that fukinolic acid may inhibit either the collagenolytic activities specific to collagenase or nonspecific to other emzymes. The present studies suggest the potential effect of fukinolic acid and cimicifugic acids of Cimicifuga rhizomes in preventing collagen degradation by collagenases or collagenolytic enzymes under pathological conditions, wound healing, or inflammation.

Caffeic Acids↗

PubMed2001

Two new pyrrolidine alkaloids, radicamines A and B, as inhibitors of alpha-glucosidase from Lobelia chinensis Lour.

Two new pyrrolidine alkaloids, radicamines A and B were isolated as inhibitors of alpha-glucosidase from Lobelia chinensis Lour. (Campanulaceae). Radicamines A and B were formulated as (2S,3S,4S,5S)-2-hydroxymethyl-3,4-dihydroxy-5-(3-hydroxy-4-methoxyphenyl)-pyrrolidine (1) and (2S,3S,4S,5S)-2-hydroxymethyl-3,4-dihydroxy-5-(4-hydroxyphenyl)-pyrrolidine (2) on the basis of spectroscopic analyses and chemical methods.

Asia↗

PubMed2001

Studies on the constituents of Broussonetia species X. Six new alkaloids from Broussonetia kazinoki Sieb.

Six new alkaloids, broussonetines W, X, M1, U1, J3, and J2 (1-6) were isolated from the branches of Broussonetia kazinoki SIEB. (Moraceae) as minor constituents. They were formulated as (2R,3R,4R,5R)-2-hydroxy-methyl-3,4-dihydroxy-5-17-(cyclohexy-2-on-1(6)-enyl)heptyllpyrrolidine (1), (2R,3S,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-17-(cyclohexy-2-on-1(6)-enyl)heptyl]pyrrolidine-4-O-beta-D-glucopyranoside (2), (2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-[(9R)-9,13-dihydroxytridecyl]- pyrrolidine (3), (2S,3S,4S)-2-hydroxymethyl-3,4-dihydroxy-5-(10-oxo-13-hydroxytridecyl)-5- pyrroline (4), (2R)-2-[(IS,2S)-1,2-dihydroxy-8-1(2R,3R,4R,5R)-5-(2-hydroxymethyl-3,4-dihydroxy-1-acetylpyrrolidinyl)loctyl]piperidine (5), (2R)-2-[(1S,2S)-1,2-dihydroxy-8-[(2R,3R, 4R,5R)-5-(2-hydroxymethy]-3,4-dihydroxypyrrolidinyl)]octyl]piperidine (6).

Alkaloids↗

PubMed2001

Studies on the constituents of Cimicifuga species. XXVIII. Cycloart-7-enol glycosides from the underground parts of Cimicifuga simplex Wormsk.

Four new cycloart-7-ene triterpenol arabinosides, bugbanosides C-F, were isolated from the underground parts of Cimicifuga simplex Wormsk. (Ranunculaceae). The structures were elucidated as 12beta-acetoxy-3beta,15alpha,-24R,25-tetrahydroxy-16,23-dione-cycloart-7-ene 3-O-alpha-arabinopyranoside, 12beta-acetoxy-24R,25-epoxy-3beta,15alpha-dihydroxy-16,23-dione-cycloart-7-ene 3-O-alpha-L-arabinopyranoside, 12beta-acetoxy-24R,25-epoxy-3beta-hydroxy-16,23-dione-cycloart-7-ene 3-O-alpha-L-arabinopyranoside, and 16,23R:16,24S-diepoxy-3beta,12beta,15alpha,25-tetrahydroxy-cycloart-7-ene 3-O-alpha-L-arabinopyranoside on the basis of spectral and chemical evidence. The circular dichroism (CD) of bugbanosides C-F showed strong negative maxima at 214-217 nm due to a cycloart-7-ene system, as well as other cycloart-7-ene triterpenes. The CD data showed to be useful in determining basic skeletons, including absolute stereostructures of cycloart-7-ene triterpenes.

Circular Dichroism↗

PubMed2001

Studies on the constituents of Broussonetia species VIII. Four new pyrrolidine alkaloids, broussonetines R, S, T, and V and a new pyrroline alkaloid, broussonetine U, from Broussonetia kazinoki Sieb.

Four new pyrrolidine alkaloids, broussonetines R, S, T, and V and a new pyrroline alkaloid, broussonetine U were isolated from the branches of Broussonetia kazinoki SIEB. (Moraceae) in low yield. Broussonetines R, S and T were formulated as (2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-[(1R)-1-hydroxy-3-[6-(4-hydroxybutyl)-cyclohexy-2-on-1(6)-enyllpropyl] pyrrolidine (1), (2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-[(1R,10S)-1,10,13-trihydroxytridecyl] pyrrolidine (2), (2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-[(1R,5S)-1,5, 13-trihydroxy-10-oxo-tridecyl] pyrrolidine (3). And broussonetines U and V were proposed to be (2S,3S,4S)-2-hydroxymethyl-3, 4-dihydroxy-5-(9-oxo-13-hydroxytridecyl)-5-pyrroline (4), (2R,3S,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-[(E)-9-oxo-13-hydroxy-3-tridecenyl] pyrrolidine (5), respectively, by spectroscopic and chemical methods.

Chromatography, High Pressure Liquid↗

PubMed2001

The biosynthesis of broussonetines: origin of the carbon skeleton.

Broussonetines are glycosidase-inhibitory alkaloids obtained from Broussonetia kazinoki. Feeding experiments using [1-13C]glucose and 13C-NMR spectroscopic studies showed that broussonetines are biosynthesized through routes similar to those of sphingosine and phytosphingosine.

Magnetic Resonance Spectroscopy↗

PubMed2000

Detection of small populations of CD59-deficient erythrocytes in patients with aplastic anemia or myelodysplastic syndrome and normal individuals.

To detect a small population of blood cells with a deficiency of glycosyl phosphatidylinositol (GPI)-anchored protein, we evaluated the expression of CD59 by flow cytometry on one million erythrocytes, which is about 100 times more than the number of erythrocytes tested by our standard immunoassay. Blood samples from healthy volunteers, patients with aplastic anemia (AA), and patients with myelodysplastic syndrome (MDS), who all showed no detectable GPI deficiency by the standard assay, were investigated. The numbers of CD59-deficient erythrocytes were 5 to 145/10(6) erythrocytes in the healthy volunteers (mean 29.2), and one of the volunteers had an increase in the deficient cells exceeding the mean + 3 SD (141.7), a normal limit. A CD59-deficient population was detected in 6 of the 21 (28.6%) patients with AA and 5 of the 18 (27.8%) patients with MDS. The new assay was performed again in 5 of these 11 patients and the normal individual who had the CD59-deficient populations at 6 and 12 months after the initial study. The number of deficient cells gradually increased in 1 patient with MDS (from 511 to 2892/10(6) erythrocytes), while the numbers of the other 4 patients showed a tendency to decline, although the deficient populations were repeatedly detected on most of the occasions. Changes in the number of the deficient cells were also seen in the healthy volunteer, but they were rather rapid; the numbers changed from 145 to 5661 and then to 18/10(6) erythrocytes within 3 months. The CD59 assay used in this study is easy to perform and enabled us to detect less than 1% GPI-deficient cells.

Adolescent↗

PubMed2000

Phylogenetic relationship of six Glycyrrhiza species based on rbcL sequences and chemical constituents.

The nucleotide sequences of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) of Glycyrrhiza glabra, G. uralensis, G. inflata, G. echinata, G. macedonica and G. pallidiflora have been determined to construct their phylogenetic tree. Based on these sequences, the six Glycyrrhiza species were divided into two groups: three, G. glabra, G. uralensis, and G. inflata, which produce glycyrrhizin as a major saponin, and the others, G. echinata, G. macedonica and G. pallidiflora, which produce macedonoside C as a major saponin. Among the three glycyrrhizin-producing species, only two nucleotide substitutions were observed between the rbcL sequences of G. glabra and G. uralensis, and the sequence of G. uralensis was identical to that of G. inflata, indicating that G. uralensis and G. inflata are closely related. Among the three macedonoside C-producing species, only one nucleotide substitution was observed between those of G. echinata and G. macedonica, indicating that these two species are also closely related.

DNA, Plant↗

PubMed2000

Studies on the constituents of Broussonetia species. VII. Four new pyrrolidine alkaloids, broussonetines M, O, P, and Q, as inhibitors of glycosidase, from Broussonetia kazinoki SIEB.

Four new pyrrolidine alkaloids, broussonetines M, O, P, and Q, were isolated from the branches of Broussonetia kazinoki SIEB, (Moraceae). Broussonetines M, O, P, and Q were formulated as (2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-[(10S)-10,13-dihydroxy-tri decyl]pyrrolidine (1), (2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-[(E)9-oxo-13-hydroxy-3- tridecenyl]pyrrolidine (2), (2R,3R,4R,5R)-2-hydroxymethyl-3,4-dihydroxy-5-[(E)10-oxo-13-hydroxy-3-++ +tridecenyl]pyrrolidine (3), and (2R,3S,4R,5R)-2-hydroxymethyl-3-hydroxy-4-(beta-D-glucopyranosyloxy++ +)-5-[10-oxo-13-(beta-D-glucopyranosyloxy)tridecyl]pyrrolidine (4) respectively, by spectroscopic and chemical methods. 1-4 inhibited beta-glucosidase, beta-galactosidase and beta-mannosidase.

Alkaloids↗

PubMed1999

Assessment of alkaline phosphatase on the surface membrane of neutrophils by immunofluorescence.

Expression of alkaline phosphatase (ALP) on the surface membrane of neutrophils (mNAP) was studied by immunofluorescence using an anti-ALP monoclonal antibody. Fluorescent intensity distribution of mNAP was analyzed using FACS (fluorescence-activated cell sorter). The mean fluorescent intensity (MFI) of the mNAP in this assay was well correlated with the neutrophil ALP (NAP) score demonstrated cytochemically (r = 0.832). mNAP levels in various hematological disorders were evaluated by % mNAP+ cells and MFI. The levels in aplastic anemia and polycythemia vera were significantly higher, and in chronic myelocytic leukemia and paroxysmal nocturnal hemoglobinuria (PNH), the levels were significantly lower compared with the levels in healthy volunteers. Two-color immunofluorescence with anti-ALP and anti-CD16 showed that the PNH clone was essentially negative for mNAP, whereas residual normal neutrophils (CD16+) had levels slightly higher than those in normal individuals. Highly reproducible results were obtained in the blood samples which were stored at 4 degrees C for at least 24 hr without any treatment prior to immunofluorescent staining. No degradation of fluorescent intensity was seen 4 days after staining and fixation. The mNAP assay is simple, without subjective evaluation for quantification, and is useful for differential diagnosis of hematological disorders.

Alkaline Phosphatase↗

PubMed1999

CD59-deficient blood cells and PIG-A gene abnormalities in Japanese patients with aplastic anaemia.

Patients with aplastic anaemia (AA) frequently develop paroxysmal nocturnal haemoglobinuria (PNH) as a late complication. We investigated the frequency of the development of PNH features including a glycosyl phosphatidylinositol (GPI) anchoring defect in 73 Japanese patients with AA. A deficient expression of CD59 was found on erythrocytes and/or granulocytes in 21/73 (28.8%) of the patients. A Ham/sugar water test was positive in 13/21 patients. We also examined mutations of the PIG-A gene in 11 patients with CD59 deficiency. A heteroduplex analysis detected PIG-A gene abnormality in 10/11 patients tested. Nucleotide sequencing was performed in six patients and identified eight mutations including three mutations in one patient. The mutations of the PIG-A gene were all different and included two single-base insertions, one single-base deletion, two two-base deletions, and one each of eight-base insertion and nine- and ten-base deletions. All mutations but one caused frameshifts. Our findings indicate that a high proportion of Japanese patients with severe AA have a GPI-anchoring defect and that the PIG-A gene is mutated in the AA patients who had a GPI deficiency. We found no significant difference in the pattern of the PIG-A gene mutation between the AA patients with a GPI deficiency and those with de novo PNH.

Adolescent↗

PubMed1998

Natural killer cell-derived large granular lymphocyte lymphoma of lung developed in a patient with hypersensitivity to mosquito bites and reactivated Epstein-Barr virus infection.

A 17-year-old female developed natural killer (NK) cell-derived large granular lymphocyte (LGL) lymphoma of the lung. She had a past history of hypersensitivity to mosquito bites (HMB). After an eight-year chronic, active Epstein-Barr virus (EBV) infection, she developed multiple lung lesions and pleural effusion. In the effusion, 60% of the cells were LGL. They were CD2+, 3-, 16+, 56+, 57+, 45RO+/RA + weak, and possessed strong NK activity. No rearrangement of T-cell-receptor genes was detected. From all these results, a diagnosis of NK-LGL lymphoma of the lung was made. EB virus DNA was detected in cells infiltrating the pleural effusion. The clonality of the LGLs was determined by Southern blot hybridization with the terminal repeat sequence of EB virus as a probe, and by chromosomal abnormalities. The patient died from respiratory failure. Necropsy of the lung revealed diffuse lymphoma composed of polymorphic cells with typical angiocentric lesions. Reportedly, lymphomas of NK lineage show predominantly extranodal involvement, and primary lung lesions are rare. In the pleural effusion of the present case, abnormally high levels of soluble Fas ligand, interleukin-10 and interferon gamma were detected. This hypercytokinemia, reflecting the microenvironment of lymphoma cells, may play a role in the progression of the lymphoma and organ injury in the lung.

Adolescent↗

PubMed1998

Phenotypical heterogeneity of CD4+CD8+ double-positive chronic T lymphoid leukemia.

Chronic T lymphoid leukemias are defined as leukemias of post-thymic T cells. The CD4+CD8+ double-positive (DP) phenotype is seen in a few cases. Since DP generally occurs in thymic T cells, whether the DP T leukemia cells represent thymic or peripheral T cells has been a matter of controversy. To address this issue, we studied phenotypical features in eight cases of DP T cell leukemia. Thymic DP T cells and peripheral CD8+ T cells have CD8 of alphabeta subunit, while CD8alphaalpha is induced in CD4+ T cells on activation with IL-4. We found that two patients with DP T large granular lymphocyte leukemia (LGLL) showed dim expression of CD8alphaalpha, identical to the phenotype on IL-4-activated DP-T cells. The leukemic cells of these patients expressed IL-4 mRNA and produced high levels of IL-4. These findings suggest that they may be derived from peripheral CD4+ T cells. Three patients with adult T cell leukemia/lymphoma (ATLL) showed CD8alphaalpha, suggestive of an activated peripheral T cell origin. One case expressed CD8alphaalpha dim and IL-4 mRNA, while the other two cases expressed no IL-4 mRNA and showed CD8alphaalpha bright phenotype, features not found in normal T cell populations. Three patients with T-prolymphocytic leukemia (T-PLL) expressed CD8alphabeta. The DP phenotype is relatively common in T-PLL, and CD4+CD8alphabeta+ is characteristic of thymic T cells. The DP T-PLL cells did not express TdT,CD1 or recombination activating gene-1 (RAG-1), which is down-regulated at the late stage of thymic T cell development. On the basis of these findings, we propose a late thymic origin for DP T-PLL. The phenotype of DP T cells differed for each entity and appeared to correlate with minor normal DP T cell population.

Adult↗

PubMed1998

Antimalarial activity and nucleoside transport inhibitory activity of the triterpenic constituents of Cimicifuga spp.

The in vitro antimalarial activity against human malaria parasite (Plasmodium falciparum, FCR-3 strain) was examined using 59 triterpenoids obtained during studies on the triterpenic constituents of Cimicifuga spp. The 50% effective concentration values (EC50) of 25 active triterpenoids were 1.0-3.0 microM, and 19 of the compounds had a common 16, 23:23, 26:24, 25-triepoxy group in the side-chain moieties. Among the active triterpenoids, 9 also showed significant inhibition of nucleoside transport in mouse splenocytes. A relationship between the antimalarial activity and the inhibition of nucleoside transport involving these triterpenoids is discussed.

Animals↗

PubMed1998

Studies on inhibitory activities of fukiic acid esters on germination, alpha-amylase and carboxypeptidase A.

Fukinolic acid (1) and cimicifugic acid A (2), caffeic acid analogs, as well as rosmarinic acid (3) and caffeic acid (4) showed inhibition on seed germination and seedling growth. The potency of 1 and 2 was comparable with that of 3. Compounds 1 and 2 also showed strong inhibitory activities as well as 3 and 4 on alpha-amylase. The activity of 1 was higher than that of acarbose used as a positive control, and its 50% inhibitory concentration (IC50) was 2.41 x 10(-5) M. Compounds 1 and 2 also showed inhibitory activities strong as 3 and stronger than 4 on carboxypeptidase A. The activities of 1 and 2 were higher than that of 1, 10-phenanthroline used as a positive control.

Caffeic Acids↗