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Biomedical subjects

N Ohno

Publications and source records attributed to N Ohno.

At least 91 records · Page 5Linked to original sources

Activation of murine kupffer cells by administration with gel-forming (1-->3)-beta-D-glucan from Grifola frondosa.

The effect of gel-forming (1-->3)-beta-D-glucan on the immunological activities of murine kupffer cells was examined. A branched type gel-forming (1-->3)-beta-D-glucan, GRN, was administered intravenously to mice. GRN associating to kupffer cells was detected by an immunohistochemical technique using anti-GRN antibody. A kinetic study of the activation of kupffer cells revealed that GRN could induce the enhanced production of cytokines and nitric oxide on 4 to 7 d after the administration. The activities are further augmented by adding GRN in the culture. The cytostatic activity of kupffer cells against murine lymphoma, EL-4, was also augmented by a time course similar to nitric oxide production. The cytostatic activity was reduced by adding an inhibitor of nitric oxide synthase, implying that the cytostatic activity of kupffer cells to EL-4 was dependent on nitric oxide. The administration of GRN increased the expression of CD11b, known as a beta-glucan receptor, on kupffer cells at day 7. The above data suggest that GRN could activate murine kupffer cells to enhance the production of cytokines and nitric oxide, and that the activation required 4 or 7 d, at least, after the administration with GRN.

Animals↗

Adjuvant effect of grifolan on antibody production in mice.

The effects of grifolan (GRN), a gel-forming (1-->6)-branched (1-->3)-beta-D-glucan, on antibody production were examined. Sera from mice that were injected with GRN and trinitrophenyl ovalbumin (TNP-OVA) intraperitoneally showed a significantly increased level of anti-TNP IgG. However, injection of TNP-OVA alone showed a lower antibody level. Two hundred fifty microg of GRN and 10 microg of TNP-OVA gave the maximum production of anti-TNP antibody. Enhanced antibody production was also observed in the culture supernatant of splenocyte obtained from GRN-administered mice. The culture supernatant contained a significant amount of nitric oxide (NO) in the case of GRN-administered mice. To observe the effect of NO on the antibody production induced by GRN, N-monomethyl arginine (NMMA), an inhibitor of NO synthetase, was added to the splenocyte cultures. The antibody level of supernatants containing NMMA was higher than that of control supernatants. These results suggest that GRN can enhance antibody production and that NO induced by stimulation with GRN concomitantly with antibody production is a negative factor on the adjuvant activity. Inhibition of NO may increase the adjuvant effect of GRN.

Adjuvants, Immunologic↗

Mitochondrial fixation for the detection of cytochrome oxidase activity using microwave irradiation.

We examined cell fixation with microwave irradiation (MWI) used in cytochemistry. MWI was applied to blocks of about 1 mm3 of mouse parotid glands at 500 W for about 5 sec in a fixative at 37 degrees C. The activities of endogenous peroxidase and mitochondrial cytochrome oxidase were demonstrated by using the DAB method with 3,3'-diaminobenzidine (DAB) and 0.01% H2O2. Under electron microscopy, peroxidase activity was localized in the nuclear envelope, endoplasmic reticulum and secretory granules. However, mitochondria cytochrome oxidase activity seemed to be rather weak against the MWI at 37 degrees C. Moreover, suspension of isolated hamster liver mitochondria was fixed by MWI and also demonstrated cytochrome oxidase activity by using the cytochemical methods with DAB, cytochrome c, catalase and sucrose. Such mitochondrial fractions were subjected to 6-second MWI given 10 or 18 times with an interval of 10 seconds with and without a chilled water bath. The final temperature of each fixative was kept at about 10 degrees C or rose to about 37 and 55 degrees C. When we took care to keep the temperature below 10 degrees C, the DAB reaction products accumulated in the mitochondrial intermembrane-intracristal space. No mitochondrial deposits were observed when the temperatures of the fixatives rose to 37 and 55 degrees C. These results indicated that peroxidase was very resistant to the heat with MWI fixation. Cytochrome oxidase is sensitive to the heat with MWI, so, a chilled water bath had to be used.

Animals↗

Granulocyte colony-stimulating factor in the combination chemotherapy for adult T-cell leukemia (ATL).

We evaluated the effect of granulocyte colony-stimulating factor (G-CSF) on the median survival of 17 patients with Adult T-cell leukemia (ATL). Standard-dose combination chemotherapy using the response-oriented cyclic multidrug (RCM) protocol with G-CSF (lenograstim 2 microg/kg/day or filgrastim 50 microg/m2/day) was administered between October 1990 and December 1994. Complete responses (CR) were achieved in 11 (64.7%) patients, and partial responses (PR) in 4 (23.5%) patients. The median duration of survival was 7.4 months, compared with 6.0 months in ATL patients treated with the RCM protocol alone (historical controls) (n.s.). Infectious complications were the cause of death in 4 (26.7%) of the 15 patients who died. The median duration of neutropenia (absolute neutrophil count < 1.0 x 10(9)/L) was 6 days. G-CSF, in the doses and schedules used here, may have shortened the duration of neutropenia and reduced the incidence of fatal infectious complications. However, concomitant use of G-CSF did not prolong the median duration of survival in patients with ATL treated according to the RCM protocol.

Adjuvants, Immunologic↗

[Autonomic nervous function of the patients with neurally mediated syncope].

Although diagnosis of neurally mediated syncope (NMS) using head-up tilt (HUT) test has been established, the exact mechanism of NMS has not yet been elucidated. We evaluated beta and alpha-adrenergic function in NMS patients by pharmacological autonomic function test. The alpha-adrenergic sensitivity of NMS patients was significantly lower than that of control subjects. The patients who need low dose isoproterenol for provocation of syncope showed higher beta-adrenergic sensitivity than patients who developed syncope without isoproterenol. Thus, pharmacological autonomic function test was useful for evaluation of NMS patients.

Adolescent↗

A cell binding domain from the alpha3 chain of type IV collagen inhibits proliferation of melanoma cells.

Our previous studies have shown that a peptide corresponding to the residue sequence 185-203 of the NC1 domain of the alpha3 chain of basement membrane collagen (type IV) inhibits the activation of polymorphonuclear leukocytes. Peptides from the same region of the alpha1, alpha2, alpha4, and alpha5(IV) chains did not exhibit this property. Because of the intimate relationship between metastasizing neoplastic cells and vascular as well as epithelial basement membranes, we measured the cell adhesion-promoting activity of peptides from the NC1 domain of type IV collagen and their effect on proliferation of human melanoma cells. We found that peptide alpha3(IV)185-203 (CNYYSNSYSFWLASLNPER) not only promotes adhesion of human melanoma cells but also inhibits their proliferation. Adhesion increased by 50-60% over control. Melanoma cell proliferation was inhibited by 40% when cells were grown in a medium containing 5 microg/ml peptide for 5 days. Studies showed that replacement of serine in position 189 or 191 by alanine resulted in significantly reduced adhesion. Similarly, serine replacement resulted in reduced ability to inhibit proliferation. Our data suggest that a region of the NC1 domain of the alpha3(IV) chain, contained within the sequence 185-203, not only specifically promotes adhesion but also inhibits proliferation of melanoma cells. These properties appear to be dependent on the presence of the triplet sequence -SNS- (residues 189-191), which is unique to the alpha3 chain and may represent an important functional epitope.

Amino Acid Sequence↗

Identification of interleukin-6 producing fibroblastoid cells in cerebrospinal fluid from patients with leukemic meningitis.

Cytokine producing native cells in cerebrospinal fluid (CSF) have not been identified. So, we investigated the cytokine producing ability of floating cells in CSF from patients with leukemic meningitis. Morphologic study revealed that established cell lines were polygonal or elongated in shape and had an abundant and irregular branched cytoplasm. Immunocytochemical analysis demonstrated positive reactivity with monoclonal anti-fibroblast antibody only. Interleukin-6 (IL-6) was constitutively produced in vitro by these cell lines; both interleukin-1 and lipopolysaccharides significantly increased its synthesis. These findings imply that these fibroblastoid cells are floating in CSF of patients with leukemic meningitis and produce IL-6 in response to various inflammatory stimulations in vivo.

Adult↗

Synthesis of cytotoxic fluorinated quassinoids.

The C-15 senecioyl side chain of brusatol was interchanged with fluorinated acyl groups, and the C-3 hydroxy group of bruceolide was esterified with fluorinated acyl chlorides. These fluorinated quassinoids 11, 12, 13, and 17 showed significant cytotoxic activity against eight human cancer cell lines including small and non-small cell lung, colon, CNS, ovarian and renal cancers, leukemia, and melanoma with 17 being about 100 times more potent than 11, 12, and 13. The activity of 17 was similar to that of bruceantin (1) in this in vitro cell line panel.

Acylation↗

Soluble selectins and ICAM-1 modulate neutrophil-endothelial adhesion and diapedesis in vitro.

We observed that normal plasma dramatically reduces neutrophil-endothelial adhesion. Therefore, we identified factors in plasma which might limit PMN adhesion in vitro. We found that the anti-adhesive effect was not mediated by vasoactive lipids present in plasma. Immunoprecipitation of soluble adhesion molecules, P and E-selectins and ICAM-1 restored PMN adhesion to control values. We further examined whether soluble adhesion molecules in plasma might also regulate PMN endothelial migration in response to fMLP (10(-6) M). Plasma significantly reduced PMN migration, and this effect was prevented only by the simultaneous removal of soluble P and E selectins and ICAM-1 together, but not individually. These data show that soluble selectins and ICAM-1 may regulate PMN adhesion and diapedesis, and that alterations in the levels of these molecules may regulate PMN-endothelial interactions in vivo.

Adult↗

Enhanced production of inducible nitric oxide synthase by beta-glucans in mice.

We have already demonstrated that various activities including NO (nitric oxide) synthesis in vivo and in vitro significantly differ between triple helical (SPG) and single helical (alkaline-treated SPG, SPG-OH) beta-glucans. It was previously suggested that the single helical conformer of beta-glucan (SPG-OH) was dominant in cytokine production and subsequent NO synthesis in vitro. In this study, we analyzed production of inducible nitric oxide synthase (iNOS) induced by beta-glucans in vitro and in vivo. The iNOS production was enhanced in proteose peptone-induced peritoneal macrophages (PMs) cultured with SPG-OH in the presence of IFN-gamma for 24 h, and SPG-OH-induced PMs. Moreover, SPG-OH was effective for iNOS production not only in isolated macrophages but also in tissue macrophages, whereas SPG was less effective. These findings suggest that a single helical conformer is essential for iNOS production, and that NO synthesis by beta-glucans is closely related to iNOS production.

Animals↗

Exogenous xanthine promotes neutrophil adherence to cultured endothelial cells.

Oxidants generated by endothelial xanthine oxidase (XO) can help trigger free radical-mediated tissue injury. An important event in oxidant-mediated tissue injury is neutrophil-endothelial adhesion. Although activation of endothelial XO increases adhesion, little is known about xanthine in the adhesive effect of XO. This study examined administered xanthine on the adhesion of neutrophils. Endothelial [human umbilical vein endothelial cells (HUVEC)] monolayers were exposed to xanthine (15 min), and neutrophils were allowed to adhere to HUVEC in an adhesion assay. Adhesion was dose dependently increased by xanthine (3-100 microM). Either catalase (1,000 U/ml), oxypurinol (XO inhibitor; 100 microM), or platelet-activating factor (PAF) receptor antagonist (WEB 2086; 10 microM) reduced neutrophil adhesion. Superoxide dismutase (1,000 U/ml) had no effect. Pretreatment of HUVEC with 50 microM tungsten also blocked xanthine-induced adherence. Adhesion was also inhibited by preincubation with 100 U/ml heparin. Finally, anti-P-selectin antibody (PB1.3; 20 micrograms/ml) attenuated adhesion. Our results indicate that xanthine may promote neutrophil-endothelial adhesion via a hydrogen peroxide- and PAF-mediated P-selectin expression.

Antioxidants↗

Nitric oxide synthesis in murine peritoneal macrophages by fungal beta-glucans.

Fungal beta-glucans have abilities to induce NO (nitric oxide) synthesis by macrophages in vivo, and the intensity of NO synthesis significantly varied dependent on the structure of beta-glucans. The molecular mechanism of NO synthesis by beta-glucans, however, has not been clarified in detail. To determine beta-glucan-mediated NO production, we used various beta-glucans (SPG-OH, GRN; Grifolan, SSG, OL-2, ZYM; zymosan A and ZYC; zymocel), which could enhance NO synthesis in vivo, and stimulated peritoneal macrophages (MPs) in vitro in the presence of interferon-gamma (IFN-gamma). Using recombinant cytokines, a minimum concentration of the cytokines for NO induction was about 20 mg/ml in the presence of IFN-gamma under the experimental conditions. Of beta-glucans tested, only SPG-OH and GRN produced high concentrations of IL-6 in the culture supernatants. SSG also induced NO synthesis in vitro, but concentrations of inflammatory cytokines were low even in the presence of IFN-gamma. In addition, there are some beta-glucans which could induce NO synthesis in vivo but not in vitro (OL-2, ZYM, ZYC). These findings suggested that NO productivity of beta-glucans in vivo is regulated by several mechanisms.

Animals↗

Inactivation of a particle beta-glucan by proteins in plasma and serum.

(1-->3)-beta-D-Glucans remained in the liver and spleen for long time, i.e. more than a month, without major structural changes/because there is no specific metabolic pathway for it in the body. However, biological activities, such as priming activity to LPS, triggered TNF-alpha synthesis, and antitumor activity was reduced more quickly. In this paper, we demonstrated the contribution of protein binding in inactivating beta-glucans. A particle beta-glucan preparation, zymosan, was treated with serum or plasma at 37 degrees C and their various biological activities were compared with zymosan alone. Such biological activities as antitumor activity, TNF-production, IL-6 production, complement activation and vascular permeability were significantly decreased by serum or plasma treatment. These results strongly suggested that the binding of serum or plasma protein(s) to beta-glucans would be a key step in inactivating a particle beta-glucan in the body.

Animals↗

Wide range of molecular weight distribution of mitogenic substance(s) in the hot water extract of a Chinese herbal medicine, Bupleurum chinense.

In this study, we examined the contribution of lignin-like materials in lower molecular weight (MW) fractions from the hot water extract of Bupleuri Radix (Bupleurum chinense) (HWE-BR) for their immunopharmacological activities. Mitogenic activity was detected in all the fractions of MW ranges: lower than 1.0 kDa, 1.0-3.5 kDa, 3.5-10 kDa, and 10-50 kDa. After NaClO2 treatment of these subfractions, UV spectra, ESR spectra, mitogenic activities on murine B-cells, and the activity of inducing nitric oxide in RAW 264.7 cells were significantly reduced, suggesting that lignin-like polyphenolic substance(s) of various MW might take part in these activities. The intensity of ESR spectra and mitogenic activities were stronger in higher MW subfractions, thus the content of stable radical species and/or the degrees of polymerization would be important for their immunopharmacological activities.

Animals↗

Analysis of mitogenic substances in Bupleurum chinense by ESR spectroscopy.

The polyphenolic substance(s) in the hot water extract of Bupleurum chinense (PSF) showed strong mitogenic activity. In this paper, we analyzed PSF by using ESR spectroscopy, and found that i) PSF showed a strong ESR signal on g = 2.005 which was similar to the commercially available lignin; ii) Sho-saiko-to, which contains an extract of B. chinense, also showed similar signals on ESR; iii) Powdered B. chinense also showed similar signals on g = 2.005. Peroxidase activity, essential for producing polyphenolic substances, was detected in the cold water extract of B. chinense. In addition, the signal intensity of the ESR spectrum of B. chinense was increased after boiling. The data of the ESR spectra of the model reactions using lignin, arginine, proline and maltose also strongly suggested that a certain chemical modification proceeded during the hot water extraction to increase the percentage of the stable free radical. These facts strongly suggested that the mitogenic substance in B. chinense is a polyphenolic substance extracted by hot water, and the structure was modified during the extraction to increase the stable free radical components.

Drugs, Chinese Herbal↗

Inhibitory effects of quassinoid derivatives on Epstein-Barr virus early antigen activation.

Short-term in vitro assays for tumor promoters and antitumor promoters (Epstein-Barr virus activation test) were carried out for semisynthetic quassinoids (3-7), which were obtained by esterification of the C-15 OH group of deacetylated isobrucein-B (2). All the ester derivatives showed higher antitumor promoting activity than that of the potent compound 2. A compound containing a fluorinated aliphatic ester showed the highest potency.

Antigens, Viral↗

Increases in hematopoietic responses caused by beta-glucans in mice.

The effects of various (1-->3)-beta-D-glucans on hematopoietic responses of mice were investigated by measuring colony stimulating activity in sera and ascites of the mice administered glucan. We have demonstrated that the hematopoietic response was increased by various structures of (1-->3)-beta-D-glucans, i.e. soluble glucans (linear, branched, single helix, triple helix) and particulate glucans. From the viewpoint of structure and activity relationships, we found several characteristic features: i) hematopoietic response induced by the particulate glucan disappeared faster than that by the soluble glucans, ii) conformation of the glucans, single vs. triple helix, are relatively independent of the response, iii) linear glucan had a weaker response, and iv) there is a strong strain-dependency of the response. These results corresponded well with the fact that branched (1-->3)-beta-D-glucans, but not linear and not particulate, are often used as biological response modifiers for cancer patients.

Animals↗