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E Kitano

Publications and source records attributed to E Kitano.

At least 19 recordsLinked to original sources
PubMed2001

Interaction of poly(2-acrylamido 2-methylpropane sulfonate)-grafted polystyrene beads with cationic complement proteins.

Influence of various biomaterials on the complement system in serum has been intensively studied by many research groups, since activation of the complement pathway in vivo has been known to give rise to some pathological conditions, such as inflammation and anaphylaxis. Much effort has been devoted to develop new materials that do not activate or deteriorate the complement system. The present work is aimed at revealing the mode of reactions of anionic poly(2-acrylamido 2-methylpropane sulfonate) grafted on polystyrene bead (PAMPS-g-bead) with serum complement. Complement activity assay, determination of complement proteins levels, and immunoblot analysis were carried out for sera pretreated with PAMPS-g-beads. The results clearly showed that, when PAMPS-g-beads were incubated with serum, those beads adsorbed several complement proteins, i.e. C1q, factor D, factor P, C6, and C8, but the generation of activation fragments of complement components was not observed. Especially, factor D was most effectively removed from serum, resulting in potential inhibition of the alternative pathway. A larger amount of PAMPS-g-beads was needed to decrease the serum CH50 level. That may be caused by removal of C6. Although some polyanions, such as dextran sulfate, were reported to activate the complement system, the obtained results indicate that the PAMPS-g-bead is not an activator of the complement pathway, but acts as an adsorbent of complement components. One possible clinical application of the PAMPS-g-beads is adsorption of serum factor D by extracorporeal treatment of patients with renal failure with a high level of factor D, because the increased quantity of factor D in serum may cause consistent activation of the alternative pathway.

Biocompatible Materials↗
PubMed2000

Study of complement activation on well-defined surfaces using surface plasmon resonance.

It has been accepted that covalent immobilization of C3b on artificial materials is the most important step to initiate the complement activation. However, there are few studies that have directly demonstrated covalent immobilization of C3b on artificial surfaces. In this study, model thin layers were prepared by the self-assembled monolayer method to produce a surface covered with hydroxyl or methyl groups using mercaptododecane (CH(3)-SAM) and mercaptoundecanol (OH-SAM). Interactions of the complement system with the model surfaces were studied using a surface plasmon resonance instrument. The OH-SAM immobilized C3b, resulting in activating of the complement system through the alternative pathway in Veronal-buffered saline, but this surface did not activate the classical pathway. However, the OH-SAM could not activate the alternative pathway in Veronal-buffered saline containing 10 mM EGTA and 2 mM MgCl(2) that is believed not to interfere with the activation of the alternative pathway. The hydrophobic CH(3)-SAM surface could not activate the classical pathway, but activated the alternative pathway, although the extent was small.

Journal Article↗
PubMed2000

Synthesis of factor D by normal human hepatocytes.

BACKGROUND: Unlike most complement proteins, complement factor D is believed to be synthesized not by the liver but exclusively by adipose tissue. METHOD: Culture supernatants obtained from primary culture of normal human hepatocytes were assayed for factor D by ELISA and analyzed by Western blotting. RESULTS: When normal hepatocytes were cultured in protein-free medium without addition of any stimulator for 5 days, factor D was detected in the supernatants at levels as high as 331.07 +/- 41.38 microgram/10(6) cells. Addition of TNF-alpha, IFN-gamma, IL-1beta or LPS to the medium did not result in any distinct effect on the amounts of secreted factor D. Reversible inhibition of factor D secretion by these cells was observed when cultured in the presence of cycloheximide. By immunoblot analysis, secreted factor D exhibited double bands, one with a molecular weight similar to factor D in normal human serum and the other with a slightly larger molecular weight. CONCLUSION: Normal human hepatocytes synthesize factor D constitutively. The liver may be a major source of plasma factor D.

Cells, Cultured↗
PubMed2000

[Complement activation in heparin--plasma inhibitory effect of anticoagulants on serum complement activation, 2nd report].

Our previous study of the inhibitory effect of EDTA and citrate in plasma on complement activation revealed that complement is activated in usual citrate plasma but not in usual EDTA plasma. The present study is a similar one using other anticoagulants containing heparin. CH50 and activities of C4 and C2 were assayed in serum or plasma containing various concentrations of anticoagulant after incubation with latex particles bearing immunoglobulin(Ig-Latex). It was found that complement activation is inhibited by heparin in a dose-dependent manner and that 10 U/ml of heparin does not inhibit CH50 reduction and C2 inactivation but inhibit C4 inactivation partially, indicating that complement activation proceeds in plasma containing usual concentration of heparin. Similar results were obtained in cases of low molecular weight heparin(LMWH) and nafamostat mesilate(Futhan). Complement activation was not inhibited by gabexate mesilate(FOY) in the tested range of concentrations. Thus, it was revealed that complement activation is not inhibited by usual concentrations of heparin, LMWH, Futhan and FOY.

Adult↗
PubMed1999

Dual effects of TNF on synthesis of complement components by a gastric cancer-derived cell line, KATO-III.

BACKGROUND: Complement components are synthesized extrahepatically, although hepatocytes are the major source of plasma complement. It is now clear that local production of complement is important in homeostasis and immune defense in tissue. METHODS: The secretion of complement components was studied in vitro with a gastric cancer-derived cell line, KATO-III (signet-ring cell carcinoma). Complement components C2 and C3 were estimated by functional assay and/or ELISA in culture medium obtained after incubation of KATO-III cells for 3 days in protein-free culture medium, with or without addition of tumor necrosis factor (TNF), in a humidified atmosphere of 5% CO2/95% air at 37 degrees C. RESULTS: (1) While a higher amount of C3 was detected in medium when KATO-III cells were cultured in the presence of TNF than in medium lacking TNF, higher C2 activity was detected when cultured in medium lacking TNF than in TNF-supplemented medium. (2) TNF suppressed C2 secretion and enhanced C3 secretion in a dose-dependent fashion. (3) C3 secretion remained less than 20 ng/10(6) cells/24 h but increased from the first day of TNF (10U/ml) addition (concentrations approached 108.6- 115.6 ng/10(6) cells/24 h on the 3rd day) and decreased on the 1st day without TNF. In contrast, C2 activity, detected when cultured in the absence of TNF, was decreased on the 2nd day of TNF addition and increased again on the 1st day without TNF. The daily secretion of C2 in the absence of TNF was 3.75-6.30x10(7) effective molecules/10(6) cells. (4) Reversible inhibition of C2 and C3 secretion was observed when the cell line was cultured in the presence of cycloheximide, indicating that both components were synthesized de novo. CONCLUSION: It appears that TNF enhances C3 secretion and suppresses C2 secretion by KATO-III.

Complement Activation↗
PubMed1999

A novel assay for serum complement activity: C42 generation assay.

BACKGROUND: In most clinics, laboratory tests for serum complement are limited to immunochemical determinations of C3 and C4 and are occasionally extended to the hemolytic titration of total complement functional activity (CH50). However, these tests are often not sufficient for the analysis of low CH50 serum. METHODS: A novel assay for serum complement activity, the C42 generation assay, has been developed. The principle of this assay is based on the hemolysis of sensitized sheep erythrocytes (EA) by complement components in two sera: C42 (the classical pathway C3 convertase) is generated on EA by C1, C4 and C2 in the first serum, followed by a second reaction leading to hemolysis by C3-C9 supplied by the addition of the second serum in the presence of EDTA. RESULTS: This methodology permits the evaluation of two distinct serum complement activities of a test serum. The combined activity of C1, C4 and C2, as well as the combined activity of C3-C9, can be estimated from the observed degrees of hemolysis. Information obtained from this assay is helpful for the analysis of test serum determined to have decreased CH50. Several clinical cases are presented in which this assay was utilized. CONCLUSIONS: The C42 generation assay is another functional assay of serum complement which can provide information beyond that obtained from the typical serum CH50 assay. Since intermediate cells or isolated complement components are not necessary, this assay can be employed rapidly and economically in a clinical setting.

Animals↗
PubMed1999

[Complement activation in citrate plasma--inhibitory effect of anticoagulants on serum complement activation].

It is generally accepted that complement activation does not proceed in EDTA- and citrate-plasma because of calcium chelation by EDTA and citrate. However, there were several cases with low CH50 level in serum and citrate plasma and normal CH50 level in EDTA plasma, suggesting that complement might be activated in citrate-plasma but not in EDTA-plasma. The present study deals with the inhibitory effect of EDTA and citrate on complement activation. CH50 was assayed in serum or plasma containing various concentrations of EDTA or citrate after incubation with latex particles bearing immunoglobulin. It was revealed that complement activation proceeded in the presence of 2.0 mmol/l of EDTA but was inhibited in the presence of 2.5 mmol/l of EDTA, while blood coagulation was inhibited in plasma containing EDTA higher than 2.0 mmol/l. As to citrate, complement activation proceeded in the presence of 25 mmol/l of citrate but was inhibited in the presence of 50 mmol/l of citrate, while blood coagulation was inhibited in plasma containing citrate higher than 6.2 mmol/l. Thus, it was indicated that, as usual plasma in clinic contains 3.5 mmol/l of EDTA or 10.9 mmol/l of citrate, complement can be activated in citrate-plasma but not in EDTA-plasma. Similar conclusion was obtained by another experiment using ordinary vacuum-type blood collection tube for EDTA-2K plasma, EDTA-2Na plasma and citrate plasma.

Anticoagulants↗
PubMed1993

Synthesis of the third component of complement (C3) by human gastric cancer-derived cell lines.

This is a study of complement components secreted by gastric cancer-derived cell lines (MKN28, MKN45, MKN74 and KATO-III), each of which has a different histological origin. Haemolytic activity of complement component was detected only in the culture supernatant of KATO-III (C2 activity) and in that of MKN45 (C5 activity). However, the third component of complement, C3, was detected by an ELISA assay in the supernatants of all cell lines. In our studies focusing on C3 production by these cell lines, we have found that: (i) tumour necrosis factor (TNF) induced an increase in the amount of secreted C3 in a dose- and time-dependent fashion; (ii) TNF (10 U/ml) stimulated C3 secretion by these cell lines to levels of 25.4-62.9 ng C3/10(6) cells per 24 hours; (iii) C3 haemolytic activity was detected in supernatants of TNF-stimulated cell lines. The mean specific activities of C3 by TNF (10 U/ml)-stimulated cell lines were 1.2-5.6 x 10(5) effective molecules/ng (e.m./ng), when that of C3 in normal human serum (NHS) was 1.7 x 10(6) e.m./ng; (iv) de novo synthesis of C3 by these cell lines was demonstrated by the effect of cycloheximide and by the incorporation of 35S-methionine into secreted C3; (v) immunoblot analysis of culture supernatants indicated that secreted C3 was mainly composed of C3 alpha and C3 beta chains, but pro-C3 was also present. These results, which show the de novo synthesis and secretion of C3 by all the tested gastric cancer-derived cell lines in response to TNF, suggest the possibility that C3 may be secreted in the gastric wall as part of its normal physiology, or as a result of tumour pathology, and thereby participate in local immune or inflammatory responses.

Complement C3↗
PubMed1993

Hemolysis of normal human erythrocytes by autologous serum complement.

Unsensitized normal human erythrocytes (E) were shown to be lysed when incubated with autologous serum in the presence of zymosan (Zy). The hemolysis proceeded slowly with a relatively constant rate for at least 24 h at 37 degrees C. It was shown that the hemolytic reaction is antibody independent and mediated by complement activation through the alternative pathway and that hemolysis is not due to the decay or inactivation of complement regulators present on the E membrane. The mechanism of the phenomenon was studied by use of several kinds of sera genetically deficient in C3, C5, C7 or C9. The reaction was found to be divided into two stages: in the first step, neither E, C5, C7 nor C9 but Zy, serum factors containing C3 and metal ions are necessary, and in the second step, neither C3 nor metal ions but E, C5, C7 and C9 are necessary. Thus, E seem to be lysed by reactive lysis induced by C5 convertase formed on Zy through alternative complement pathway activation.

Complement Activation↗
PubMed1990

Plant constituents biologically active to insects. VI. Antifeedants for larvae of the yellow butterfly, Eurema hecabe mandarina, in Osmunda japonica. (2).

Three antifeedants for larvae of the yellow butterfly, Eurema hecabe mandarina de l'Orza, were isolated from Osmunda japonica Thunb. and identified as osmundalin, parasorboside and methyl (3S,5S)-5-hydroxy-3-(beta-D-glucopyranosyloxy)hexanoate. In the course of isolation of the antifeedants, a new glycoside, dihydroisoosmudalin (9), was isolated together with maltol beta-D-glucopyranoside, 2-deoxy-L-ribopyranolactone, 5-hydroxymethyl.2-furfural and glycerin. The structure of 9 was elucidated as (4R,5S)-5-(beta-D-glucopyranosyloxy)hexan-4-olide on the basis of chemical and spectroscopic evidence.

Animals↗
PubMed1982

Nature and distribution of mucosubstances in human mature enamel identified by enzyme electron microscopy.

Longitudinal slices of 12 freshly extracted human third molars fixed in 10 per cent neutral formalin were demineralized with 1M lactic acid. Ultrathin sections of slices were stained with ruthenium red or silver methenamine. The reaction products were mainly found in the prism sheath regions. To identify the mucosubstances, the digestion tests with chondroitinase ABC, AC, hyaluronidase and neuraminidase were performed. It was concluded that the major component of the organic matrix of inner human dental enamel is chondroitin 4-sulphate or chondroitin 6-sulphate localized in prism sheath regions and inter-crystalline spaces.

Chondroitin Sulfates↗