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Biomedical subjects

T Akaike

Publications and source records attributed to T Akaike.

At least 163 records · Page 9Linked to original sources
PubMed1996

Difference in adhesion and proliferation of fibroblast between Langmuir-Blodgett films and cast surfaces of poly (gamma-benzyL L-glutamate)/poly(ethylene oxide) diblock copolymer.

Block copolymers consisting of poly (gamma-benzyl L-glutamate) and poly (ethylene oxide) as the hydrophobic and hydrophilic components, respectively were prepared. Cell attachment onto the surfaces of block copolymers fabricated either as well-defined ordered Langmuir-Blodgett (LB) films or solvent cast microphase-separated structures was studied. On ordered LB surfaces, adherent fibroblasts were larger than on the microphase-separated cast surfaces within 15 min. The difference in cell adhesion between LB films and cast surfaces increased with increasing PEO contents in block copolymer. Adherent cells increased with an increase in surface pressure of LB films in any polymer. Phase-contrast microphotographs of adherent cells showed rapid and extensive morphologic changes associated with the LB surface as compared to cast film surfaces. The number of cells grown on the LB surface is greater than that on the cast film.

3T3 Cells↗
PubMed1996

Bradykinin and nitric oxide in infectious disease and cancer.

Vascular pathophysiology at the sites of bacterial infection and cancerous tissues share numerous common events similar to inflammatory tissue. Among them enhanced vascular permeability is the universal and hallmark event mediated by bradykinin. All 16 or more bacterial or fungal proteases we have examined activated one or more steps of the kinin generating Hageman-factor-kallikrein cascade. In the meantime, most of the microbial proteases rapidly inactivated various plasma inhibitors such as alpha 1-protease inhibitor and alpha 2-macroglobulin. In addition to the extracellular proteases, bacterial cell wall components (negatively charged LPS) of gram-negative bacteria and teichoic acid moieties of gram-positive bacteria activate the Hageman-factor-kallikrein system and exert hypotensive effects via kinin generation. Endotoxin (LPS) also induces nitric oxide synthase (NOS) which appears to exhibit a rather slow, but significant, effect in relaxing the vascular tone of the infected animal (thus hypotension). Furthermore, bacterial proteases can activate the matrix metalloproteinase (collagenase) resulting in exacerbation of tissue injury in the diseased animal. Many tumor cells or tissues excrete plasminogen activator, and hence activate plasminogen. The plasmin thus generated activates procollagenases, as well as the Hageman-factor-kallikrein system, resulting in pronounced extravasation. Fluid accumulation in pleural and ascitic carcinomatoses is largely due to the activated bradykinin-generating system. We can also demonstrate and control enhanced vascular permeability using kallikrein inhibitors, especially the polymer-conjugated soybean trypsin inhibitor which exhibits a prolonged plasma t1/2, kinin antagonists, NOS inhibitors, NO scavengers, inhibitors of prostaglandins and others. Bacterial proteases induce shock in mice which can be prevented by the soybean trypsin inhibitor by blocking the kallikrein-kinin cascade. Therapeutic use of kinin antagonists and a kallikrein inhibitor has been made for infectious diseases such as septicemia and in tumor pathology.

Animals↗
PubMed1996

Further evidence of bradykinin involvement in septic shock: reduction of kinin production in vivo and improved survival in rats by use of polymer tailored SBTI with longer t1/2.

Involvement of bradykinin in septic shock and its therapeutic endeavor using soybean trypsin inhibitor (SBTI, Kunitz type) were investigated in an in vivo model of septic shock induced by pseudomonal elastase. Pseudomonal elastase injection at 0.5 mg/kg i.v. to guinea pigs resulted in elevation level of bradykinin in the blood from < 1 ng/ml to 25 ng/ml which was accompanied by a drop of mean arterial blood pressure (MABP) (about 45 mmHg). When native soybean trypsin inhibitor (SBTI, Kunitz type, 20 kDa) was injected, into this model, induction of bradykinin generation and hypotension by the bacterial protease treatment was completely obliterated as judged by the both levels of bradykinin and MABP. Specifically, by the treatment with SBTI, bradykinin levels did not increase and the drop of the blood pressure was minimal (< 10 mmHg) in this time frame (< 30 min). We designed and prepared succinylated gelatin-conjugated SBTI (suc-gel SBTI) with enlarged molecular mass (M(r) approximately 110,000) and higher area under the curve of the plasma concentration, which exhibits about 6 times longer plasma half-life (t1/2) and about 4 times larger area under the curve of plasma concentration. Suc-gel-SBTI suppressed the pseudomonal protease-induced shock much more effectively than native SBTI, the conjugate exhibited its effect for more than 3 h, while the native SBTI showed the effect only within 2 h after i.v. injection.

Animals↗
PubMed1996

Development of CCD-based direct observation equipment for biological samples.

Equipment for the direct observation of biological samples has been developed. The equipment comprises a charge coupled device (CCD) unit and a light-emitting diode (LED), which is placed above the light-sensing face of the CCD. The biological sample is positioned just onto the CCD with no lens system interposed. Because of its small size, the equipment can be operated in an incubator, allowing the continuous observation of biological samples under appropriate temperature, humidity and gas concentration conditions. The equipment is operated periodically at 5-minute periods to reduce the influence of heat generated by the equipment and to maintain a constant sample temperature. E. coli colony formation is observed continuously for 70 hours without any adverse effects occurring during the observation. Hepatocyte morphological change and hepatocyte pattern formation are also observed.

Animals↗
PubMed1996

Concanavalin A induces perforin-mediated but not Fas-mediated hepatic injury.

Concanavalin A (Con A) induces T-cell-mediated hepatic injury in vivo, although Con A-stimulated lymphocytes are not cytotoxic to normal hepatocytes in vitro. This contradiction makes the mechanism of Con A-induced hepatitis elusive. In this study, we demonstrate that Con A but not tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), or actinomycin D (ActD) induced the susceptibility of hepatocytes to activated autologous lymphocyte cytotoxicity. Con A sensitized hepatocytes within 30 minutes after the stimulation in a dose-dependent fashion. The cytotoxicity was dose-dependently inhibited by either a Con A ligand, alpha-methyl mannoside, or a perforin inhibitor, concanamycin A (CMA), but not by anti-Fas ligand antiserum. In addition, Con A-treated hepatocytes were not sensitive to autologous activated lymphocytes from a perforin-deficient mouse, while hepatocytes from lpr mice were sensitized by Con A. In fact, Con A did not induce liver injury in perforin-deficient mice within the concentration employed in this study. Therefore, we conclude that the cytotoxicity was mediated through perforin/granzymes but not through the Fas/Fas ligand pathway. The cytotoxicity was inhibited by anti-intercellular adhesion molecule-1 (ICAM-1)/LFA-1 antibodies, but not by anti-VCAM-1/VLA-4 antibodies, both in vitro and in vivo. The cytotoxicity appears to be caused by CD8+ T cells; however, the cytokines from activated CD4+ T cells play a critical role in the pathogenesis of the hepatitis in vivo, because administration of anti-IFN-gamma antibodies inhibited the occurrence of the hepatitis. In conclusion, Con A-induced hepatitis is thought to be dominantly mediated by a perforin-dependent pathway through ICAM-1/LFA-1 interaction.

Animals↗
PubMed1996

Bradykinin generation triggered by Pseudomonas proteases facilitates invasion of the systemic circulation by Pseudomonas aeruginosa.

To elucidate the mechanism of bacterial exoprotease in promotion of the intravascular dissemination of Pseudomonas aeruginosa, we examined the possible involvement of bradykinin (whose generation is induced by pseudomonal proteases in septic foci) in the invasion by bacteria, and in access of bacterial toxins to systemic blood circulation. P. aeruginosa 621 (PA 621), which produces very little protease, was injected intraperitoneally into mice together with pseudomonal exoproteases (elastase/alkaline protease). Dissemination of bacteria from the peritoneal septic foci to the blood was assessed by counting viable bacteria in the blood and spleen by use of the colony-forming assay. The results showed that pseudomonal proteases markedly enhanced (10- to 100-fold) intravascular dissemination of bacteria in mice. This enhancement was induced not only by pseudomonal proteases but also by bradykinin. More importantly, the increased spread of PA 621 induced by pseudomonal protease and bradykinin was significantly augmented by the addition of kininase inhibitors, indicating the direct involvement of bradykinin in bacterial dissemination. Similarly, bradykinin caused effective dissemination of pseudomonal toxins such as endotoxin (lipopolysaccharide) and exotoxin A when the toxins were injected into the peritoneal cavity with bradykinin. Furthermore, the lethality of the infection with PA 621 was strongly enhanced by pseudomonal proteases given i.p. simultaneously with PA 621. On the basis of these results, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are involved in facilitation of bacterial dissemination in vivo.

Animals↗
PubMed1996

Effect of ligand orientation on hepatocyte attachment onto the poly(N-p-vinylbenzyl-o-beta-D-galactopyranosyl-D-gluconamide) as a model ligand of asialoglycoprotein.

The orientation effect of galactose ligand on hepatocyte attachment was investigated. Poly(N-p-vinylbenzyl-o-beta-D-galactopyranosyl-D-gluconamide )(PVLA), a beta-galactose-carrying styrene homopolymer, was used as a model ligand for the asialoglycoprotein receptors on hepatocytes. PVLA was transferred onto the poly(gamma-benzyl L-glutamate) (PBLG) or PBLG/poly(ethylene glycol) (PEG)PBLG Langmuir-Blodgett (LB) films as the monolayer level. The dichroic fluorescence values of the confocal microscope indicated that the PVLA transferred onto the LB films was located with a preferential orientation of its molecular axes with regard to the direction of the alpha-helix of polypeptide. Hepatocyte recognized well-oriented galactose moieties of the surface of PVLA through asialoglycoprotein receptors.

Animals↗
PubMed1996

Potentiation of nitric oxide-mediated vasorelaxation by xanthine oxidase inhibitors.

Nitric oxide (NO), now almost synonymous with endothelium-derived relaxing factor (EDRF), reacts with superoxide anion radical (O2-) and forms a potentially toxic molecular species, peroxynitrite (ONOO-). Because xanthine oxidase (XO) seems to be a major O2- -producing enzyme in the vascular system, it is important to clarify the mechanism of XO regulation of NO/EDRF. We first characterized the inhibition of XO in vitro by three types of pyrazolopyrimidine derivatives. Kinetic studies indicated that 4-amino-6-hydroxpyrazolo[3,4-d]pyrimidine (AHPP) and allopurinol competitively inhibited the conversion of xanthine to uric acid catalyzed by XO, with apparent Ki values of 0.17 +/- 0.02 and 0.50 +/- 0.03 micro M respectively; alloxanthine inhibited this conversion in a noncompetitive manner with an apparent Ki value of 3.54 +/- 1.12 microM. O2- generation in the xanthine/XO system assayed by lucigenin-dependent chemiluminescence was suppressed most strongly by AHPP in a dose-dependent fashion; allopurinol itself appears to reduce the enzyme by transfer of an electron to O2, thus generating O(2-). AHPP significantly augmented EDRF-mediated relaxation of aortic rings from both rabbits and spontaneously hypertensive rats (SHR) in a dose-dependent manner, whereas allopurinol did not affect the relaxation and only marginal potentiation of the vasorelaxation was observed with alloxanthine. Finally, iv injection of AHPP (50.4 mg/kg; 100 micromol/300 g rat) reduced the blood pressure of SHR rats to 70% of the initial pressure; this pressure is almost the blood pressure of normal rats. Allopurinol (100 micromol/300 g rat; iv) showed transient decrease in blood pressure and moderate reduction of hypertension of SHR (10%) was observed with iv injection of alloxanthine (100 mumol/300 g rat). On the basis of these results, it seems that XO regulates EDRF/NO via production of O2-.

Allopurinol↗
PubMed1996

Pharmacological advantages of conjugation of Cu, Zn-superoxide dismutase with succinylated keratin fragment: improvement of biological properties and resistance to oxidative damage.

To improve the in vivo pharmacological potential of Cu, Zn-superoxide dismutase (Cu,Zn-SOD), human recombinant Cu,Zn-SOD was conjugated with succinylated keratin fragment (Suc-ker). Suc-ker-conjugated Cu,Zn-SOD (Suc-ker-SOD) was formed by attachment of about 3.7 mol of Suc-ker to 1 mol of Cu,Zn-SOD and exhibited an apparent mean molecular weight of 107 kDa. Suc-ker-SOD exhibited 74.1% SOD activity on a molar basis. When Suc-ker-SOD was administered i.v. into mice, its plasma half-life was prolonged to 20.5 min compared with 4.7 min for native SOD. After i.v. administration of 51Cr-labeled proteins into mice, native SOD was excreted rapidly into urine and no significant accumulation was observed in organs other than the kidneys. On the other had, Suc-ker-SOD was taken up rapidly by the liver. Furthermore, Suc-ker-SOD exhibited much lower immunogenicity and much better therapeutic effects than native and poly(styrene-co-maleic acid)-conjugated SOD against paraquat toxicity in mice. Moreover, the inactivation rate of Suc-ker-SOD by H2O2 was less than those of native SOD and polyethylene glycol-conjugated SOD. The effect of H2O2 on liganded Cu++ in the active site of Suc-ker-SOD was much less than those of other SOD derivatives. These results indicate that Suc-ker-SOD has advantages over the other SOD derivatives tested.

Animals↗
PubMed1996

Expression of inducible nitric oxide synthase and its involvement in pulmonary granulomatous inflammation in rats.

Two types of pulmonary granulomatosis were produced in rats by intratracheal instillation of zymosan or silica. In both models, immunostaining with anti-rat monoclonal antibody for inducible nitric oxide synthase (iNOS), ANOS11, showed that the intensity of iNOS immunoreactivity in the inflammatory lesions peaked at 3 days and declined thereafter. Immunohistochemical double staining and in situ hybridization demonstrated the expression of iNOS in neutrophils, monocyte-derived macrophages, and bronchiolar epithelial cells in the pulmonary lesions. Electron spin resonance spectroscopy revealed the production of an excessive amount of nitric oxide (NO) in the pulmonary lesions. Immunostaining with a polyclonal antibody against nitrotyrosine indicated the formation of nitrotyrosine residues in the granulomatous lesions, particularly in the periphery of the lesions, providing indirect evidence for the generation of peroxynitrite anion in the zymosan- or silica-instilled lungs. Administration of N omega-nitro-L-arginine methyl ester or S-methylisothiourea sulfate, which significantly suppressed NO production, resulted in marked reduction of monocyte/macrophage infiltration as well as in inhibition of induction of monocyte chemoattractant protein-1 in the lesions. These data indicate that NO and its more reactive product peroxynitrite anion may be important mediators of granuloma formation in the lung.

Animals↗
PubMed1995

Regulation of hepatic genes and liver transcription factors in rat hepatocytes by extracellular matrix.

Culturing hepatocytes on different extracellular matrix (ECM) substrata including tissue culture plastic, type I collagen, Engelbreth-Holm-Swarm (EHS) gel and poly-N-p-vinylbenzyl-D-lactonamide (PVLA) regulated levels of mRNAs for cytoskeleton and liver-specific genes. In hepatocytes on EHS gel, the ratio of albumin/beta-actin in mRNA levels was high and serially increased during the culture period, while the ratio was low and declined in cells on plastic substratum, collagen or PVLA. The changes in cellular levels of albumin mRNA which were regulated by ECM corresponded with those in two liver-specific transcription factors, hepatocyte nuclear factors-1 and -4, which control the transcription of liver-specific genes. These results suggest that cell-matrix interaction may determine and maintain the differentiated phenotype of hepatocytes by regulating liver-specific transcription factors.

Actins↗
PubMed1995

Tumor necrosis factor alpha and interleukin-1 beta but not interferon gamma induce vascular cell adhesion molecule-1 expression on primary cultured murine hepatocytes.

Inflammatory cytokines such as tumor necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) and interleukin-1 beta (IL-1 beta) play important roles in the mechanisms of hepatitis. The effects of these cytokines on the expression of vascular cell adhesion molecule-1 (VCAM-1) in hepatocytes were examined. TNF alpha and IL-1 beta but not IFN gamma or IL-6 induced VCAM-1 expression on primary cultured murine hepatocytes in a dose- and a time-dependent fashion. TNF alpha is significantly more effective than IL-1 beta on the induction of VCAM-1 expression. The results of RT-PCR demonstrate that these cytokines regulate VCAM-1 expression at mRNA level. These results suggest that TNF alpha and IL-1 beta participate in the pathogenesis of hepatitis via induction of VCAM-1 molecules on hepatocytes.

Animals↗
PubMed1995

Structural effect of galactose residue in synthetic glycoconjugates on interaction with rat hepatocytes.

N-p-Vinylbenzyl-O-beta-D-galactopyranosyl-(1,4)-D-gluconamide++ + (PVLA) has been used as an asialoglycoprotein model polymer. Rat hepatocytes expressing asialoglycoprotein receptors are capable of binding to hydrophobic plastic dishes coated with PVLA. PVLA, water-soluble polystyrene derivatives bearing galactose residues preferentially adsorb to plastic plates made of polystyrene rather than those of poly(methyl methacrylate). Hence, we modified chitosan beads with linear chains composed of a long alkyl or phenyl moiety to study the effect of structural variations of adsorbed PVLA, and investigated the extent of hepatocyte attachment to the hydrophobic beads coated with PVLA. The attachment was independent of the amount of immobilized PVLA; rather, it was dependent on the hydrophobicity of the beads with PVLA. To simplify the surface of the hydrophobic beads with PVLA, galactopyranoses were covalently linked to chitosan beads via hydrophobic spacer arms, and hepatocyte attachment was compared among the prepared beads. The beads with spacer arms containing phenylalanine and a phthalic moiety showed increased hepatocyte attachment, which was elicited by galactose residues on the beads. These results suggest that rotational restriction or stiffness and hydrophobicity due to the phenyl moiety are essential to enhance the specificity of terminal galactose in PVLA. This analysis contributes to the design and optimization of an artificial ligand for cellular receptors recognizing sugar moieties.

Animals↗
PubMed1995

Receptor-mediated interaction of the low-density lipoprotein complex with dextran sulfate: potential as a multi-biological coupler.

We have investigated the receptor-mediated binding and uptake of low-density lipoprotein (LDL) and its insoluble complex with dextran sulfate to determine the contribution of positively charged sites in LDL to receptor-mediated interactions. Rabbit plasma LDL derivatized with FITC retained its sedimentation ability with dextran sulfate, as well as intact LDL, and binding of fluorescent LDL and its complex to liver cells was assayed by flow cytometry. Flow cytometry revealed that the binding of complex LDL with dextran sulfate, as well as that of pure LDL, increased on rat liver parenchymal cells treated with estrogen, which enhanced the expression of LDL receptors, and decreased on Hep G2 and Chang Liver cells treated with a monoclonal antibody against LDL receptors. The binding of pure LDL and complex LDL to hepatocytes was depressed by pretreatment with unlabeled LDL in a similar manner, but not with asialoglycoprotein, a ligand of asialoglycoprotein receptors on liver cells. Furthermore, we carried out a stable primary culture of rat hepatocytes, and then pure LDL and complex LDL were applied to the cultured hepatocytes. Hepatic-differentiated functions such as albumin and bile acid secretion decreased on the uptake of pure LDL and complex LDL in a similar manner. Consequently, comparative studies using pure LDL and complex LDL allowed us to determine that the complex formation with dextran sulfate had no influence on the receptor-mediated binding or uptake of LDL, and that LDL possessed binding domains for LDL receptors and sulfonic carbohydrates, containing positively charged amino acids.

Animals↗
PubMed1995

Direct evidence of macrophage differentiation from bone marrow cells in the liver: a possible origin of Kupffer cells.

Controversy has surrounded origin and differentiation of tissue macrophages. We directly demonstrate the differentiation of bone marrow cells into macrophages in the liver in vivo using a cell-labeling fluorescence dye, PKH-26. Bone marrow cells labeled with PKH26 were intravenously injected into syngenic mice, and these cells were tracked by flow cytometric analysis. The majority of the labeled cells were detected only in the liver after 4 days. Interestingly, antigens specific for macrophage lineage cells (F4/80, Fc gamma RII, and CD14) were detected on the liver-accumulated cells only 4 h after the injection. The pattern of the antigen expression changed to that of Kupffer cells (F4/80+, Fc gamma RII+, Mac-1-) after 4 days and remained so thereafter. These labeled cells in the liver were esterase staining-positive and showed phagocytic activity at day 7. The number of labeled cells among the Kupffer cells in the liver increased with days after injection. This indicates that bone marrow cells accumulate in the liver and differentiate into liver macrophages on site. Roles of factors secreted from hepatocytes are also discussed.

Animals↗