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Biomedical subjects

T Akaike

Publications and source records attributed to T Akaike.

At least 37 records · Page 2Linked to original sources
PubMed2001

Galactosylated chitosan-graft-poly(ethylene glycol) as hepatocyte-targeting DNA carrier.

Lactobionic acid bearing galactose group was coupled with chitosan for liver specificity, and poly(ethylene glycol) (PEG) was grafted to galactosylated chitosan (GC) for stability in water and enhanced cell permeability. Complex formation of galactosylated chitosan-graft-PEG (GCP)/DNA complexes was confirmed by agarose gel electrophoresis. Compared to GC/DNA complex, the stability of GCP/DNA complex could be enhanced. Particle sizes of GCP/DNA complexes decreased as the charge ratio of GCP to DNA increased and had a minimum value around 27 nm at the charge ratio of 5. Conformational change of DNA did not occur after complex formation with GCP compared to conformation of DNA itself. GCP/DNA complexes were only transfected into Hep G2 having asialoglycoprotein receptors (ASGR), indicative of specific interaction of ASGR on cells and galactose ligands on GCP.

Chitin↗
PubMed2001

Regulation of asialoglycoprotein receptor expression in the proliferative state of hepatocytes.

It is necessary to proliferate hepatocytes and to increase the number of hepatocytes for development of bioartificial liver (BAL) and reconstitutive therapy. But usually the cell has a precarious balance between proliferation and differentiation: as the cell proliferation increases, functional differentiation decreases. Therefore, it is desirable for the hepatocytes to be functional by differentiation as a material for such clinical use not to be proliferative. In this study, we investigated the background of hepatocyte proliferation for the springboard of control between proliferation and differentiation of hepatocytes, and we focused attention to the asialoglycoprotein receptors (ASGP-R) of the hepatocytes. Partially hepatectomized (PH) rats were used as a model animal. When the isolated hepatocytes were plated onto the artificial extracellular matrix of poly-(N-p-vinylbenzyl-O-beta-d-galactopyranosyl-d-gluconamide) (PVLA) having galactose residues as cell-specific ligand, the rate of adhesion was decreased along with liver regeneration. Interestingly, the release of the ASGP-R from hepatocytes in serum after PH in vivo and reduction of ASGP-R of the hepatocytes in the proliferative state occurred due to cell growth in vitro. It is suggested that the ASGP-R on the hepatocyte surface during the differentiation was released in the proliferative state.

Animals↗
PubMed2001

Direct production of an activated matrix metalloproteinase-9 (gelatinase B) from mammalian cells.

Matrix metalloproteinase-9 (MMP-9) is produced by the inactive proform and activated by a proteolytic process. However, it has not been reported to produce the active form directly from cells, which has hindered the research to elicit the physiological roles of this enzyme. In this study, we prepared mutant MMP-9 containing the furin-recognizing sequence in the prodomain and showed that the mutant MMP-9 was secreted as the active form directly from CHO-K1 cells and primary hepatocytes after the gene was transfected. The secreted MMP-9 showed proteolytic activity without further activation and degraded collagen IV in vitro. In addition, the transfection of the gene into the liver resulted in the efficient expression of active MMP-9 in the liver and the serum in vivo.

Animals↗
PubMed2001

Low asialoglycoprotein receptor expression as markers for highly proliferative potential hepatocytes.

Development of a reliable method to isolate highly proliferative potential hepatocytes will provide insight into the molecular mechanisms of liver regeneration, as well as proving crucial for the development of a biohybrid artificial liver. The aim of this study is to isolate highly proliferative, e.g., progenitor-like, hepatocytes. To this end, we fractionated hepatocytes expressing low and high levels of the asialoglycoprotein receptor (ASGP-R) based on the difference in their adhesion to poly[N-p-vinylbenzyl-O-beta-d-galactopyranosyl-(1-->4)-d-gluconamide] (PVLA), and examined the proliferative activity and gene expression of these fractionated hepatocytes. The results showed that approximately 0.5 to 1% of the total number of hepatocytes, which showed low adhesion to PVLA, expressed low levels of the ASGP-R, while the rest of hepatocyte population with high adhesion to PVLA expressed high levels of the ASGP-R. Interestingly hepatocytes with low ASGP-R expression levels had much higher DNA synthesizing activity (i.e., are much more proliferative) than those with high ASGP-R expression levels. Moreover, hepatocytes with low ASGP-R expression levels expressed higher levels of epidermal growth factor receptor (EGF-R), CD29 (beta1 integrin) and CD49f (alpha6 integrin) and lower levels of glutamine synthetase than those with high ASGP-R expression. These findings suggested that hepatocytes with low adhesion to PVLA due to their low ASGP-R expression could be potential candidates for progenitor-like hepatocytes due to their high proliferative capacity; hence, the low expression of the ASGP-R could be a unique marker for progenitor hepatocytes. The isolation of hepatocytes with different functional phenotypes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease.

Animals↗
PubMed2001

Activation of matrix metalloproteinases by peroxynitrite-induced protein S-glutathiolation via disulfide S-oxide formation.

Oxidative stress may cause tissue injury through activation of the precursors of matrix metalloproteinase (proMMPs). In this study, we observed glutathione (GSH)-dependent proMMP activation induced by peroxynitrite, a potent oxidizing agent formed during inflammatory processes. Peroxynitrite strongly activated all three types of purified human proMMPs (proMMP-1, -8, and -9) in the presence of similar concentrations of GSH. Of the potential reaction products between peroxynitrite and GSH, only S-nitroglutathione (GSNO(2)) caused proMMP activation. Extensive S-glutathiolation of the proMMP protein occurred during activation of proMMP by peroxynitrite and GSH, as shown by radiolabeling studies with [(35)S]GSH or [(3)H]GSH. Evidence of appreciable S-glutathiolation persisted even after dithiothreitol and protein-denaturing treatment, however, suggesting that some S-glutathiolation did not occur through formation of simple mixed disulfide. Matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry indicated that not only peroxynitrite plus GSH but also synthetic GSNO(2) produced dithiothreitol-resistant S-glutathiolation of the synthetic peptide PRCGVPD, which is a well conserved Cys-containing sequence of the propeptide autoinhibitory domain of proMMPs. PRCGVPD S-glutathiolation is presumed to be formed through glutathione disulfide S-oxide (GS(O)SR), based on the m/z 1064. Our results illustrate a unique mechanism of oxidative proMMP activation and oxidative tissue injury during inflammation.

Amino Acid Sequence↗
PubMed2001

Specific binding of glucose-derivatized polymers to the asialoglycoprotein receptor of mouse primary hepatocytes.

In this study, we designed a novel amphiphilic poly-(p-N-vinylbenzyl-D-glucuronamide) (PV6Gna) modified at the 6-OH position of glucose for hepatocyte recognition to address the mechanism of the interaction between mouse primary hepatocytes and the PV6Gna. PV6Gna bound to lectins specific for glucose but not galactose as did other glucose-derivatized polymers. However, hepatocyte adhesion onto the PV6Gna surface was inhibited in the presence of galactose and its analogues but not in the presence of glucose and its analogues. We also showed that hepatocyte adhesion to the PV6Gna surface was inhibited dose dependently by asialofetuin (ASF). Interactions between soluble PV6Gna and hepatocytes were inhibited by GalNAc, ASF, and EGTA in flow cytometry analysis using fluorescein isothiocyanate-conjugated PV6Gna. Hepatocyte adhesion to the PV6Gna surface was inhibited more effectively by GalNAc than by methyl beta-D-galactose. In flow cytometry analysis and cell adhesion assay, ASF competed for the inhibition of interaction between PV6Gna and hepatocytes 0.5-4 x 10(5)-fold more effectively than did GalNAc. These results demonstrate involvement of asialoglycoprotein receptors (ASGPRs) in the interaction between PV6Gna and hepatocytes. Furthermore, to clarify the mechanism of the interaction between glycopolymers modified at the 6-OH position of glucose and the hepatocyte, we prepared a gel particle containing 6-O-methacryloyl-d-glucose (PMglc) synthesized by an enzymatic method. ASGPRs could be detected using Western blot analysis following precipitation with PMglc in hepatocyte cell lysate. The precipitation of ASGPRs was inhibited in the presence of galactose, ASF, PV6Gna, and EGTA. The precipitation was inhibited more effectively by GalNAc than by methyl beta-d-galactose. ASGPRs were rarely precipitated by PMglc in the cell lysate that had been treated with ASF-conjugated Sepharose. Taken together, we suggest that mouse primary hepatocytes adhere to the PV6Gna surface mediated by ASGPRs, which specifically interacted with the glycopolymers modified at the C-6 position of glucose.

Animals↗
PubMed2001

Intracerebroventricular infusion of fibroblast growth factor-1 increases Fos immunoreactivity in periventricular astrocytes in rat hypothalamus.

When released into the third ventricle of the brain, fibroblast growth factor (FGF)-1 acts as a feeding suppressor. To identify the cells that respond to FGF-1 in this process, we investigated the effects of intracerebroventricular administration of FGF-1 on Fos protein expression in the rat hypothalamus. Two hours after infusion of FGF-1, significantly more Fos signals were observed in the periventricular zone of the third ventricle and its surroundings. Double immunohistochemical studies using antibodies against Fos, glial fibrillary acidic protein, and neuronal nuclei revealed that the FGF-1-induced Fos signals in the periventricular zone were in astrocytes, not in neurons. FGF-1-induced Fos expression was not found in neurons in such hypothalamic nuclei as the lateral hypothalamic area, paraventricular nucleus, ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, or arcuate nucleus. These results suggest a possible involvement of periventricular astrocytes in the early stages of FGF-1-induced feeding suppression.

Animals↗
PubMed2001

Comb-type cationic copolymer expedites DNA strand exchange while stabilizing DNA duplex.

The accelerating effect of cationic substances on the DNA strand exchange reaction between a 20 bp DNA duplex and its complementary single strand was studied. A polycationic comb-type copolymer, that consists of a poly(L-lysine) backbone and a dextran graft chain (PLL-g-Dex) and known to stabilize triplex DNA, expedites the strand exchange reaction under physiological relevant conditions. Electrostatically a small excess of the copolymer let to a 300-1500-fold increase in the DNA strand exchange while large excess of spermine or cetyltrimethylammonium bromide, a cationic detergent known to promote markedly hybridization of complementary DNA strands, shows only a slight effect. The efficacy of the copolymer was not affected by a 10 mM Mg2+ concentration. Notably the copolymer promotes the strand exchange reaction while it stabilizes double-stranded DNA. The stabilization of strand exchange intermediates consisting of the parent duplex and the single strand by the copolymer is believed to be responsible for the observed acceleration behavior.

Cations↗
PubMed2001

Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro.

The pathogenesis of pseudomonal keratitis was investigated by focusing on induction and activation of matrix metalloproteinases (MMPs) by pseudomonal virulence factors and proinflammatory cytokines. Corneal lesions and MMP induction in vivo were evaluated in rabbit corneas infected with a clinical isolate of Pseudomonas aeruginosa. Effects of pseudomonal virulence factors [elastase, alkaline protease, exotoxin A and lipopolysaccharide (LPS)], tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta on MMP induction and activation were further examined in vitro in rabbit corneal fibroblasts (RCF) and human fibrosarcoma (HT1080) cells using reverse transcriptase-polymerase chain reaction (RT-PCR), zymography and immunoblotting. Corneal ulcers with typical ring abscesses were observed 12-24 h after infection, and MMPs, particularly MMP-9, were upregulated in infected corneas. Pseudomonal elastase caused the most extensive damage to both cell types. RCF treated with pseudomonal exoproteases or LPS expressed and secreted MMP-9. Exotoxin A had no effect on MMP expression. Both IL-1beta and TNF-alpha augmented MMP-9 expression in HT1080 cells. Pseudomonal elastase proteolytically activated MMP-2 and MMP-9 released from the cells. In conclusion, corneal destruction seen with P. aeruginosa infections may result from enhanced expression of MMPs by corneal stromal cells stimulated with pseudomonal exoproteases and proinflammatory cytokines and the proteolytic activation of MMPs by pseudomonal elastase.

ADP Ribose Transferases↗
PubMed2001

Receptor-mediated cell modulator delivery to hepatocyte using nanoparticles coated with carbohydrate-carrying polymers.

Cell modulators such as colchicine (CO), cytochalasin B (CY) and taxol (TX) loaded nanoparticles coated with carbohydrate-carrying polymers, as hepatocyte-specific targeting material using galactose ligands as recognition signals to asialoglycoprotein receptors were prepared by the diafiltration method. Effects of cell modulators from their loaded nanoparticles on morphology of hepatocytes were studied. Receptor-mediated endocytosis of the nanoparticles were examined by fluorescence and confocal laser microscopy. It was found that the shapes of most hepatocytes were changed for the CY-loaded, TX-loaded, or CO-loaded nanoparticles whereas their shapes were not changed in comparison with control when CY, TX, or CO were mixed with the nanoparticles. From the fluorescence and confocal laser microscopic studies, it is suggested that the nanoparticles coated with sugar-carrying polymers were internalized by the hepatocytes through the receptor-mediated mechanism.

Animals↗
PubMed2001

Feeding suppression by fibroblast growth factor-1 is accompanied by selective induction of heat shock protein 27 in hypothalamic astrocytes.

It has been suggested that fibroblast growth factor (FGF)-1 serves as a physiological satiety factor in the hypothalamus, although the molecular mechanism underlying such a function is poorly understood. To gain additional insight into this issue, we used a Sendai virus (SeV) gene expression system in rats to explore genes differentially expressed subsequent to expression of FGF-1. Using cDNA arrays, we determined that infusion of FGF-1/SeV into one lateral ventricle induced selective expression of heat shock protein (HSP) 27 in the hypothalamus. Whereas FGF-1 expression was restricted to the ependymal cell layer of the cerebral ventricles, HSP27 was more widely expressed in astrocytes residing in the surrounding periventricular region. Similarly, infusion of FGF-1 polypeptide into a lateral ventricle induced dose-dependent HSP27 expression in periventricular astrocytes surrounding the third ventricle, with maximum mRNA levels being attained 6 h after infusion. This induction of HSP27 was accompanied by a significant suppression of feeding behaviour. Interestingly, suppression of feeding caused by intracerebro ventricular infusion of ciliary neurotrophic factor was also accompanied by induction of HSP27 in periventricular astrocytes, but suppression of feeding caused by infusion of leptin was not. It therefore appears that suppression of feeding by FGF-1 is accompanied by selective induction of HSP27 expression in hypothalamic astrocytes surrounding the third ventricle, and that this response may be a key component of the mechanism by which appetite is regulated by FGF-1.

Animals↗
PubMed2001

Dexamethasone impairs pulmonary defence against Pseudomonas aeruginosa through suppressing iNOS gene expression and peroxynitrite production in mice.

To elucidate the in vivo mechanisms involved in the impairment in pulmonary defence as the result of treatment with glucocorticoids, we established fatal pneumonia with bacteraemia in dexamethasone (DEX)-treated mice by means of an intratracheal challenge of Pseudomonas aeruginosa. An increased neutrophil influx was observed in bronchoalveolar lavage (BAL) fluids from both untreated and DEX-treated mice. The complete suppression of an inducible isoform of nitric oxide synthase (iNOS) mRNA expression and tumour necrosis factor alpha (TNF-alpha) production during the early phase of pneumonia, but not CXC chemokine production, were found in the case of the DEX-treated mice. An immunohistochemical study with a specific antibody also revealed negative staining for nitrotyrosine in the lung tissue of DEX-treated mice, while the formation of nitrotyrosine, which indirectly indicates the generation of peroxynitrite with a potent bactericidal activity, was detected clearly in the bronchial epithelium as well as alveolar phagocytic cells of lung tissue from untreated mice. Furthermore, an intraperitoneal administration of S-methyl-isothiourea (SMT), a potent inhibitor of NOS, significantly decreased the survival and increased bacterial density in the case of untreated mice. In contrast, no significant effects on the survival and bacterial density in the lung and blood were found as the result of treatment with SMT in DEX-treated mice. Collectively, a complete repression of iNOS gene expression and a lack of the generation of peroxynitrite as well as an inhibition of TNF-alpha production in the lung appeared to be responsible for the progression of the fatal pneumonia due to P. aeruginosa in DEX-treated mice.

Animals↗
PubMed2001

Establishment of heterotropic liver tissue mass with direct link to the host liver following implantation of hepatocytes transfected with vascular endothelial growth factor gene in mice.

One of the major goals of tissue engineering is to establish an integrated organ in vivo. We have previously shown that transfection of vascular endothelial growth factor (VEGF) gene into hepatocytes promotes tissue formation by engrafted cells. Here we show that tissue growth was significantly enhanced by co-transplantation of hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF alpha) gene transfected hepatocytes with VEGF-gene transfected cells, but tissue islands were scattered nonspecifically in the abdomen of mice. The result brought us forward to the next step to establish an integrated mass and structural formation of liver tissue. We entrapped VEGF gene transfected hepatocytes in a nylon mesh bag and intraperitoneally engrafted close to the liver. Three weeks later, the bag was covered by a thick network of blood vessels, compared to the control. Histological examination showed that the blood vessels penetrated the parenchyma of the engrafted bag and formed a well-developed vessel network in the region. The use of hepatocytes from lacZ transgenic mice and PCR analysis demonstrated survival and albumin production by hepatocytes in the engrafted bag. Our model can potentially be developed into a heterotropic artificial liver with direct access to the host blood circulation.

Actins↗
PubMed2001

Polycation graft copolymers accelerating DNA strand exchange: involvement of ionic interaction.

In the previous study (Chem. Eur. J., 7, 176 (2001)) we demonstrated that the comb-type polycationic copolymer (PLL-g-Dex) which is composed of poly (L-lysine) backbone and dextran graft chains expedited the DNA strand exchange reaction. In this study, fluorescence resonance energy transfer (FRET) was employed to explore the copolymer-mediated DNA strand exchange with higher time-resolution. To initiate strand exchange reaction the duplex prepared from 3'-fluorescein isothiocyanate (FITC)--and 5'-carboxytetramethylrhodamine(TAMRA)-labeled complementary DNAs was added to its non-labeled complementary single strand. DNA strand exchange was monitored by observing the recovery of the FITC quenching. More than 20,000 times increase in strand exchange rate at 37 degrees C by the copolymer was estimated. To investigate the accelerating mechanisms of the copolymer, the same reactions but at various ionic strenghts were studied. With increasing ionic strengths the strand exchange rate in the absence of the copolymer increased, suggesting that ionic repulsion among DNAs is unfavorable for the strand exchange to occur. Hence, alleviation of the electrostatic repulsion through interpolyelectrolyte complex formation is probably a role of the copolymer for accelerating the strand exchange reaction.

DNA↗
PubMed2001

Synergistic stabilization of triplex by combination of comb-type cationic copolymer and oligo-N3'-->P5' phosphoramidates.

In the present study, we describe rapid formation of stable pyrimidine motif triplex at physiological pH. The triplex formation was achieved by the synergistic effect of poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO). Either the PLL-g-Dex copolymer or the PN modification alone increased the binding constant by nearly two orders of magnitude for the triplex formation at neutral pH. The combination of both stabilizing factors that was the triplex formation with the PN TFO in the presence of the copolymer increased the binding constant by nearly four orders of magnitude. The kinetic study indicated that the copolymer increased the association rate constant, whereas the PN modification decreased the dissociation rate constant. No negative interference between these stabilizing effects was observed. The kinetically orchestrated effects in which the copolymer and the PN TFO contribute to distinct ingredients in triplex equilibrium achieved the rapid formation of the stable triplex.

Amides↗
PubMed2001

Protein/oligonucleotide conjugates as a cell specific PNA carrier.

We have focused on proteineus ligand conjugate with oligonucleotides (ODNs) as a cell-specific delivery vector for peptide nucleic acids (PNAs). Asialofetuin (AF), a hepatocyte-specific proteineus ligand, was conjugated with ODNs that served as binding sites for PNAs. Succinimidyl-transe-4(N-maleimidylmethyl)-cyclohexane-1-carboxylate (SMCC) modified AF was coupled with 5'-thiolated oligodeoxynucleotide (HS-ODN). The resulting conjugate held PNAs with sequence-specific manner. The PNA/DNA conjugate complex has resistance against nucleases in serum. The efficient release of PNA from the complex was observed when the complex was made in contact with a target nucleotide. PNA uptake to hepatocytes was greatly enhanced when hepatocytes was incubated with PNA/conjugate complex. Free AF thoroughly inhibited PNA uptake with the conjugate, evidencing asialoglycoprotein receptor (ASGP-R) mediated endocytosis to be a major-route for the cellular uptake.

Animals↗
PubMed2001

Enhanced vascular permeability in solid tumor involving peroxynitrite and matrix metalloproteinases.

Peroxynitrite (ONOO(-)), which is generated from nitric oxide (NO) and superoxide anion (O(2)(.-)) under pathological conditions, plays an important role in pathophysiological processes. Activation of matrix metalloproteinases (MMPs) contributes to tumor angiogenesis and metastasis. NO mediates the enhanced vascular permeability and retention (EPR) effect in solid tumors, and ONOO(-)activates proMMP to MMP in vitro. In this study, we examined the role of ONOO(-)in the EPR effect in solid tumors and normal tissues as related to MMP activation. Authentic ONOO(-), at 50 nmol or higher concentrations, induced the enhanced vascular permeability in normal dorsal skin of mice. ONOO(-)scavengers ebselen and uric acid significantly suppressed the EPR effect in mouse sarcoma 180 (S-180) tumors. Indirect evidence for formation of ONOO(-)in S-180 and mouse colon adenocarcinoma (C-38) tumors included strong immunostaining for nitrotyrosine in the tumor tissue, predominantly surrounding the tumor vessels. MMP inhibitor BE16627B (66.6 mg / kg i.v., given 2 times) or SI-27 (10 mg / kg i.p., given 2 times) significantly suppressed the ONOO(-)-induced EPR effect in S-180 tumors and in normal skin. Soybean trypsin inhibitor (Kunitz type), broad-spectrum proteinase inhibitor ovomacroglobulin, and bradykinin receptor antagonist HOE 140 also significantly suppressed the ONOO(-)-induced EPR effect in normal skin tissues. These data suggest that ONOO(-)may be involved in and promote the EPR effect in tumors, which could be mediated partly through activation of MMPs and a subsequent proteinase cascade to generate potent vasoactive mediators such as bradykinin.

Adenocarcinoma↗