Showing papers by "Hiroshi Maeda published in 2015"

Photodynamic therapy and imaging based on tumor-targeted nanoprobe, polymer-conjugated zinc protoporphyrin.

Abstract: Aim: To evaluate the potential of tumor-targeted nanoprobe, N-(2-hydroxypropyl)methacrylamide copolymer-conjugated zinc protoporphyrin (PZP) for photodynamic therapy (PDT) and tumor imaging. Materials & Methods: Different tumor models including carcinogen-induced cancer were used, PZP was intravenously injected followed by irradiation with xenon or blue fluorescent light on tumor. Results: One PZP 20 mg/kg (ZnPP equivalent) dose with two or three treatments of light at an intensity of ≥20 J/cm2 caused necrosis and disappearance of most tumors (>70%) in different tumor models. We also confirmed PZP-based tumor imaging in carcinogen-induced breast tumor and colon cancer models. Conclusion: These findings support the potential application of PZP as a tumor-selective nanoprobe for PDT as well as tumor imaging, by virtue of the enhanced permeability and retention effect.

Poly-S-nitrosated human albumin enhances the antitumor and antimetastasis effect of bevacizumab, partly by inhibiting autophagy through the generation of nitric oxide

Abstract: Autophagy is one of the major causes of drug resistance. For example, the angiogenesis inhibitor bevacizumab shows only transient and short-term therapeutic effects, whereas long-term therapeutic benefits are rarely observed, probably due to hypoxia-induced autophagy. Nitric oxide (NO) is an important molecule with multiple functions, and it has recently been reported to function as a regulator of autophagy. Therefore, a reasonable therapeutic strategy for overcoming drug resistance by NO would involve it being directly delivered to the tumor. Here, we investigated the inhibitory effect of NO on autophagy by using a macromolecular NO donor S-nitrosated human serum albumin (SNO-HSA) with a high degree of NO loading and tumor targeting potential. In colon 26 (C26) cells, SNO-HSA significantly suppressed hypoxia-induced autophagy by inhibiting the phosphorylation of JNK1 and the expression of its downstream molecule Beclin1. The effect of SNO-HSA was also confirmed in vivo by combining it with Bev. In C26-bearing mice, significant suppression of tumor growth as well as lung metastasis was achieved in the combination group compared to the SNO-HSA or bevacizumab alone group. Similar to the in vitro experiments, the immunostaining of tumor tissues clearly showed that SNO-HSA inhibited the autophagy of tumor cells induced by bevacizumab treatment. In addition to other known antitumor effects of SNO-HSA, that is, the induction of apoptosis and the inhibition of multidrug efflux pumps, these data may open alternate strategies for cancer chemotherapy by taking advantage of the ability of SNO-HSA to suppress autophagy-mediated drug resistance and enhance the efficacy of chemotherapy.

Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1

Abstract: Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co-adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA-CutL1 interaction by using site-directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N-terminus of RolA, play crucial roles in the RolA-CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 (K(D) = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger KD values, particularly in the interaction between the double variant RolA-H32S/K34S and the triple variant CutL1-E31S/D142S/D171S (K(D) = 78.0 nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface.

Analysis of the relationship between periodontal disease and atherosclerosis within a local clinical system: a cross-sectional observational pilot study

Abstract: It has been revealed that atherosclerosis and periodontal disease may have a common mechanism of “chronic inflammation”. Several reports have indicated that periodontal infection is related to atherosclerosis, but none have yet reported such an investigation through the cooperation of local clinics. This study was performed in local Japanese clinics to examine the relationship between periodontal disease and atherosclerosis under collaborative medical and dental care. A pilot multicenter cross-sectional study was conducted on 37 medical patients with lifestyle-related diseases under consultation in participating medical clinics, and 79 periodontal patients not undergoing medical treatment but who were seen by participating dental clinics. Systemic examination and periodontal examination were performed at baseline, and the relationships between periodontal and atherosclerosis-related clinical markers were analyzed. There was a positive correlation between LDL-C level and plasma IgG antibody titer to Porphyromonas gingivalis. According to the analysis under adjusted age, at a cut-off value of 5.04 for plasma IgG titer to Porphyromonas gingivalis, the IgG titer was significantly correlated with the level of low-density lipoprotein cholesterol (LDL-C). This study suggested that infection with periodontal bacteria (Porphyromonas gingivalis) is associated with the progression of atherosclerosis. Plasma IgG titer to Porphyromonas gingivalis may be useful as the clinical risk marker for atherosclerosis related to periodontal disease. Moreover, the application of the blood examination as a medical check may lead to the development of collaborative medical and dental care within the local medical clinical system for the purpose of preventing the lifestyle-related disease.

Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E

Abstract: Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes

Abstract: Background and Objective Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from −550 to −487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. Material and Methods To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. Results Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the −550 to −487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. Conclusion We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression.

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate

Abstract: Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

Mounting tool and mounting method of vehicular seat

Abstract: PROBLEM TO BE SOLVED: To obtain a mounting tool for a vehicular seat, with which mounting work efficiency of divided seats that are plurally divided in a vehicle width direction can be improved.SOLUTION: A mounting tool 1 of a vehicular seat for mounting, on a vehicle body, divided seats 70to 70which are plurally divided in the vehicle width direction comprises a holding member 10 which connects and holds the back surfaces of seat backs 71 in a state that the respective seat backs 71 are laid. At the holding member 10, a vehicle width direction position restriction part which restricts the vehicle width direction positions of respective brackets 80 of the respective divided seats 70to 70and a turning restriction part which restricts turning of the brackets 80 around a hinge shaft 81, are provided. By the vehicle width direction position restriction part, the positions of the respective brackets 80 are restricted to the positions at which the brackets can be fitted to the corresponding vehicle body side fitting part while matching the vehicle width direction respectively, and, by the turning restriction part, the turning positions of the respective brackets 80 are restricted all together to the turning positions at which the brackets can be fitted to the corresponding vehicle body side fitting surface.