Yasuhiko Hashida

Photothermal ablation of tumor cells using a single-walled carbon nanotube-peptide composite.

Single-walled carbon nanotubes (SWCNTs) are known to have great potential for biomedical applications such as photothermal ablation of tumor cells in combination with near-infrared (NIR) irradiation. In this study, the photothermal activity of a novel SWCNTs composite with a designed peptide having a repeated structure of H-(-Lys-Phe-Lys-Ala-)7-OH [(KFKA)7] against tumor cells was evaluated in vitro and in vivo. The SWCNT-(KFKA)7 composite demonstrated high aqueous dispersibility that enabled SWCNTs to be used in tumor ablation. The NIR irradiation of SWCNT-(KFKA)7 solution resulted in a rapid temperature increase dependent on the SWCNTs concentration up to 50μg/ml. Three minutes of NIR irradiation of a colon 26 or HepG2 cell culture incubated with SWCNT-(KFKA)7 resulted in remarkable cell damage, while that by single treatment with SWCNT-(KFKA)7 or NIR irradiation alone was moderate. The intratumoral injection of SWCNT-(KFKA)7 solution followed by NIR irradiation resulted in a rapid increase of the temperature to 43°C in the subcutaneously inoculated colon 26 tumor based on thermographic observation and remarkable suppression of tumor growth compared with treatment with only SWCNT-(KFKA)7 injection alone or NIR irradiation alone. These results suggest the a great potential of an SWCNT-peptide composite for use in photothermal cancer therapy.

Engineering, expression, purification, and production of recombinant thermolysin

Thermolysin [EC 3.4.24.27] is a thermostable neutral zinc metalloproteinase originally identified in the culture broth of Bacillus thermoproteolyticus Rokko. Since the discovery in 1962, the enzyme has been extensively studied regarding its structure and catalytic mechanism. Today, thermolysin is a representative of zinc metalloproteinase and an attractive target in protein engineering to understand the catalytic mechanism, thermostability, and halophilicity. Thermolysin is used in industry, especially for the enzymatic synthesis of N-carbobenzoxy L-Asp-L-Phe methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame. Generation of genetically engineered thermolysin with higher activity in the synthesis of ZDFM has been highly desired. In accordance with the expansion of studies on thermolysin, various strategies for its expression and purification have been devised and successfully used. In this review, we aim to outline recombinant thermolysins associated with their engineering, expression, purification, and production.

Preparation of immunostimulatory single-walled carbon nanotube/CpG DNA complexes and evaluation of their potential in cancer immunotherapy.

Carbon nanotubes (CNTs) have many interesting properties. In particular, their photohyperthermic effect by near-infrared (NIR) irradiation could be used to kill cancer cells, and could thus be applied in photohyperthermic therapy. However, the solubility of CNTs must be improved before they can be used in biological applications. As DNA is reported to disperse the CNTs in aqueous solution with π-π interactions, we hypothesis that immunostimulatory CpG DNA may also disperse the CNTs in aqueous solution. In this study, we used CpG DNA to disperse single-walled CNTs (SWCNTs) in aqueous solution, in order to combine photohyperthermic effect and immunoactivation together to achieve a more effective cancer therapy. As expected, CpG DNA effectively dispersed the SWCNTs in aqueous solution via the formation of SWCNT/CpG DNA complexes. Moreover, the immunoreactivity of the SWCNT/CpG DNA complexes was investigated. The results showed that intratumoral administration of the SWCNT/CpG DNA complexes in mice enhanced the production level of inflammatory cytokines in tumor tissues. Finally, we evaluated the antitumor effects of the SWCNT/CpG DNA complexes in tumor-bearing mice. The result indicated that intratumoral administration of the SWCNT/CpG DNA complexes combined with NIR irradiation was a more effective approach to prevent the proliferation of tumor growth.

Development of a Novel Composite Material with Carbon Nanotubes Assisted by Self-Assembled Peptides Designed in Conjunction with β-Sheet Formation

A novel composite material is developed with single-walled carbon nanotubes (SWCNTs) and artificially designed peptides, and its chemical and physicochemical characteristics are evaluated with an aim toward biomedical application. The peptides were designed to form a β-sheet structure that would be suitable for wrapping SWCNTs. The complex of SWCNTs and peptide (SWCNT-peptide) showed good dispersibility in aqueous media and was considerably stable even in the absence of an excess amount of peptide in the media. The formation of SWCNT-peptide was confirmed by its performance in water, atomic force microscopy and transmission electron microscopy observation, and molecular modeling. The possibility of introducing various functions to SWCNT-peptide was also demonstrated by several methods, such as introduction of special amino acids, chemical modification, and additional complex formation based on electrostatic interaction. These results suggest the potential of the SWCNT-peptide complex as a molecular platform on which a desirable structure and/or function can be constructed for biomedical and industrial application.

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k(cat) values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190±10, 840±30, and 500±10 s(-1), respectively. The k(cat) values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240±60, 1450±30, and 2250±80 s(-1), respectively, at 1.0M. On the other hand, the k(cat) values at 0.05-1.0M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s(-1). These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K(m) values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K(m) value at 1.0M diethanolamine (0.83±0.15 mM) was 12-fold higher than that at 0.05M (0.07±0.01 mM), and that at 1.0M N-methylethanolamine (2.53±0.20 mM) was 14-fold higher than that at 0.2M (0.18±0.02 mM), while that at 1.0M triethanolamine (0.31±0.01 mM) was similar as that at 0.2M (0.25±0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.

High-density lipoprotein mutant eye drops for the treatment of posterior eye diseases

ABSTRACT Age‐related macular degeneration (AMD), in which choroidal neovascularization (CNV) affects the center of the retina (macula), leads to the irreversible visual loss. The intravitreal injection of anti‐angiogenesis antibodies improved the prognosis of AMD, but relatively less invasive therapies should be explored. In the present study, we show that a high‐density lipoprotein (HDL) mutant is a therapeutically active drug carrier capable of treating a posterior eye disease in mice via instillation. Various HDL mutants were prepared with apoA‐I proteins fused with different cell‐penetrating peptides (CPPs) and phospholipids with different alkyl chain lengths; their sizes were further controlled in the range of 10–25 nm. They were screened based on the efficiency of fluorescent dye delivery to the inner retinal layer in mice. The best mutant was found to have penetratin (PEN) as a CPP, 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC), and a size of 15 nm. In preclinical studies on a laser‐induced CNV murine model, 1 week of instillation of the best mutant carrying the anti‐angiogenesis drug pazopanib had dramatic therapeutic effects in reducing the CNV size. Importantly, the HDL mutant by itself contributed to the therapeutic effects. Future clinical trials for treating AMD with instillation of the HDL mutant are expected. Graphical abstract Figure. No caption available.

Asp3Gly polymorphism affects fatty acid-binding protein 3 intracellular stability and subcellular localization

Fatty acid‐binding proteins (FABP) play a crucial role in intracellular fatty acid transportation and metabolism. In this study, we investigate the effects of the FABP3 Asp3Gly (D3G) polymorphism on protein structure and function. Although the mutation did not alter protein secondary structure or the ability to bind 1‐anilinonaphthalene‐8‐sulfonic acid and palmitate, the intracellular stability of the D3G mutant was significantly decreased. Immunocytochemical analysis reveals that the mutation alters FABP3 subcellular localization. Our results suggest that the D3G polymorphism may impact energy metabolism and physiological functions.

Effects of Conversion of the Zinc-Binding Motif Sequence of Thermolysin, HEXXH, to That of Dipeptidyl Peptidase III, HEXXXH, on the Activity and Stability of Thermolysin

Most zinc metalloproteinases have the consensus zinc-binding motif sequence HEXXH, in which two histidine residues chelate a catalytic zinc ion. The zinc-binding motif sequence of thermolysin, H142ELTH146, belongs to this motif sequence, while that of dipeptidyl peptidase III (DPP III), H450ELLGH455, belongs to the motif sequence HEXXXH. In this study, we examined effects of conversion of HEXXH to HEXXXH in thermolysin on its activity and stability. Thermolysin variants bearing H142ELLGH146 or H142ELTGH146 (designated T145LG and T145TG respectively) were constructed by site-directed mutagenesis and were produced in Escherichia coli cells by co-expressing the mature and pro domains separately. They did not exhibit hydrolyzing activity for casein or N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but exhibited binding ability to a substrate analog glycyl-D-phenylalanine (Gly-D-Phe). The apparent denaturing temperatures based on the ellipticity at 222 nm of T145LG and T145TG were 85 ± 1 °C and 86 ± 1 °C respectively, almost the same as that of wild-type thermolysin (85 ± 1 °C). These results indicate that conversion of HEXXH to HEXXXH abolishes thermolysin activity, but does not affect its binding ability to Gly-D-Phe or its stability. Our results are in contrast to ones reported previously, that DPP III variants bearing H450ELGH455 exhibit activity.

Stabilization of Bovine Intestine Alkaline Phosphatase by Sugars

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T 50, of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0–55.5 °C, 4.7–5.2 °C higher than without sugar (50.3±0.1 °C). The T 50 of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the k cat of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP.