- [Show abstract] [Hide abstract] ABSTRACT: The olfactory system of living organisms can accurately discriminate numerous odors by recognizing the pattern of activation of several odorant receptors (ORs). Thus, development of an odorant sensor array based on multiple ORs presents the possibility of mimicking biological odor discrimination mechanisms. Recently, we developed novel odorant sensor elements with high sensitivity and selectivity based on insect OR-expressing Sf21 cells that respond to target odorants by displaying increased fluorescence intensity. Here we introduce the development of an odorant sensor array composed of several Sf21 cell lines expressing different ORs. In this study, an array pattern of four cell lines expressing Or13a, Or56a, BmOR1, and BmOR3 was successfully created using a patterned polydimethylsiloxane film template and cell-immobilizing reagents, termed biocompatible anchor for membrane (BAM). We demonstrated that BAM could create a clear pattern of Sf21 sensor cells without impacting their odorant-sensing performance. Our sensor array showed odorant-specific response patterns toward both odorant mixtures and single odorant stimuli, allowing us to visualize the presence of 1-octen-3-ol, geosmin, bombykol, and bombykal as an increased fluorescence intensity in the region of Or13a, Or56a, BmOR1, and BmOR3 cell lines, respectively. Therefore, we successfully developed a new methodology for creating a cell-based odorant sensor array that enables us to discriminate multiple target odorants. Our method might be expanded into the development of an odorant sensor capable of detecting a large range of environmental odorants that might become a promising tool used in various applications including the study of insect semiochemicals and food contamination.
- [Show abstract] [Hide abstract] ABSTRACT: Phase-change nano-droplets (PCNDs) are sub-micron particles that are coated with phospholipid and contain liquid-state perfluorocarbons such as perfluoropentane (boiling point = 29 °C) and perfluorohexane (boiling point = 57 °C), which can vapourise upon application of ultrasound. The bubbles generated by such reactions can serve as ultrasound contrast agents or HIFU sensitisers. However, the lifetime of bubbles generated from PCNDs on μs-order is not well known. Knowledge of the condition of PCND-derived bubbles on μs-order is essential for producing bubbles customised for specific purposes. In this study, we use an optical measurement system to measure the vapourisation and stability of the bubbles (bubble-lifetime) as well as the stability-controlling method of the nucleated bubbles on μs-order while changing the internal composition of PCNDs and the ambient temperature. PCND-derived bubbles remain in a bubble state when the boiling point of the internal composition is lower than the ambient temperature, but lose their optical contrast after approximately 10 μs by re-condensation or dissolution when the boiling point of the internal composition is higher than the ambient temperature. We reveal that the superheating condition significantly affects the fate of vapourised PCNDs and that the bubble-lifetime can be controlled by changing both the ambient temperature conditions and the internal composition of PCNDs.
- [Show abstract] [Hide abstract] ABSTRACT: Photolytic protein aggregates are developed as a facile and versatile platform for light-induced release of active proteins. The proteins modified with biotin through a photo-cleavable linker rapidly form aggregates with streptavidin and biotinylated functional molecules simply by mixing. Light irradiation releases active proteins from the aggregates in high yields, and light-induced uptake of drug-modified transferrin into living cells is successfully demonstrated.
- [Show abstract] [Hide abstract] ABSTRACT: Knowledge of the relationship between the activity and structural features of enzymes is critical for designing conjugation materials. Nonetheless, few studies have addressed the decrease in enzyme activity after modifications of the corresponding enzymes. In cytochrome P450cam, the structural changes around the heme active center can be easily evaluated by spectrophotometric analysis. Thus, with the use of P450cam, we evaluated the relationship between enzyme activity and structural changes after its modification. Our study revealed the existence of a new mode for P450 activity downregulation, which was distinct from a well-known inactivation mode for the conversion of P450 to the inactive P420 form. In this new mode, the activity decreased due to a reduction in binding or accessibility of a substrate to the heme pocket of P450cam, while the enzymatic tertiary structure was retained after conjugations.
- [Show abstract] [Hide abstract] ABSTRACT: Transductions of exogenous proteins into cells enable the precise study of the effect of the transduced proteins on cellular functions. Accordingly, the protein transduction technique, which can control the release of proteins into the cytosol with certainty and high-throughput, is highly desired in various research fields. In this study, streptavidin (SA) labeled with a photosensitizer and cell-permeable peptides (CPP) was proposed as a nano-carrier for light-controlled protein transduction. SA was modified with biotinylated oligo-arginine peptides (Rpep), which were functionalized with Alexa Fluor 546 (AF546), to achieve cell penetrating and endosomal escape functionalities. The SA-Rpep complex was efficiently internalized into living HeLa cells corresponding to the length and the modification number of Rpep. SA conjugated with more than three equimolar AF546-modified Rpep consisting of fifteen arginine residues was achieved to diffuse throughout the cytosol without cytotoxicity by irradiation of the excitation light for AF546. The optimized nano-carrier was confirmed to transduce a biotinylated model cargo protein, enhanced green fluorescent protein fused with thioredoxin (tEGFP) into the cytosol at the light-irradiated area. The results provided proof-of-principle that SA possessing multiple AF546-modified Rpep has the potential to be a versatile and facile carrier for light-controlled protein transduction into the cytosol of mammalian cells. Copyright © 2015. Published by Elsevier B.V.
- [Show abstract] [Hide abstract] ABSTRACT: Specific ligand-grafted cyclic peptides are promising drug candidates that can modulate protein-protein interactions (PPIs) with increased proteolytic stability. In this study, we aimed to demonstrate that Sortase A (SrtA)-mediated peptide transpeptidation can be applied to produce bioactive sequence-grafted, stable, cyclic peptides. A naturally occurring cyclic peptide, sunflower trypsin inhibitor 1 (SFTI-1), was selected as the scaffold, and a tetrapeptide motif, Glu-Ser-Asp-Val (ESDV), was grafted into the scaffold as a model ligand. The linear precursor of the grafted peptide with SrtA-recognition motifs at the N- and C-termini was cyclized in good yield simply by co-incubation with SrtA. The ESDV-grafted cyclic SFTI-1 obtained was confirmed to have high stability against proteolysis by human serum and bound to the target PDZ2 domain of postsynaptic density-95 protein. An optimized sequence-grafted cyclic SFTI-1 could competitively suppress the interaction of PDZ2 with its natural ligand, the C-terminal peptide of the NR2B subunit of the N-methyl-D-aspartate receptor. These results show that a strategy combining peptide grafting into the SFTI-1 scaffold with SrtA-catalyzed cyclization can be a simple and effective method for producing stable peptide drugs. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- [Show abstract] [Hide abstract] ABSTRACT: Alkyl imidazolium-based polymer hydrogels were prepared by copolymerizing N-allylimidazolium chloride monomers (AlImCls) bearing four types of alkyl groups with N-isopropylacrylamide and N, N'-methylenebisacrylamide. The conditions for preparing the anion-responsive hydrogels were screened by gelation test before and after the addition of tetrafluoroborate anions. The sol-gel phase transition was observed in N-dodecyl and N-octadecyl AlImCls-based copolymer solutions. Considering the anion selectivity in the gelation test, this phase transition was assumed to be derived from the formation of water-immiscible ion pairs of N-allylimidazolium cations and large anions on the polymers. Microscopic observations revealed that spherical domains with micro-sized diameters were formed in the hydrogels. A hydrophobized fluorescent dye was localized to the microdomains. Observations using time-lapse confocal microscopy and electron microscopy indicated that large microdomains were captured by the gel meshes consisting of thin sub-micron fibers. Based on the data collected during the analysis of the copolymer solutions, the spherical microdomains and the thin gel meshes were presumably formed through the anion-induced aggregation of small copolymer assemblies. Furthermore, the high fluidity of the microdomains was confirmed by measuring fluorescence recovery after photobleaching. In summary, anion-responsive hydrogels with hydrophobic microdomains were developed using ionic salt-based copolymers. These hydrogels are promising as a unique carriers for the use in biotechnology.
- [Show abstract] [Hide abstract] ABSTRACT: Cell patterning on photo-responsive materials are a promising tool for preparing unique single-cell arrays. However, most conventional single-cell arrays on such smart materials can be applied only to adherent cells and limit cellular functions such as extension and migration within the patterned adhesive surfaces. In this study, a versatile single cell array that works with both non-adherent and adherent cells was constructed using a photo-cleavable polyethylene glycol (PEG)-lipid/collagen surface. On this single-cell array, cells behaved similar to their native functions without limitation from the patterned surface. Furthermore, quantitative imaging analyses of cellular motility and morphological changes were performed in a high-throughput manner.
- [Show abstract] [Hide abstract] ABSTRACT: G2A (from G2 accumulation) receptor is a member of the proton-sensing G-protein coupled receptor (GPCR) family and induces signal transduction events that regulate the cell cycle, proliferation, oncogenesis, and immunity. The mechanism by which G2A-mediated signal transduction is regulated by the extracellular pH remains unresolved. Here, we first visualize the pH-dependent G2A distribution change in living cells by a sortase A-mediated pulse labeling technology: the short-peptide tag-fused human G2A on human embryo kidney HEK293T cell surfaces was labeled with a small fluorescent dye in the presence of lysophosphatidylcholine, and the labeled G2A was chased at acidic and neutral pHs in real time by microscope time course observations. G2A internalization from cell surfaces into intracellular compartments was observed to be inhibited under acidic pH conditions, and this inhibition was relieved at neutral pH. Additionally, the internalized G2A was redistributed onto cell surfaces by jumping from a neutral to an acidic pH. From quantitative image analysis data, we conclude the amount of G2A on the cell surface was controlled by suppressing the G2A internalization rate by one-tenth in response to the extracellular acidic pH, and this acidic pH-induced G2A accumulation on cell surfaces may be explained by proton-induced dissociation of G2A from endocytic machinery.- Lan, W., Yamaguchi, S., Yamamoto, T., Yamahira, S., Tan, M., Murakami, N., Zhang, J., Nakamura, M., Nagamune, T. Visualization of the pH-dependent dynamic distribution of G2A in living cells.
- [Show abstract] [Hide abstract] ABSTRACT: Cell micropatterning methods with stimuli-responsive dynamic surfaces are getting a lot of attention in a wide variety of research fields, ranging from cell engineering to fundamental studies in cell biology. The surface of a slide coated with photo-cleavable poly(ethylene glycol) (PEG)-lipid can be used to spatiotemporally control cell immobilization and release by light irradiation. On the basis of this surface, it is easy to design simple methods for making a fine micropattern of any kind of cell. Furthermore, target cells can be selectively and rapidly released from this surface by light irradiation. In this review, we first describe how to obtain the photo-cleavable PEG-lipid from commercially available compounds through a facile four-step synthesis. Next, as a cell-patterning method, the protocols of coating substrates with the PEG-lipid, irradiating a pattern of light onto the coated substrate, and loading cells onto the irradiated surface are described. These protocols require no expensive equipment and potentially apply to any substrates that can adsorb serum albumin or chemically expose amine moieties on their surfaces. Finally, as an advanced method, cell release from the PEG-lipid surface in microfluidic devices is introduced. We also discuss the advantages and the possible applications of the present dynamic cell-patterning method.
- [Show abstract] [Hide abstract] ABSTRACT: We have developed a novel technique for constructing microarrays of transfected mammalian cells on or in extracellular matrix (ECM) hydrogels by transfer printing from patterned poly(ethylene glycol) (PEG)-oleyl surfaces. A mixed solution of small interfering RNA (siRNA) and a transfection reagent was spotted on PEG-oleyl-coated glass slides using an inkjet printer, and the cells were then transiently immobilized on the patterned transfection mixtures. After overlaying an ECM hydrogel sheet onto the immobilized cells, the cells sandwiched between the glass slide and the hydrogel sheet were incubated at 37°C for simultaneous transfection of siRNA into cells and adhesion of cells to the hydrogel sheet. Transfer of the adhered, transfected cells was completed by peeling off the hydrogel sheet. The knockdown of a model gene in the transferred cell microarray by the transfected siRNA was successfully confirmed. Transfected cell microarrays were also embedded within three-dimensional ECM hydrogels. In the three-dimensional hydrogel, the inhibition effect of siRNA on cancer cell invasion was evaluated by quantifying the size of cell clusters on the microarrays. These results indicate that transfection of cell microarrays on or in a biological matrix is a promising technique for high-throughput screening of disease-related genes by direct observation of cellular phenomena in a physiologically relevant environment. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
- [Show abstract] [Hide abstract] ABSTRACT: Small enough to permeate through tumor blood vessel, and can be detect by ultrasound, phase change nano droplet (PCND) have been studied as contrast agents and therapeutic sensitizer for career. To investigate performance for these purpose, we investigated physical behavior of PCND, especially a lifetime of microbubbles generated by ultrasound stimulation. To investigate bubble's behavior after phase change, we observed a time-lapse change of bubbles population with following method. Focused transducer with a frequency of 3.3 MHz was placed in a water bath of 37 degrees. At the focal point, polyacrylamide gel including PCND was placed. Focused hydrophone was placed perpendicularly to the direction of ultrasound propagation. Two kind of ultrasound pulse wave was used; phase change pulse at the beginning and observation pulse at every 500 μs. The amplitude of scattering signal (SS) reflects the sum of scattering cross-section of bubbles. The time lapse observation of PCND after phase change showed two kinds of behavior, quick and slow decay of scattering signal from the bubbles. The unique increase of SS from the phase-changed bubbles in the monitoring phase was observed. We improved the experimental setup to measure bubble's population properly from various directions using 1-D array transducer and will present the result.
- [Show abstract] [Hide abstract] ABSTRACT: Cell surface display of functional proteins is a powerful and useful tool for regulating and reinforcing cellular functions. Direct incorporation of site-specifically lipidated proteins from the extracellular medium is more rapid, easily controllable and reliable in displaying active proteins than expression through gene transfer. However, undesirable amphiphilic reagents such as organic cosolvents and detergents were required for suppressing aggregation of ordinary lipidated proteins in solution. We report here sortase A-catalyzed modification of proteins with a poly(ethylene glycol)(PEG)-lipid in situ on the surface of living cells. Proteins fused with a recognition tag were site-specifically ligated with the PEG-lipid which was preliminary incorporated into cell membranes. Accordingly, target proteins were successfully displayed on living cells without aggregation under an amphiphilic reagent-free condition. Furthermore, to demonstrate the availability of the present method, Fc domains of immunoglobulin G were displayed on cancer cells, and the phagocytosis of cancer cells with dendritic cells were enhanced through the Fc-Fc receptor interaction. Thus, the present facile chemoenzymatic method for protein display can be utilized for modulating cell-cell interactions in cell and tissue engineering fields. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
- [Show abstract] [Hide abstract] ABSTRACT: We site-specifically conjugated biotin-PEG derivatives with spacer arms of different lengths to mutant P450cam (3mD) and evaluated the activity of and structural changes in the conjugates as a first step toward clarifying the mechanism whereby the activity of the 3mD conjugate is inhibited. 3mD was prepared by site-specific mutation to inhibit its enzymatic activity artificially, after which the derivative compounds were conjugated to the enzyme. 3mD has one cysteine on its surface with a reactive thiol group that can react with compounds near the active site, where a conformational change will be induced after conjugation. The activity of 3mD was retained in the biotin-PEG2-3mD conjugate, but was dramatically reduced in the biotin-PEG11-3mD conjugate. To investigate the effect of poly(ethylene glycol) (PEG) length on the enzymatic activity after conjugation, PEGs of different lengths, exceeding that in biotin-PEG11, and whose termini were not biotin, were conjugated to 3mD. The activity of 3mD decreased in all these conjugates. This indicates that the activity of 3mD in these conjugates decreased after its conjugation with PEG molecules that exceeded a certain length. The biotin-PEG2-3mD, which retains enzymatic activity after conjugation, showed avidin responsiveness; the enzymatic activity decreased after avidin binding.
- [Show abstract] [Hide abstract] ABSTRACT: Streptococcus pyogenes sortase A (SpSrtA) was transduced into mammalian cells. Then SpSrtA-mediated intracellular circularization of a model protein was confirmed in a time and dose-dependent manner by Western blotting analysis. Direct transduction of SpSrtA is expected to be a strong tool for more conditional specific protein modification in mammalian cells.
- [Show abstract] [Hide abstract] ABSTRACT: A method for caging a target protein with steric regulator molecules was developed to enable simple, strict, light-induced control of protein activity.
- [Show abstract] [Hide abstract] ABSTRACT: We conjugated a molecular recognition moiety, biotin, with an enzyme site-specifically near to its active site and succeeded in inactivating the enzyme by binding the specific target biomolecule avidin to biotin. Bacterial P450 was used as a model enzyme, which has attracted much attention in several fields. Site-directed mutagenesis was conducted to produce a mutant P450 that could attach biotin site-specifically. The activity of the conjugate decreased markedly to one tenth of that of biotinylated P450 after binding to avidin. Ultraviolet-visible spectroscopy of the carbon monoxide-bound P450, circular dichroism data, and the ratio of the active form to the sum of the active form and the inactive form indicated that this decrease in activity was because of a conformational change in the tertiary structure surrounding the active center after avidin binding, while the secondary structure of P450 remained unchanged.
- [Show abstract] [Hide abstract] ABSTRACT: Analysis of the structural features of rhodopsin-type G-protein-coupled receptors (GPCRs) revealed the existence of an additional α-helix, termed helix 8, in the C-terminal tail. Furthermore, these GPCRs were determined to possess several conserved residues in their transmembrane domains. The functional deficiencies of receptors in which these domains or residues have been mutated have not been examined in living cells due to their accumulation in the endoplasmic reticulum (ER), although the ligand affinities of these receptors have been tested in membrane preparations. Recent studies have demonstrated that ER-accumulated receptors are effectively exported from ER using membrane permeable ligands as pharmacological chaperones. Here, we identified several residues of the platelet-activating factor receptor and leukotriene B(4) type-II receptor that are crucial for export from ER. Moreover, we used their specific ligands as pharmacological chaperones to traffic ER-accumulated GPCRs to the cell surface in order to examine the functional deficiencies of each mutant receptor. Here, we introduce the novel technique of site-specific N-terminal labeling of cell surface proteins in living cells with Sortase-A, a transpeptidase isolated from Staphylococcus aureus, to evaluate the trafficking of receptors after agonist stimulation.
- [Show abstract] [Hide abstract] ABSTRACT: In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science and engineering. Inclusion-body-based protein production is a promising method because high yields are achieved in the upstream process, although the refolding of solubilized, unfolded proteins in downstream processes often leads to significantly lower yields. The most challenging problem is that the effective condition for refolding is protein dependent and is therefore difficult to select in a rational manner. Accordingly, considerable time and expense using trial-and-error approaches are often needed to increase the final protein yield. Furthermore, for certain target proteins, finding suitable conditions to achieve an adequate yield cannot be obtained by existing methods. Therefore, to convert such a troublesome refolding process into a routine one, a wide array of methods based on novel technologies and materials have been developed. These methods select refolding conditions where productive refolding dominates over unproductive aggregation in competitive refolding reactions. This review focuses on synthetic refolding additives and describes the concepts underlying the development of reported chemical additives or chemical-additive-b.
- [Show abstract] [Hide abstract] ABSTRACT: A simple method for attaching immunoglobulin G (IgG) on the cell surface was successfully developed for enhancing phagocytosis of apoptotic tumor cells (ATCs) by dendritic cells (DCs) ex vivo. By conjugating with a poly(ethylene glycol) (PEG)-lipid, named the biocompatible anchor for the membrane (BAM), arbitrary IgG could be incorporated into the cell membrane. In particular, when IgG-BAM conjugates were prepared at the optimal molar ratio of IgG to BAM (1 to 20), almost all cells were efficiently modified with IgG by treatment with IgG-BAM. This simple method was successfully applied to four types of mammalian cells. Furthermore, treatment of ATCs with the IgG-BAM conjugate increased the phagocytosis ratio of ATCs by DCs two-fold when compared to no treatment. This phagocytosis-enhancing effect was nearly identical to treatment with a tumor-specific IgG. Thus, without employing the tumor-specific IgG, which is difficult to obtain for any tumor cells and is expensive, the present method could opsonize ATC with the use of arbitrary IgG. The results strongly indicate that IgG-BAM treatment represents a promising method for opsonizing ATC with human serum IgG, and that this approach will lead to objective clinical responses in DC vaccines.
The University of Tokyo白山, Tōkyō, Japan