Chikashi Nakamura

High-efficiency DNA injection into a single human mesenchymal stem cell using a nanoneedle and atomic force microscopy

We describe a low-invasive gene delivery method that uses an etched atomic force microscopy (AFM) tip or nanoneedle that can be inserted into a cell nucleus without causing cellular damage. The nanoneedle is 200 nm in diameter and 6 mum in length and is operated using an AFM system. The probabilities of insertion of the nanoneedle into human mesenchymal stem cells (MSCs) and human embryonic kidney cells (HEK293) were higher than those of typical microinjection capillaries. A plasmid containing the green fluorescent protein (GFP) gene was adsorbed on a poly-L-lysine-modified nanoneedle surface, which was then inserted into primary cultured single human MSCs. A highly efficient gene delivery of over 70% was achieved in human MSCs, which compared very favorably with other major nonviral gene delivery methods (lipofection approximately 50%, microinjection approximately 10 %). The single cells expressing GFP were collected and the amount of delivered DNA in each cell was analyzed. The highest rate of expressed GFP per delivered DNA was achieved using the nanoneedle, because the nanoneedle could be inserted into the nucleus directly without causing significant cell damage.

Stretching the α-helix: a direct measure of the hydrogen-bond energy of a single-peptide molecule

Abstract Atomic force microscopy was used to measure the force required to stretch individual molecules of the peptide cysteine 3 –lysine 30 –cysteine from the α-helical state into a linear chain (approximately 200 pN). The measured force versus peptide elongation was used to calculate the work done in breaking the hydrogen bonds which give rise to the helical structure. The average experimental value of the hydrogen-bond energy (20.2 kJ/mol) is in good agreement with reported theoretical calculations. In addition, the stiffness of individual peptides was measured directly using a force modulation technique and found to vary from approximately 0.005–0.012 N/m during elongation.

Immobilized liposome chromatography to study drug–membrane interactions: Correlation with drug absorption in humans

For rapid screening of drug-membrane interactions and predicting drug absorption in vivo, unilamellar liposomes were stably immobilized in the pores of gel beads by avidin-biotin binding. Interactions of a diverse set of well-described drugs with the immobilized liposomal membranes were reflected by their elution profiles. The membrane partitioning coefficients (KLM) of the drugs were determined from the retention volumes. The drug retentions on egg phosphatidylcholine (EPC)-phosphatidylserine (PS)-cholesterol (chol) and EPC-PS-phosphatidylethanolamine (PE)-chol columns intended to mimic small intestine membranes were similar, although the positively-charged drugs were more strongly retarded on the negatively-charged liposomes than the negatively-charged drugs. The relationship between log KLM with the drug fraction absorbed in humans showed that the log KLM values obtained with unilamellar liposomes can be used to predict drug passive transcellular absorption, similarly to that previously shown for entrapped multilamellar liposomes. The immobilized liposome chromatography method should be useful for screening compounds at an early stage of the drug discovery process. The avidin-biotin immobilization of the liposomes prolongs the lifetime of the columns.

Rapid and specific detection of herbicides using a self-assembled photosynthetic reaction center from purple bacterium on an SPR chip

In this study, a direct detection system for herbicides inhibiting photosynthetic electron transfer was developed using the photosynthetic reaction center (RC) from the purple bacterium, Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus. The heavy-subunit-histidine-tagged RCs (HHisRCs) were immobilized on an SPR sensor chip via nickel chelation chemistry as a binder for one of the triazine herbicides, atrazine. Immediately after injection of atrazine solution on the HHisRCs-immobilized chip, the SPR responses increased and reached plateaus within 1 min. The SPR signals were proportional to the sample concentrations of atrazine in the range 1-100 microg/ml. To evaluate the binding specificity to atrazine, chlorinated aromatic herbicides, DCMU and MCPP, were investigated using the HHisRCs-immobilized chip. An RC inhibitor, DCMU, could also be detected with a higher detection limit of 20 microg/ml than atrazine (1 microg/ml). MCPP showed no signals because its inhibition mechanism against plants is different from that of atrazine and DCMU. These results indicated that the sensor chip immobilized RCs could be used for the specific detection of photosynthetic inhibitors.

Metal-mediated coordination polymer nanotubes of 5,10,15,20-tetrapyridylporphine and tris(4-pyridyl)-1,3,5-triazine at the water–chloroform interface

Coordination polymer nanotubes have been prepared by using the Hg2+-mediated co-assembly of two ligands, tetrapyridylporphine (TPyP) and tris(4-pyridyl)-1,3,5-triazine (TPyTa), at the water-chloroform interface.

Design and Activity of Antimicrobial Peptides against Sporogonic-Stage Parasites Causing Murine Malarias

ABSTRACT Insects produce several types of peptides to combat a broad spectrum of invasive pathogenic microbes, including protozoans. However, despite this defense response, infections are often established. Our aim was to design novel peptides that produce high rates of mortality among protozoa of the genus Plasmodium, the malaria parasites. Using existing antimicrobial peptide sequences as templates, we designed and synthesized three short novel hybrids, designated Vida1 to Vida3. Each has a slightly different predicted secondary structure. The peptides were tested against sporogonic stages of the rodent malaria parasites Plasmodium berghei (in vitro and in vivo) and P. yoelii nigeriensis (in vitro). The level of activity varied for each peptide and according to the parasite stage targeted. Vida3 (which is predicted to have large numbers of β sheets and coils but no α helices) showed the highest level of activity, killing the early sporogonic stages in culture and causing highly significant reductions in the prevalence and intensity of infection of P. berghei after oral administration or injection in Anopheles gambiae mosquitoes. The secondary structures of these peptides may play a crucial role in their ability to interact with and kill sporogonic forms of the malaria parasite.

Spectroscopic studies of the multiporphyrin arrays at the air–water interface and in Langmuir–Blodgett films

Abstract We describe the spectroscopic studies of the monolayers and Langmuir–Blodgett (LB) films of tetrapyridylporphyrin (TPyP), its mixture with a phospholipid (DPPTE) and the (2DPPTE–)Cd 2+ –TPyP multiporphyrin arrays. Red-shift of the porphyrin Soret band was 23–27 nm in the monolayers and LB films of TPyP or the TPyP–2DPPTE mixture; while it was only 3–11 nm in the monolayers and LB films of the (2DPPTE–)Cd 2+ –TPyP multiporphyrin arrays. This difference has been attributed to the weakened TPyP–TPyP interaction in the multiporphyrin arrays. Immersion of an intercalation phospholipid layer in the matrix of the monolayers and LB films of TPyP or the Cd 2+ –TPyP multiporphyrin array further reduced this interaction and made it possible for the fabrication of three-dimensional layered multiporphyrin arrays. The fluorescence spectra of TPyP in the LB films of the (2DPPTE–)Cd 2+ –TPyP multiporphyrin array were also quite different from those in the solutions and TPyP(–2DPPTE) LB films. The average molecular orientation angle θ between the mean porphyrin plane and the substrate surface was approximately 26–31° for the LB films of the TPyP and the Cd 2+ –TPyP multiporphyrin array, and approximately 35–38° for the LB films of the TPyP–2DPPTE and 2DPPTE–Cd 2+ –TPyP multiporphyrin array.

The principle and applications of piezoelectric crystal sensors

Abstract The principle, construction, and applications of piezoelectric crystal sensors as universal sensor are reviewed. A historical overview and basic piezoelectric crystal physics as well as design considerations for different sensor system are reviewed. Most of previous reviews were treated with gas phase application, but this review is focused mainly on the liquid phase application, such as monitoring the dynamic microrheology phenomena of liquid crystal, lipid thin films, and electrochemical polymerized polypyrrole film.

A hydrogen biosensor made of clay, poly(butylviologen), and hydrogenase sandwiched on a glass carbon electrode

A hydrogen gas (H(2)) biosensor was developed in which hydrogenase (H(2)ase) was immobilized and sandwiched between two layers of a montmorillonite clay and poly(butylviologen) (PBV) mixture on a glass carbon electrode. The immobilized PBV efficiently enhanced the electron transfer among the electrode, H(2)ase, and methyl viologen in solution. Both PBV and methyl viologen acted as the electron carrier in the clay-PBV-H(2)ase modified electrode. The clay-PBV-H(2)ase electrode catalyzed the oxidation of H(2) to protons (H(+)) with the electrons being transferred by viologen groups to the electrode. The activation energy of this process was 38+/-2 kJ/mol at pH 7. The catalytic current of the clay-PBV-H(2)ase electrode increased linearly when exposed to increasing concentrations of H(2) gas. In contrast, this electrode showed no activity when exposed to three combustible compounds, namely, carbon monoxide, methane and methanol. The optimum pH range for the oxidation of H(2) by the clay-PBV-H(2)ase electrode was from 7 to 10. Electron transfer process in the clay-PBV-H(2)ase electrode is discussed.

Insight into conformational changes of a single α-helix peptide molecule through stiffness measurements

Stiffness variations during the conformational change of a single α-helix polylysine peptide molecule were measured in a liquid environment using atomic force microscopy (AFM) with magnetic cantilever modulation. At the initial stage of the stretching process the stiffness decreased due to the breaking of hydrogen bonds and then increased due to the stretching of the helix backbone. These changes were reversible on reversal of the stretching motion. Below pK, the stiffness did not show increase on reversal, indicating that the reforming of hydrogen bonds did not take place. Conformational changes in the molecule were examined via these changes in stiffness.