Biomedical subjects

Kenichi Yoshikawa

Publications and source records attributed to Kenichi Yoshikawa.

At least 19 recordsLinked to original sources

PubMed2007

Weak interaction induces an ON/OFF switch, whereas strong interaction causes gradual change: folding transition of a long duplex DNA chain by poly-L-lysine.

A large-scale conformational change in genomic DNA is an essential feature of gene activation in living cells. Considerable effort has been applied to explain the mechanism in terms of key-lock interaction between sequence-specific regulatory proteins and DNA, in addition to the modification of DNA and histones such as methylation and acetylation. However, it is still unclear whether these mechanisms can explain the ON/OFF switching of a large number of genes that accompanies differentiation, carcinogenesis, etc. In this study, using single-molecule observation of DNA molecules by fluorescence microscopy with the addition of poly-L-lysine with different numbers of monomer units (n = 3, 5, 9, and 92), we found that an ON/OFF discrete transition in the higher-order structure of long duplex DNA is induced by short poly-L-lysine, whereas a continuous gradual change is induced by long poly-L-lysine. On the other hand, polycations with a lower positive charge have less potential to induce DNA compaction. Such a drastic difference in the conformational transition of a giant DNA between short and large oligomers is discussed in relation to the mechanisms of gene regulation in a living cell.

Biophysics↗

PubMed2006

Chemical reaction-inspired crystal growth of a coordination polymer toward morphology design and control.

This paper reports a novel crystal growth system of a coordination framework {[Cu3(CN)3{hat-(CN)3(OEt)3}]}n (1) (hat-(CN)3(OEt)3 = 2,6,10-tricyano-3,7,11-triethoxy-1,4,5,8,9,12-hexaazatriphenylene). The coordination polymer is crystallized through the reaction of 2,3,6,7,10,11-hexacyano-1,4,5,8,9,12-hexaazatriphenylene (hat-(CN)6), ethanol, and copper(I) complex, involving the breaking and forming of covalent bonds. The crystal morphologies obtained in the present system contain dumbbells, cogwheels, and superlattices. Moreover, in the growth perpendicular to the c-axis, periodic ramification at regular interval is observed, affording superlattice morphologies. Observation of the growth of dumbbell crystals shows that the growth rates parallel and perpendicular to the crystallographic c-axis are quite different: the former shows a drastic change with the reaction duration, while the latter is almost constant. These results are reproduced as a simple reaction-diffusion system, indicating that chemical reactions on crystal surfaces play an important role in determining the macroscopic crystal morphologies.

Journal Article↗

PubMed2006

Transcription of giant DNA complexed with cationic nanoparticles as a simple model of chromatin.

We prepared complexes of giant double-stranded DNA with cationic nanoparticles of 10-40 nm in diameter as an artificial model of chromatin and characterized the properties of changes in their higher-order conformation. We measured the changes in transcriptional activity that accompanied the DNA conformational transitions. Complete inhibition was found at excess concentrations of nanoparticles. In contrast, at intermediate stages of DNA binding with nanoparticles, the transcription activity of DNA survived, and this strongly depended on the size of the nanoparticles. For large nanoparticles of 40 nm, a decrease in transcriptional activity can be caused by the addition of only a small amount of nanoparticles. On the other hand, there was almost no inhibition of DNA transcriptional activity with the addition of small nanoparticles (10 nm) until very high concentrations, even under conditions that induced DNA compaction as revealed by single-DNA observation. At higher concentrations of 10-nm nanoparticles, DNA transcription activity decreased abruptly until it was completely inhibited. These results are discussed in relation to the actual size of the histone core, together with the mechanism of switching of transcriptional activity in eukaryotic cells.

Bacteriophage T4↗

PubMed2006

Spontaneous transfer of phospholipid-coated oil-in-oil and water-in-oil micro-droplets through an oil/water interface.

We studied the evolution of oil-in-oil (O/O) and water-in-oil (W/O) phospholipid-coated micro-droplets at an oil/water interface. We found that, in both cases, micro-droplets spontaneously transferred from the oil phase to the water phase. O/O micro-droplets transformed into oil-in-water micro-droplets, while W/O micro-droplets led to the formation of liposomes.

Chemical Phenomena↗

PubMed2006

Regular self-motion of a liquid droplet powered by the chemical marangoni effect.

We describe here our recent work on spontaneous regular motion of liquid droplet powered by the chemical Marangoni effect under spatially symmetric conditions. It is shown that a spontaneously crawling oil droplet on a glass substrate with a nonequilibrium chemical condition of cationic surfactant exhibits regular rhythmic motion in a quasi-one-dimensional vessel, whereas irregular motion is induced in a two-dimensionally isotropic environment. Such behavior of a droplet demonstrates that spontaneous regular motion can be generated under fluctuating conditions by imposing an appropriate geometry. As another system, we introduce alcohol droplet moving spontaneously on water surface. The droplet spontaneously forms a specific morphology depending on its volume, causing specific mode of translational motion. An alcohol droplet with a smaller volume floating on water surface moves irregularly. On the other hand, a droplet with a larger volume undergoes vectorial motion accompanied by deformation into an asymmetric shape. This result suggests a scenario on the emergence of regular motion coupled with geometrical pattern formation under far-from-equilibrium conditions.

Alcohols↗

PubMed2006

Rhythmic motion of a droplet under a dc electric field.

The effect of a stationary electric field on a water droplet with a diameter of several tens micrometers in oil was examined. Such a droplet exhibits repetitive translational motion between the electrodes in a spontaneous manner. The state diagram of this oscillatory motion was deduced; at 0-20 V the droplet is fixed at the surface of the electrode, at 20-70 V the droplet exhibits small-amplitude oscillatory motion between the electrodes, and at 70-100 V the droplet shows large-amplitude periodic motion between the electrodes. The observed rhythmic motion is explained in a semiquantitative manner by using differential equations, which includes the effect of charging the droplet under an electric field. We also found that twin droplets exhibit synchronized rhythmic motion between the electrodes.

Journal Article↗

PubMed2006

On the formation of rings-on-a-string conformations in a single polyelectrolyte chain: A possible scenario.

Recent single-molecular observations have revealed that a single giant DNA molecule assumes (micro) phase separated structures upon the addition of condensing agents. Electron and atomic force microscopy have clearly shown the coexistence of ordered tori and disordered coil structures within a single DNA molecule. Motivated by these experimental findings, we theoretically investigated the collapse transition of a single polyelectrolyte chain driven by the addition of condensing guest molecules. We found that the transition behavior critically depends on the degree of the surviving charge inside the torus. When the torus is charged, even slightly, "rings-on-a-string" structures are expected for a sufficiently long chain, owing to the combinational entropy of segment state distribution along the chain and the unique property of the stability of charged torus.

Journal Article↗

PubMed2006

Cell-free protein synthesis at high temperatures using the lysate of a hyperthermophile.

Systems for cell-free protein synthesis available today are usually based on the lysates of either Escherichia coli, wheat germ or rabbit reticulocyte, and protein synthesis reactions using these extracts are performed at moderate temperatures (20-40 degrees C). We report here the development of a novel system for cell-free protein synthesis that can be operated at high temperatures using a lysate of the hyperthermophilic archaeon, Thermococcus kodakaraensis. With the system, cell-free protein synthesis of ChiADelta4, a derivative of T. kodakaraensis chitinase (ChiA), was observed within a temperature range of 40-80 degrees C, with an optimum at 65 degrees C. Corresponding chitinase activity was also detected in the reaction mixtures after cell-free protein synthesis, indicating that the synthesized ChiADelta4 folded in a proper tertiary structure. The maximum concentration of active ChiADelta4 synthesized was determined to be approximately 1.3 microg/mL. A time course experiment indicated that the amount of synthesized ChiADelta4 saturated within 30 min at 65 degrees C, and energy depletion was suggested to be the main cause of this saturation. We further developed a system for transcription and translation-coupled protein synthesis at high temperatures using a combination of T. kodakaraensis lysate and thermostable T7 RNA polymerase.

Archaea↗

PubMed2006

Hyaluronic acid and its derivative as a multi-functional gene expression enhancer: protection from non-specific interactions, adhesion to targeted cells, and transcriptional activation.

Hyaluronic acid (HA), a natural anionic mucopolysaccharide, can be deposited onto the cationic surface of DNA/polyethyleneimine (PEI) complexes to recharge the surface potential and reduce nonspecific interactions with proteins. HA can also be used as a ligand to target specific cell receptors. Furthermore, HA-coating enhanced the transcriptional activity of the plasmid/PEI complexes, probably by loosening the tight binding between DNA and PEI, which facilitated the approach of transcription factors. Amphoteric HA derivative having spermine side chains (Spn-HA) with a structure similar to HMG protein showed higher transcription-enhancing activity than HA. Plasmid/PEI/Spn-HA ternary complex exhibited 29-fold higher transgene expression efficiency than naked plasmid/PEI complexes in CHO cells.

Animals↗

PubMed2006

Real-time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells.

Notch signaling components such as the basic helix-loop-helix gene Hes1 are cyclically expressed by negative feedback in the presomitic mesoderm (PSM) and constitute the somite segmentation clock. Because Hes1 oscillation occurs in many cell types, this clock may regulate the timing in many biological systems. Although the Hes1 oscillator is stable in the PSM, it damps rapidly in other cells, suggesting that the oscillators in the former and the latter could be intrinsically different. Here, we have established the real-time bioluminescence imaging system of Hes1 expression and found that, although Hes1 oscillation is robust and stable in the PSM, it is unstable in the individual dissociated PSM cells, as in fibroblasts. Thus, the Hes1 oscillators in the individual PSM cells and fibroblasts are intrinsically similar, and these results, together with mathematical simulation, suggest that cell-cell communication is essential not only for synchronization but also for stabilization of cellular oscillators.

Animals↗

PubMed2005

Compaction of single-chain DNA by histone-inspired nanoparticles.

We elaborated a versatile experimental model of chromatin which consists of a single chain of long duplex DNA that interacts with well-defined cationic nanoparticles of various sizes. We found that the DNA compaction by nanoparticles is stepwise and progressive at the single-chain level. It is controlled by the ability of DNA to wrap nanoparticles, which is more efficient for larger particles and, similar to DNA-histone interaction, is optimal at a physiological salt concentration.

Chromatin↗

PubMed2005

Protective effect of vitamin C against double-strand breaks in reconstituted chromatin visualized by single-molecule observation.

Direct attack to genomic DNA by reactive oxygen species causes various types of lesions, including base modifications and strand breaks. The most significant lesion is considered to be an unrepaired double-strand break that can lead to fatal cell damage. We directly observed double-strand breaks of DNA in reconstituted chromatin stained by a fluorescent cyanine dye, YOYO (quinolinium, 1,1'-[1,3- propanediylbis[(dimethyliminio)-3,1- propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-, tetraiodide), in solution, where YOYO is known to have the ability to photo-cleave DNAs by generating reactive oxygen species. Reconstituted chromatin was assembled from large circular DNA (106 kbp) with core histone proteins. We also investigated the effect of vitamin C (ascorbic acid) on preventing photo-induced double-strand breaks in a quantitative manner. We found that DNA is protected against double-strand breaks by the addition of ascorbic acid, and this protective effect is dose dependent. The effective kinetic constant of the breakage reaction in the presence of 5 mM ascorbic acid is 20 times lower than that in the absence of ascorbic acid. This protective effect of ascorbic acid in reconstituted chromatin is discussed in relation to the highly compacted polynucleosomal structure. The results highlight the fact that single-molecule observation is a useful tool for studying double-strand breaks in giant DNA and chromatin.

Ascorbic Acid↗

PubMed2005

Chemosensitive running droplet.

Chemical control of the spontaneous motion of a reactive oil droplet moving on a glass substrate under an aqueous phase is reported. Experimental results show that the self-motion of an oil droplet is confined on an acid-treated glass surface. The transient behavior of oil-droplet motion is also observed with a high-speed video camera. A mathematical model that incorporates the effect of the glass surface charge is built based on the experimental observation of oil-droplet motion. A numerical simulation of this mathematical model reproduced the essential features concerning confinement of oil droplet motion within a certain chemical territory and also its transient behavior. Our results may shed light on physical aspects of reactive spreading and a chemotaxis in living things.

Journal Article↗

PubMed2005

Enhancement and inhibition of DNA transcriptional activity by spermine: a marked difference between linear and circular templates.

We compared the transcriptional activities of the circular and linear forms of short (4kbp) and giant (106kbp) DNA molecules in the presence of a polyamine, spermine (4+). With an increase in the spermine concentration, transcriptional activity was enhanced, followed by an inhibitory effect, and complete inhibition was observed in the sole case of long, linear templates. A difference between the transcriptional properties of circular and linear conformations is found for giant DNA molecules. These results are discussed in relation to DNA conformational transitions.

DNA↗

PubMed2005

Reversible photoswitching in a cell-sized vesicle.

A photosensitive amphiphilic molecule can switch the shape of an assembled vesicle as determined by microscopic observation. Photoisomerization induces a change in membrane fluctuation behavior or a morphological transition between ellipsoid and bud shapes, depending on the asymmetrical degree of the initial shape. The mechanism of this reversible photoswitching in the vesicle morphology is interpreted in terms of a change in the effective cross-sectional area of the photosensitive molecule.

Azo Compounds↗

PubMed2005

Specific formation of beads-on-a-chain structures on giant DNA using a designed polyamine derivative.

Fluorescence microscopy was used to study the folding transition of giant DNAs, T4 DNA (ca. 166 kbp), and lambda DNA (ca. 48 kbp), which proceeds through intermediates with intramolecular segregation induced by pteridine-polyamine conjugates, i.e., 2-amino-6,7-dimethyl-4-(4,9,13-triazatridecylamino)pteridine and -4-(3-(aminopropyl)amino)pteridine. According to the results of DNA denaturation, UV and fluorescent spectroscopy, and transmission electron microscopic observations, it became clear that DNA folding induced by the polyamine derivative is not a continuous shrinking process but a combination of discontinuous processes.

Bacteriophage T4↗

PubMed2005

Dramatic change in the tertiary structure of giant DNA without distortion of the secondary structure caused by pteridine-polyamine conjugates.

Pteridine-polyamine conjugates, such as 2-polyamine (1,3-diaminopropane and spermine) substituted 6,7-dimethyl-3H-pteridine-4-one, induce a folding transition of a giant DNA molecule more effectively than the corresponding polyamines. However, since neither a DNA high-temperature shift of denaturation (Tm) curve nor distortion of the UV/fluorescence spectra is observed in a mixture of these compounds with DNA, they do not interact with the DNA duplex strongly.

Bacteriophage T4↗

PubMed2005

Helical superstructures of fullerene peapods and empty single-walled carbon nanotubes formed in water.

Aqueous dispersions of fullerene C70-filled carbon nanotubes (C70@SWNTs or peapods) and empty single-walled carbon nanotubes (empty SWNTs) were prepared with the aid of trimethyl-(2-oxo-2-pyrene-1-yl-ethyl)-ammonium bromide (1), which is a carbon nanotube solubilizer. This is the first report describing the preparation and characterization of the transparent dispersion/dissolution of the peapods. The UV-vis-near-IR spectra of C70@SWNTs-1 and empty SWNTs-1 were almost identical. We found by means of transmission electron microscopy and atomic force microscopy that the empty SWNTs and C70-peapods form helical nanostructures in the shapes of rings, irregular rings, lassos, handcuffs, catenanes, pseudorotaxanes, and figure-eight structures. The mechanism of the superstructure formation has been discussed in relation to the unique characteristics of stiff polymer chains with the aid of an off-lattice Monte Carlo simulation.

Journal Article↗