Biomedical subjects

Akihiko Kikuchi

Publications and source records attributed to Akihiko Kikuchi.

At least 19 recordsLinked to original sources

PubMed2003

Induction of apoptosis by depletion of DNA topoisomerase IIalpha in mammalian cells.

Inactivation of topoisomerase (topo) IIalpha arrests murine embryonic development. In topo IIalpha-depleted embryos, nuclei were partitioned to daughter cells without complete separation and formed an interconnecting droplet-like structure. The present study examined the fates of topo IIalpha-depleted cells with the droplet-like nuclear structure. When the embryos with abnormal nuclei were further incubated, apoptosis was induced along with the formation of fragmented and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive nuclei. ICRF-193 treatment of embryos activated caspases. Apoptosis induced by ICRF-193 was suppressed by z-VAD-fmk, a caspase inhibitor, and pifithrin-alpha, a p53 inhibitor. Moreover, when mitosis was blocked by nocodazole, ICRF-193-induced nuclear abnormalities and apoptosis were abolished. These data suggest that cycling through the M-phase is essential for ICRF-193-induced apoptosis. Nuclear abnormalities similar to those of topo IIalpha-depleted embryos were induced in HeLa cells in which topo IIalpha was knocked down by transfection with short interfering RNA (siRNA) against topo IIalpha, followed by induction of apoptosis. Our results suggest that topo IIalpha-depleted cells with the droplet-like nuclear structure induce apoptosis, which is dependent on caspase and p53 activity during the G1 phase in mammalian cells.

Animals↗

PubMed2003

Cross-linked thermoresponsive anionic polymer-grafted surfaces to separate bioactive basic peptides.

Cross-linked, thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) [poly(IPAAm-co-AAc-co-tBAAm)] thin hydrogel layers on silica beads were used as new column matrix modifiers for LC separation of basic bioactive peptides, angiotensin subtypes I, II, and III. Terpolymer poly(IPAAm-co-AAc-co-tBAAm) showed both phase transition and apparent carboxylate pKa shifts in water, depending on temperature. Polymer-grafted silica bead surfaces exhibited simultaneous thermally modulated changes in hydrophilic/hydrophobic properties and charge densities. More effective separation of angiotensin peptide subtypes was achieved on columns of these terpolymer thin hydrogel grafted surfaces, as compared to an uncharged control binary copolymer of IPAAm and tBAAm. Although hydrophobic interactions effect separation of angiotensin subtypes, combined electrostatic and hydrophobic interaction resulted in more pronounced retention. At temperature below the terpolymer phase transition, hydrophobic interactions predominated, and minimal changes in electrostatic interactions were supported by little shift in the apparent AAc carboxylate pKa values. Above the phase transition temperature, electrostatic interactions were dramatically reduced as a result of the decreased charge densities of the polymer grafted surfaces. Therefore, peptide retention times were also reduced, exhibiting a maximum at near 30-35 degrees C. Interestingly, column retention behavior of angiotensins is dramatically modulated by applied step temperature gradients. Thermoresponsive surface property alteration is a very rapid, reversible phenomenon, allowing step temperature gradients on thermoresponsive columns to enable the analogous performance advantages as gradient elution in reversed-phase HPLC. More importantly, injected peptides were recovered completely from the columns from calculation of peak area. In conclusion, these anionic thermoresponsive polymer-modified surfaces are good candidates for improved separation of bioactive peptides under exclusively aqueous conditions.

Amino Acid Sequence↗

PubMed2003

Genome-wide expression analysis of NAP1 in Saccharomyces cerevisiae.

Nap1 is a nucleosome assembly protein which is necessary to keep proper nucleosome structures in transcription and replication in vitro. In Saccharomyces cerevisiae, additional functions have been ascribed to Nap1, as it has been shown to interact with Clb2 (B type cyclin) and Gin4 (septum formation). In this study, we investigate genome-wide expression in the Deltanap1 cells using DNA microarrays. About 10% of all yeast open reading frames changed the transcription level more than 2-fold in the Deltanap1 strain, compared with wild-type strain, in one experiment. Interestingly, these genes, whose expressions are up- or down-regulated in the Deltanap1 cells, are clustered. This result suggests that yeast NAP1 is required for the maintenance of cumulative nucleosome formation in vivo and the loss of Nap1 leads to a change in the gene expression level in a cluster.

Cell Cycle Proteins↗

PubMed2003

Affinity chromatography with collapsibly tethered ligands.

We introduce a novel affinity chromatography mode in which affinity ligands are secured to the media surface via collapsible tethers. In traditional affinity chromatography, the immobilized ligands act passively, and their local concentration is static. In collapsibly tethered affinity chromatography, the ligand can move dynamically in response to external stimuli, a design that enables marked changes in both the local concentration of the ligand and its surrounding environment without exchange of solvent. Using the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) as a scaffold for ligand and hapten attachment, we were able to achieve controlled mobility and microenvironment alteration of the affinity ligand Ricinus communis agglutinin (RCA120). The glycoprotein target, asialotransferrin, was loaded onto a column in which PIPAAm was partially substituted with both RCA120 and lactose. At 5 degrees C, the column retained the glycoprotein, but released most (95%) of the asialotransferrin upon warming to 30 degrees C. This temperature-induced elution was much greater than can be explained by temperature dependency of sugar recognition by RCA120. The simplest explanation is that upon thermally induced dehydration and collapse of the PIPAAm chains, coimmobilized RCA120 ligand and lactose hapten are brought into closer proximity to each other, enabling immobilized lactose to displace affinity-bound asislotransferrin from the immobilized RCA120 lectin.

Asialoglycoproteins↗

PubMed2003

Function of the loop residue Thr792 in human DNA topoisomerase II alpha.

We studied the mutation effect of one of the putative loop residues Thr792 in human DNA topoisomerase II alpha (TOP2 alpha). Thr792 mutants were expressed from high or low copy plasmids in a temperature sensitive yeast strain deficient in TOP2 (top2-1). When expressed from a high copy plasmid, mutants with small side chains complemented the yeast defect; however, from a low copy plasmid, only wild-type, Ser, and Cys substitution mutants complemented the yeast defect. Interestingly, at the permissive temperature other mutants (e.g., Val, Gly, and Glu substitutions) showed the dominant negative effect to the top2-1 allele, which was not observed by the control alpha 4-helix mutants. T792E mutant was 10-fold less active than wild-type and the T792P had no decatenation activity in vitro. These results suggest that Thr792 in human TOP2 alpha is involved in enzyme catalysis.

Antigens, Neoplasm↗

PubMed2003

Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes.

Fabrication of functional tissue constructs from designed three-dimensional structures of cells using the layered method of cultured cell sheets could prove to be an attractive approach to tissue engineering. Rapid recovery of cell sheets is considered to be important as a basic technology for practical assembly of tissue-mimicking structures. To accelerate required culture substrate hydrophilic/hydrophobic functional changes according to the hydrated/dehydrated structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted with poly(ethylene glycol) (PEG) onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform infrared and electron spectroscopy for chemical analysis revealed that PIPAAm and PEG were successfully grafted to surfaces of porous membranes. PIPAAm-grafted porous membranes (PIPAAm-PM) were compared with porous membranes co-grafted with various amounts of PEG and PIPAAm (PIPAAm(PEG)-PM) for cell sheet detachment experiments. Approximately 35min incubation at 20 degrees C was required to completely detach cell sheets from PIPAAm-PM in a static condition, while only 19min to detach cell sheets from PIPAAm(PEG0.5%)-PM, which is co-grafted with PIPAAm and 0.5wt% of PEG. With porous membranes, water molecules were accessed by the PIPAAm molecules grafted on the surfaces from both underneath and peripheral to the attached cell sheet, resulting in more rapid hydration of grafted PIPAAm molecules and detachment of cell sheet than that for nonporous tissue culture polystyrene (TCPS) dish. With PIPAAm(PEG)-PMs, grafted PEG chains should accelerate the diffusion of water molecules to PIPAAm grafts, showing more rapid detachment of cell sheet compare to PIPAAm-PMs.

Acrylic Resins↗

PubMed2003

Cell sheet engineering for myocardial tissue reconstruction.

Myocardial tissue engineering has now emerged as one of the most promising treatments for the patients suffering from severe heart failure. Tissue engineering has currently been based on the technology using three-dimensional (3-D) biodegradable scaffolds as alternatives for extracellular matrix. According to this most popular technique, several types of 3-D myocardial tissues have been successfully engineered by seeding cardiomyocytes into poly(glycolic acid), gelatin, alginate or collagen scaffolds. However, insufficient cell migration into the scaffolds and inflammatory reaction due to scaffold biodegradation remain problems to be solved. In contrast to these technologies, we now propose novel tissue engineering methodology layering cell sheets to construct 3-D functional tissues without any artificial scaffolds. Confluent cells on temperature-responsive culture surfaces can be harvested as a viable contiguous cell sheet only by lowering temperature without any enzymatic digestions. Electrical communications are established between layered cardiomyocyte sheets, resulting in simultaneous beating 3-D myocardial tissues. Layered cardiomyocyte sheets in vivo present long survival, macroscopic pulsation and characteristic structures of native heart tissue. Cell sheet engineering should have enormous potential for fabricating clinically applicable myocardial tissues and should promote tissue engineering research fields.

Animals↗

PubMed2003

PCR-based identification of common dermatophyte species using primer sets specific for the DNA topoisomerase II genes.

BACKGROUND: We have determined nucleotide sequences of the DNA topoisomerase II genes of the dermatophyte species, and conducted a PCR-based identification system using species-specific primers for the nucleotide sequences. OBJECTIVE: To identify the major dermatophytes, Trichophyton rubrum, T. mentagrophytes, T. violaceum, M. gypseum, M. canis and E. floccosum, by PCR amplifications at the species level, without determining the nucleotide sequence. METHODS: For PCR-based identification of the major dermatophyte species, a common primer set (dPsD1) for these species and species-specific primer sets (PsT and PsME) for each species were designed based on the genomic sequences of the DNA topoisomerase II genes of the dermatophytes, and tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by the common primer set, followed by a second PCR with the primer sets consisting of species-specific primers for each dermatophyte species. RESULTS: Using dPsD1, a DNA fragment of 3390 bp was amplified from the genomic DNA of all the dermatophyte species. In the subsequent nested PCR using species-specific primer sets (PsT and PsME), both sets amplified unique sizes of PCR products, all of which corresponded to a species of the dermatophytes even in the presence of other fungal DNA. CONCLUSION: We demonstrate that the PCR-based identification targeting the DNA topoisomerase II gene is rapid and simple, and is available as a tool for the identification of the major dermatophyte species.

Arthrodermataceae↗

PubMed2003

Evidence for the presence of neutralizing antibodies against human papillomavirus type 6 in infants born to mothers with condyloma acuminata.

Despite human papillomavirus type 6 or 11 (HPV6/11) being often vertically transmitted from mothers with condyloma acuminata (CA) to their infants, HPV-related neonatal mucosal diseases are rare. The role of maternal anti-HPV6/11 neutralizing antibodies in preventing the vertical transmission remains to be unknown because of lack of the neutralization assay system of HPV infection. We experienced two cases of HPV6-positive CA during pregnancy. Neutralizing antibodies against HPV6 in maternal, umbilical, and infantile sera were determined using a surrogate assay system to monitor HPV6 pseudo-infections. The neutralizing antibodies were detected in maternal and umbilical sera and in serum of one of the infants tested at 5 weeks old. In the infant exposed to HPV6 at birth, viral DNA was not detectable in the oral cavity 5 weeks after birth. This is the first report to describe that neutralizing antibodies against HPV6 in mothers with CA go through the placenta and enter the circulation of their infants. These data may provide a mechanistic paradigm for the prevention of its vertical transmission.

Adult↗

PubMed2003

Involvement of nucleocytoplasmic shuttling of yeast Nap1 in mitotic progression.

Nucleosome assembly protein 1 (Nap1) is widely conserved from yeasts to humans and facilitates nucleosome formation in vitro as a histone chaperone. Nap1 is generally localized in the cytoplasm, except that subcellular localization of Drosophila melanogaster Nap1 is dynamically regulated between the cytoplasm and nucleus during early development. The cytoplasmic localization of Nap1 is seemingly incompatible with the proposed role of Nap1 in nucleosome formation, which should occur in the nucleus. Here, we have examined the roles of a putative nuclear export signal (NES) sequence in yeast Nap1 (yNap1). yNap1 mutants lacking the NES-like sequence were localized predominantly in the nucleus. Deletion of NAP1 in cells harboring a single mitotic cyclin gene is known to cause mitotic delay and temperature-sensitive growth. A wild-type NAP1 complemented these phenotypes while nap1 mutant genes lacking the NES-like sequence or carboxy-terminal region did not. These and other results suggest that yNap1 is a nucleocytoplasmic shuttling protein and that its shuttling is important for yNap1 function during mitotic progression. This study also provides a possible explanation for Nap1's involvement in nucleosome assembly and/or remodeling in the nucleus.

Active Transport, Cell Nucleus↗

PubMed2002

Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes.

Confluent human aortic endothelial cells (HAECs) cultured on thermo-responsive culture dish grafted with poly (N-isopropylacrylamide) were recovered as a contiguous cell sheet. The double-layered co-culture was achieved by placing the recovered HAEC sheet onto the rat hepatocyte layer directly. The double-layered structure of HAEC and hepatocytes remained in tight contact during culture. Hepatocytes in the layered co-culture system with the HAEC sheet maintained the differentiated cell shape and the albumin expression for over 41 days of culture, whereas the functions disappeared within 10 days of culture in control hepatocytes without the HAEC sheet. The layered co-culture of hepatocytes and the HAEC sheets, which allows for the expression of differentiated functions of hepatocyte continuously, such as liver lobule, offers a major advancement in liver tissue engineering.

Coculture Techniques↗

PubMed2002

Three-dimensional electron microscopy of the reverse gyrase from Sulfolobus tokodaii.

Reverse gyrase is a type IA topoisomerase, found in various hyperthermophiles and promotes ATP-dependent positive supercoiling of DNA. Electron microscopy combined with single particle analyses revealed the three-dimensional structure of the DNA-free Sulfolobus tokodaii reverse gyrase and two-dimensional average images of both the protein alone and that complexed with double-stranded DNA. The 23A resolution map exhibited a parallelogrammatic morphology of 110 x 87 x 43A, which is in good agreement with the crystal structure of the Archaeoglobus fulgidus reverse gyrase. The average image of the complex revealed that the monomeric enzyme binds DNA duplex. Together with this average image of the complex, the three-dimensional map implies that, at the beginning of the supercoiling reaction, DNA is bound within a 10-20A wide cleft in the helicase-like domain. We also speculate that DNA may pass through a 20A wide hole at the end of the cleft.

DNA Topoisomerases, Type I↗

PubMed2002

Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer.

A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column.

Chromatography, Affinity↗

PubMed2002

Aqueous chromatography utilizing hydrophobicity-modified anionic temperature-responsive hydrogel for stationary phases.

A new pH-/temperature-responsive poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) (poly(IPAAm-co-AAc-co-tBAAm)) hydrogel grafted on silica beads was evaluated as column matrix for a cation-exchange thermoresponsive chromatography. The stationary phase showed simultaneous changes in temperature-responsive surface charge density and hydrophobicity by incorporation of anionic AAc and hydrophobic tBAAm into IPAAm sequences. Thermoresponsive polymer property alterations were confirmed by temperature-responsive phase transition and shift in apparent pKa values. Catecholamine derivatives were retained on poly(IPAAm-co-AAc-co-tBAAm)-modified column at pH 7.0. Analyte retention was primarily due to the electrostatic interaction. It was noted that the temperature-induced phase transition of poly(IPAAm-co-AAc-co-tBAAm) hydrogel layer on the stationary phases was evidenced by the apparent inflection point in van't Hoff plots around 36 degrees C. This suggests that solute interactions should be changed below and above the stationary phase transition temperature, reducing electrostatic interaction above the transition temperature.

Anions↗

PubMed2002

Time-programmed pulsatile release of dextran from calcium-alginate gel beads coated with carboxy-n-propylacrylamide copolymers.

Time-programmed release of macromolecular drugs was achieved by utilization of calcium-alginate gel beads modified with coated copolymer layers. Modified calcium-alginate gel beads coated with poly(carboxy-n-propylacrylamide-co-dimethylacrylamide) [poly(CNPAAm-co-DMAAm)] (22.7 mol% of CNPAAm) of varying coating thickness from 25 to 125 microm were developed as drug carriers. Model macromolecular drugs used were fluorescein isothiocyanate (FITC)-labeled dextrans with different molecular weights ranging from 9400 to 145000. FITC-dextran release was strongly dependent on both copolymer coating thicknesses and the dextran molecular weights. Release of FITC-dextran (MW 9400) followed Fickian diffusion according to t(1/2) dependence, indicating that the drug diffusion is the main driving force for release of dextran MW 9400. Release of higher molecular weight FITC-dextrans (71,00 and 145,00) exhibited a burst-effect preceded by a preset lag time. These release profiles were governed by the dissociation of calcium ions from polyguluronate sequences in alginate molecules along with the diffusion of sodium ions into the gel bead core. This created osmotic pressure inside the gel, inducing breakage of the coated copolymer layer and accelerated drug release. Burst release of macromolecular drugs thus occurred after a certain lag period. The lag time was regulated by the copolymer coat thickness. A pulsatile release of FITC-dextran was demonstrated by combining a series of modified alginate gel beads in a single batch.

Acrylic Resins↗

PubMed2002

Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces.

Recent progress in cell transplantation therapy to repair impaired hearts has encouraged further attempts to bioengineer 3-dimensional (3-D) heart tissue from cultured cardiomyocytes. Cardiac tissue engineering is currently pursued utilizing conventional technology to fabricate 3-D biodegradable scaffolds as a temporary extracellular matrix. By contrast, new methods are now described to fabricate pulsatile cardiac grafts using new technology that layers cell sheets 3-dimensionally. We apply novel cell culture surfaces grafted with temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), from which confluent cells detach as a cell sheet simply by reducing temperature without any enzymatic treatments. Neonatal rat cardiomyocyte sheets detached from PIPAAm-grafted surfaces were overlaid to construct cardiac grafts. Layered cell sheets began to pulse simultaneously and morphological communication via connexin43 was established between the sheets. When 4 sheets were layered, engineered constructs were macroscopically observed to pulse spontaneously. In vivo, layered cardiomyocyte sheets were transplanted into subcutaneous tissues of nude rats. Three weeks after transplantation, surface electrograms originating from transplanted grafts were detected and spontaneous beating was macroscopically observed. Histological studies showed characteristic structures of heart tissue and multiple neovascularization within contractile tissues. Constructs transplanted into 3-week-old rats exhibited more cardiomyocyte hypertrophy and less connective tissue than those placed into 8-week-old rats. Long-term survival of pulsatile cardiac grafts was confirmed up to 12 weeks. These results demonstrate that electrically communicative pulsatile 3-D cardiac constructs were achieved both in vitro and in vivo by layering cardiomyocyte sheets. Cardiac tissue engineering based on this technology may prove useful for heart model fabrication and cardiovascular tissue repair. The full text of this article is available at http://www.circresaha.org.

Actin Cytoskeleton↗

PubMed2002

Pulsatile drug release control using hydrogels.

Current research in the field of drug delivery devices, by which pulsed and/or pulsatile release is achieved, has been intensified. In this article several types of drug delivery systems using hydrogels are discussed that showed pulsed and/or pulsatile drug delivery characteristics. As is frequently found in the living body, many vital functions are regulated by pulsed or transient release of bioactive substances at a specific site and time. Thus it is important to develop new drug delivery devices to achieve pulsed delivery of a certain amount of drugs in order to mimic the function of the living systems, while minimizing undesired side effects. Special attention has been given to the thermally responsive poly(N-isopropylacrylamide) and its derivative hydrogels. Thermal stimuli-regulated pulsed drug release is established through the design of drug delivery devices, hydrogels, and micelles. Development of modified alginate gel beads with pulsed drug delivery characteristic is also described in this article.

Animals↗

PubMed2002

Adhesion behavior of monocytes, macrophages, and foreign body giant cells on poly (N-isopropylacrylamide) temperature-responsive surfaces.

Monocyte and macrophage adhesion and foreign body giant cell (FBGC) formation has been observed on surfaces with a wide range of properties. In this study we have utilized novel, temperature-responsive surfaces (TRS) with dynamic surface properties to investigate inflammatory cell adhesion behavior. With temperature changes, grafted chains of poly-N-isopropylacrylamide pass through their lower critical solution temperature (LCST) and can either extend (hydrate), creating a hydrophilic surface at 20 degrees C, or contract (dehydrate), creating a hydrophobic surface at 37 degrees C. Isolated human monocytes and monocyte-derived macrophages were able to adhere, spread, and form FBGC on the hydrophobic surface. Decreasing the temperature below the lower critical solution temperature induced a change in the surface wettability, creating a hydrophilic surface, that induced a differential detachment of adherent cells that decreased with time, ranging from 98% after 2 h of culture to 30% at day 10. These detached cells remained viable, and were recultured onto TCPS for 3, 7, and 10 days. These novel surfaces allow investigation of the adhesive behavior of adherent inflammatory cells in a temporal manner, and the effects of surface conformation and wettability changes on cell adhesion and detachment.

Cell Adhesion↗