Biomedical subjects

Akihiko Kikuchi

Publications and source records attributed to Akihiko Kikuchi.

At least 19 recordsLinked to original sources

PubMed2004

Fractal analysis of surface growth patterns in endometrioid endometrial adenocarcinoma.

OBJECTIVE: A fractal is a shape made of parts similar to the whole in some way. The objective of this study was to determine whether surface growth patterns in endometrioid endometrial adenocarcinoma are fractal, and the mean fractal dimension differs according to histologic grades and depth of myometrial invasion. METHODS: After the images of photographs of 120 resected uteri with endometrial cancers were digitized, the fractal dimensions of surface of tumors were measured using a fractal analysis software. RESULTS: The mean fractal dimensions of surface growth patterns in G1, G2, and G3 adenocarcinoma were 2.318, 2.303, and 2.383, respectively. These values were significantly greater than the topological dimension of a surface (= 2). The value was significantly higher in G3 than in G2 (p = 0.03). And although not statistically significant, the value of G3 was greater than G1 (p = 0.10) and than (G1 and G2) group (p = 0.06). No significant difference nor tendency was found in the fractal dimension of the surface of the tumor according to depth of invasion. CONCLUSION: This study shows that the surface of endometrioid endometrial adenocarcinoma has a fractal structure, and the mean fractal dimension may differ according to histologic grades. Our report proposes a new way of looking at endometrial cancer pathology. We believe that fractal geometry gives insights into tumor morphology and becomes a useful tool for analyzing complex and irregular tumor growth patterns mathematically.

Adult↗

PubMed2004

Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces.

The hydrophobic monomer, n-butyl methacrylate (BMA) has been incorporated into thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) to lower PIPAAm phase transition temperatures necessary for systematically regulating cell adhesion on and detachment from culture dishes at controlled temperatures. Poly(IPAAm-co-BMA)-grafted dishes were prepared by electron beam irradiation methods, systematically changing BMA content in the feed. Copolymer-grafted surfaces decreased grafted polymer transition temperatures with increasing BMA content as shown by water wettabilities compared to homopolymer PIPAAm-grafted surfaces. Bovine endothelial cells readily adhered and proliferated on copolymer-grafted surfaces above collapse temperature at 37 degrees C, finally reaching confluence. Cell sheet detachment behavior from copolymer-grafted surfaces depended on the culture temperature and BMA content. In conclusion, cell attachment/detachment can be controlled to an arbitrary temperature by varying the content of hydrophobic monomer incorporated into PIPAAm grafted to culture surfaces.

Animals↗

PubMed2004

Temperature- and pH-responsive aminopropyl-silica ion-exchange columns grafted with copolymers of N-isopropylacrylamide.

We have designed copolymers of N-isopropylacrylamide, environmentally-responsive polymers, which respond to temperature and other external stimuli. In this study, we designed and synthesized copolymers that introduced ion-exchange groups. These copolymers responded to the temperature and the pH, and the copolymer-grafted aminopropyl silica beads were used as HPLC packing materials. This stationary phase altered the properties from hydrophilic to hydrophobic and from charge to non-charge by temperature and pH changes. We studied the separations of organic acids and phenylthiohydantoin-amino acids using environmentally-responsive chromatography, and confirmed the effects of the ion-exchange groups. The elution behaviors of these samples were controlled by the temperature changes without organic solvents in the mobile phase. It was confirmed that the interactions between the solute and stationary phase could be freely controlled by the temperature and the pH. Environmentally-responsive chromatography is expected to be applicable to the separation of pharmaceuticals and biomolecules, such as peptides, proteins and nucleic acids.

Acrylamides↗

PubMed2004

The alpha4 residues of human DNA topoisomerase IIalpha function in enzymatic activity and anticancer drug sensitivity.

We introduced a series of Pro substitutions within and near the alpha4 helix, a part of the breakage/rejoining region, in human DNA topoisomerase IIalpha, and analyzed if this region is involved in determination of anti-cancer drug sensitivity in a temperature- sensitive yeast strain (top2-4 allele). Among the 19 mutants generated, H759P and N770P showed resistance to etoposide and doxorubicin at the non-permissive temperature, where cell growth depends on activity of the human enzyme. For these residues, mutants with an Ala substitution were further created, in which H759A also showed resistance to etoposide. H759P, H759A and N770P were expressed, purified and subjected to in vitro measurement of drug sensitivity. They generated lower amounts of the etoposide-induced cleavable complexes, and were also found to have lower decatenation activity than the wild-type. In the crystal structure, the yeast equivalent of His759 is found in the vicinity of the Arg713, a putative anchoring residue of the 3'-side of cleaved DNA strands. These results suggest that His759 and the other alpha4 helix residues are involved in the enzymatic activity and drug sensitivity of human DNA topoisomerase IIalpha, via interaction with cleaved DNA.

Amino Acid Sequence↗

PubMed2004

Functional compatibility between isoform alpha and beta of type II DNA topoisomerase.

DNA topoisomerase II (topo II) plays a crucial role in controlling the conformation of both DNA and whole chromosomes. This activity is essential for several cellular events such as DNA replication, transcription, chromosome condensation and segregation. In mammals, two genes code for isoforms of topo II, termed alpha and beta. They are similar in primary structure and have almost identical catalytic properties in vitro. We transfected HeLa cells with small interfering RNAs (siRNAs) targeted against either topo IIalpha or IIbeta, and succeeded in knocking down the expression of the corresponding protein. Chromosomes were condensed and aligned at metaphase in topo IIalpha-knockdown cells. Although some lagging chromosomes were observed, they were still segregated at anaphase despite the absence of topo IIalpha. When both topo IIalpha and topo IIbeta were removed, the segregation of chromosomes was severely arrested, suggesting that topo IIbeta could partially substitute for topo IIalpha. Double-knockdown experiments also revealed that topo II was required for shortening of the chromosome axis.

Antigens, Neoplasm↗

PubMed2004

Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface.

BACKGROUND: Limbal stem-cell deficiency by ocular trauma or diseases causes corneal opacification and visual loss. Recent attempts have been made to fabricate corneal epithelial graft constructs, but the technology is still evolving. We have developed a novel cell-sheet manipulation technology using temperature-responsive culture surfaces to generate functional, cultivated corneal epithelial cell sheet grafts. METHODS: Human or rabbit limbal stem cells were cocultured with mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes at 37 degrees C. Cell sheets were harvested from the dishes after 2 weeks by reducing temperature to 20 degrees C. Histologic analyses, immunoblotting, and colony-forming assay were performed to characterize the cell sheets. Autologous transplantation was undertaken to reconstruct the corneal surfaces of rabbits with experimentally induced limbal stem cell deficiencies. RESULTS: Multilayered corneal epithelial sheets were harvested intact simply by reducing the temperature, without the use of proteases. Cell-cell junctions and extracellular matrix on the basal side of the sheet, critical to sheet integrity and function, remained intact. A viable population of corneal progenitor cells, close in number to that originally seeded, was found in the sheets. Harvested sheets were easily manipulated, transplantable without any carriers, and readily adhesive to corneal stroma so that suturing was not required. Corneal surface reconstruction in rabbits was highly successful. CONCLUSIONS: Cell sheet engineering technology allows us to create intact, transplantable corneal epithelial cell sheets that retain stem cells from limbal stem cells expanded ex vivo. Our research indicates highly promising clinical capabilities for our bioengineered corneal epithelial sheet.

3T3 Cells↗

PubMed2004

Cell micropatterning using photopolymerization with a liquid crystal device commercial projector.

Photopolymerization has been widely used for surface micropatterning. The technique often requires photomasks and light sources with appropriate energies or filters. For rapid prototyping of surface photo-micropatterning, we have developed a novel device by modifying a commercially available liquid crystal device projector. In place of the image expansion unit of the projector, we attached an image reduction unit, an adjustable stage, and an optical monitoring unit. The device projected computer-generated images onto surfaces and subjected these patterns to photopolymerization. Micropatterned images can be easily prepared with various software run on personal computers. With the developed photopolymerization device, micropatterning of poly(ethylene glycol) (PEG) was achieved with PEG-diacrylate and a visible light photopolymerization initiator, camphorquinone. Selective cell adhesion control was also achieved on the micropatterned surfaces.

Animals↗

PubMed2004

PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis.

BACKGROUND: We have focused on the DNA topoisomerase II genes of several pathogenic fungi, and developed polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods targeting this gene for identification of dermatophytes. OBJECTIVE: To assess the availability of the PCR-based identification for an etiologic study of dermatophytosis, by testing these PCR and PCR-RFLP methods for stability and reproducibility. METHODS: Three hundred and fifty-six dermatophyte strains were isolated from 305 patients with tinea, and their genomic DNAs were used as templates for the PCR using primer mixes (PsT, PsME, dPsD1 or dPsD2) composed of gene-specific primers for identification of dermatophytes to the species level. The genomic DNAs of Trichophyton rubrum were further subjected to subrepeat element analysis of the nontranscribed spacer (NTS) of ribosomal DNA (rDNA). RESULTS: In this study, six dermatophyte species (T. rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum) were obtained. In all cases, the identifications obtained from the PCR and PCR-RFLP targeting the DNA topoisomerase II gene coincided with those from the conventional morphological features-based identification technique. The sensitivity of the PCR-based identification was found to be a colony of approximately 3mm in diameter. Furthermore, T. rubrum was divided into three groups (17 types) on the basis of the sizes and numbers of the products generated from the TRS-1 region, and three types from the TRS-2 region. CONCLUSION: The PCR and PCR-RFLP targeting the DNA topoisomerase II gene were rapid, stable, and reproducible for species identification of dermatophytes, and thus are convenient tools for an etiologic study of dermatophytosis.

Adolescent↗

PubMed2004

A case of laryngeal atresia (congenital high airway obstruction syndrome) with chromosome 5p deletion syndrome rescued by ex utero intrapartum treatment.

The authors report a case of laryngeal atresia (congenital high airway obstruction syndrome [CHAOS]) that was diagnosed prenatally. The patient underwent successfully tracheostomy by ex utero intrapartum treatment (EXIT). The fetal ultrasonography and magnetic resonance imaging MRI showed a typical CHAOS pattern with expanded hyperechogenic lungs, inverted diaphragms, and a dilated trachea. Recently, 3 cases of prenatally diagnosed CHAOS were reported to be treated successfully by EXIT. The clinical manifestation and course of this case was not similar to these 3 cases. The 3 previous patients did not fare as well during gestation and were delivered earlier than that in our case. In our case, fetal hydrops was seen at 23 gestational weeks, but it gradually subsided and disappeared at 30 gestational weeks. The fetus was stable and well. After delivery at 39 weeks, the baby received respiratory assistance by ventilator assistance. After 3 days, she could breath well on her own. The patient also had chromosome 5p deletion syndrome and perineal groove. More experience in treating CHAOS cases with EXIT to fully estimate its clinical course and prognosis is needed.

Abnormalities, Multiple↗

PubMed2003

Nanofabrication for micropatterned cell arrays by combining electron beam-irradiated polymer grafting and localized laser ablation.

Most methods reported for cell-surface patterning are generally based on photolithography and use of silicon or glass substrates with processing analogous to semiconductor manufacturing. Herein, we report a novel method to prepare patterned plastic surfaces to achieve cell arrays by combining homogeneous polymer grafting by electron beam irradiation and localized laser ablation of the grafted polymer. Poly(N-isopropylacrylamide) (PIPAAm) was covalently grafted to surfaces of tissue culture-grade polystyrene dishes. Subsequent ultraviolet ArF excimer laser exposure to limited square areas (sides of 30 or 50 microm) produced patterned ablative photodecomposition of only the surface region (approximately 100-nm depth). Three-dimensional surface profiles showed that these ablated surfaces were as smooth and flat as the original tissue culture-grade polystyrene surfaces. Time-of-flight secondary ion mass spectrometry analysis revealed that the ablated domains exposed basal polystyrene and were surrounded with PIPAAm-grafted chemistry. Before cell seeding, fibronectin was adsorbed selectively onto ablated domains at 20 degrees C, a condition in which the non-ablated grafted PIPAAm matrix remains highly hydrated. Hepatocytes seeded specifically adhered onto the ablated domains adsorbed with fibronectin. Because PIPAAm, inhibits cell adhesion and migration even at 37 degrees C when the grafted density is > 3 microg/cm2, all the cells were confined within the ablated domains. A 100-cell domain array was achieved by this method. This surface modification technique can be utilized for fabrication of cell-based biosensors as well as tissue-engineered constructs.

Acrylic Resins↗

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↗