Biomedical subjects

Kazuo Harada

Publications and source records attributed to Kazuo Harada.

At least 19 recordsLinked to original sources

PubMed2007

Effect of pore size on in vitro cartilage formation using chitosan-based hyaluronic acid hybrid polymer fibers.

In this study, we successfully developed three-dimensional scaffolds fabricated from the chitosan-based hyaluronic acid hybrid polymer fibers, which can control the porous structure. To determine the adequate pore size for enhancing the chondrogenesis of cultured cells, we compared the behaviors of rabbit chondrocytes in scaffolds comprising different pore sizes (100, 200, and 400 microm pore size). Regarding the cell proliferation, there was no significant difference among the three groups. On the other hand, glycosaminoglycan contents in the 400 microm group significantly increased during the culture period, compared with those in the other groups. The ratio of type II to type I collagen mRNA level was also significantly higher in the 400 microm group than in the other groups. These results indicate that our scaffold with 400 microm pore size significantly enhances the extracellular matrix synthesis by chondrocytes. Additionally, the current scaffolds showed high mechanical properties, compared with liquid and gel materials. The data derived from this study suggest great promise for the future of a novel fabricated material with relatively large pore size as a scaffold for cartilage regeneration. The biological and mechanical advantages presented here will make it possible to apply our scaffold to relatively wide cartilaginous lesions.

Animals↗

PubMed2006

Time-course metabolic profiling in Arabidopsis thaliana cell cultures after salt stress treatment.

Salt stress is one of the most important factors limiting plant cultivation. Many investigations of plant response to high salinity have been performed using conventional transcriptomics and/or proteomics approaches. However, transcriptomics and proteomics techniques are not all-encompassing methods that can achieve exclusive insights into the metabolite networks contributing to biochemical reactions. Hence, the functions of the complex stress response pathways are yet to be determined, especially at the metabolic level. A time-course metabolic profiling with Arabidopsis thaliana cell cultures after the imposition of salt stress is reported in this study. Analyses of primary metabolites, especially small polar metabolites such as amino acids, sugars, sugar alcohols, organic acids, and amines, was performed by GC/MS and LC/MS at 0.5, 1, 2, 4, 12, 24, 48, and 72 h after a salt-stress treatment with 100 mM NaCl being the final concentration. The mass chromatographic data were converted into matrix data sets, which were subjected to data mining processes, including principal component analysis (PCA) and batch-learning self-organizing mapping analysis (BL-SOM). The mining results suggest that the methylation cycle for the supply of methyl groups, the phenylpropanoid pathway for lignin production, and glycinebetaine biosynthesis are synergetically induced as a short-term response against salt-stress treatment. The results also suggest the the co-induction of glycolysis and sucrose metabolism as well as co-reduction of the methylation cycle as long-term responses to salt stress.

Amines↗

PubMed2006

An efficient thermally induced RNA conformational switch as a framework for the functionalization of RNA nanostructures.

RNA offers a variety of interactions and dynamic conformational switches not available with DNA that may be exploited for the construction of nanomolecular structures. Here, we show how the RNA loop-loop, or "kissing", interaction can be used to construct specific circular RNA arrangements that are capable of thermal isomerization to alternative structures. We also show how this thermally induced structural rearrangement can be used to unmask a functional RNA structure, in this case, a peptide-binding RNA structure, the Rev-response element (RRE) of HIV, thereby acting as a functional peptide-binding switch. The relative ease with which the RRE could be engineered into the RNA substrates suggested that a variety of functional RNA structures may be introduced. In addition, the structural rearrangement was extremely efficient, showing that the "kissing" complexes described in this study may provide a useful framework for the construction of functional RNA-based nanostructures, as well as aid in our understanding of the way RNA functions in biological systems.

Base Sequence↗

PubMed2006

Identification of antisense RNA stem-loops that inhibit RNA-protein interactions using a bacterial reporter system.

A bacterial reporter system for the identification of antisense RNA stem-loops that inhibit RNA-protein interactions was constructed. RNA stem-loops were chosen since they have been shown to act as triggers in a variety of natural antisense systems. An RNA stem-loop library was screened for sequences that bind to the U1 hpII and inhibit U1A protein binding using a bacterial system based on phage lambda N mediated antitermination. As a result, antisense RNA stem-loops that appeared to be binding to the boxA region, which is important for the formation of functional antitermination complex, and not to the hpII loop were identified. In vivo and in vitro mutagenic analysis showed that the inhibitor RNA stem-loops were indeed binding to the boxA region through Watson-Crick base pairing and disrupting binding to host factors NusG, NusB and S10. The results show that the bacterial reporter system may be of general use for the identification of antisense RNA stem-loops that disrupt RNA-protein interactions.

Bacteria↗

PubMed2006

Pressure-assisted capillary electrophoresis mass spectrometry using combination of polarity reversion and electroosmotic flow for metabolomics anion analysis.

We have developed a method based on capillary electrophoresis (CE) coupled to mass spectrometry (MS) that is a powerful tool for metabolome analysis. In this paper, a simple method for the simultaneous analysis of anionic metabolites such as sugar phosphates, organic acids, nucleotides and CoA compounds based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS) is described. The feature of this method is that CE polarity, where the inlet of the capillary is at the anode and the outlet at the cathode, is inverted from conventional CE analysis for anions. Moreover, an ordinary fused silica capillary was chosen instead of a specific cationic polymer-coated capillary (SMILE (+) capillary). A robust and inexpensive analytical method was established from the above-mentioned CE polarity and noncoated fused silica capillary. A trimethylamine acetate electrolyte, pH 10.0, provides a high resolution between isomers. Electrolyte flow using an air pump after electrophoresis enables the comprehensive and simultaneous analyses of sugar phosphates, organic acids, nucleotides and a CoA compound that to date have been impossible to analyze. Our method can be used as a de facto standard for metabolome analysis.

Anions↗

PubMed2005

Novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering.

To clarify the feasibility of using novel chitosan-based hyaluronan hybrid polymer fibers as a scaffold in ligament tissue engineering, their mechanical properties and ability to promote cellular adhesion, proliferation, and extracellular matrix production were studied in vitro. Chitosan fibers and chitosan-based 0.05% and 0.1% hyaluronan hybrid fibers were developed by the wet spinning method. Hyaluronan coating significantly increased mechanical properties, compared to the chitosan fibers. Rabbit fibroblasts adhesion onto hybrid fibers was significantly greater than for the control and chitosan fibers. For analysis of cell proliferation and extracellular matrix production, a three-dimensional scaffold was created by simply piling up each fiber. At 1 day after cultivation, the DNA content in the hybrid scaffolds was higher than that in the chitosan scaffold. Scanning electron microscopy showed that the fibroblasts had produced collagen fibers after 14 days of culture. Immunostaining for type I collagen was clearly predominant in the hybrid scaffolds, and the mRNA level of type I collagen in the hybrid scaffolds were significantly greater than that in the chitosan scaffold. The present study revealed that hyaluronan hybridization with chitosan fibers enhanced fiber mechanical properties and in vitro biological effects on the cultured fibroblasts.

Animals↗

PubMed2005

A novel crude oil emulsifier excreted in the culture supernatant of a marine bacterium, Myroides sp. strain SM1.

A marine bacterium, Myroides sp. SM1, can grow on weathered crude oil and show emulsification of it. The biosurfactant able to emulsify crude oil was excreted in culture supernatant of Myroides sp. SM1 grown on marine broth, which was extracted with chloroform/methanol (1:1) at pH 7 and purified by normal and reverse phase silica gel column chromatographies. The compound was ninhydrin-positive, and the chemical structure was elucidated by nuclear magnetic resonance (NMR), infrared spectroscopy (IR), fast atom bombardment mass spectrometry, and gas chromatography-mass spectrometry (GC-MS) to be a mixture of L: -ornithine lipids, which were composed of L: -ornithine and a different couple of iso-3-hydroxyfatty acid (C(15)-C(17)) and iso-fatty acid (C(15) or C(16)) in a ratio of 1:1:1. The critical micelle concentration for a mixture of ornithine lipids was measured to be approximately 40 mg/l. A mixture of ornithine lipids exhibited emulsifying activity for crude oil in a broad range of pH, temperature, and salinity and showed higher surface activity for oil displacement test than other several artificial surfactants and a biosurfactant, surfactin.

Culture Media, Conditioned↗

PubMed2005

Evolvability of the mode of peptide binding by an RNA.

The HIV Rev-response element (RRE) RNA binds strongly to two unrelated peptides, the HIV Rev peptide and an RRE-binding aptamer, the RSG-1.2 peptide, at a similar site, but using distinct sets of interactions. In this study, the nucleotide base requirements for the binding of the RRE to the Rev and RSG-1.2 peptides were determined by selection of Rev- and RSG-1.2-binding RRE variants using a bacterial reporter system. As a result, distinct differences in the bases necessary for binding the two peptides were found in the upper stem of the RRE. Strikingly, single nucleotide changes in this region were found to switch the peptide-binding specificity of the RRE from a bifunctional Rev- and RSG-1.2-binding mode to either a Rev-specific or a RSG-1.2- specific mode, demonstrating how an RNA can evolve alternative binding strategies in discrete steps without intermediate loss of function. This evolvability of the mode of peptide binding by an RNA presumably reflects the multidimensionality of conformational space that a given RNA has available for ligand recognition, which may have been utilized in the evolution of RNA-polypeptide complexes.

Electrophoresis, Polyacrylamide Gel↗

PubMed2005

Application of tissue engineering techniques for rotator cuff regeneration using a chitosan-based hyaluronan hybrid fiber scaffold.

BACKGROUND: The current surgical procedures for irreparable rotator cuff tears have considerable limitations. Tissue engineering techniques using novel scaffold materials offer potential alternatives for managing these conditions. HYPOTHESIS: A chitosan-based hyaluronan hybrid scaffold could enhance type I collagen products with seeded fibroblasts and thereby increase the mechanical strength of regenerated tendon in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: The scaffolds were created from chitosan-based hyaluronan hybrid polymer fibers. Forty-eight rabbit infraspinatus tendons and their humeral insertions were removed to create defects. Each defect was covered with a fibroblast-seeded scaffold (n = 16) or a non-fibroblast-seeded scaffold (n = 16). In the other 16 shoulders, the rotator cuff defect was left free as the control. At 4 and 12 weeks after surgery, the engineered tendons were assessed by histological, immunohistochemical (n = 2), and biomechanical (n = 6) analyses. RESULTS: Type I collagen was only seen in the fibroblast-seeded scaffold and increased in the regenerated tissue. The tensile strength and tangent modulus in the fibroblast-seeded scaffold were significantly improved from 4 to 12 weeks postoperatively. The fibroblast-seeded scaffold had a significantly greater tangent modulus than did the non-fibroblast-seeded scaffold and the control at 12 weeks. CONCLUSION: This scaffold material enhanced the production of type I collagen and led to improved mechanical strength in the regenerated tissues of the rotator cuff in vivo. CLINICAL RELEVANCE: Rotator cuff regeneration is feasible using this tissue engineering technique.

Animals↗

PubMed2005

Alginate and chitosan polyion complex hybrid fibers for scaffolds in ligament and tendon tissue engineering.

Selecting the material for a scaffold is critically important for the success of tissue engineering. To simplify complicated biosynthetic matrices and achieve a novel class of potential materials, a model of polyion complex fibers was prepared from alginate and chitosan. In the current in vitro study, we thought that alginate-based chitosan hybrid biomaterials could provide excellent supports for fibroblast adhesion. In the current study, alginate polymer fiber (alginate group) and alginate-based chitosan hybrid polymer fibers (alginate with 0.05% chitosan, alginate-chitosan 0.05% group; alginate with 0.1% chitosan, alginate-chitosan 0.1% group) were originally prepared. We investigated the adhesion behavior of rabbit tendon fibroblast onto alginate polymer fibers versus the adhesion of the fibroblast onto alginate-based chitosan hybrid polymer fibers. Furthermore, mechanical properties and synthesis of the extracellular matrix were investigated. Mechanically, the novel fiber has considerable tensile strength of more than 200 MPa. We demonstrated that the alginate-based chitosan hybrid polymer fibers showed much improved adhesion capacity with fibroblast compared with alginate polymer fiber. Additionally, morphologic studies revealed the dense fiber of the type I collagen produced by the fibroblast in the hybrid polymer fibers. We concluded that an alginate-based chitosan hybrid polymer fiber has considerable potential as a desirable biomaterial scaffold for tendon and ligament tissue engineering.

Alginates↗

PubMed2005

Feasibility of chitosan-based hyaluronic acid hybrid biomaterial for a novel scaffold in cartilage tissue engineering.

In this study, we hypothesized that hyaluronic acid could provide superior biological effects on the chondrocytes in a three-dimensional culture system. To test this hypothesis, we investigated the in vitro behavior of rabbit chondrocytes on a novel chitosan-based hyaluronic acid hybrid polymer fiber. The goal of the current study was to show the superiority of this novel fiber as a scaffold biomaterial for cartilage tissue engineering. Chitosan polymer fibers (chitosan group) and chitosan-based hyaluronic acid hybrid polymer fibers (HA 0.04% and HA 0.07% groups, chitosan coated with hyaluronic acid 0.04% and 0.07%, respectively) were originally developed by the wetspinning method. Articular chondrocytes were isolated from Japanese white rabbits and cultured in the sheets consisting of each polymer fiber. The effects of each polymer fiber on cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were analyzed by quantitative a cell attachment test, DNA quantification, light and scanning electron microscopy, semi-quantitative RT-PCR, and immunohistochemical analysis. Cell adhesivity, proliferation and the synthesis of aggrecan were significantly higher in the hybrid fiber (HA 0.04% and 0.07%) groups than in the chitosan group. On the cultured hybrid polymer materials, scanning electron microscopic observation showed that chondrocytes proliferated while maintaining their morphological phenotype and with a rich extracellular matrix synthesis around the cells. Immunohistochemical staining with an anti-type II collagen antibody demonstrated rich production of the type II collagen in the pericellular matrix from the chondrocytes. The chitosan-based hyaluronic acid hybrid polymer fibers show great potential as a desirable biomaterial for cartilaginous tissue scaffolds.

Animals↗

PubMed2005

Regulation of the structure of RNA assemblies through RNA tertiary interactions.

We have previously shown that RNA building blocks containing two or three stem-loops based on the dimerization initiation site (DIS) connected by short linkers can be assembled into various one- and two-dimensional structures. Here, in an attempt to regulate the conformation of the RNA assemblies by adjusting the rigidity of the RNA building block, we have introduced the triple-helical scaffold (THS) into the linker region connecting two DIS-based stem-loops. As a result, linear and circular assemblies could be selectively formed with RNA building blocks containing the wild-type and mutant THS sequences, respectively. The results demonstrate how the conformation of RNA assemblies may be regulated by external factors, thereby expanding the utility of RNA-based nanostructures.

Dimerization↗

PubMed2005

An isotope effect on the comparative quantification of flavonoids by means of methylation-based stable isotope dilution coupled with capillary liquid chromatography/mass spectrometry.

Ionization suppression is a serious problem in liquid chromatography/mass spectrometry-based metabolomics, and stable isotope dilution-based comparative quantification is one of the most important methods of overcoming this problem. Herein, the use of [(13)C]-methylation-based stable isotope dilution for comparative quantification of flavonoids is demonstrated. This is in contrast to the equivalent deuterium labeling methylation method, which has an adverse isotope effect on reverse phase chromatography.

Artifacts↗

PubMed2005

Stable isotope dilution-based accurate comparative quantification of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo (15)N-isotope enrichment.

Stable isotope dilution-based comparative quantification of nitrogen-containing metabolites for highly sensitive and selective metabolomics was developed using liquid chromatography/mass spectrometry (LC/MS) and (15)N-isotope enrichment. We produced metabolically stable isotope-labeled Arabidopsis T87 cells by culturing with (15)N-labeled medium. We found that the growth of cells maintained in (15)N-labeled medium is very similar to the growth in normal medium, as evidenced by cell morphology, doubling time, and measurement of chlorophyll and carotenoid contents. Complete incorporation of (15)N in folate, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) in T87 cells was accomplished after culturing for 21 d. Accurate comparative quantification of folate, SAM, and SAH was established by means of LC/MS using the isotopomers of the target metabolites as internal standards. The within- and between-run assay coefficients of variation for the folate, SAM, and SAH levels were all less than 8.5%. Stable isotope labeling by nitrogen source in Arabidopsis T87 cell culture provided simple, inexpensive, and accurate amino acid profiling. This interesting new protocol is valuable for the study of dynamic changes in N-compound pools in cultured cells.

Arabidopsis↗

PubMed2004

Formation of dendrimeric RNA assemblies though RNA loop-loop interactions.

We describe the construction of nano-scale dendritic assemblies using RNA building blocks based on dimerization initiation site (DIS) RNA of the human immunodeficiency virus type 1 (HIV-1) that forms stable base pairing through loop-loop ("kissing") interactions. RNA building blocks containing three DIS-like hairpins connected by a two nucleotide linker self-assembled in a magnesium-dependent manner to form dendrimeric structures as observed by non-denaturing polyacrylamide gel electrophoresis (PAGE).

Base Sequence↗

PubMed2003

RNA LEGO: magnesium-dependent formation of specific RNA assemblies through kissing interactions.

The high affinity and specificity of nucleic acid base complementarity has been proven to be a powerful method for constructing specific molecular assemblies. On the other hand, recent structural studies of RNA have revealed the wide range of tertiary interactions utilized in RNA folding, which may potentially be used as tools for the design of specific macromolecular assemblies. Here, RNA building blocks containing two hairpin loops, based on the dimerization initiation site (DIS) of HIV RNA, connected by a short linker were used to construct large RNA assemblies through hairpin loop-loop ("kissing") interactions. We show that specific linear and circular assemblies can be constructed in a magnesium-dependent manner using several non-self-complementary loop-loop interactions designed in this study. These results show that the use of RNA tertiary interactions may broaden the repertoire of nucleic acid-based nanostructures.

Circular Dichroism↗

PubMed2003

The variability of the peptide-binding specificity of RNA.

Variants of the HIV Rev-response element (RRE) that bind to HIV Rev peptide or to RSG-1.2, a selected RRE-binding peptide, were selected using an in vivo assay system for RNA-polypeptide interactions. The selected RRE variants could be classified into three groups based on their specificity towards the Rev and RSG-1.2 peptide. The class I RRE variants showed specificities similar to wild-type RRE, while class II variants specifically bound RSG-1.2 peptide, and class III variants bound specifically to the Rev peptide. Examination of the class II and class III RNAs revealed distinct nucleotide and structural requirements in the upper stem region, and suggested that single nucleotide changes may dramatically alter the peptide-binding specificity of the RRE.

Amino Acid Sequence↗

PubMed2003

Selection of RRE RNA binding peptides using a kanamycin antitermination assay.

The arginine-rich domains of several RNA-binding proteins have been shown to bind their cognate RNAs with high affinities and specificities as isolated peptides, adopting different conformations within different complexes. The sequence simplicity and structural diversity of the arginine-rich motif has made it a good framework for constructing combinatorial libraries and identifying novel RNA-binding peptides, including those targeted to the HIV Rev response element (RRE). Here we describe a modified transcription antitermination reporter assay engineered with kanamycin resistance that enables larger in vivo screens (approximately 10(9) sequences) than previously possible. We show that the assay detects only specific RNA-protein complexes, and that binders are enriched at least 300-fold per round of selection. We screened a large peptide library in which amino acids with charged, polar, and small side chains were randomly distributed within a polyarginine framework and identified a set of high affinity RRE-binding peptides. Most contain glutamine at one particular peptide position, and the best peptides display significantly higher antitermination activities than Rev or other previously described high-affinity RRE-binding peptides. The kanamycin antitermination (KAN) assay should be useful for screening relatively large libraries and thereby facilitate identification of novel RNA binders.

Biological Assay↗