Electrochemical Deposition of Copper on Bioactive Porous Titanium Dioxide Layer: Antibacterial and Pro-Osteogenic Activities.

Ti surfaces must exhibit antibacterial activity without cytotoxicity to promote bone reconstruction and prevent infection simultaneously. In this study, we employed a two-step electrochemical treatment process, namely, microarc oxidation (MAO) and cathodic electrochemical deposition (CED), to modify Ti surfaces. During the MAO step, a porous TiO2 (pTiO2) layer with a surface roughness of approximately 2.0 µm was generated on the Ti surface, and in the CED step, Cu was deposited onto the pTiO2 layer on the Ti surface, forming Cu@pTiO2. Cu@pTiO2 exhibited a similar structure, adhesion strength, and crystal phase to pTiO2. Moreover, X-ray photoelectron spectroscopy (XPS) confirmed the presence of Cu in Cu@pTiO2 at an approximate concentration of 1.0 atom %. Cu@pTiO2 demonstrated a sustained release of Cu ions for a minimum of 28 days in a simulated in vivo environment. In vitro experiments revealed that Cu@pTiO2 effectively eradicated approximately 99% of Staphylococcus aureus and Escherichia coli and inhibited biofilm formation, in contrast to the Ti and pTiO2 surfaces. Moreover, Cu@pTiO2 supported the proliferation of osteoblast-like cells at a rate comparable to that observed on the Ti and pTiO2 surfaces. Similar to pTiO2, Cu@pTiO2 promoted the calcification of osteoblast-like cells compared with Ti. In summary, we successfully conferred antibacterial and pro-osteogenic activities to Ti surfaces without inducing cytotoxic effects or structural and mechanical alterations in pTiO2 through the application of MAO and CED processes. Moreover, we found that the pTiO2 layer promoted bacterial growth and biofilm formation more effectively than the Ti surface, highlighting the potential drawbacks of rough and porous surfaces. Our findings provide fundamental insights into the surface design of Ti-based medical devices for bone reconstruction and infection prevention.

Rapid removal of detergent in glycolipids using ionic liquids.

Detergent removal in glycolipid after sample preparation, such as enzymatic reaction or isolation of detergent-resistant membrane microdomain, is indispensable for further structural characterization. We previously established the rapid and effective method of detergent removal in glycolipid samples from glass test tube using 1,2-dichloroethane (DCE) washing. However, the use of DCE has several drawbacks, such as environmental risks, harmful effects (potentially carcinogenic), and high vaporability and flammability. To solve the issue, we used ionic liquids to remove detergents from glycolipid samples, and found 1-butyl-3-methylimidazolium iodide was a suitable alternative for DCE.

Effect of cold rolling on the magnetic susceptibility of Zr-14Nb alloy.

The magnetic susceptibility of cold-rolled Zr-14Nb was evaluated to apply a new metallic medical device used for magnetic resonance imaging (MRI). The magnetic susceptibility of cold-rolled Zr-14Nb decreased up to the reduction ratio of 30%, then gradually decreased up to the ratio of 90%. Transmission electron microscopic observation revealed the strain-induced formation of ω phase after cold rolling at the reduction ratio of 5%, indicating that the initial decrease in magnetic susceptibility was caused by the formation of the ω phase. The ω phase was saturated at the reduction ratio of 30%. The formation of the ω phase could be explained on the basis of the increase in the Young's modulus and Vickers hardness of cold-rolled Zr-14Nb. The effect of texture formation on these properties was not obvious in the cold-rolled Zr-14Nb. Because of the strain-induced formation of the ω phase, the magnetic susceptibility of Zr-14Nb can be reduced by cold rolling to as low as that of as-cast Zr-9Nb, which is one-third that of Ti and Ti alloys. Therefore, cold-workable Zr-14Nb with low magnetic susceptibility could be a promising alloy for medical devices under MRI.

Differential expression of three flavanone 3-hydroxylase genes in grains and coleoptiles of wheat.

Flavonoid pigments are known to accumulate in red grains and coleoptiles of wheat and are synthesized through the flavonoid biosynthetic pathway. Flavanone 3-hydroxylase (F3H) is a key enzyme at a diverging point of the flavonoid pathway leading to production of different pigments: phlobaphene, proanthocyanidin, and anthocyanin. We isolated three F3H genes from wheat and examined a relationship between their expression and tissue pigmentation. Three F3Hs are located on the telomeric region of the long arm of chromosomes 2A, 2B, and 2D, respectively, designated as F3H-A1, F3H-B1, and F3H-D1. The telomeric regions of the long arms of the chromosomes of homoeologous group 2 of wheat showed a syntenic relationship to the telomeric region of the long arm of rice chromosome 4, on which rice F3H gene was also located. All three genes were highly activated in the red grains and coleoptiles and appeared to be controlled by flavonoid regulators in each tissue.

Bone healing of commercial oral implants with RGD immobilization through electrodeposited poly(ethylene glycol) in rabbit cancellous bone.

Immobilization of RGD peptides on titanium (Ti) surfaces enhances implant bone healing by promoting early osteoblastic cell attachment and subsequent differentiation by facilitating integrin binding. Our previous studies have demonstrated the efficacy of RGD peptide immobilization on Ti surfaces through the electrodeposition of poly(ethylene glycol) (PEG) (RGD/PEG/Ti), which exhibited good chemical stability and bonding. The RGD/PEG/Ti surface promoted differentiation and mineralization of pre-osteoblasts. This study investigated the in vivo bone healing capacity of the RGD/PEG/Ti surface for biomedical application as a more osteoconductive implant surface in dentistry. The RGD/PEG/Ti surface was produced on an osteoconductive implant surface, i.e. the grit blasted micro-rough surface of a commercial oral implant. The osteoconductivity of the RGD/PEG/Ti surface was compared by histomorphometric evaluation with an RGD peptide-coated surface obtained by simple adsorption in rabbit cancellous bone after 2 and 4 weeks healing. The RGD/PEG/Ti implants displayed a high degree of direct bone apposition in cancellous bone and achieved greater active bone apposition, even in areas of poor surrounding bone. Significant increases in the bone to implant contact percentage were observed for RGD/PEG/Ti implants compared with RGD-coated Ti implants obtained by simple adsorption both after 2 and 4 weeks healing (P<0.05). These results demonstrate that RGD peptide immobilization on a Ti surface through electrodeposited PEG may be an effective method for enhancing bone healing with commercial micro-rough surface oral implants in cancellous bone by achieving rapid bone apposition on the implant surface.

Development of PCR markers for Tamyb10 related to R-1, red grain color gene in wheat.

The grain color of wheat affects not only the brightness of flour, but also tolerance to preharvest sprouting. Grain color is controlled by dominant R-1 genes located on the long arm of hexaploid wheat chromosomes 3A, 3B, and 3D (R-A1, R-B1, and R-D1, respectively). The red pigment of the grain coat is composed of catechin and proanthocyanidin (PA), which are synthesized via the flavonoid biosynthetic pathway. We isolated the Tamyb10-A1, Tamyb10-B1, and Tamyb10-D1 genes, located on chromosomes 3A, 3B, and 3D, respectively. These genes encode R2R3-type MYB domain proteins, similar to TT2 of Arabidopsis, which controls PA synthesis in testa. In recessive R-A1 lines, two types of Tamyb10-A1 genes: (1) deletion of the first half of the R2-repeat of the MYB region and (2) insertion of a 2.2-kb transposon belonging to the hAT family. The Tamyb10-B1 genes of recessive R-B1 lines had 19-bp deletion, which caused a frame shift in the middle part of the open reading frame. With a transient assay using wheat coleoptiles, we revealed that the Tamyb10 gene in the dominant R-1 allele activated the flavonoid biosynthetic genes. We developed PCR-based markers to detect the dominant/recessive alleles of R-A1, R-B1, and R-D1. These markers proved to be correlated to known R-1 genotypes of 33 varieties except for a mutant with a single nucleotide substitution. Furthermore, double-haploid (DH) lines derived from the cross between red- and white-grained lines were found to necessarily carry functional Tamyb10 gene(s). Thus, PCR-based markers for Tamyb10 genes are very useful to detect R-1 alleles.

Duplicate polyphenol oxidase genes on barley chromosome 2H and their functional differentiation in the phenol reaction of spikes and grains.

Polyphenol oxidases (PPOs) are copper-containing metalloenzymes encoded in the nucleus and transported into the plastids. Reportedly, PPOs cause time-dependent discoloration (browning) of end-products of wheat and barley, which impairs their appearance quality. For this study, two barley PPO homologues were amplified using PCR with a primer pair designed in the copper binding domains of the wheat PPO genes. The full-lengths of the respective PPO genes were cloned using a BAC library, inverse-PCR, and 3'-RACE. Linkage analysis showed that the polymorphisms in PPO1 and PPO2 co-segregated with the phenol reaction phenotype of awns. Subsequent RT-PCR experiments showed that PPO1 was expressed in hulls and awns, and that PPO2 was expressed in the caryopses. Allelic variation of PPO1 and PPO2 was analysed in 51 barley accessions with the negative phenol reaction of awns. In PPO1, amino acid substitutions of five types affecting functionally important motif(s) or C-terminal region(s) were identified in 40 of the 51 accessions tested. In PPO2, only one mutant allele with a precocious stop codon resulting from an 8 bp insertion in the first exon was found in three of the 51 accessions tested. These observations demonstrate that PPO1 is the major determinant controlling the phenol reaction of awns. Comparisons of PPO1 single mutants and the PPO1PPO2 double mutant indicate that PPO2 controls the phenol reaction in the crease on the ventral side of caryopses. An insertion of a hAT-family transposon in the promoter region of PPO2 may be responsible for different expression patterns of the duplicate PPO genes in barley.

Differences in the bone differentiation properties of MC3T3-E1 cells on polished bulk and sputter-deposited titanium specimens.

The roughness and cleanness of a titanium surface must be controlled in order to investigate the expression mechanism of hard tissue compatibility on titanium. In this study, osteogenic MC3T3-E1 cells were cultured and differentiation-induced on bulk and sputter-deposited titanium specimens, and the osteogenesis were investigated. For the preparation of bulk specimens, titanium discs were mirror-polished. On the other hand, titanium was sputter-deposited on smooth and clean cover glasses as sputter-deposited specimens. As a result, no significant difference was observed in the cell morphology and attached number. On the other hand, the time showing maximum activity in the alkaline phosphatase and gene expressions, which are related to bone differentiation on the bulk titanium, were superior to those on the sputter-deposited titanium. From the surface observation of the specimens with a scanning electron microscope and a scanning probe microscope, the surface on the sputter-deposited titanium was more uniform and cleaner than that on the bulk titanium. According to X-ray photoelectron spectroscopy, the thickness of surface oxide film on the sputter-deposited titanium was smaller than that on the bulk titanium. In addition, the proportions of TiO and Ti(2)O(3) in the surface oxide film on the sputter-deposited titanium were larger than those on the bulk titanium. These differences might influence the differentiation of osteoblastic cells.

Polyphenol oxidase from wheat bran is a serpin.

Polyphenol oxidase (PPO; EC 1.10.3.2) was isolated from wheat bran by a procedure that included ammonium sulfate fractionation, batch adsorption by DEAE-cellulofine, CM-cellulofine column chromatography, DEAE-cellulofine column chromatography, preparative isoelectric focusing, adsorption on the membrane of a Vivapure Q Maxi H spin column, and heat treatment. These procedures led to 150-fold purification with 4.2% recovery. The PPO was homogeneous by SDS/PAGE. The relative molecular weight of the PPO was estimated to be 37,000 based on its mobility in SDS/PAGE. The isoelectric point of the PPO was 4.4. The K(m) values of the PPO for caffeic acid, chlorogenic acid, pyrocatechol, 4-methyl catechol and l-DOPA as substrates were 0.077, 0.198, 1.176, 1.667 and 4.545 mM. The PPO was strongly inhibited by tropolone. The K(i) value for tropolone is 2.2 x 10(-7) M. The sequence of the 15 N-terminal amino-acid residues was determined to be ATDVRLSIAHQTRFA, which was identical to those of serpin from Triticum aestivum and protein Z from Hordeum vulgare. The PPO strongly inhibited the activity of trypsin, which is an enzyme of serine proteases; 50% inhibition was observed with 1.5 x 10(-7) M PPO. The K(i) value for PPO is 2.3 x 10(-8) M. The wheat bran PPO should be a very important protein for protecting wheat against disease, virus, insect and herbivore damages by both the activities of PPO and protease inhibitor.

Structural and functional properties of Viviparous1 genes in dormant wheat.

Viviparous 1 (Vp1) of maize is known to encode a transcription factor VP1 that controls seed germination. Hexaploid wheat possesses three Vp1 homoeologues (TaVp1): TaVp-A1, TaVp-B1 and TaVp-D1. In this study, we attempted to characterize the molecular properties of TaVp1 in a highly dormant wheat cultivar, Minamino-komugi (Minamino). The seeds of Minamino showed much higher sensitivity to the inhibitory effect of ABA on germination than those of non-dormant cultivars, Sanin-1 and Tozan-18. The sequence analyses of cDNAs also revealed that some of TaVp-A1 transcripts and TaVp-D1 transcripts were spliced incorrectly, presumably resulting in production of truncated or deleted proteins. Most TaVp-B1 transcripts were spliced correctly, but some had an additional 3-bp (AAG) insertion in the B3 domain, which may not affect their function. RT-PCR analyses showed that TaVp1 was highly expressed in Minamino embryos in maturing seeds but much less in roots and leaves of seedlings. The level of TaVp1 mRNA was high when the embryos were treated with ABA but markedly decreased in water-imbibed mature embryos whose dormancy had been broken. Expression analyses of the individual homoeologues showed that the level of TaVp-A1 transcripts was highest in embryos of DAP 20 but much lower in the matured embryos. TaVp-B1 was highly expressed in developing and maturing seed embryos, while TaVp-D1 mRNA existed at lower levels in developing embryos but increased as the seeds were matured. These results suggest that the majority of TaVp1, especially TaVp-B1, are properly spliced and may function as a transcription factor playing an important role on dormancy in Minamino. By employing an efficient transient expression system using diploid wheat seeds, we confirmed the dual function of TaVP-B1: the activation of Em expression and the repression of alpha-amylase expression.

Differential expression of three ABA-insensitive five binding protein (AFP)-like genes in wheat.

Abscisic acid (ABA) signaling includes positive and negative regulators in the signaling pathway. ABA-insensitive five (ABI5) binding protein (AtAFP), one of the negative regulators found in Arabidopsis, is involved in the proteolysis of a positive regulator, ABI5 (bZIP-type transcription factor). Three wheat orthologs (TaAFPs) of AtAFP were isolated. TaAFPs have a nuclear localization domain in the middle of the deduced amino acid sequence and an ABI5 binding domain in the C-terminal region as AtAFP. Three TaAFPs were located on the short arms of chromosomes 2A, 2B, and 2D of wheat, and based on their chromosomal locations, they were named TaAFP-A, TaAFP-B, and TaAFP-D. In comparison to AtAFP, which was activated in developing seeds and the early stage of germination, TaAFPs were expressed in a greater variety of tissues, such as flag leaves, roots, and leaves of seedlings, and developing grains. TaAFP-B was expressed predominantly in all tissues examined; TaAFP-A and TaAFP-D responded to ABA and stresses, such as salt and dehydration. These three TaAFPs may differentiate their roles in ABA signaling during wheat evolution.

Effect of active hydroxyl groups on the interfacial bond strength of titanium with segmented polyurethane through gamma-mercapto propyl trimethoxysilane.

The objective of this study was to investigate the effect of active hydroxyl groups on a titanium (Ti) surface on the bond strength between Ti and segmented polyurethane (SPU) composite through gamma-mercapto propyl trimethoxysilane (gamma-MPS). Active hydroxyl groups on Ti surface oxide were controlled by immersion in hydrogen peroxide (H2O2) with different lengths of immersion time, and the resulting concentrations of active hydroxyl groups were evaluated using a zinc-complex substitution technique. For the H2O2-treated Ti, it was characterized using X-ray photoelectron spectroscopy and scanning electron spectroscopy. For the bond strength of Ti/ gamma-MPS/SPU interface, it was determined using a shear bond test. Results showed that the bond strength increased with increase in the concentration of active hydroxyl groups. In terms of durability after immersion in water at 310 K for 30 days, it was found that bond strength was improved with increase in active hydroxyl groups. Based on the results obtained, active hydroxyl groups on the surface oxide film were clearly one of the causes governing the interfacial bond strength.

Effect of UV irradiation on the shear bond strength of titanium with segmented polyurethane through gamma-mercapto propyl trimethoxysilane.

The objective of this study was to investigate the effect of UV irradiation on shear bond strength between a titanium (Ti) and a segmented polyurethane (SPU) composite through gamma-mercapto propyl trimethoxysilane (gamma-MPS). To this end, the shear bond strength of Ti/SPU interface of Ti-SPU composite under varying conditions of ultraviolet ray (UV) irradiation was evaluated by a shear bond test. The glass transition temperatures of SPU with and without UV irradiation were also determined using differential scanning calorimetry. It was found that the shear bond strength of Ti/SPU interface increased with UV irradiation. However, excessive UV irradiation decreased the shear bond strength of Ti/SPU interface. Glass transition temperature was found to increase during 40-60 seconds of UV irradiation. In terms of durability after immersion in water at 37 degrees C for 30 days, shear bond strength was found to improve with UV irradiation. In conclusion, UV irradiation to a Ti-SPU composite was clearly one of the means to improve the shear bond strength of Ti/SPU interface.

Colour genes (R and Rc) for grain and coleoptile upregulate flavonoid biosynthesis genes in wheat.

Pigmentation of wheat grain and coleoptile is controlled by the R gene on chromosomes of the homoeologous group 3 and the Rc gene on chromosomes of the homoeologous group 7, respectively. Each of these genes is inherited monogenically. The pigment of grain has been suggested to be a derivative of catechin-tannin and that of coleoptile to be anthocyanin. These polyphenol compounds are known to be synthesized through the flavonoid biosynthesis pathway. We isolated 4 partial nucleotide sequences of the early flavonoid biosynthesis genes (CHS, CHI, F3H, and DFR) in wheat. The expression of these genes was examined in the developing grain of red-grained and white-grained wheat lines. CHS, CHI, F3H, and DFR were highly upregulated in the grain coat tissue of the red-grained lines, whereas there was no significant expression in the white-grained lines. These results indicate that the R gene is involved in the activation of the early flavonoid biosynthesis genes. As for coleoptile pigmentation, all 4 genes were expressed in the red coleoptile; however, DFR was not activated in the white coleoptile. The Rc gene appears to be involved in DFR expression. The possibility that wheat R and Rc genes might be transcription factors is discussed.

Isolation and location of three homoeologous dihydroflavonol-4-reductase (DFR) genes of wheat and their tissue-dependent expression.

DFR is involved in an important step in the flavonoid biosynthesis pathway upstream of anthocyanin, proanthocyanidin, and phlobaphene production, which contributes to the pigmentation of various plant tissues. Full genomic sequences of three DFRs were isolated in hexaploid wheat. Loci of TaDFRs were found in a more proximal region of the long arm of chromosomes of homoeologous group 3 than the R gene for red grain colour of wheat. These DFRs were designated TaDFR-A, TaDFR-B, and TaDFR-D on chromosome 3A, 3B, and 3D, respectively. In the 5' upstream region of DFR genes, two or three combinations of a G box core element and a putative binding site for a Myb-type transcription factor, P, of maize were found. Expression of DFR reached a maximal level in red grain of wheat cv. Chinese Spring (CS) at 5 d post-anthesis (DPA) and decreased gradually in the grain coat tissue from 10 to 20 DPA, in contrast to a very low expression level of DFR in white wheat grain during the same period. These DFRs differed in their expression. TaDFR-B and -D were expressed predominantly in grains. In developing leaves, DFR expression was light-responsive, and TaDFR-B was more up-regulated in leaves and roots than the other two.

Effect of grain colour gene (R) on grain dormancy and sensitivity of the embryo to abscisic acid (ABA) in wheat.

The level of grain dormancy and sensitivity to ABA of the embryo, a key factor in grain dormancy, were examined in developing grains of a white-grained wheat line, Novosibirskaya 67 (NS-67), and its red-grained near-isogenic lines (ANK-1A to -1D); a red-grained line, AUS 1490, and its white-grained mutant line (EMS-AUS). ANK lines showed higher levels of grain dormancy than NS-67 at harvest maturity. AUS 1490 grain also showed higher dormancy than EMS-AUS grain. These results suggest that the R gene for grain colour can enhance grain dormancy. However, the dormancy effect conferred by the R gene was not large, suggesting that it plays a minor role in the development of grain dormancy. Water extracts of AUS 1490 and EMS-AUS bran contained germination inhibitors equivalent to 1-10 microM ABA, although there was no difference in the amount of inhibitors between AUS 1490 and EMS-AUS. Thus, the grain colour gene of AUS 1490 did not appear to enhance the level of grain dormancy by accumulating germination inhibitors in its bran. Sensitivity to ABA of embryos was higher in grains collected around harvest-maturity for ANK lines and AUS 1490, compared with NS-67 and EMS-AUS. The R gene might enhance grain dormancy by increasing the sensitivity of embryos to ABA.

Deviations of capacitive and inductive loops in the electrochemical impedance of a dissolving iron electrode.

The electrochemical impedance of an iron electrode often shows the capacitive and inductive loops on the complex plane. The capacitive loop originates from the time constant of the charge transfer resistance and the electric double layer capacitance. The inductive loop is explained by Faradaic processes involving the reaction intermediate. In some cases, these loops deviate from a true semicircle. In this paper, the origins and curve-fitting methods for the deviated loops of electrochemical impedance are discussed. The constant phase element (CPE) was used to present the deviation of the capacitive loop instead of electric double layer capacitance. The reaction rate constants, which are a function of the frequency, are proposed for the Faradaic impedance to present the deviated inductive loop.

Two-gene systems of vernalization requirement and narrow-sense earliness in einkorn wheat.

The genetic segregation of the heading trait was analyzed using a recombinant inbred line (RIL) of einkorn wheat, RILWA-1, derived from cultivated Triticum monococcum L., and wild-type T. boeoticum Boiss. The latency to heading was examined in 115 lines under controlled environmental conditions, as well as in the field, and the degrees of narrow-sense earliness and vernalization requirement were evaluated for quantitative trait locus (QTL) analysis. Single-marker analysis using 107 RFLP markers segregating in RILWA-1 detected 20 linking markers for heading factors. In all marker loci, the alleles for early heading were conferred by T. monococcum. In interval analysis of chromosome 5Am, two vernalization genes, Vrn-Am1 and Vrn-Am2, were precisely mapped to the Xcdo504-Xpsr426 interval on the central region of the long arm and to the Xwg114-Xwec87 interval on its distal region, respectively. Interval analysis also showed that two genes for narrow-sense earliness, designated Nse-3Am and Nse-5Am, were located on chromosome 3Am and 5Am, respectively. It was noticed that heading time in the field was determined mainly by Nse-3Am, suggesting that narrow-sense earliness is critical for heading in the field in einkorn wheat.