Takayuki Uchihashi

Common mutations F310L and T1559del in the tissue-nonspecific alkaline phosphatase gene are related to distinct phenotypes in Japanese patients with hypophosphatasia.

A total of 22 Japanese patients with hypophosphatasia were included in a study analysing the relationship between mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene and the severity of the phenotype in Japanese patients with hypophosphatasia. The enzymatic activity of some of the identified mutant TNSALP proteins was also examined using corresponding expression vectors. Eighteen mutations, including 6 novel ones, were identified in the patients. Among them, the common mutations were F310L and T1559del. Of note, five patients with F310L mutation in one of the alleles exhibited a relatively mild phenotype without respiratory complications despite its perinatal onset. In contrast, the T1559del mutation is associated with perinatal lethal and infantile forms when not found in patients with the F310L mutation. The genotype-phenotype relationship was, to some extent, consistent with the enzymatic activity of the mutant ALP proteins; mutations K207E and G409C found in a surviving case of infantile hypophosphatasia, as well as F310L, retained some residual activities, whereas T1559del caused a complete loss of activity. Conclusion:In Japanese patients, the common mutations F310L and T1559del are associated with the relatively mild and lethal forms of hypophosphatasia, respectively. Our results may enhance the importance of genotyping patients with hypophosphatasia to predict their prognosis.

True atomic resolution imaging of surface structure and surface charge on the GaAs(110)

We demonstrated a novel method to detect the van der Waals and the electrostatic force interactions simultaneously on an atomic scale, which is based on frequency modulation detection method. For the first time, the surface structure and the surface charge at atomic-scale point defects on the GaAs(110) surface have been simultaneously resolved with true atomic resolution under ultra-high vacuum condition. From the bias voltage dependence of the image contrast, we can verify that the sign of the atomically resolved surface charge at the point defect was positive.

Carbon-Nanotube Tip for Highly-Reproducible Imaging of Deoxyribonucleic Acid Helical Turns by Noncontact Atomic Force Microscopy

A carbon nanotube (CNT) was used as a tip for a noncontact-mode atomic force microscope (NC-AFM). A CNT tip was attached to an Au/Si tip by a well-controlled procedure in a scanning-electron-microscope (SEM) chamber. The NC-AFM with the CNT tip produced highly reproducible images of right-handed helical turns of linear deoxyribonucleic acid (DNA) with a spacing of 3.5 ±1.0 nm. The full-width at half-maximum (FWHM) of the cross section of DNA measured was 3.1 ±0.6 nm.

Distance dependence of noncontact-AFM image contrast on Si(111)×–Ag structure

Abstract Atomic resolution imaging of the Si(111) 3 × 3 R30°–Ag surface was investigated using a noncontact atomic force microscopy (NC-AFM) in ultrahigh vacuum. NC-AFM images showed three types of contrasts depending on the distance between an AFM tip and a sample surface. When the tip–sample distance was about 1–3 A, the images showed the honeycomb arrangement with weak contrast. When the tip–sample distance was about 0–0.5 A, the images showed the periodic structure composed of three bright spots with relatively strong contrast. On the other hand, the contrasts of images measured at the distance of 0.5–1 A seemed to be composed of the above-mentioned two types of contrasts. By comparing the site of bright spots in the AFM images with honeycomb-chained trimer (HCT) model, we suggested the following models: when the tip is far from the sample surface, tip–sample interaction force contributing to imaging is dominated by physical bonding interaction such as Coulomb force and/or van der Waals (vdW) force between the tip apex Si atoms and Ag trimer on the sample surface. On the other hand, just before the contact, tip–sample interaction force contributing to imaging is dominated by chemical bonding such as the force due to hybridization between the dangling bond out of the tip apex Si atom and the orbit of Si–Ag covalent bond on the sample surface.

Imaging of chemical reactivity and buckled dimers on Si(100)2×1 reconstructed surface with noncontact AFM

Abstract We have investigated the force interactions between the Si tip and the Si(100)2×1 reconstructed surface in the noncontact atomic-force microscopy (AFM) measurement. We observed two types of frequency shift curves without and with discontinuity, similar to the Si(111)7×7 surface. The image contrast changes drastically whether the frequency shift curve shows discontinuity or not. In the case of the frequency shift curves without discontinuity, the noncontact AFM images almost reflect the surface topography including dimers and adsorbates. In the case of the frequency shift curves with discontinuity, they reflect strongly the chemical reactivity of surface. Furthermore, in the case of the frequency shift curves without discontinuity, for the first time, the stabilize-buckling of dimers induced by a defect can be observed. This suggests that the force interactions during the noncontact AFM measurement hardly influence the surface dynamics.

Involvement of phosphoinositide 3-kinase signaling pathway in chondrocytic differentiation of ATDC5 cells: application of a gene-trap mutagenesis.

Gene‐trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. Here, we applied this method to murine mesenchymal ATDC5 cells, which differentiate into mature chondrocytes in the presence of insulin. As the trap vector we used pPT1‐geo, which lacks its own promoter and enhancer, but contains a lacZ–neo fusion gene as a reporter and selection marker driven by the promoter of the trapped gene. After pPT1‐geo was introduced into ATDC5 cells by electroporation, the neomycin‐resistant clones were screened for β‐galactosidase activity. The selected clones were cultured in differentiation medium to evaluate the chondrogenic phenotype. The clones no. 6–30 and 6–175, which exhibited impaired and accelerated mineralization, respectively, were subjected to further analysis. In clone no. 6–30 in which the gene coding for the p85α subunit of phosphoinositide 3‐kinase (PI3K) was trapped, the expression of marker genes of early chondrocytes including collagen type II, aggrecan, and PTH/PTHrP receptor was delayed. The insulin‐induced stimulation of growth was reduced in clone no. 6–30 compared with the parental ATDC5 cells. Moreover, treatment of parental ATDC5 cells with a specific inhibitor of PI3K, LY294002, phenocopied clone no. 6–30, suggesting the involvement of PI3K signaling in the chondrogenic differentiation of ATDC5 cells. Clone no. 6–175 with accelerated mineralization was revealed to have a gene homologous to human KIAA0312 trapped, whose function remains unclear. Taken together, the gene‐trap in ATDC5 cells might be useful to identify the molecules involved in chondrogenic differentiation.

Non‐contact AFM images measured on Si(111)√3×√3‐Ag and Ag(111) surfaces

We investigated the force interactions between a Si tip and a Si(111)√3 × √3-Ag surface, as well as between a Si tip and an Ag(111) surface, using non-contact atomic force microscopy (AFM) operating in ultrahigh vacuum (UHV). The AFM images on the Si(111)√3 × √3-Ag surface showed three types of contrast that depended on the distance between a tip and a sample surface: at a tip-sample distance of 0.1-03 nm the AFM image showed a honeycomb arrangement, at a tip-sample distance of 0-0.05 nm the image showed the periodic structure of a triangle, consisting of three bright spots with relatively strong contrast, but at a distance of 0.05-0.1 nm the image contrast seemed to be intermediary between the other two types of contrast. When the tip is far from the sample surface, the tip-sample interaction force is dominated by physical bonding interactions such as Coulomb and/or van der Waals forces between the tip apex Si atom and the Ag trimer on the sample surface. On the other hand, just before contact, the tip-sample interaction force is dominated by chemical bonding interaction due to the onset of hybridization between the dangling bond of the tip apex Si atom and the orbital of the Si-Ag covalent bond on the surface. Furthermore, atomic resolution imaging of a pure metallic surface of Ag(111) was achieved, suggesting that non-contact AFM has potential for the investigation of a pure metallic surface.