Relationship between degree of dynamic morphological change and proliferative potential of murine embryonic stem cells

The degree of dynamic morphological change of murine embryonic stem cells is investigated through direct observation by microscopy. As a result, we find that the degree of dynamic morphological change is proportional to the increase in the ratio of the cellular population in subculture.     

Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering

This article reviews the properties and characterization of an intelligent thermoresponsive surface, which is a key technology for cell sheet-based tissue engineering. Intelligent thermoresponsive surfaces grafted with poly(N-isopropylacrylamide) exhibit hydrophilic/hydrophobic alteration in response to temperature change. Cultured cells are harvested on thermoresponsive cell culture dishes by decreasing the temperature without the use of digestive enzymes or chelating agents. Our group has developed cell sheet-based tissue engineering for therapeutic uses with single layer or multilayered cell sheets, which were recovered from the thermoresponsive cell culture dish. Using surface derivation techniques, we developed a new generation of thermoresponsive cell culture dishes to improve culture conditions. We also designed a new methodology for constructing well-defined organs using microfabrication techniques.     

Electrochemical telomerase assay with ferrocenylnaphthalene diimide as a tetraplex DNA-specific binder

Spectroscopic studies revealed that ferrocenylnaphthalene diimide (1) can bind to tetraplex DNA at high potassium ion concentration. The tetraplex DNA was stabilized by the binding of 1, and this effect was larger than that of any other tetraplex stabilizers, which are known as a telomerase inhibitor. Quantitative analysis with circular dichroism and a quartz crystal microbalance strongly suggested a 3:1 binding stoichiometry of 1 to the tetraplex DNA. The telomere sequence could be extended by telomerase with the telomerase substrate primer on the surface of an electrode as proven by an increased current signal of 1 bound to the tetraplex DNA formed on the electrode. This is the first example of electrochemical detection of telomerase activity without relying on PCR.     

Ozonation of PC in ethanol: separation and identification of a novel ethoxyhydroperoxide

The product of the ozonolysis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in ethanol-containing solvent was analyzed by chemiluminescence detection-HPLC with on-line electrospray MS, and characterized on the basis of NMR spectroscopy and MS in high-resolution fast atom bombardment mode. The reaction yielded a large amount of a novel ethoxyhydroperoxide compound [1-palmitoyl-2-(9-ethoxy-9-hydroperoxynonanoyl)-sn-glycero-3-phosphocholine]. In addition to a structural analysis, we speculate on the reaction pathway and discuss the possibility of ethoxyhydroperoxide as a potentially reactive ozonized lipid in food and biological materials.     

In vitro expansion of hematopoietic progenitor cells induces functional expression of Fas antigen (CD95)

Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis. To investigate the role of both molecules in hematopoiesis, we evaluated the expression of Fas Ag and Bcl-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured in iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 1% bovine serum albumin, 50 ng/mL stem cell factor, 50 ng/mL interleukin-3 (IL-3), 50 ng/mL IL-6, 100 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin for 7 days. Colony-forming unit of granulocytes/macrophages (CFU-GM) and burst-forming unit of erythroids (BFU-E) were expanded 6.9-fold and 8.8-fold in number at day 5 of culture, respectively. Freshly isolated CD34+ cells did not express Fas Ag, whereas approximately half of them expressed Bcl-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag and rapidly lost Bcl-2 expression. Furthermore, apoptosis was induced in the cultured CD34+ population when anti-Fan antibody (IgM; 1 microgram/mL) was added, as shown by significant decrease in the number of viable cells, morphologic changes, induction of DNA fragmentation, and significant decrease in the number of clonogenic progenitor cells including CFU. GM and BFU-E. These results indicate that functional expression of Fas Ag is induced on CD34+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and downregulation of Bcl-2 may be expressed as part of the differentiation program of hematopoietic cells and may be involved in the regulation of hematopoiesis.     

High power and high focusing CWCO2 laser using anunstable resonator with a phase-unifying output coupler

A high-power and high-focusing continuous-wave (CW) CO₂ laser with a novel unstable resonator was developed. This resonator contains a step-wise variable reflecting output coupler named the phase-unifying output coupler and a total reflector. The laser was excited by a capacitive-barriered AC discharge called a silent discharge. A linearly polarized laser beam with a diffraction-limited divergence angle of 0.55 mrad was obtained. Very stable CW laser operation at an output power of 5 kW was achieved with a threshold discharge power of 10 kW and a slope efficiency of 18%     

13.3 GHz YBCO microstrip bandpass filter

The design and development of a 13.3 GHz three-pole Chebyshev bandpass filter using a YBa/sub 2/Cu/sub 3/O/sub 7- delta / thin film on an MgO substrate is described. An insertion loss of 0.2 dB at the centre frequency and an unloaded Q of 2800 are obtained at 77 K. The measured characteristics are better than other high-temperature superconductor planar filters so far reported.<>     

A new method for preparing plan-view TEM specimen of multilayered films using focused ion beam

A new method is proposed for preparing plan-view specimens of a CeO(2)/Gd(2)Zr(2)O(7) multilayer on a metal substrate using focused ion beam milling. In the plan-view specimen, a membrane from the surface region of the CeO(2) to the Gd(2)Zr(2)O(7) layer was thinned to electron transparence so that the entire span of the multilayer can be observed in a single sample. The in-plane alignments of the CeO(2) layer and the Gd(2)Zr(2)O(7) layer were analysed using selected-area diffraction patterns (SADPs). The boundaries between the CeO(2) grains were also examined using SADPs.     

Cathepsins in the osteoclast

The mechanism by which bone collagen and other organic components are degraded by the osteoclast during osteoclastic bone resorption was unclear until the 1980s. Studies conducted since the early 1990s have identified lysosomal proteases, mainly cathepsins that are active at low pH, involved in osteoclastic bone resorption. Several cathepsins, such as cathepsins C, D, B, E, G and L, were initially demonstrated to take part in the degradation of organic bone matrix in osteoclasts. Cathepsin K, which has high proteolytic activity and localizes primarily in osteoclasts, was discovered in 1995. This first tissue-specific cathepsin was associated with pycnodysostosis, a genetic disorder observable as an osteopetrotic phenotype in cathepsin K-deficient mice. Cystatin C, an endogenous inhibitor of cysteine proteases, regulates the activity of cathepsin K. However, detailed morphological observations suggest that the organic bone matrix is degraded by not only cathepsin K, but also by matrix metalloproteinases or other cathepsins. The osteoclast possesses a unique endocytotic/exocytotic structure and each cathepsin is specifically localized in the osteoclast, which implies that each cathepsin contributes cooperatively to the process of osteoclastic bone resorption. Further studies may clarify the regulation of cathepsin activities and the roles of cathepsins during bone remodelling.