Rapid Photoinduced Single Cell Detachment from Gold Nanoparticle-Embedded Collagen Gels with Low Denaturation Temperature.

Cell Separation is important in various biomedical fields. We have prepared gold nanoparticle (AuNP)-embedded collagen gels as a visible-light-responsive cell scaffold in which photoinduced single cell detachment occurs through local thermal denaturation of the collagen gel via the photothermal effect of AuNP. Physicochemical properties of collagen materials depend on the origin of the collagen and the presence of telopeptides. In this study, we prepared various AuNP-embedded collagen gels by using different collagen materials with and without the telopeptides to compare their thermal denaturation properties and photoinduced single cell detachment behaviors. Cellmatrix type I-C without telopeptides exhibited a lower denaturation temperature than Cellmatrix type I-A and Atelocell IAC, as examined by Fourier transform infrared (FTIR) spectroscopy, rheological analysis, and sol-gel transition observation. Three-dimensional (3D) laser microscopic imaging revealed that collagen fibers shrank in Cellmatrix type I-A upon heating, but collagen fibers disappeared in Cellmatrix type I-C upon heating. Cells cultured on the Cellmatrix type I-C-based AuNP-embedded collagen gel detached with shorter photoirradiation than on the Cellmatrix type I-A-based AuNP-embedded collagen gel, suggesting that collagen gels without telopeptides are suitable for a photoinduced single cell detachment system.

Peripheral N- and C-terminal domains determine deactivation kinetics of HCN channels.

Among four subtypes of mammalian HCN channels, HCN1 has the fastest activation and deactivation kinetics while HCN4 shows the slowest. We previously showed that the activation kinetics are determined mainly by S1, S1-S2, and the S6-cyclic nucleotide binding domain. However, the effects of those regions on the deactivation kinetics were relatively small. Therefore, we investigated the structural basis for deactivation kinetics. Substitution of the core region (from S3 to S6) between HCN1 and HCN4 did not affect deactivation kinetics. This suggests that the peripheral regions (outside of S3 to S6) determine subtype-specific deactivation kinetics. Furthermore, we examined whether peripheral regions determined the deactivation kinetics across species by introducing the core region of DMIH (Drosophila homologue) into both HCN1 and HCN4. The DMIH core with HCN1 activated and deactivated more than threefold faster than that with HCN4. Taken together, the peripheral domains are diversified to create distinct kinetics.

Effects of perfluorooctane sulfonate on action potentials and currents in cultured rat cerebellar Purkinje cells.

Recently, PFOS was reported to be ubiquitously detected in the environment, as well as in human serum, raising concerns regarding its health risks. We investigated the effects of PFOS on action potentials and currents in cultured rat cerebellar Purkinje cells using whole-cell patch-clamp recording. In current-clamp experiments, PFOS significantly decreased the action potential frequency during current injection, the maximum rate of fall and the threshold of action potential, and negatively shifted the resting membrane potential at doses over 30microM. In voltage-clamp experiments, PFOS shifted the half-activation and inactivation voltages of I(Ca), I(Na), and I(K) toward hyperpolarization at 30microM. I(HCN1) expressed in Xenopus oocytes was similarly affected. Incorporation of PFOS into the cell membrane probably increased the surface negative charge density, thereby reducing the transmembrane potential gradient and resulting in hyperpolarizing shifts of both the activation and inactivation of ionic channels. These findings indicate that PFOS may exhibit neurotoxicity.

A novel Ca2+ indicator protein using FRET and calpain-sensitive linker.

Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of alpha-spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP-EYFP fusion protein linked by a calpain-sensitive sequence of alpha-spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca(2+) indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480nm/535nm was repeatedly increased when the intracellular Ca(2+) concentration ([Ca(2+)](i)) was raised. F2C had a Ca(2+) sensitivity with an apparent dissociation constant (K(d) for Ca(2+)) of 150nM, and demonstrated kinetics that paralleled Fura-2 when [Ca(2+)](i) was measured simultaneously. These properties of F2C are useful to be a Ca(2+) indicator.

Occurrence of fructosyl-amino acid oxidase-reactive compounds in fungal cells.

Fructosyl-amino acid oxidase (FAOD)-reactive fraction (FRY) was found in commercial yeast extract. FRY showed very hydrophilic property and was adsorbed to phenylboronate silica gel, indicating that it contained the Amadori compound. TLC and amino acid analyses revealed that glucosone, lysine, and arginine were produced from FRY after incubation with FAOD. TOF-MS analysis confirmed that FRY is a mixture of fructosyl lysine and fructosyl arginine. These compounds were also detected in mycelial extract of an FAOD-producer, Aspergillus terreus GP1, grown on the minimum medium, suggesting that a glycation reaction occurs in fungal cells and that FAOD acts toward the resultant Amadori compounds.

Overexpression of RFT induces G1-S arrest and apoptosis via p53/p21(Waf1) pathway in glioma cell.

The regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) has been reported to be a transcriptional repressor of FGF-2 and induce glioma cell death by its overexpression. Here we report that RFT regulates cell cycle as well as apoptosis by a novel mechanism. RFT expressed in some glioma cell lines, U138MG and T98G, but neither in U87MG nor U251MG. Overexpressed RFT-induced apoptosis in U87MG and U138MG with functioning-type p53 but neither in U251MG nor T98G with non-functioning-type p53. Administration of FGF-2 failed to prevent RFT-induced apoptosis. Overexpression of RFT caused G1-S arrest and upregulated both the phosphorylation of p53 at Ser-15 and the expression level of p21(Waf1). Furthermore, RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG. Taken together, our results support that RFT regulates G1-S transition and apoptosis via p53/p21(Waf1) pathway.

Roles of the bHLH gene Hes1 in retinal morphogenesis.

During retinal development, common precursors give rise to various types of cells in a time course specific to each cell type. Previously, we demonstrated that the bHLH gene Hes1 inhibits neuronal differentiation whereas, in Hes1-null retina, precursors prematurely differentiate into neurons and form abnormal rosette-like structures. Thus, Hes1 is essential for maintenance of precursors and morphogenesis of the neural retina. However, the precise causal link between premature differentiation and abnormal structures remains to be determined. Here, we found that misexpression of Hes1 in the developing retina promotes formation of undifferentiated precursor-like cells, whereas in Hes1-null retina, precursors are not properly maintained and prematurely differentiate into ganglion cells. Strikingly, those prematurely differentiated ganglion cells erupt into the subretinal space through the regions where precursors and the outer limiting membrane are lost. These results indicate that Hes1 maintains precursors and the outer limiting membrane and thereby regulates retinal morphogenesis.