Investigation on light-addressable potentiometric sensor as a possible cell-semiconductor hybrid

This article reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible biological cell-semiconductor hybrid that will enable us to make an interface between the physical and biological system. To increase the surface potential sensitivity, we used a LAPS structure with single insulator (SiO2) coated with poly-L-ornithine and laminin (PLOL) on Si. Efficient culturing of PC-12 and nerve cells of Lymnaea stagnalis on PLOL-coated Si3N4 and SiO2 was achieved. The thickness of the PLOL layer was found to be about 4 nm by the atomic force microscope (AFM) measurement. Using the advantage of this thin layer of PLOL, we compared the performance of a novel structure to the previously reported "PLOL-coated Si3N4/SiO2/Si" structure. Due to high insulating capacitance, the photocurrent response of the novel LAPS was found to be very steep. As a result, higher sensitivity was achieved. This steepness did not degrade during 10 days when the sensor surface was kept in contact with the cell culture medium and environment. The thickness of PLOL layer, its ability to improve the biological cell adhesion, enhanced sensitivity, and experiment with simulated neural action potential (AP) applied to the novel LAPS show a good promise for LAPS to be a biological cell-semiconductor hybrid.     

The rates of deceleration of nuclear and organellar DNA syntheses differ in the progenitor cells of the apical meristems during carrot somatic embryogenesis

 The synthesis of DNA in nuclei and organellar nucleoids at the various stages of somatic embryogenesis in carrot (Daucus carota L. cv. Kurodagosun) was analyzed using anti-5-bromo-2′-deoxyuridine (BrdU) immunofluorescence microscopy. The active syntheses of both nuclear and organellar DNA started in the cells forming the embryo proper 3 d after the initiation of embryogenesis, but not in cells forming suspensor-like cell aggregates. In the early globular embryo, active DNA syntheses were continuously observed in the whole embryo proper, except for the progenitor cells of the root apical meristem (RAM) and shoot apical meristem (SAM). These were recognized as slowly cycling cells with a non-BrdU-labelled nucleus and strongly BrdU-labelled organellar nucleoids. At the heart- and torpedo-shaped embryo stages, both nuclear and organellar DNA syntheses were inactive in the presumptive RAM and SAM. Thus, slowing down of organellar DNA synthesis is not coupled with, but is later than, that of nuclear DNA synthesis in the progenitor cells of the embryonic RAM and SAM. These findings clearly indicate that the timing of DNA synthesis is similar in the progenitor cells of both the RAM and SAM in the early stages of somatic embryogenesis. Received: 18 January 2000 / Accepted: 2 March 2000     

Surface plasmon field-enhanced fluorescence spectroscopy apparatus with a convergent optical system for point-of-care testing

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) is a promising methodology for point-of-care (POC) testing. The SPFS devices that have been reported are equipped with an angle rotating stage to adjust the surface plasmon resonance (SPR) angle. In a clinical setting, however, the SPR angle determination is a tedious and time-consuming process. In this study, we employed an SPFS instrument with a convergent optical system that allows the omission of this procedure. We demonstrated that this instrumentation allowed the sensitive determination of low concentrations of α-fetoprotein in serum and reduced the variation effect caused by the protein concentrations in samples. The SPFS with a convergent optical system is suitable for POC testing.     

Physiological properties of rod photoreceptor cells in green-sensitive cone pigment knock-in mice

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was approximately 11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 x 10(-7) s(-1), about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.     

Detection of insulin-releasing cells using in situ immunoblotting

Embryonic stem (ES) cells hold promise as a source for cell transplantation treatment of diseases such as type I diabetes. Further, cells releasing bioactive substances from ES cell progeny may be concentrated and purified for clinical applications. Although ES cell lines that express reporter genes have been established to isolate cells releasing bioactive substances, other difficulties must be overcome before these genetically modified cells can be used for gene therapy in human patients. Fluorescence- or magnetic-activated cell sorters are commonly used to isolate specific cells using antibodies against cell surface antigens. However, for some cells, such as insulin-producing beta cells, specific surface antigens have not yet been identified. In this study, we developed a simple and efficient method to identify and purify insulin- and alpha-fetoprotein-producing cells. A nitrocellulose membrane treated with anti-insulin or anti-alpha-fetoprotein antibodies was placed on a cell layer to trap insulin or alpha-fetoprotein released from the cells. The location of specific substance-producing cells was identified by immunostaining the membrane. The insulin-releasing cells were selectively collected from the culture dish using a cloning ring and transferred to another culture plate.