Akiko Hisada

Light-Induced Nuclear Translocation of Endogenous Pea Phytochrome A Visualized by Immunocytochemical Procedures

Although the physiological functions of phytochrome A (PhyA) are now known, the distribution of endogenous PhyA has not been examined. We have visualized endogenous PhyA apoprotein (PHYA) by immunolabeling cryosections of pea tissue, using PHYA-deficient mutants as negative controls. By this method, we examined the distribution of PHYA in different tissues and changes in its intracellular distribution in response to light. In apical hook cells of etiolated seedlings, PHYA immunolabeling was distributed diffusely in the cytosol. Exposure to continuous far-red (cFR) light caused a redistribution of the immunolabeling to the nucleus, first detectable after 1.5 hr and greatest at 4.5 hr. During this time, the amounts of spectrally active phytochrome and PHYA did not decline substantially. Exposure to continuous red (cR) light or to a brief pulse of red light also resulted in redistribution of immunolabeling to the nucleus, but this occurred much more rapidly and with a different pattern of intranuclear distribution than it did in response to cFR light. Exposures to cR light resulted in loss of immunolabeling, which was associated with PHYA degradation. These results indicate that the light-induced intracellular location of PHYA is wavelength dependent and imply that this is important for PhyA activity.

Phytochrome A requires jasmonate for photodestruction

The plant photoreceptor phytochrome is organised in a small gene family with phytochrome A (phyA) being unique, because it is specifically degraded upon activation by light. This so called photodestruction is thought to be important for dynamic aspects of sensing such as measuring day length or shading by competitors. Signal-triggered proteolytic degradation has emerged as central element of signal crosstalk in plants during recent years, but many of the molecular players are still unknown. We therefore analyzed a jasmonate (JA)-deficient rice mutant, hebiba, that in several aspects resembles a mutant affected in photomorphogenesis. In this mutant, the photodestruction of phyA is delayed as shown by in vivo spectroscopy and Western blot analysis. Application of methyl-JA (MeJA) can rescue the delayed phyA photodestruction in the mutant in a time- and dose-dependent manner. Light regulation of phyA transcripts thought to be under control of stable phytochrome B (phyB) is still functional. The delayed photodestruction is accompanied by an elevated sensitivity of phytochrome-dependent growth responses to red and far-red light.

A Pt layer/Pt disk electrode configuration to evaluate respiration and alkaline phosphatase activities of mouse embryoid bodies.

A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E(1/2)) for [Fe(CN)(6)](4-) oxidation and O(2) reduction measured versus the internal Pt pseudo reference was shifted by about -0.2V, compared with the E(1/2) measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at -0.7 V versus the Pt pseudo reference and compared with those monitored at -0.5 V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O(2))) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R(2)=0.90). The slope of the least squares method was 1. In a 1.0mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E(1/2) for PAP oxidation measured versus the Pt layer was not shifted, compared with the E(1/2) versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3 V versus the Pt layer and compared with those monitored at +0.3 V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier.

Noninvasively measuring respiratory activity of rat primary hepatocyte spheroids by scanning electrochemical microscopy

Construction of an in vitro drug screening method for evaluating drug metabolism and toxicity by using cells is required instead of the conventional in vivo one that uses animals. In order to realize the in vitro study, analyzing the cellular activity or viability noninvasively in advance of the screening is essential. The aim of the current study is to establish a method that can evaluate the cellular activity depending on spheroid sizes by means of oxygen consumption and to determine the valid diameter of hepatocyte spheroids. To measure the respiratory activity of the spheroids, which were formed on a nanopillar sheet, we applied scanning electrochemical microscopy (SECM). From the viewpoint of high respiratory activity and its small variation, we determined that spheroids with 70 μm in diameter were adequate. We then performed a gene expression analysis by using a real-time PCR to evaluate the correlation with respiratory activity. As a result, a higher expression level of Hnf4α, which is essential for hepatocytes to fulfill many liver functions and is the indicator of well-differentiated hepatocytes, showed relatively higher respiratory activity. We concluded that the noninvasive SECM technique could evaluate the cellular activity of a single spheroid. Noninvasively measuring cellular activity by SECM makes it possible to evaluate the cellular activity prior to a nonclinical test and enables the continued monitoring of the drug response by using single spheroid. SECM becomes a powerful tool to satisfying the increasing demand for an in vitro system in the course of new drug development.

In Vivo X‐Ray Fluorescence Microtomographic Imaging of Elements in Single‐Celled Fern Spores

We have observed in vivo three‐dimensional distributions of constituent elements of single‐celled spores of the fern Adiantum capillus‐veneris using an X‐ray fluorescence computed microtomography method. The images of these distributions are generated from a series of slice data, each of which is acquired by a sample translation‐rotation method. An incident X‐ray microbeam irradiates the sample with a spot size of 1 μm. The high Ca concentration in the testa and the localized and overlapping Fe and Zn concentrations inside the spore are shown in three‐dimensional images. The K concentration is high throughout the cell, and there are localized regions of higher density. The atomic number densities of these elements in the testa and inside the cell in a tomographic slice are estimated with a resolution of about 1 μm.

Higher Expressions of Cytochrome P450, UDP-Glucuronosyltransferase,and Transporter Genes in Nanopillar-Cultured Rat Hepatocyte Spheroids

Primary hepatocytes have been widely explored as cell sources for the study of in vitro drug metabolism and pharmacokinetics (DMPK). Aiming toward establishing an in vitro drug screening method, the objective of the current study is to illustrate a comprehensive increase in the DMPK-related gene expression of nanopillar (NP)-cultured 3D-spheroids. To examine the expressional changes in DMPK-related genes under four different conditions, namely, NP-, sandwich (SW)-, monolayer (ML)-cultured rat hepatocytes, and freshly isolated hepatocytes, genome-wide gene-expression analysis using a DNA microarray was performed. Among the DMPK-related genes, cytochrome P450, UDP-glucuronosyltransferase, and transporter genes were focused on. Principal component analysis showed that the global gene expression profile in a sample from an NP culture was closer to that from freshly isolated hepatocytes than to that from an SW culture. The expressions of almost all Cyp 1 to 3 and Ugt genes of NP-cultured 3-D spheroids were higher than those of ML and SW. The expression of the Abcc2 gene, whose translation product has a critical role in the excretion of metabolized bile acids in hepatocyte to bile canaliculi, was three times higher in NP than in ML. From these results, 3-D spheroids formed by the NP culture were suggested to possess a higher ability of metabolism and excretion than does 2-D tissue formed by the conventional monolayer culture.

Electron tomography of whole cultured cells using novel transmission electron imaging technique

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research.

Whole-Cell Tomography Using a Conventional Scanning Electron Microscope

Traditionally, ultrastructure of biological samples has been elucidated predominantly using transmission electron microscopy (TEM). For TEM observation, the samples are embedded in resin and cut into ultrathin sections using an ultramicrotome. These sample preparation procedures, however, require skills to make adequate electron-transparent thin sections on a small grid, which is obstacle to TEM examination. To overcome the difficulty, we developed plate-transmission electron microscopy (PlateTEM), by which internal structure of biological samples is observed using a transmitted electron (TE) mode of scanning electron microscopy (SEM) with a scintillator plate [1,2]. Furthermore, whole-cell tomography was conducted using this technique in order to image the three-dimensional (3D) structure of the samples.

Simplified Biological Tissue Analysis with Combination of Atmospheric Scanning Electron Microscope and Light Microscope

Correlative light and electron microscopy has recently been considered to be a powerful tool for analyzing cell biology. For any observation that uses different types of microscopes in succession, specimens should be processed appropriately. Viable cells or fixed cells maintained in liquid can be observed with a light microscope. When those cells are successively observed with a conventional electron microscope, they should be processed so that they remain intact when placed in a high vacuum and irradiated with an electron beam. However, cells easily change shape if processed using drying. To keep the focus on cell samples that are processed differently, various procedures to create a trace of the visual field under a light and electron microscope have been proposed.