Inhibitory effects of fucoidan on NMDA receptors and l-type Ca2+ channels regulating the Ca2+ responses in rat neurons.

CONTEXT: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities. OBJECTIVE: The effects of fucoidan on Ca2+ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined. MATERIALS AND METHODS: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca2+ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5 mg/mL or 1.5 mg/mL was applied for 3 min to determine its effects on Ca2+ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5 mg/mL for 3 h. RESULTS: The Ca2+ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca2+ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca2+ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca2+ channels, PR1/PR2. DISCUSSION AND CONCLUSIONS: Our data indicate that fucoidan suppresses the intracellular Ca2+ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca2+ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.

Fucoidan inhibits Ca2+ responses induced by a wide spectrum of agonists for G­protein­coupled receptors.

Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has been used in traditional Chinese herbal medicine to treat thyroid tumors for many years. Although a number of its cellular effects have been investigated, the role of fucoidan in molecular signaling, particularly in Ca2+ signaling, remains largely unknown. In the present study, the effects of fucoidan on Ca2+ responses in HeLa cells, human umbilical vein endothelial cells and astrocytes were investigated using a wide range of receptor agonists. Fucoidan inhibited the increase in intracellular free calcium concentration that was induced by histamine, ATP, compound 48/80 and acetylcholine. The responses induced by the same agonists in the absence of extracellular Ca2+ were also markedly suppressed by fucoidan. Reverse transcription­polymerase chain reaction demonstrated that 0.5 and 1.0 mg/ml fucoidan treatment for 3 h decreased histamine receptor 1 expression in HeLa cells. Similarly, the expressions of purinergic receptor P2Y, G­protein coupled (P2YR)1, P2YR2 and P2YR11 were significantly downregulated within cells pretreated with 1.0 mg/ml fucoidan for 3 h, and 0.5 mg/ml fucoidan significantly inhibited P2YR1 and P2YR11 expression. The results demonstrated that fucoidan may exert a wide spectrum of inhibitory effects on Ca2+ responses and that fucoidan may inhibit a number of different G­protein coupled receptors associated with Ca2+ dynamics.

Suppression of ciliary movements by a hypertonic stress in the newt olfactory receptor neuron.

Olfactory receptor neurons isolated from the newt maintain a high activity of the ciliary beat. A cilium of neuron is so unique that only little is known about regulatory factors for its beat frequency. We examined the olfactory receptor neuron immersed in various extracellular media under the video-enhanced differential interference contrast microscope. The activation of voltage-gated Ca2+ channels by K+ depolarization or by application of Ca2+ to membrane-permeabilized olfactory cells did not affect the ciliary movement, suggesting that Ca2+ influx through the cell membrane has no direct effect on the movement. However, when an extracellular medium contained NaCl or sucrose at concentrations only 30% higher than normal levels, ciliary movement was greatly and reversibly suppressed. In contrast, a hypotonic solution of such a solute did not change the ciliary movement. The hypertonic solutions had no effect when applied to permeabilized cells. Suction of the cell membrane with a patch pipette easily suppressed the ciliary movement in an isotonic medium. Application of positive pressure inside the cell through the same patch pipette eliminated the suppressive effect. From these findings, we concluded that the hypertonic stress suppressed the ciliary movement not by disabling the motor proteins, microtubules, or their associates in the cilia, but rather by modifying the chemical environment for the motor proteins. The ciliary motility of the olfactory receptor cell is directly sensitive to the external environment, namely, the air or water on the nasal epithelium, depending on lifestyle of the animal.

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Photodynamic Treatment of Tumor with Bacteria Expressing KillerRed.

Photodynamic therapy (PDT) is a cancer treatment modality in which a photosensitizing dye is administered and exposed to light to kill tumor cells via the production of reactive oxygen species (ROS). A fundamental obstacle for PDT is the low specificity for staining solid tumors with dyes. Recently, a tumor targeting system guided by anaerobic bacteria was proposed for tumor imaging and treatment. Here, we explore the feasibility of the genetically encoded photosensitizer KillerRed, which is expressed in Escherichia coli, to treat tumors. Using nitroblue tetrazolium (NBT), we detected a lengthy ROS diffusion from the bodies of KillerRed-expressing bacteria in vitro, which demonstrated the feasibility of using bacteria to eradicate cells in their surroundings. In nude mice, Escherichia coli (E. coli) expressing KillerRed (KR-E. coli) were subcutaneously injected into xenografts comprising CNE2 cells, a human nasopharyngeal carcinoma cell line, and HeLa cells, a human cervical carcinoma cell line. KR-E. coli seemed to proliferate rapidly in the tumors as observed under an imaging system. When the intensity of fluorescence increased and the fluorescent area became as large as the tumor one day after KR-E. coli injection, the KR-E. coli-bearing tumor was irradiated with an orange light (λ = 540-580 nm). In all cases, the tumors became necrotic the next day and were completely eliminated in a few days. No necrosis was observed after the irradiation of tumors injected with a vehicle solution or a vehicle carrying the E. coli without KillerRed. In successfully treated mice, no tumor recurrence was observed for more than two months. E. coli genetically engineered for KillerRed expression are highly promising for the diagnosis and treatment of tumors when the use of bacteria in patients is cleared for infection safety.

High-resolution, label-free imaging of living cells with direct electron-beam-excitation-assisted optical microscopy.

High spatial resolution microscope is desired for deep understanding of cellular functions, in order to develop medical technologies. We demonstrate high-resolution imaging of un-labelled organelles in living cells, in which live cells on a 50 nm thick silicon nitride membrane are imaged by autofluorescence excited with a focused electron beam through the membrane. Electron beam excitation enables ultrahigh spatial resolution imaging of organelles, such as mitochondria, nuclei, and various granules. Since the autofluorescence spectra represent molecular species, this microscopy allows fast and detailed investigations of cellular status in living cells.

Blockage of LMP1-modulated store-operated Ca(2+) entry reduces metastatic potential in nasopharyngeal carcinoma cell.

Epstein-Barr virus (EBV)-encoded latent membrane proteins (LMPs) expedite progression of EBV-relevant cancers. Of the full set of LMPs, latent membrane protein 1 (LMP1) was identified to uniquely augment store-operated Ca(2+) entry (SOCE). Previously, we reported that the suppression of SOCE exhibited inhibitory effects on cell migration and the extravasation from vasculature in EBV-negative nasopharyngeal carcinoma (NPC) cells. In this follow-up study, we aimed to expand our understanding of the modulation of SOCE by LMP1 and test the possibility that blockage of LMP1-modulated SOCE affects the LMP1-promoted metastatic potential. Here we showed that suppressions of the LMP1-boosted SOCE blunted the LMP1-promoted cell migration, VEGF-mediated angiogenesis and permeabilization in vitro. Blockage of SOCE inhibited vasculature-invasion of circulating cells and distant metastatic colonization in vivo. Notably, utilizing VEGFR2-EGFP-tag zebrafish we revealed that the LMP1-expressing cells arrested in a small-caliber vessel mobilized surrounding endothelial cells to facilitate vasculature-invasion. Thus, the LMP1-boosted SOCE promotes metastatic potential of NPC cells by solidifying their collaborations with the nearby non-cancer cells through the manipulation of oncogenic Ca(2+) signaling. Our study highlights the advantage of using both conventional mammal and transgenic zebrafish for developing a novel therapeutic strategy targeting the multiple steps of invasion-metastasis cascade.

Cell-to-cell propagation of intracellular signals fluorescently visualized with acridine orange in the gastric glands of guinea pigs.

Secretion from the gastric gland involves the activation of various types of cells in a coordinated manner. In order to elucidate the mechanisms underlying the coordination of secretion, we studied live fluorescence images of guinea pig gastric glands stained with acridine orange (AO). On 2 µM AO staining, individual cells were characterized by metachromatic colors and various intensities of fluorescence. When the gland was stimulated with 100 µM of histamine, green fluorescence was transiently increased in parietal cells and intermediate cells and propagated along the gland for a long distance over many cells. Local stimulation in a couple of cells with histamine in the presence of suramin also induced propagation. However, the fluorescence response was suppressed by the addition of H-89, a protein kinase A inhibitor. These findings suggest that a cAMP-dependent signal propagates intercellularly through a variety of cells to induce coordinated secretion in the entire gastric gland.

Dynamic autofluorescence imaging of intracellular components inside living cells using direct electron beam excitation.

We developed a high-resolution fluorescence microscope in which fluorescent materials are directly excited using a focused electron beam. Electron beam excitation enables detailed observations on the nanometer scale. Real-time live-cell observation is also possible using a thin film to separate the environment under study from the vacuum region required for electron beam propagation. In this study, we demonstrated observation of cellular components by autofluorescence excited with a focused electron beam and performed dynamic observations of intracellular granules. Since autofluorescence is associated with endogenous substances in cells, this microscope can also be used to investigate the intrinsic properties of organelles.

An adaptive approach for uniform scanning in multifocal multiphoton microscopy with a spatial light modulator.

We propose high-quality generation of uniform multiple fluorescence spots (MFS) with a spatial light modulator (SLM) and demonstrate uniform laser scanning in multifocal multiphoton microscopy (MMM). The MFS excitation method iteratively updates a computer-generated hologram (CGH) using correction coefficients to improve the fluorescence intensity distribution in a dye solution whose consistency is uniform. This simple correction method can be applied for calibration of the MMM before observation of living tissue. We experimentally demonstrate an improvement of the uniformity of a 10 × 10 grid of MFS by using a dye solution. After the calibration, we performed laser scanning with two-photon excitation to observe fluorescent polystyrene beads, as well as the gastric gland of a guinea pig specimen.

Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation.

Multi-color, high spatial resolution imaging of fluorescent nanodiamonds (FNDs) in living HeLa cells has been performed with a direct electron-beam excitation-assisted fluorescence (D-EXA) microscope. In this technique, fluorescent materials are directly excited with a focused electron beam and the resulting cathodoluminescence (CL) is detected with nanoscale resolution. Green- and red-light-emitting FNDs were employed for two-color imaging, which were observed simultaneously in the cells with high spatial resolution. This technique could be applied generally for multi-color immunostaining to reveal various cell functions.

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads.

Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish.

The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies.

Inhibition of store-operated Ca2+ entry suppresses EGF-induced migration and eliminates extravasation from vasculature in nasopharyngeal carcinoma cell.

Store-operated Ca(2+) entry (SOCE) mediates Ca(2+) responses evoked by extracellular signaling molecules to promote increases in cytosolic Ca(2+), thereby triggering downstream signal transduction. Here we demonstrated that either the pharmacological blockage of Ca(2+) influx through SOCE or the knockdown of Orai1, a key molecule of SOCE, suppressed the epidermal growth factor-induced migration by disturbing Ca(2+) signaling in nasopharyngeal carcinoma (NPC) cell. Furthermore, Orai1 depletion led to a delayed cell attachment to the extracellular matrix surface in vitro and eliminated the extravasation of microinjected cells from vasculature in a zebrafish hematogenous metastasis model. Our findings thus indicate that SOCE acts as a predominant Ca(2+) signaling involved in NPC cell metastasis, and may serve as a candidate target for anti-metastasis therapy in NPC.

Multi-beam confocal microscopy based on a custom image sensor with focal-plane pinhole array effect.

Multi-beam confocal microscopy without any physical pinhole was demonstrated. As a key device, a custom CMOS image sensor realizing a focal-plane pinhole array effect by special pixel addressing and discarding of the unwanted photocarriers was developed. The axial resolution in the confocal mode measured by FWHM for a planar mirror was 8.9 µm, which showed that the confocality has been achieved with the proposed CMOS image sensor.

Dynamic and high-resolution live cell imaging by direct electron beam excitation.

We propose a direct electron-beam excitation assisted optical microscope with a resolution of a few tens of nanometers and it can be applied for observation of dynamic movements of nanoparticles in liquid. The technique is also useful for live cell imaging under physiological conditions as well as observation of colloidal solution, microcrystal growth in solutions, etc. In the microscope, fluorescent materials are directly excited with a focused electron beam. The direct excitation with an electron beam yields high spatial resolution since the electron beam can be focused to a few tens of nanometers in the specimens. In order to demonstrate the potential of our proposed microscope, we observed the movements of fluorescent nanoparticles, which can be used for labelling specimens, in a water-based solution. We also demonstrated an observation result of living CHO cells.

Diversity of neural-hemodynamic relationships associated with differences in cortical processing during bilateral somatosensory activation in rats.

The neural-hemodynamic relationships may vary depending on cortical processing patterns. To investigate how cortical hemodynamics reflects neural activity involving different cortical processing patterns, we delivered electrical stimulation pulses to rat hindpaws, unilaterally or bilaterally, and simultaneously measured electrophysiological (local field potential, LFP < 100 Hz; multiunit activity, MUA>300 Hz) and optical intrinsic signals associated with changes in cerebral blood volume (CBV). Unilateral stimulation evoked neural and optical signals in bilateral primary somatosensory cortices. Ipsilateral optical responses indicating an increased CBV exhibited a peak magnitude of ~30% and mediocaudal shifts relative to contralateral responses. Correlation analyses revealed different scale factors between contralateral and ipsilateral responses in LFP-MUA and LFP-CBV relationships. Bilateral stimulation at varying time intervals evoked hemodynamic responses that were strongly suppressed at 40-ms intervals. This suppression quantitatively reflected suppressed LFP responses to contralateral testing stimulation and not linear summation, with slowly fluctuating LFP responses to ipsilateral conditioning stimulation. Consequently, in the overall responses to bilateral stimulation, CBV-related responses were more linearly correlated with MUA than with LFPs. When extracting high-frequency components (>30 Hz) from LFPs, we found similar scale factors between contralateral and ipsilateral responses in LFP-MUA and LFP-CBV relationships, resulting in significant linear relationships among these components, MUA, and cortical hemodynamics in overall responses to bilateral stimulation. The dependence of LFP-MUA-hemodynamic relationships on cortical processing patterns and the LFP temporal/spectral structure is important for interpreting hemodynamic signals in complex functional paradigms driving diverse cortical processing.

Fucoidan suppresses endocytosis in cultured HeLa cells.

OBJECTIVE: To evaluate the effects of fucoidan on endocytosis in cultured HeLa cells: in vitro using live cell imaging. METHODS: A confocal scanning system and an incubation imaging system were used to: observe the effects of fucoidan on the initial (6 h) stages of endocytosis using the fl uorescent probe FM1-43 and inorganic fl uorescent quantum dot (Q-dots). RESULTS: According to the time-lapse images, fucoidan inhibited the: formation of endocytic vesicles in HeLa cells, in which the FM1-43 dye was entrapped. Fucoidan also had an inhibitory effect on the uptake of the Q-dots by the cell membranes of HeLa cells. CONCLUSION: It was concluded: that fucoidan suppresses Ca(2+)-dependent endocytosis in HeLa cells, which may be caused by its inhibitory -effects on agonist-induced Ca(2+) responses.

Intravital oxygen radical imaging in normal and ischemic rat cortex.

OBJECTIVE: We examined reactive oxygen species (ROS) generation on cerebral ischemia/reperfusion by intravital fluorescence imaging. METHODS: In anesthetized adult rats, a fluorescent dye (5 microL), MitoSOX (5 micromol/L) for superoxide radical (.O2-), and hydroxyphenyl fluorescein (20 micromol/L) for hydroxyl radical (.OH), was injected into cortices by a pressurized bolus. Through a closed cranial window, fluorescent images were taken with a confocal microscope on 10-minute forebrain ischemia. Because hemoglobin absorbs excitation and emission lights, ischemia may affect the change in fluorescence intensity (FI) inside the brain. To examine the effects of ischemia on the FI change, fluoromicrospheres (0.2-microm diameter) were used to mimic a dye and FI was analyzed in the same manner as when using ROS indicators. Their FI increased to 129% during ischemia (n=3/mimicking each dye), and based on the results, FI of ROS indicators was corrected. RESULTS: After correcting the FI of MitoSOX and hydroxyphenyl fluorescein, they showed no change during ischemia, whereas the raw data showed the increase. In the early period of reperfusion, FI significantly (n=5/each, P<.01) increased (to 183% in MitoSOX and to 189% in hydroxyphenyl fluorescein), and these increases were significant in the areas adjacent to the arteries. To test the feasibility of our imaging, edaravone (3.0 mg/kg) was used. The treatment completely scavenged .OH, but did not do so in .O2- generation. CONCLUSION: ROS production increased in the early period of reperfusion but not during ischemia, which was location selective, being significant in the areas adjacent to the arteries. Our method was useful for investigating intracellular in situ ROS production.

Use of genetically engineered bone marrow-derived mesenchymal stem cells for glioma gene therapy.

In our previous study, we successfully treated an established C6 brain tumor using neural stem cells transduced with the herpes simplex virus-thymidine kinase gene (HSVtk) and ganciclovir in the rat. In the present study, we investigated the use of mesenchymal stem cells (MSCs), obtained from adult rats and transduced with HSVtk (MSCtk cells), instead of neural stem cells because MSCs are much easier to obtain from the adult subjects. Those cells were used for in vitro co-culture study and in vivo co-implantation study with C6 rat glioma cells to examine bystander tumoricidal effect, which revealed a sufficient bystander effect and only 1/32 MSCtk cells were needed for complete tumor eradication. In vitro bystander effect was also observed in a real-time fashion using a culture microscope and it was shown that only tumor cells that had contact with MSCtk cells died. In vivo treatment study of an established C6 brain tumor with an intratumoral injection of MSCtk cells followed by systemic ganciclovir administration demonstrated a significant reduction of the tumor size and a significant survival prolongation. The treatment strategy using MSCtk and ganciclovir (MSCtk therapy) is more feasible and practical for clinical application than the method using neural stem cells.