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.

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.

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.

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.

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.

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.

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.

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.

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.

Slow rise of intracellular Ca(2+) concentration in rat primary sensory neurons triggered by loureirin B.

In the present study, the intracellular free calcium concentration ([Ca(2+)](i)) in acutely isolated rat dorsal root ganglia (DRG) neurons modulated by loureirin B, an active component of "dragon's blood" which is a kind of Chinese herbal medicine, was determined by the means of Fura-2 based microfluorimetry. It was found that loureirin B could evoke the elevation of [Ca(2+)](i) in a dose-dependent manner. However, the elevation of [Ca(2+)](i) evoked in the calcium free solution was much smaller than that in the standard external cell solution, suggesting that most change of [Ca(2+)](i) was generated by the influx of extracellular Ca(2+), not by the activities of intracellular organelles like Ca(2+) stores and mitochondria. In addition, the mixture of loureirin B and caffeine also induced [Ca(2+)](i) rise, but the peak of [Ca(2+)](i) rise induced by the mixture was significantly lower than that by caffeine alone, which means the triggering pathway and the targets of caffeine are probably involved in loureirin B-induced [Ca(2+)](i) rise. Moreover, compared to the transients induced by caffeine, KCl and capsaicin, the loureirin B-induced [Ca(2+)](i) rise is much slower and more stable. These results indicate that the capability of loureirin B of inducing the [Ca(2+)](i) rise is solid and unique.

Photodynamic therapy using talaporfin sodium for synovial membrane from rheumatoid arthritis patients and collagen-induced arthritis rats.

We investigated the efficacy of photodynamic therapy (PDT) using talaporfin sodium as a new method of synovectomy for rheumatoid arthritis (RA). We first used RA synovial membrane (RASM) for in vitro and in vivo study. The RASM was obtained from patients with RA during total knee replacement. In the in vitro study, RA fibroblast-like synoviocytes (RASCs) obtained from the RASM were examined by fluorescent microscopy to measure the intracellular localization of talaporfin sodium. The cells were then subjected to PDT, and their viability was examined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium inner salt assay. In the in vivo assay, RASM was obtained as described above, grafted onto severe combined immunodeficiency (SCID) mice and subjected to PDT. The damaged area of RASM was evaluated histologically at 1 day after PDT. Next, we performed a separate experiment using rats with collagen-induced arthritis (CIA). After intra-articular injection of talaporfin sodium, the concentration of talaporfin sodium accumulated in the CIA synovial membrane (CIASM) was compared with that in cartilage, periarticular muscle, and skin. We then performed PDT with intra-articular injection of talaporfin sodium and intra-articular irradiation. The damaged area of the CIASM was measured at 1 day after the PDT, and the articular histological and radiological changes of the ankle were observed at 56 days after the PDT. In RASM, talaporfin sodium accumulated in lysosomes in vitro, and the phototoxicity to RASCs in vitro and to RASM grafted onto SCID mice in vivo depended on the concentration of talaporfin sodium and the laser energy. In CIA rats, there was a greater accumulation of talaporfin sodium in the CIASM than in normal tissue. The CIASM was selectively damaged at 1 day after the PDT, and the bone and cartilage destruction were ameliorated at 56 days after the PDT. In conclusion, PDT using talaporfin sodium might be a new method for synovectomy in patients with RA.

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.

Genetically engineered neural stem cells migrate and suppress glioma cell growth at distant intracranial sites.

Our previous study demonstrated successful treatment of an established rat brain tumor through the bystander effect by intra-tumoral injection of neural stem cells transduced with herpes simplex virus-thymidine kinase gene (NSCtk) followed by systemic ganciclovir (GCV) administration (NSCtk therapy). Since glioma has a strong tendency to infiltrate into surrounding brain tissue and that is one of the main causes of local treatment failure, we, in the present study, injected NSCtk cells at distant sites of rat brain tumors and evaluated migratory potential of NSCtk toward the tumor and anti-tumor effects of the NSCtk therapy of this experimental setting. NSCtk cells were intracranially implanted either at 2mm medial in the ipsilateral hemisphere or at the mirror point in the contralateral hemisphere to the C6 rat glioma cell implantation. Active migration of NSCtk cells toward C6 cells was observed even when NSCtk cells were implanted in the contralateral hemisphere. When GCV was systemically administered, growth of intracranial tumor was markedly inhibited and the survival was significantly prolonged through the bystander effect by NSCtk cells migrated from distant injection sites of the tumor. The results of the present study suggest that NSCtk therapy is still effective in the area far from the NSCtk injection site and, therefore, suitable for treatment of malignant gliomas that deeply infiltrate and widely disseminate in the brain.

Three-dimensional measurement endoscope system with virtual rulers.

Conventional endoscopic images do not provide quantitative 3-D information. We present an endoscope system that can measure the size and position of an object in real time. Our endoscope contains four laser beam sources and a camera. The procedural steps for 3-D measurements are as follows. First, to obtain the function that maps 2-D coordinates of an image point to its 3-D coordinates in 3-D space, we observe a standard chart with the endoscope lens and determine the correspondence between the image and object height. In addition to the mapping, this function can correct barrel-shaped distortion of endoscopic images. The system detects laser spots on an object surface automatically using a template matching method, and maps the 2-D coordinates of the laser spots to the 3-D coordinates by the triangulation method. Then the system calculates the magnification ratio on the object plane, which is perpendicular to the optical axis and passes the laser spot, so that the system can superimpose a ruler whose scale fits the 3-D coordinates of the object. Thus, physicians can measure the size and position of objects in real time on undistorted images similar to placing rulers on the surface of an organ.

Monitoring of singlet oxygen is useful for predicting the photodynamic effects in the treatment for experimental glioma.

PURPOSE: Singlet oxygen ((1)O(2)) generated in photodynamic therapy (PDT) plays a very important role in killing tumor cells. Using a new near-IR photomultiplier tube system, we monitored the real-time production of (1)O(2) during PDT and thus investigated the relationship between the (1)O(2) production and photodynamic effects. EXPERIMENTAL DESIGN: We did PDT in 9L gliosarcoma cells in vitro and in an experimental tumor model in vivo using 5-aminolevulinic acid and nanosecond-pulsed dye laser. During this time, we monitored (1)O(2) using this system. Moreover, based on the (1)O(2) monitoring, we set the different conditions of laser exposure and investigated whether they could affect the tumor cell death. RESULTS: We could observe the temporal changes of (1)O(2) production during PDT in detail. At a low fluence rate the (1)O(2) signal gradually decreased with a low peak, whereas at a high fluence rate it decreased immediately with a high peak. Consequently, the cumulative (1)O(2) at a low fluence rate was higher, which thus induced a strong photodynamic effect. The proportion of apoptosis to necrosis might therefore be dependent on the peak and duration of the (1)O(2) signal. A low fluence rate tended to induce apoptotic change, whereas a high fluence rate tended to induce necrotic change. CONCLUSIONS: The results of this study suggested that the monitoring of (1)O(2) enables us to predict the photodynamic effect, allowing us to select the optimal laser conditions for each patient.

Nicotine exposure, mimicked smoking, directly and indirectly enhanced protein kinase C activity in isolated canine basilar artery, resulting in enhancement of arterial contraction.

Cigarette smoking is a significant risk factor in the incidence of cerebrovascular disorders. Among the many compounds in cigarette smoke, nicotine is considered to most significantly affect cerebral arterial tone. The purpose of this study is to investigate precise pharmacological effects of nicotine on the regulation of cerebral arterial tone. To mimic smoking, a low concentration of nicotine (10(-6) mol/L), which is equivalent to the serum level of habitual smokers, was treated for 1 hour in an isometric tension study and for 24 hours in a study using cultured vascular endothelial cells (VECs). Using the canine basilar artery, the effect of nicotine on uridine 5'-triphosphate (UTP)-induced vasoconstriction was examined in the isometric tension study. Protein kinase C (PKC) activity in the canine basilar artery was measured by enzyme immunoassay. Endothelial function was assessed by endothelium-dependent vasodilatation and endogenous nitric oxide (NO) synthesis in VECs using a fluorescent indicator, diaminofluorescein-FM diacetate (DAF-FM/DA). Nicotine significantly enhanced UTP-induced contraction and PKC activity in the artery, and attenuated endothelium-dependent vasodilatation and NO synthesis in VECs. Because PKC activity was increased by de-endothelialization itself, endothelial dysfunction by nicotine enhances PKC activity. Because PKC was further activated by nicotine even in the de-endothelialized artery, nicotine directly affects PKC activities in smooth muscle. These results indicate that nicotine potentiates contractile response through direct and indirect PKC activation in the canine basilar artery.

Selective accumulation and strong photodynamic effects of a new photosensitizer, ATX-S10.Na (II), in experimental malignant glioma.

We investigated the feasibility of a novel photosensitizer, ATX-S10.Na (II), in photodynamic therapy (PDT) for glioma. First, PDT was performed in various brain tumor cell lines in vitro. Cytotoxicity depended upon both drug concentration and laser energy and the 50% inhibitory concentration ranged from 3.5 to 20 microg/ml. Next, PDT was performed in the subcutaneous and intracranial 9L tumor models in Fischer rats using ATX-S10.Na (II) and light from a 670-nm diode laser delivered by intratumoral insertion of an optical fiber. The effect of PDT on brain tumors was evaluated using magnetic resonance imaging. Sequential changes of the ATX-S10.Na (II) concentrations were also measured quantitatively by fluorospectrometry up to 12 h after intravenous administration in rats with intracranial and subcutaneous tumors. The concentration of ATX-S10.Na (II) in the brain tumor reached a maximum at 2 h after administration and the tumor/normal brain concentration ratio was as high as 131 at 8 h. Intratumoral PDT for intracranial tumors irradiated at this timing showed an obvious anti-tumor effect without severe side effects. The present study demonstrated the highly selective accumulation of ATX-S10.Na (II) in tumor tissue and its potent photodynamic effect in an experimental malignant glioma model.

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.

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.