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S Terakawa

Publications and source records attributed to S Terakawa.

At least 19 recordsLinked to original sources
PubMed2000

Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event.

The classical model of secretory vesicle recycling after exocytosis involves the retrieval of membrane (the omega figure) at a different site. An alternative model involves secretory vesicles transiently fusing with the plasma membrane (the 'kiss and run' mechanism) [1,2]. No continuous observation of the fate of a single secretory vesicle after exocytosis has been made to date. To study the dynamics of fusion immediately following exocytosis of insulin-containing vesicles, enhanced green fluorescent protein (EGFP) fused to the vesicle membrane protein phogrin [3] was delivered to the secretory vesicle membrane of INS-1 beta-cells using an adenoviral vector. The behaviour of the vesicle membrane during single exocytotic events was then examined using evanescent wave microscopy [4-6]. In unstimulated cells, secretory vesicles showed only slow Brownian movement. After a depolarizing pulse, most vesicles showed a small decrease in phogrin-EGFP fluorescence, and some moved laterally over the plasma membrane for approximately 1 microm. In contrast, secretory vesicles loaded with acridine orange all showed a transient (33-100 ms) increase in fluorescence intensity followed by rapid disappearance. Simultaneous observations of phogrin-EGFP and acridine orange indicated that the decrease in EGFP fluorescence occurred at the time of the acridine orange release, and that the lateral movement of EGFP-expressing vesicles occurred after this. Post-exocytotic retrieval of the vesicle membrane in INS-1 cells is thus slow, and can involve the movement of empty vesicles under the plasma membrane ('kiss and glide').

Acridine Orange↗
PubMed2000

Exocytosis in the dissociated pancreatic acinar cells of the guinea pig directly visualized by VEC-DIC microscopy.

To elucidate the detailed process of exocytosis at the highest possible accuracy, we dissociated the pancreatic acinus of the guinea pig and observed zymogen granules under a video-enhanced contrast differential interference contrast (VEC-DIC) microscope. The preparation was thin enough to resolve each zymogen granule with the best clarity. When acinar cells were stimulated with ACh (20 microM), many zymogen granules near the lumen showed an abrupt light intensity change. For a period of 10 s immediately before exocytosis, zymogen granules neither shifted their position nor altered their shape within an accuracy of 38 nm. The time required for individual granules to change the light intensity (the releasing time) ranged from 0.15 to 0.70 s. After each response, the granule maintained its altered contrast for a few seconds until it was retrieved to a planar membrane. No compound exocytosis including granule-granule fusion was observed. We concluded that the exocytosis is not directly initiated by any supramolecular change but by a purely molecular event.

Acetylcholine↗
PubMed2000

Calicheamicin-conjugated humanized anti-CD33 monoclonal antibody (gemtuzumab zogamicin, CMA-676) shows cytocidal effect on CD33-positive leukemia cell lines, but is inactive on P-glycoprotein-expressing sublines.

Calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, CMA-676, has recently been introduced to clinics as a promising drug to treat patients with acute myeloid leukemia (AML) in relapse. However, the mechanism of action of CMA-676 has not been well elucidated. The cytotoxic effect of CMA-676 on HL60, NOMO-1, NB4, NKM-1, K562, Daudi, and the multidrug-resistant sublines, NOMO-1/ADR and NB4/MDR, was investigated by cell cycle distribution and morphology. These studies were done by a video-microscopic system, DNA fragmentation, dye exclusion and 3H-thymidine uptake after analysis of CD33, CD34, P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein and lung-related protein on these cells. A dose-dependent, selective cytotoxic effect of CMA-676 was observed in cell lines that expressed CD33, and was dependent on the amount of CD33 and the proliferative speed of the cells. Sensitive cells were temporally arrested at the G2/M phase before undergoing morphological changes. CMA-676 is not effective on P-gp-expressing multidrug-resistant sublines compared with parental cell lines. MDR modifiers, MS209 and PSC833, restored the cytotoxic effect of CMA-676 in P-gp-expressing sublines. CMA-676 is a promising agent in the treatment of patients with AML that expresses CD33. The combined use of CMA-676 and MDR modifiers may increase the selective cytotoxic effect in multidrug-resistant AML.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Exocytosis and movement of zymogen granules observed by VEC-DIC microscopy in the pancreatic tissue en bloc.

The dynamic aspects of exocytosis, especially in the normal acinar tissue en bloc, have remained unclear. We visualized exocytosis directly in the tissue of the exocrine pancreas of rodents by video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy to investigate various exocytosis-related rates and the relationship between the movement of granules and exocytotic responses. Stimulation of the tissue with bethanechol or cholecystokinin caused many of the zymogen granules in the apical pole to disappear abruptly. The exocytotic transients of individual granules were completed in 0.48-0.65 s. Granules destined to participate in the exocytotic response moved randomly at velocities of approximately 0.06 microm/s or less during stimulation. In the tissue preparation, granules located far from the apical pole frequently moved back and forth for 1-7 microm without showing exocytosis. Colchicine suppressed this movement and the late phase of the secretory response. Real-time (VEC-DIC) observation of granule dynamics revealed that the initial step of exocytosis was not coupled directly with the microtubule-dependent translocation but with a continuous, slow Brownian fluctuation of granules.

Animals↗
PubMed2000

[Distribution in normal subjects of performance in Token test: a basic study for the diagnosis of learning disabilities].

For the diagnosis of specific reading disorder (SRD) we studied the distribution in 187 elementary school children of the scores of Token test. Token test was performed under two conditions: listening and reading by presenting the same sentences. The diagnosis required a normal score under the listening condition, an abnormally low score under the reading condition and significantly large discrepancy between them. This test is valid and convenient for the diagnosis of SRD.

Acoustic Stimulation↗
PubMed1999

Pathophysiological role of endothelins in pulmonary microcirculatory disorders due to intestinal ischemia and reperfusion.

BACKGROUND: This study was conducted to investigate pulmonary microcirculatory disorders caused by intestinal ischemia reperfusion (IIR), and the pathophysiological roles of endothelin (ET) in acute lung injury (ALI). METHODS: Male rats were pretreated with normal saline or a nonselective ET receptor antagonist (TAK-044) and subjected to IIR (60 min of intestinal ischemia and 180 min of reperfusion). The right upper lobe of the lung was examined by intravital confocal microscopy. RESULTS: The size of arterioles and venules was not significantly reduced during IIR, but the functional capillary density (FCD) decreased significantly. TAK-044 improved the pulmonary microhemodynamics, inhibiting the accumulation of leukocytes, the pulmonary edema, and the decrease of FCD. CONCLUSIONS: In the early stage of IIR, pulmonary microhemodynamics seemed more likely to be disturbed by the decrease of FCD, than by arteriolar or venular vasoconstriction. ETs decrease the FCD, promoting the interaction between leukocytes and pulmonary vessels.

Animals↗
PubMed1999

Intravital laser confocal microscopy of pulmonary edema resulting from intestinal ischemia-reperfusion injury in the rat.

OBJECTIVE: To observe pulmonary edema resulting from intestinal ischemia-reperfusion injury. We used a newly developed laser confocal microscope to observe the subpleural capillary network and the superficial alveoli under intravital conditions, and created three-dimensional images of the pulmonary microcirculation to analyze the time course and spatial pattern of pulmonary exudative changes during intestinal ischemia-reperfusion injury in vivo. DESIGN: Prospective, randomized, unblinded study. SETTING: Laboratory of a university hospital. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: The rats were injected intravenously with bovine serum albumin labeled with fluorescein isothiocyanate and subjected to 60 mins of intestinal ischemia, followed by 180 mins of reperfusion. During mechanical ventilation, the upper lobe of the right lung was examined in the intravital state using a high-speed confocal fluorescence microscope. MEASUREMENTS AND MAIN RESULTS: Interstitial edema and alveolar leakage were recognized as changes of interstitial fluorescence in the subpleural capillary network and as changes of alveolar fluorescence in the alveolar cross-sectional view. Although exudative changes in the interstitium and alveoli were observed during intestinal ischemia, there was a marked increase in both interstitial edema and alveolar leakage after intestinal reperfusion. CONCLUSION: We observed pulmonary edema under intravital conditions and demonstrated the utility of a newly developed laser confocal microscope. This system not only enabled us to analyze the development of pulmonary edema three-dimensionally, but also allowed us to evaluate the pulmonary microcirculation.

Analysis of Variance↗
PubMed1999

Fenestration nodes and the wide submyelinic space form the basis for the unusually fast impulse conduction of shrimp myelinated axons.

Saltatory impulse conduction in invertebrates is rare and has only been found in a few giant nerve fibres, such as the pairs of medial giant fibres with a compact multilayered myelin sheath found in shrimps (Penaeus chinensis and Penaeus japonicus) and the median giant fibre with a loose multilayered myelin sheath found in the earthworm Lumbricus terrestris. Small regions of these nerve fibres are not covered by a myelin sheath and serve as functional nodes for saltatory conduction. Remarkably, shrimp giant nerve fibres have conduction speeds of more than 200 m s-1, making them among the fastest-conducting fibres recorded, even when compared with vertebrate myelinated fibres. A common nodal structure for saltatory conduction has recently been found in the myelinated nerve fibres of the nervous systems of at least six species of Penaeus shrimp, including P. chinensis and P. japonicus. This novel node consists of fenestrated openings that are regularly spaced in the myelin sheath and are designated as fenestration nodes. The myelinated nerve fibres of the Penaeus shrimp also speed impulse conduction by broadening the gap between the axon and the myelin sheath rather than by enlarging the axon diameter as in other invertebrates. In this review, we document and discuss some of the structural and functional characteristics of the myelinated nerve fibres of Penaeus shrimp: (1) the fenestration node, which enables saltatory conduction, (2) a new type of compact multilayered myelin sheath, (3) the unique microtubular sheath that tightly surrounds the axon, (4) the extraordinarily wide space present between the microtubular sheath and the myelin sheath and (5) the main factors contributing to the fastest impulse conduction velocity so far recorded in the Animal Kingdom.

Animals↗
PubMed1998

Video-enhanced microscopic visualization of apoptotic cell death caused by anti-Fas antibody in living human glioma cells.

To study the morphological changes of anti-Fas antibody-mediated apoptosis in living U251-SP human glioma cells, we employed video-enhanced contrast differential interference contrast (VEC-DIC) microscopy. In our previous study, we investigated the susceptibility of human glioma cell lines to anti-Fas Immunoglobulin M (IgM) antibody. U251-SP cells express Fas antigen on their surface. The cells exposed to anti-Fas antibody underwent apoptotic cell death, as reported previously. In this study, morphological changes of apoptosis characterized by bleb formation, shrinkage of cells, and nuclear condensation were observed under VEC-DIC microscopy in U251-SP human glioma cells treated with anti-Fas antibody. These results demonstrate the usefulness of VEC-DIC microscopy to study the process of apoptotic cell death.

Antibodies↗
PubMed1997

Video-rate dynamics of exocytotic events associated with phagocytosis in neutrophils.

Exocytotic responses associated with phagocytosis were investigated in a single neutrophil with a special reference to their dynamic properties and their spatiotemporal relationships with ionic and chemical responses during phagocytosis. The real-time sequence of phagocytosis-exocytosis was directly visualized by video-enhanced contrast differential interference contrast (VEC-DIC) microscopy. The actual release of contents from such a granule was proven by examining a cell loaded with quinacrine with a dual imaging system that allowed us to observe DIC and fluorescence images simultaneously at a high magnification. During the process of phagosome formation in a neutrophil engulfing an opsonized zymosan, the exocytotic response was observed first in a granule located near the cell surface initially attached to the zymosan, and then in other granules sequentially along pseudopodia surrounding the zymosan. When the phagocytosis was induced in a medium containing luminol, a chemiluminescence due to active oxidants was detected exclusively in the region of phagosome, suggesting that exocytosis took place on the phagosomal membrane and not on the plasma membrane. Changes in cytosolic free calcium concentration ([Ca2+]i) were further measured using fura-2 under the dual imaging system. [Ca2+]i transients were more closely related to the extension of pseudopodia for engulfing zymosan and not directly to the exocytosis. These findings lead to a conclusion that exocytosis associated with phagocytosis is initiated by attachment of the cell membrane to the invading organism and mediated by local activation of the phagosomal membrane.

Animals↗
PubMed1996

Optical responses evoked by white matter stimulation in rat visual cortical slices and their relation to neural activities.

To characterize optical responses (ORs) evoked by white matter (WM) stimulation in slices of rat visual cortex (VC) stained with voltage sensitive dyes, time course of ORs in each layer was investigated by recording ORs with a linearly aligned photodiode array, and the spatial patterns of the ORs at specified time after stimulation were investigated by a CCD camera in combination with stroboscopic illumination. The ORs recorded by the photodiode array were an increase in absorption at 700 nm and a decrease in the wavelength below 650 nm, suggesting that the ORs were dye related. The ORs were compared with field potentials (FPs) to clarify that neural events were represented by the ORs, and in support of this view, we found that the first order spatial differentials of ORs and that of FPs were in good agreement. We further compared ORs with intracellular responses, and found that the ORs mainly represent postsynaptic potentials (PSPs) of VC neurons except for the deeper part of layer VI, where a component representing action potentials in fibers stimulated directly was observed. The time-lapse imaging of ORs showed that excitation first propagated vertically up to layer I and subsequently in the horizontal direction along layers II-III and V-VI as in previous investigations. Spatio-temporal patterns of ORs under blockade of synaptic transmission were also investigated to reveal activity of fibers evoked by WM stimulation which produced such patterns of propagation.

Action Potentials↗
PubMed1996

Nuclear disintegration as a leading step of glutamate excitotoxicity in brain neurons.

Recent studies on ischemic brain disease in vivo and glutamate excitotoxicity in vitro suggest that apoptosis may play a role in excitotoxic neuronal death. To examine the possible involvement of apoptosis in glutamate excitotoxicity, we studied an early process of morphological changes in rat cortical neurons exposed to 1 mM glutamate. Observations under Nomarski optics combined with a digital image processor revealed a rapid change in the nucleus followed by a cellular swelling. The nucleus increased in granularity and swelled in 5 min, then became liquefied in 30 min. The cell body swelled slowly in 15-45 min. These changes could be prevented by treatment of the neuron with MK-801 (dizocilpine maleate), a blocker of N-methyl-D-aspartate (NMDA) receptor-coupled ion channel. However, treatment of the neurons with N(G)-nitro-L-arginine (N-NORG), a nitric oxide synthase inhibitor, had no significant effect. Use of the in situ end-labeling technique for the demonstration of free 3'-hydroxyl ends revealed that DNA fragmentation took place within 1 hr after glutamate exposure. A change in intracellular Ca(2+) concentration was examined with fluo-3 under a confocal laser microscope. Application of 1 mM glutamate induced rapid Ca transients in the nucleus as well as in the cytoplasm. Both of these Ca responses were blocked by MK-801. These results indicate that glutamate excitotoxicity in the brain neuron does not fulfill morphological criteria of apoptosis, but suggest that the nuclear disintegration associated with DNA fragmentation is involved as a leading step in glutamate excitotoxicity.

Animals↗
PubMed1996

Fenestration in the myelin sheath of nerve fibers of the shrimp: a novel node of excitation for saltatory conduction.

Giant nerve fibers of the shrimp family Penaeidae conduct impulses at the velocity highest among all animal species (approximately 210 m/s; highest in mammals = 120 m/s). We examined these giant and other small nerve fibers morphologically using a differential interference contrast microscope as well as an electron microscope, and found a very specialized form of excitable membrane that functions as a node for saltatory conduction of the impulse. This node appeared under the light microscope as a characteristic pattern of concentrically aligned rings in a very small spot of the myelin sheath. The diameter of the innermost ring of the node was about 5 microns, and the distance between these nodes was as long as 12 mm. Via an electron microscope, these nodes were characterized by a complete lack of the myelin sheath, forming a fenestration that has a tight junction with an axonal membrane. Voltage clamp measurements by a sucrose gap technique demonstrated that the axonal membrane at these fenestration nodes is exclusively excitable and that the large submyelinic space is a unique conductive pathway for loop currents for saltatory conduction through such fenestration nodes.

Animals↗
PubMed1996

Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity.

Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthus martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V8 protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus, functional and structural similarities suggest that BmK I and BmK II belong to group 3 of scorpion alpha-type toxins.

Amino Acid Sequence↗