Visualization of ultraviolet absorption distribution beyond the diffraction limit of light by electron-beam excitation-assisted optical microscope.

We demonstrated that the high spatial resolution absorption contrast imaging of the crystal of vitamin B9 has absorption at ultraviolet wavelengths. The absorption wavelength matches with the wavelength of the emission of the fluorescent thin film of an electron-beam excitation-assisted (EXA) optical microscope. The fine crystal structure was imaged beyond the optical diffraction limit. The image contrast corresponded with the thickness of the crystal. The illumination light is absorbed with the vitamin B9 crystal and the intensity of the transmitted light depends on the thickness of the vitamin B9 crystal. The EXA optical microscope is useful for analysis of growth of a crystal, bioimaging and so on.

Stroboscopic near-field imaging for the analysis of contraction of muscle cells.

We have developed a stroboscopic near-field optical microscope for observation of biological specimens and observed glycerinated muscles before and after muscle contraction with the developed system. In the system, the optical field distribution localized near the specimen is recorded as the surface topographic distribution of a photosensitive film surface. Our system is very useful for observing living biological specimens with high resolutions, because it is possible to get stroboscopic image by using a photosensitive film as detecting optical distributions instead of a scanning of probes. We have succeeded in observing inner structures of muscle cells with sub-wavelength resolution and achieved higher contrast than an ordinary optical microscope.

Mechanical fragmentation and transportation of calcium phosphate substrate by filopodia and lamellipodia in a mature osteoclast.

The functions of filopodia and lamellipodia in mature osteoclasts are not well known in the process of bone resorption. We investigated the function of filopodial/lamellipodial movement in mature osteoclasts by video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy. Mature osteoclasts, which were isolated from Japanese white rabbits, were cultured on calcium phosphate (CP)-coated quartz coverslips to observe filopodial/lamellipodial movement and the formation of CP-free areas precisely. Filopodia broke the CP substrate into pieces and transported them to the cell body by capturing them at the tip. Lamellipodia destroyed the CP substrate, and transported it to the cell body by capturing small particles in a mass. This study suggests two functions of filopodia and lamellipodia in mature osteoclasts, i.e., the mechanical fragmentation of the CP substrate and the transportation of the CP particles to the cell body.

An Na+/H+ exchanger inhibitor suppresses cellular swelling and neuronal death induced by glutamate in cultured cortical neurons.

We examined the effects of a selective Na+/H+ exchanger inhibitor, SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate), on neuronal death induced by glutamate in rat cortical neurons. Morphological changes in neurons were observed with a differential interference contrast microscope, and cellular swelling was analysed. Neuronal death was assessed by staining the cell with propidium iodide. The intracellular calcium concentration ([Ca2+]i) and the intracellular pH were measured by fluorescence imaging with fluo-3/AM as an indicator for [Ca2+]i and BCECF/AM for pH, respectively. SM-20220 (0.3 to 30 nM) dose-dependently attenuated glutamate (300 microM)-induced neuronal death in a dose-dependent fashion over 6 hours, and inhibited acute cellular swelling following glutamate (100 microM) exposure. SM-20220 suppressed the persistent [Ca2+]i increase following glutamate (500 microM) exposure, and inhibited intracellular acidification induced by glutamate (1 mM). The activation of the Na+/H+ exchanger system may enhance the progress of cerebral damage and oedema following glutamate exposure. SM-20220, a Na+/H+ exchanger inhibitor, suppressed neuronal death and cellular swelling induced by glutamate through inhibition of both Ca2+ influx and acidification in neurons.

Tissue-type plasminogen activator is involved in the process of neuronal death induced by oxygen-glucose deprivation in culture.

Effect of tissue-type plasminogen activator (tPA) on oxygen-glucose deprivation (OGD) was studied in cultured cortical neurons prepared from tPA gene knockout (tPA-KO) and wild-type (Wt) mice. Three hours of OGD induced 45% and 23% of neuronal death in Wt and tPA-KO mice, respectively. Neuronal death in tPA-KO mice was increased to 42% by additional tPA. Six hours of OGD induced 80% and 40% of neuronal death in Wt and tPA-KO mice, respectively, whereas the addition of tPA increased to 62% in tPA-KO mice. These results suggest that tPA is directly involved in the process of neuronal death induced by ischemia-mimic stress without involving vascular or circulatory components.

Protein kinase C-dependent supply of secretory granules to the plasma membrane.

To elucidate the mechanism for supplying secretory granules to the cell membrane, chromaffin cells isolated from the bovine adrenal medulla were observed by the evanescent wave microscopy after staining their granules with acridine orange. The secretory granules showed only a very small fluctuation, indicating their docking to the plasma membrane. The rate and range of movement increased greatly by application of botulinum toxin A or C. The number of secretory granules docked to the plasma membrane significantly decreased by botulinum toxin C. Conversely, the number increased greatly by activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu). In the presence of an anti-actin reagent cytochalasin D, no increasing effect of PDBu on the number of docked granules was observed. While in the presence of an anti-mitotic reagent, colchicine, a clear increasing effect of PDBu was observed. The final step for supplying granules to the plasma membrane in endocrine cells is concluded to be mediated by a phosphorylation-dependent and actin-based transport system.

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').

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.

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.

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.

[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.

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.

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.

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.

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.

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.