Search PubMed⌕ Search

Biomedical subjects

S Terakawa

Publications and source records attributed to S Terakawa.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1996

Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth.

The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur. Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear. We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth. Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesicle-rich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite. The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth. The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1. The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or alpha-SNAP, supported the results using neurotoxin C1. Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way.

Animals↗
PubMed1996

Swift transformation and locomotion of polymorphonuclear leukocytes and microglia as observed by VEC-DIC microscopy (video microscopy).

The detailed assembly used by us for video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy (video microscopy is first described. Employing such video microscopy, we then examined the morphological changes occurring during locomotion and activation processes of polymorphonuclear leukocytes (PMNL) and microglia at an almost electron microscopic magnification. Upon contacting the substratum, PMNL transformed into a polarized ameboid shape and crawled extending pseudopodia, as has been well documented previously. The PMNL sometimes displayed a peculiar locomotion as if they were stepping on "tiny legs", or sliding on a treadmill of cell membrane. Cultured microglia were observed to exist in 4 forms; ramified, reactive, villous, and ameboid. Microglia in the reactive form pivoted, circled and crawled on the astroglial cell layer using their transparent lamellipodia with no morphological changes in their cell body. Unlike PMNL, reactive microglia exhibited no agitated movements of their intracellular organelles, including granules and cytosol, during locomotion. Lamellipodia on the undersurface of the cell body touching the cell layer adhesively, appeared to serve as the locomotive apparatus. When activated, both floating PMNL and microglia of villous form assumed an ameboid shape within a few seconds. Microglia occasionally swam in the medium waving their lamellipodia towards a target object (e.g. zymosan A particles), remodelling to an amorphous ameboid form and covering up the target. We attempt to discuss such swift morphological changes from the standpoint of thermodynamic potential of Gibbs free energy which is stored within the cells.

Cell Movement↗
PubMed1995

Purification and partial amino acid sequences of a new presynaptic toxin and a cytotoxin from venom of pit veper Agkistrodon blomhoffii brevicaudus.

The technique of the reverse-phase performance liquid chromatography (RP-HPLC) was employed to separate and purify the toxic proteins from the venom of Agkistrodon blomhoffii brevicaudus collected in China. 3 toxic proteins marked as AgTx-1, AgTx-2 and AgTx-3 consisting of about 122 amino acid residues were screened. The toxicities (LD50) of the AgTx-1, AgTx-2 and AgTx-3 were 0.075, 0.51 and 6.6 mg per kg weight of mice respectively. Toxicological experiment in the chick biventer cervicis nerve-muscle preparation showed that the acetylcholine (Ach) sensitivity of the preparation was unchanged after the total failure of the indirect contraction caused by AgTx-1 and AgTx-2, suggesting that they were presynaptic blockers, namely beta-type of snake toxins. However, the amplitude of indirect contraction of the preparation was gradually reduced due to its incomplete relaxation caused by AgTx-3, indicating that it should belong to the category of cytotoxins. The partial amino acid sequences of 3 toxins have been established. It was found in ref. [1] that the sequences of the first 32 N-terminal amino acid residues of AgTx-1 and AgTx-2, as well as beta-agkistrodotoxin (beta-AgTx) reported previously were identical (the residue at the position 30 of beta-AgTx should be Trp). In view of the similarity in toxicities, and the amounts in the venom and other properties, it was concluded that AgTx-1 should be beta-AgTx and consequently was renamed beta 1-AgTx. AgTx-2 should be the isoform of beta 1-AgTx, and correspondingly named beta 2-AgTx.

Agkistrodon↗
PubMed1994

Differential behavior of glial and neuronal cells exposed to hypotonic solution.

The comparative modes of swelling of glial and neuronal cells were examined by video-enhanced differential interference contrast (VEC) microscopy. When exposed to hypotonic solution, C6 cells swelled slowly to 5 times their normal volume and burst, whereas N18 cells swelled more rapidly, forming blebs, and then burst partially. The time to burst was 410.6 +/- 45.7 s (mean +/- SD, n = 5) for C6, and 69.3 +/- 10.4 s (n = 5, p < 0.01) for N18, respectively. The present findings suggest a great difference in physical strength of the cell membrane between glial and neuronal cells: the former cell membrane appears to be elastic and tolerant to high tension, while the latter cell membrane is relatively weak.

Animals↗
PubMed1994

Rapid sprouting of filopodia in nerve terminals of chromaffin cells, PC12 cells, and dorsal root neurons induced by electrical stimulation.

Rapid morphological changes induced by direct electrical stimulation of nerve terminals were studied by using video-enhanced differential interference contrast microscopy at a very high magnification (12,000x). We used mainly cultured bovine chromaffin cells, which developed neurite-like processes, and PC12 cells, which showed neuronal differentiation upon NGF treatment. In a few cases, primary neurons of the rat dorsal root ganglion were also examined. Brief pulse stimulation of the terminals and varicosities induced exocytosis accompanied by rapid formation of filopodia. These filopodia, 0.1-0.2 micron in diameter and up to 10 microns in length, formed within a few hundreds of milliseconds and then retracted within tens of seconds. They could also be induced by K depolarization. This rapid filopodial sprouting strongly depended on the presence of extracellular Ca2+ and could be abolished in a medium containing a Ca chelator (EGTA) or La2+. Anti-cytoskeletal agents colchicine and cytochalasin B failed to block this response completely but lidocaine fully suppressed it. Quantitative analysis of exocytosis and filopodial sprouting showed that they were independent events, not directly linked to each other, having different thresholds usually higher for filopodial formation. In PC12 cells, the extent of filopodial sprouting varied with the state of differentiation of the cells, suggesting a functional role of rapid sprouting during a particular phase of their differentiation. Filopodia could be induced with greater ease by repetitive stimulation. The same responses may occur at growth cones approaching the target cells or even at mature synapses particularly after repetitive electrical activity, possibly playing a role in use-dependent synapse formation or plasticity.

Aging↗
PubMed1994

Molecular characteristics of four new depressant insect neurotoxins purified from venom of Buthus martensi Karsch by HPLC.

Suitable pattern and high yield were obtained when the reverse-phase performance liquid chromatography (RP-HPLC) was used to separate neurotoxins from venom of Chinese scorpion Buthus martensi Karsch. Using this technique, the venom was first separated to two main regions. The toxicity tests show that the insect-selective neurotoxical components are concentrated in the latter region, from which five insect-selective neurotoxins designated by BmK IT1-IT5 were obtained. According to the results of the toxicity test as well as the amino acid composition and N-terminal analyses, BmK IT1 is the excitatory insect neurotoxin as reported in a previous paper, and the others are the newly found depressant insect-selective neurotoxins. The molecules of all the four toxins are single-chain minipeptides of about 60 amino acids. Their isoelectric points (pI) are between 8.3 and 8.5. The fact that BmK IT2 loses completely its insect toxicity after being modified by fluorochrome shows that the positive charges on the molecular surface of this kind of toxins are important to maintaining the bioactivity of the molecules.

Amino Acid Sequence↗
PubMed1994

Amino acid sequence of an excitatory insect-selective toxin (BmK IT) from venom of the scorpion Buthus martensi Karsch.

The insect-selective neurotoxin (BmK IT) of scorpion Buthus martensi Karsch was first reduced and S-alkylated, and then digested by TPCK-trypsin and Staphylococcus aureus V-8 Protease. The enzymatic peptides were purified on TLC-plastic sheet and submitted to determine their amino acid compositions and sequences. The sequence of the 70 amino acid residues of BmK IT was established with reference to the primary structure of AaH IT, another excitatory insect-selective toxin from the venom of North African scorpion Androctonus australis Hector. About 75% of the homologous sequence was found in the molecules of BmK IT and AaH IT. It is obvious that the results contribute toward better understanding of the molecular structure characteristics, structure/activity relationship of scorpion insect-selective toxins, and they can serve as the molecular basis for utilizing the toxins as a tool to clarify molecular mechanism involved in channel gating, and to infer the possibility of developing them as new selective bioinsecticides.

Amino Acid Sequence↗
PubMed1993

Evidence against the swelling hypothesis for initiation of exocytosis in terminals of chromaffin cell processes.

Exocytosis of transmitter-containing granules was directly visualized in neurite terminals of cultured chromaffin cells under a video-enhanced contrast microscope. The granule diameter did not change immediately before their exocytotic responses. Large granules responded as early as small ones. These findings are inconsistent with the swelling hypothesis for initiation of exocytosis.

Adrenal Medulla↗
PubMed1993

Neurotransmitter-induced exocytosis in goblet and acinar cells of rat nasal mucosa studied by video microscopy.

To investigate the mechanism and neural control of the nasal secretion, we observed the isolated rat nasal mucosa by video-enhanced differential interference contrast microscopy. This technique allowed us to visualize abrupt changes of the individual granules leading to degranulation in the acinar cells and in epithelial goblet cells during secretory stimulation. This image provided evidence that exocytosis is the major mode for regulated secretion in the nasal acinar cells and goblet cells. Acetylcholine (ACh, 0.1-100 microM), substance P (SP, 0.1-10 microM), and vasoactive intestinal peptide (VIP, 0.1-1 microM) induced exocytotic responses and shrinkage of the acinus in a concentration-dependent manner. The effects of ACh (10 microM) on the acinus were clearly inhibited by atropine (5 microM), but the effects of SP (1 microM) and VIP (1 microM) were not. The acinar shrinkage always started before exocytosis, suggesting that the fluid secretion precedes the mucus release. In goblet cells, SP (1 microM) and ACh (10 microM) increased the frequency of exocytotic responses significantly, suggesting that these substances truly play the role of a neurotransmitter for nasal secretion. Histamine (HIST) induced no visible response. The effect of HIST on secretory cells may be neuronally mediated in vivo.

Acetylcholine↗
PubMed1993

Mastoparan evokes exocytosis without Ca2+ movement in adrenal chromaffin cells.

The secretagogue action of mastoparan, a tetradecapeptide from wasp venom, was studied in adrenal chromaffin cells. Pulsatile stimulation with mastoparan evoked a sharp transient release of CA in a dose-dependent manner, as measured by real-time monitoring system. The secretagogue action of mastoparan in these cells was apparently independent of extracellular Ca2+, and may not require the intracellular mobilization of Ca2+ since BAPTA failed to block its secretagogue action. Video-imaging data also suggested that mastoparan evokes CA release without an increase in the intracellular Ca2+. In addition, visualization of exocytotic events with video-enhanced light microscopy demonstrated that CA release evoked by mastoparan at relatively low concentration was indeed mediated by exocytotic process, not by cell lysis.

Animals↗
PubMed1993

Exocytosis in the growth cone of differentiated chromaffin cells induced by electrical stimulation.

In order to elucidate the mechanism involved in the specialized function of the nerve terminals, we studied the growth cone of cultured chromaffin cells using a video-enhanced Nomarski microscope. At a very high magnification, we found rapid and discrete morphological changes of small granules induced by electrical stimulation. Since they shared properties similar to those of the exocytotic responses found in granules in the cell body region, we conclude that they also reflect exocytosis of transmitter-containing granules. The frequency of the exocytotic responses was highest in the growth cone, lower in the cell body region, and none in the thin shaft of the neurite, suggesting a functional differentiation in the growth cone. Similar differentiation may be instrumental in physiological functions of the neuronal growth cone in vivo.

Animals↗
PubMed1993

Rapid filopodial sprouting induced by electrical stimulation in nerve terminals.

Direct electrical stimulation of terminals of neurite-like extensions of cultured chromaffin cells and neuronally differentiated PC12 cells induced rapid sprouting of filopodia in a Ca dependent manner. Repetitive stimulation could produce filopodia with greater ease. Their formation could not be significantly suppressed by treatment with cytoskeletal blocking agents. Filopodial sprouting was localized to the terminal region only. This response may occur at the growth cone in vivo playing a role in neuronal development.

Animals↗
PubMed1993

Saltatory conduction and a novel type of excitable fenestra in shrimp myelinated nerve fibers.

The findings of saltatory conduction in the invertebrate giant nerve fibers were mentioned, and the experiments for analyzing the mechanism of impulse conduction in the giant myelinated nerve fibers of Penaeus orientalis and Penaeus japonicus were reviewed. Saltatory conduction was also found in many middle- and small-sized myelinated nerve fibers of, at least, 6 species of Penaeus shrimps. Saltatory conduction with its morphological basis in myelinated nerve fibers of vertebrates and invertebrates were compared, and it was concluded that the myelination of the nerve fibers in vertebrates and invertebrates has occurred independently.

Animals↗