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71.
72.
Inoue I Tsutsui I Bone Q 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2002,172(6):541-546
In the locomotor muscle of the pelagic tunicate Doliolum, both the sarcoplasmic reticulum (SR) and the transverse-tubular (T-tubular) system are absent. The mechanism of excitation-contraction (E-C) coupling was studied in single muscle fibres enzymatically dissociated from Doliolum denticulatum. Whole cell voltage clamp experiments demonstrated an inward ionic current associated with membrane depolarisation. This current was blocked by 5 mmol.l(-1)Co(2+), a calcium current blocker, and suppressed by nifedipine, a specific L-type calcium channel blocker. An increase in the external K(+) concentration to 200 mmol.l(-1) (K(+)-depolarisation) induced a rise in the intracellular Ca(2+) level detected with fluo-3, a Ca(2+)-sensitive dye. However, when 5-10 mmol.l(-1) Co(2+) or 10-15 micro mol.l(-1) nifedipine was present in the external solution, K(+)-depolarisation did not induce a rise in the intracellular Ca(2+) level. Externally applied 5-10 mmol.l(-1) caffeine or 20 micro mol.l(-1) ryanodine had no effect on the intracellular Ca(2+) level. K(+)-depolarisation induced a rise in the intracellular Ca(2+) level in the presence of caffeine or ryanodine. Replacement of external Na(+) with Li(+) increased intracellular Ca(2+) levels. Our results show that contraction of the locomotor muscle in Doliolum is solely due to the influx of Ca(2+) through L-type calcium channels, and that relaxation is due to extrusion of Ca(2+) by Na(+)/Ca(2+) exchange across the sarcolemma. 相似文献
73.
Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding 下载免费PDF全文
A variety of cellular stresses activate the stress-responsive mitogen-activated protein (MAP) kinases p38 and JNK. In this study, we studied the activation mechanism of a human MAP kinase kinase kinase, MTK1 (also known as MEKK4), which mediates activation of both p38 and JNK. MTK1 has an extensive N-terminal noncatalytic domain composed of approximately 1,300 amino acids. Full-length or near full-length MTK1 is catalytically inactive when expressed in Saccharomyces cerevisiae cells, as it is in mammalian cells. Deletion of a segment including positions 253 to 553 activates kinase, indicating that this segment contains the autoinhibitory domain. In the autoinhibited conformation, the MTK1 kinase domain cannot interact with its substrate, MKK6. By a functional complementation screening with yeast cells, GADD45 proteins (GADD45alpha, beta, and gamma) were identified as MTK1 activators. GADD45 proteins bind a site in MTK1 near the inhibitory domain and relieve autoinhibition. Mutants of full-length MTK1 were isolated that can interact with MKK6 in the absence of the activator GADD45 proteins. These MTK1 mutants are constitutively active, in both yeast and mammalian cells. A model of MTK1 autoinhibition by the N-terminal inhibitory domain and activation by GADD45 binding is presented. 相似文献
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Suzuki Y Tasumi S Tsutsui S Okamoto M Suetake H 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2003,136(4):723-730
Among lectins in the skin mucus of fish, primary structures of four different types of lectin have been determined. Congerin from the conger eel Conger myriaster and AJL-1 from the Japanese eel Anguilla japonica were identified as galectin, characterized by its specific binding to β-galactoside. Eel has additionally a unique lectin, AJL-2, which has a highly conserved sequence of C-type lectins but displays Ca2+-independent activity. This is rational because the lectin exerts its function on the cutaneous surface, which is exposed to a Ca2+ scarce environment when the eel is in fresh water. The third type lectin is pufflectin, a mannose specific lectin in the skin mucus of pufferfish Takifugu rubripes. This lectin showed no sequence similarity with any known animal lectins but, surprisingly, shares sequence homology with mannose-binding lectins of monocotyledonous plants. The fourth lectin was found in the ponyfish Leiognathus nuchalis and exhibits homology with rhamnose-binding lectins known in eggs of some fish species. These lectins, except ponyfish lectin, showed agglutination of certain bacteria. In addition, pufflectin was found to bind to a parasitic trematode, Heterobothrium okamotoi. Taken together, these results demonstrate that skin mucus lectins in fish have wide molecular diversity. 相似文献
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Kaya T Torisawa YS Oyamatsu D Nishizawa M Matsue T 《Biosensors & bioelectronics》2003,18(11):1379-1383
The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors. 相似文献
79.
Hirano Y Mitsumori Y Oyamatsu D Nishizawa M Matsue T 《Biosensors & bioelectronics》2003,18(5-6):587-590
Scanning chemiluminescence microscopy (SCLM) with electrophoretic injection was developed and applied to visualize enzyme reactions localized in an enzyme microspot. The SCLM uses a tapered glass capillary as a probe for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The electrophoretic injection by application of a constant current between the inside and outside of the capillary enabled the continuous and controllable injection of a minute quantity of luminol in the range of 0.1 pmol/s. The image of enzyme activity in a monolayer spot of horseradish peroxidase was obtained by using the electrophoretic injection-based SCLM system. 相似文献
80.
Tsutsui K Sakamoto H Ukena K 《The Journal of steroid biochemistry and molecular biology》2003,85(2-5):311-321
The brain is considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have established that the brain itself also synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To understand neurosteroid actions in the brain, we need data on the specific synthesis in particular sites of the brain at particular times. Therefore, our studies for this exciting area of brain research have focused on the biosynthesis and action of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently, genomic and nongenomic actions of neurosteroids have become clear by a series of our studies using an excellent Purkinje cellular model. On the basis of these findings, we summarize the advances made in our understanding of biosynthesis and action of neurosteroids in the cerebellar Purkinje cell. 相似文献