Biomedical subjects

Y Miyashita

Publications and source records attributed to Y Miyashita.

At least 19 recordsLinked to original sources

PubMed1997

Characterization of an extremely thermophilic and oxygen-stable membrane-bound hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus.

The membrane-bound hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus was characterized as highly oxygen-tolerant, extremely thermophilic and thermostable in its membrane-bound form. The optimum temperatures for H2 oxidation and H2 evolution were 90 and 80 degrees C, respectively. The enzyme retained 90% of its activity after heating at 70 degrees C for 50 min under air and retained full activity at 90 degrees C for 80 min under hydrogen. The optimum pH values were 5.5 (H2 evolution) and 9.4 (H2 oxidation), and the activity ratio (H2 evolution/H2 oxidation) was high (50.3) at pH 5.5. The hydrogen evolution from reduced methyl viologen continued at high temperatures and acidic pH with the continuous addition of sodium dithionite.

Cell Membrane↗

PubMed1997

Kinetic diversity in the fusion of exocytotic vesicles.

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense-core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+-dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.

Acetylcholine↗

PubMed1997

Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells.

Agonists induce Ca2+ spikes, waves and oscillations initiating at a trigger zone in exocrine acinar cells via Ca2+ release from intracellular Ca2+ stores. Using a low affinity ratiometric Ca2+ indicator dye, benzothiazole coumarin (BTC), we found that high concentrations of agonists transiently increased Ca2+ concentrations to the micromolar range (>10 microM) in the trigger zone. Comparison with results obtained with a high affinity Ca2+ indicator dye, fura-2, indicated that fura-2 was in fact saturated with Ca2+ during the agonist-induced Ca2+ spikes in the trigger zone. We further revealed that the micromolar Ca2+ spikes were necessary for inducing exocytosis of zymogen granules investigated using capacitance measurements. In contrast, submicromolar Ca2+ spikes selectively gave rise to sequential activation of luminal and basal ion channels. These results suggest new functional diversity in Ca2+ spikes and a critical role for the micromolar Ca2+ spikes in exocytotic secretion from exocrine acinar cells. Our data also emphasize the value of investigating the Ca2+ signalling using low affinity Ca2+ indicators.

Acetylcholine↗

PubMed1997

Cytoplasmic Ca2+ gradients evoked by acetylcholine and peptides in pancreatic acinar cells of the guinea-pig.

Cytoplasmic Ca2+ levels ([Ca2+]i) in guinea-pig pancreatic acinar cells stimulated with acetylcholine (ACh) as well as cholecystokinin octapeptide (CCK-8), its analogue CCK-JMV-180, bombesin and substance P (SP) have been compared by confocal Ca2+ imaging. We found that at intermediate and high concentrations all agonists evoked [Ca2+]i waves emanating from the secretory pole of the cell. The [Ca2+]i gradients evoked by ACh and SP were often unidirectional whereas CCK-8- and bombesin-elicited [Ca2+]i responses consisting of [Ca2+]i waves were normally followed by a secondary phase with reversed [Ca2+]i gradients. At low agonist concentrations the [Ca2+]i responses appeared diffuse or localized to the granular part of the cells. The lag times to the [Ca2+]i rises were shorter in response to ACh and SP than for other peptides. Thus, we found that agonists induced different spatio-temporal patterns of Ca2+ waves in pancreatic acinar cells of the guinea-pig. The reactions evoked by SP and bombesin resembled those evoked by ACh and CCK-8, respectively.

Acetylcholine↗

PubMed1997

Thyroid hormone stimulates renin synthesis in rats without involving the sympathetic nervous system.

The present study was performed to examine whether renal expression of the renin gene is regulated by thyroid hormone. Thirty male Sprague-Dawley rats were divided into hypothyroid, control, and hyperthyroid groups by use of daily intraperitoneal administration of methimazole, saline vehicle, or thyroxine, respectively. Each group was further subdivided into sympathetic innervated and sympathetic denervated subgroups by use of intraperitoneal administration of saline vehicle or 6-hydroxydopamine. Plasma renin activity and renal levels of renin were measured by radioimmunoassays after 8 wk. Renal expression of renin mRNA was evaluated by a semiquantitative reverse transcriptase-polymerase chain reaction. Compared with control animals, plasma renin activity, renal level of renin, and renal expression of renin mRNA were reduced (82, 94, and 71%, respectively) in hypothyroid animals and elevated (155, 1,182, and 152%, respectively) in hyperthyroid animals. Sympathetic denervation had no independent effect on these renin values. Our results indicate that thyroid hormone stimulates renin synthesis without involving the sympathetic nervous system.

Animals↗

PubMed1997

Redistribution of intracellular Ca2+ stores after beta-adrenergic stimulation of rat tail artery SMC.

beta-Adrenergic agonists induce the relaxation of vascular smooth muscle by a mechanism that activates the extrusion of Na+ and Ca2+ from the cell. A primary source of contractile Ca2+ resides in the sarcoplasmic reticulum (SR), which releases Ca2+ in response to vasoactive agents through inositol trisphosphate-mediated channels. To determine if smooth muscle relaxation induced by beta 2-adrenergic agonists involves the redistribution of intracellular Ca2+, we studied the effects of isoproterenol (Iso) on freshly isolated, single rat tail artery smooth muscle cells loaded with fura 2, using digital ratiometric fluorescence imaging. Stimulation with 1 microM phenylephrine (PE) or norepinephrine produced phasic and tonic increases in cytoplasmic intracellular Ca2+ concentration ([Ca2+]i) associated associated with cell shortening. Exposure to caffeine and to Ca2(+)-free solutions eliminated the phasic and tonic components, respectively, from the Ca2+ signal. Intermittent superfusion with PE or caffeine was used to evaluate SR Ca2+ stores after stimulation by Iso. Exposure to 1 microM Iso induced a time-dependent decrease in PE-activated peak and tonic [Ca2+]i without any change in resting [Ca2+]i. Intermittent stimulation with 10 mM caffeine revealed a similar decline in peak [Ca2+]i, indicating Iso-dependent depletion of SR Ca2+ stores. The Ca2+ that remained in the SR after prolonged exposure to Iso (30% of the pre-Iso level by 80 min at 22 degrees C) failed to elicit a contractile response. The cells, perfused with a Na(+)- and Ca2(+)-free medium to block Na+/ Ca2+ exchange, prevented depletion of the SR Ca2+ stores by Iso. We propose that Iso inhibits agonist-mediated Ca2+ influx through sarcolemmal Ca2+ channels and activates Ca2+ redistribution from storage sites in the SR to the extracellular compartment by a mechanism that involves Na+/Ca2+ exchange. These combined effects of Iso facilitate smooth muscle relaxation (and reduce vascular tonus) by reducing the increase in cytoplasmic Ca2+ evoked by vasoconstrictors.

Adrenergic beta-Agonists↗

PubMed1997

Clinical efficacy of the direct assay method using polymers for serum high density lipoprotein cholesterol.

The clinical efficacy and accuracy of the homogeneous assay method for the serum high density lipoprotein (HDL)-cholesterol determination were evaluated. The principle is as follows: low density lipoproteins (LDL) and very low density lipoproteins (VLDL) were coated by polymers and polyanion to be blocked from cholesterol esterase and cholesterol oxidase. The reaction of these enzymes for HDL cholesterol was enhanced with a detergent, and HDL cholesterol was selectively measured. Both within-run (n = 3, 20 times) and between-run (n = 3, 7 days) CVs were < 2%. The repeated freezing and thawing (4 times) of three distinct sera resulted in no changes of HDL cholesterol values. Additions of lipid emulsion (Triglyceride = 100 mg/dl) and free bilirubin (20 mg/dl) gave no effect. Linearity was found up to 300 mg/dl. Increases in HDL cholesterol values by the addition of VLDL (total cholesterol (TC) = 300 mg/dl) or LDL (TC = 300 mg/dl) to the tested sera were < 0.5%. The correlation coefficient of the new method with a precipitation method was 0.995 (n = 64). HDL-C values for patients with hyperlipidemia (Type IIa, IIb, or III, IV, and V) by this method were comparable with those obtained by the precipitation method. From these results, we concluded that the new method meets the requirements for accuracy, precision, ease of handling massive samples, and was clinically useful.

Adult↗

PubMed1996

Transient brain activity used in magnetic resonance imaging to detect functional areas.

Functional areas were detected with short stimuli eliciting transient brain activity using the method of 'transient' regions of interest (ROIs) and functional magnetic resonance imaging (fMRI). This method was validated by comparing the results with sustainedly activated areas identified conventionally. Eighty-eight and 89% of the total areas of transient ROIs derived from 0.2 and 2 s stimulation, respectively, were identified at 5-7 s and 5-9 s, respectively, after stimulus onset. Eighty-eight and 76%, respectively, of these areas overlapped 'conventional' ROIs derived from 20 s stimulation. These results suggest that the delineation of transient ROIs, by targeting a period approximately 7 s after transient neural activity, can be useful for fMRI studies of cognitive functions.

Adult↗

PubMed1996

Preference for background color of the Xenopus laevis tadpole.

The background color preferences of three age groups of Xenopus laevis tadpoles (young-stages tadpoles [stages 44-46], premetamorphic tadpoles [stages 54-56], and metamorphic tadpoles [stages 58-60]) were examined. Young tadpoles selected a white background, metamorphic tadpoles preferred a black background, and premetamorphic tadpoles selected a white or black background consistent with the background black or white of the test box on which they previously were conditioned. If premetamorphic tadpoles were conditioned in the checkerboard-pattern (white and black) box or white box which was placed a small black square at the center of the box, they preferred to stay in the white area just after transfer to the test box, then shifted their preference for background color to black strongly. Premetamorphic tadpoles conditioned on a white background lost this preference if kept in the dark for 12 hours. Blinding of premetamorphic tadpoles by severance of the optic nerves resulted in loss of preference for a specific background matching the white or black background to which they had been adapted while sighted. Given a choice 3 days postblinding, they tended to congregate on a white background. Injection of MSH into premetamorphic tadpoles conditioned to white shifted their preference to black. In contrast, injection of melatonin stimulated black adapted tadpoles to select a white background. Young frogs showed a preference for a black background.

Animals↗

PubMed1996

Light-sensitive response in melanophores of Xenopus laevis: II.Rho is involved in light-induced melanin aggregation.

Melanophores of the isolated tail fin of the Xenopus tadpole aggregate melanin granules in response to light. This aggregation was found to be inhibited by subcutaneous injection of exoenzyme C3 of Clostridium botulinum. A 26 kDa protein in homogenate obtained from the Xenopus tail fin was ADP-ribosylated by exoenzyme C3. This reaction was inhibited effectively by a monoclonal antibody, anti-Rho mab A5. raised against the small GTP-binding protein Rho. The extent of ADP-ribosylation depended on light and guanine nucleotide. Incubation under illumination partly reduced ADP-ribosylation and the reduction was restored by addition of guanine nucleotide during incubation. These findings suggest that Rho is involved in the photo-sensitive melanophore response as a signal transducer linking photo-stimuli to melanin granule translocation with Xenopus melanophores.

ADP Ribose Transferases↗

PubMed1996

Light-sensitive response in melanophores of Xenopus laevis: I. Spectral characteristics of melanophore response in isolated tail fin of Xenopus tadpole.

Melanophores in the isolated tail from the amphibian larvae Xenopus laevis, Hyla japonicus, Rana pirica, and Hynobius retardatus aggregated melanin granules in response to light and dispersed them when placed in darkness. The spectral characteristics for the melanin-aggregation response were examined by irradiating the Xenopus tail-fin locally (diameter, 2.1 mm) with monochromatic light (380-1,020 nm). The spectral region of wave length which induced melanosome aggregation depended on the light intensity but was limited to the visible spectrum. At low light intensity (1.59 microW/cm2, delta lambda = 5 nm), the aggregation response occurred in the spectral region between 400 and 600 nm and the maximum response was observed at 500 nm. This range is very close to the absorption spectrum of rhodopsin in the visual rod cell. Hypodermic injection of cGMP into isolated tail-fin induced a marked melanin-dispersion in spite of light-stimuli. When the tail-fin was treated with isobutylmethylxanthine (IBMX; phosophodiesterase inhibitor) in darkness and then was re-exposed to light, the aggregation response was inhibited. The photo-sensitive melanin aggregation was independent of a requirement for Ca2+ ions but melanosome dispersion in darkness was Ca(2+)-dependent. K(+)-rich Hanks' solution, ouabain (inhibitor of Na(+)-K(+)-ATPase) or nonactin (cation ionophore), which induced a change of the membrane potential of melanophores, inhibited the aggregation response when the melanophores were re-exposed to light after a period in darkness. These results suggest that the molecular mechanism of photoreception in melanophores of amphibian tadpoles is similar to that in visual cells.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Ca2+-dependent exocytotic pathways in Chinese hamster ovary fibroblasts revealed by a caged-Ca2+ compound.

Ca2+-dependent exocytosis and endocytosis of Chinese hamster ovary (CHO) fibroblasts were investigated using capacitance measurement and rapid photolysis of a caged-Ca2+ compound, dimethoxynitrophenamine tetrasodium salt. CHO cells exhibited large and fast increases in membrane capacitance (1.9 +/- 1 picofarads, or 13 +/- 7% of total membrane area, mean +/- S.D., n = 37) upon Ca2+ jumps to [Ca2+]i larger than 20 microM. The fast exocytosis occurred with a delay (20-80 ms), and exhibited a rate constant that was strongly dependent on [Ca2+]i. The maximal rate constant of exocytosis was 2.8/s, and a half-maximal rate was achieved at 30 microM. The fast exocytosis was followed by rapid endocytosis in 28% of the cells. The endocytosis often began after a delay of 0.5-2 s. Ca2+ jumps also induced stepwise increases in membrane capacitance of 10-134 femtofarads in 40% of the cells, indicating fusion of large vesicles with diameters of 0.4-1.5 micron. The exocytosis of the large vesicles could selectively be induced with smaller Ca2+ jumps (6-20 microM), and occurred slowly with a rate constant of 0. 3/s. These data indicate that CHO fibroblasts possess Ca2+-dependent exocytotic mechanisms. Moreover, two parallel exocytotic pathways may exist reminiscent of those of neurons and endocrine cells. A kinetic model was constructed to account for the fast exocytosis of CHO cells.

Acetates↗

PubMed1996

Two components of exocytosis and endocytosis in phaeochromocytoma cells studied using caged Ca2+ compounds.

1. Changes in membrane capacitance evoked by the rapid photolysis of a caged Ca2+ compound, DM-nitrophen or nitrophenyl-EGTA, were investigated in undifferentiated PC12 cells. They were interpreted as representing exocytosis and endocytosis. 2. The Ca2+ jumps evoked two components of exocytosis. Slow exocytosis was selectively evoked with small increases in intracellular Ca2+ concentration between 5 and 10 microM, while fast exocytosis preceded the slow one at [Ca2+]i greater than 10 microM. 3. The release rates of the two components of exocytosis depended steeply on [Ca2+]i. A half-maximal release rate was achieved at 8 and 24 microM for the slow and fast exocytoses, respectively. 4. Prior Ca2+ rises did not augment the fast exocytosis. 5. The fast exocytosis was often followed by a rapid decrease in membrane capacitance, representing endocytosis, after a delay of 0.5-2 s. The speed and delay in the fast endocytosis were Ca2+ dependent. Amounts of the fast endocytosis tended to balance with those of the fast exocytosis evoked by the same Ca2+ jumps. 6. The slow exocytosis was followed by a sluggish endocytosis that was associated with large capacitance steps indicative of secretory processes involving large dense-core vesicles. The onset of the slow endocytosis exhibited a complex Ca2+ dependence. The amounts of the slow endocytosis appeared to parallel those of the slow exocytosis. Prior induction of the slow exocytosis gave rise to selective excess retrieval of membrane during the slow endocytosis. 7. These data indicate the existence of two distinct populations of secretory vesicles in PC12 cells. They seem to couple selectively with specific endocytotic mechanisms. Our data suggest that the two vesicles belong to two distinct secretory pathways.

Animals↗

PubMed1996

Characteristics of 45Ca2+ release induced by quinolidomicin A1, a 60-membered macrolide from skeletal muscle sarcoplasmic reticulum.

Quinolidomicin A1, a 60-membered macrolide purified from an actinomycete Micromonospora sp. markedly induced 45Ca2+ release from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR), but induced only slightly from the light fraction of sarcoplasmic reticulum (LSR), showing a lack of the ionophoretic activity even at a high concentration (300 microM). This was also confirmed by measuring the 45Ca2+ transport activity of quinolidomicin A1 across an organic solvent barrier. Quinolidomicin A1 (3-300 microM) increased 45Ca2+ release from HSR with an EC50 value of approx. 20 microM. The potency of quinolidomicin A1 was approx. 100-fold higher than that of caffeine. The bell-shaped profile of Ca2+ dependence for quinolidomicin A1 was different from that for caffeine. Blockers of Ca2+ release channels such as Mg2+ (10 mM), procaine (10 mM) and ruthenium red (10 microM) partially blocked quinolidomicin A1 (30 microM)-induced 45Ca2+ release from HSR. At 0 degrees C, quinolidomicin A1-induced 45Ca2+ release was ascertained not to be due to the inhibition of Ca2+ ATPase by the ATPase assay. Quinolidomicin A1 potentiated [3H]ryanodine binding to HSR with a decrease in KD but without a change in Bmax. These results suggest that quinolidomicin A1-induced Ca2+ release from HSR is consisted of two components, which are both sensitive and insensitive to blockers of Ca2+ release channels, and that the former component is associated with the ryanodine receptor.

Actinomycetales↗

PubMed1996

Melatonin. A possible role in pathogenesis of adolescent idiopathic scoliosis.

STUDY DESIGN: The serum melatonin levels during 24-hour periods were compared between patients with idiopathic and age-matched normal control subjects. OBJECTIVE: To find if the melatonin deficiency may have some role for progression or etiology of idiopathic scoliosis in humans. SUMMARY OF BACKGROUND DATA: Experimentally induced scoliosis in chicken by pinealectomy can be attributed to the defect in melatonin metabolism. METHOD: Blood samples were correlated every 3 hours during 24-hour periods, and serum melatonin levels were measured and statistically analyzed. RESULTS: The level of melatonin, integrated concentration through 24 hours and night time (0:00 am-6:00 am), in the patients who had progressive curve (more than 10 degrees of progression in the previous 12 months) was significantly lower than the level in the patients who had a stable curve (less than 10 degrees of progression in the previous 12 months) or in the control subjects (P < 0.05). CONCLUSION: The study suggests that normal melatonin synthesis or metabolism may have crucial role in regulating normal spine growth. The level of melatonin appears to be a useful predictor for progression of spine curvature in idiopathic scoliosis.

Adolescent↗

PubMed1996

Activity of primate inferotemporal neurons related to a sought target in pair-association task.

Visual long-term memory in primates has been assessed by using the pair-association (PA) task, in which a subject retrieves and chooses the paired associate of a cue picture. Our previous studies on single neurons in the anterior inferotemporal (AIT) cortex suggested their roles in representing paired associates in the mind. To test the possibility that the delay activity of AIT neurons is related to a particular picture as a sought target, we devised the PA with color switch (PACS) task. In the PACS task, the necessity for memory retrieval and its initiation time were controlled by a color switch in the middle of the delay period. A control task, in which there is no color switch, corresponds to the conventional delayed matching-to-sample (DMS) task where the monkey chooses the same picture as a cue. We found that AIT neurons started to respond just after the color switch in the PACS task, when the cue-optimal picture's associate was presented as a cue. In contrast, they showed no response change in the DMS task. We confirmed that this effect is not due to the visual response to colors. Furthermore, when the cue-optimal picture was presented as a cue, these neurons showed suppression after the color switch in the PACS task. These results suggest that the activity of AIT neurons mediates gating mechanisms that preferentially pass information about a sought target, even when the sought target is retrieved from long-term memory.

Animals↗

PubMed1996

Formation of mnemonic neuronal responses to visual paired associates in inferotemporal cortex is impaired by perirhinal and entorhinal lesions.

Functional roles of the cortical backward signal in long-term memory formation were studied in monkeys performing a visual pair-association task. Before the monkeys learned the task, the anterior commissure was transected, disconnecting the anterior temporal cortex of each hemisphere. After training with 12 pairs of pictures, single units were recorded from the inferotemporal cortex of the monkeys as the control. By injecting a grid of ibotenic acid, we unilaterally lesioned the entorhinal and perirhinal cortex, which provides massive direct and indirect backward projections ipsilaterally to the inferotemporal cortex. After the lesion, the monkeys fixated the cue stimulus normally, relearned the preoperatively learned set (set A), and learned a new set (set B) of paired associates. Then, single units were recorded from the same area as for the prelesion control. We found that (i) in spite of the lesion, the sampled neurons responded strongly and selectively to both the set A and set B patterns and (ii) the paired associates elicited significantly correlated responses in the control neurons before the lesion but not in the cells tested after the lesion, either for set A or set B stimuli. We conclude that the ability of inferotemporal neurons to represent association between picture pairs was lost after the lesion of entorhinal and perirhinal cortex, most likely through disruption of backward neural signals to the inferotemporal neurons, while the ability of the neurons to respond to a particular visual stimulus was left intact.

Animals↗

PubMed1996

Feedback signal from medial temporal lobe mediates visual associative mnemonic codes of inferotemporal neurons.

Functional roles of the cortical backward signal in long-term memory formation were studied in monkeys performing a visual pair-association task. Before learning of the task, the anterior commissure of the monkeys was transected, disconnecting the anterior temporal cortex of each hemisphere. After training with 12 pairs of pictures, we injected a grid of ibotenic acid unilaterally into the entorhinal and perirhinal cortex that provide massive backward projections ipsilaterally to the inferotemporal cortex. According to the histological examination, the lesions covered medial and lateral banks of the rhinal sulcus completely and most the entorhinal and perirhinal cortex. After the injections, the monkeys fixated the cue stimulus normally, relearned the preoperatively learned set (set-A) and learned a new set (set-B) of paired associates. Then single units were recorded from the same area as that for the prelesion control. We found that (i) in spite of the lesion, the sampled neurons responded strongly and selectively to both the set-A and set-B patterns, and that (ii) the paired associates elicited significantly correlated responses in the control neurons but not in the cells tested after the lesion either for set-A or set-B stimuli. We conclude that the ability of inferotemporal neurons to represent association between picture pairs was lost after disruption of backward neural signals from the limbic cortex to the inferotemporal neurons, while the ability of the neurons to respond to a particular visual stimulus was left intact.

Analysis of Variance↗