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Meiotic maturation is essential for the reproduction procedure of many animals. During this process an oocyte produces a large egg cell and tiny polar bodies by highly asymmetric division. In this study, to fully understand the sophisticated spatiotemporal regulation of accurate oocyte meiotic division, we focused on the global and local changes in the tension at the surface of the starfish (Asterina pectinifera) oocyte in relation to the surface actin remodeling. Before the onset of the bulge formation, the tension at the animal pole globally decreased, and started to increase after the onset of the bulge formation. Locally, at the onset of the bulge formation, tension at the top of the animal pole began to decrease, whereas that at the base of the bulge remarkably increased. As the bulge grew, the tension at the base of the bulge additionally increased. Such a change in the tension at the surface was similar to the changing pattern of actin distribution. Therefore, meiotic cell division was initiated by the bulging of the cortex, which had been weakened by actin reduction, and was followed by contraction at the base of the bulge, which had been reinforced by actin accumulation. The force generation system is assumed to allow the meiotic apparatus to move just under the membrane in the small polar body. Furthermore, a detailed comparison of the tension at the surface and the cortical actin distribution indicated another sophisticated feature, namely that the contraction at the base of the bulge was more vigorous than was presumed based on the actin distribution. These features of the force generation system will ensure the precise chromosome segregation necessary to produce a normal ovum with high accuracy in the meiotic maturation.

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In starfish oocytes, microtubules (MTs) form a spindle, which plays an important role in contributing to the selective loss of chromosomes and centrosomes to the polar bodies (PBs) during meiosis. When Taxol was locally injected near the germinal vesicle (GV) or the mitotic apparatus during meiosis I, PB formation was inhibited as mentioned below. In the oocytes, which were injected with Taxol after spindle formation, the spindle became large, and then the volume of the first PB also increased more than that of the control. In contrast, in the oocytes injected with Taxol before the spindle formation, chromosome capture and alignment were inhibited. These oocytes did not form PB, but only a bulge at the cell cortex was occasionally observed. Moreover, in the oocytes injected with Taxol before GV breakdown, the chromosomes did not gather in one place, and then two asters were observed at distant positions from the cell cortex. These results suggested that MTs lost not only the ability to obtain the bipolar attachment of chromosomes by Taxol injection but also the aster closer to the cell cortex lost its interaction with the cell cortex of the animal pole.

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Depression is associated with vascular disease, such as myocardial infarction and stroke. Pharmacological treatments may contribute to this association. On the other hand, Mg(2+) deficiency is also known to be a risk factor for the same category of diseases. In the present study, we examined the effect of imipramine on Mg(2+) homeostasis in vascular smooth muscle, especially via melastatin-type transient receptor potential (TRPM)-like Mg(2+) -permeable channels. The intracellular free Mg(2+) concentration ([Mg(2+) ](i) ) was measured using (31) P-nuclear magnetic resonance (NMR) in porcine carotid arteries that express both TRPM6 and TRPM7, the latter being predominant. pH(i) and intracellular phosphorus compounds were simultaneously monitored. To rule out Na(+) -dependent Mg(2+) transport, and to facilitate the activity of Mg(2+) -permeable channels, experiments were carried out in the absence of Na(+) and Ca(2+) . Changing the extracellular Mg(2+) concentration to 0 and 6 mM significantly decreased and increased [Mg(2+) ](i) , respectively, in a time-dependent manner. Imipramine statistically significantly attenuated both of the bi-directional [Mg(2+) ](i) changes under the Na(+) - and Ca(2+) -free conditions. This inhibitory effect was comparable in influx, and much more potent in efflux to that of 2-aminoethoxydiphenyl borate, a well-known blocker of TRPM7, a channel that plays a major role in cellular Mg(2+) homeostasis. Neither [ATP](i) nor pH(i) correlated with changes in [Mg(2+) ](i) . The results indicate that imipramine suppresses Mg(2+) -permeable channels presumably through a direct effect on the channel domain. This inhibitory effect appears to contribute, at least partially, to the link between antidepressants and the risk of vascular diseases.

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Magnesium is associated with several important cardiovascular diseases. There is an accumulating body of evidence verifying the important roles of Mg(2+)-permeable channels. In the present study, we estimated the intracellular free Mg(2+) concentration ([Mg(2+)](i)) using (31)P-nuclear magnetic resonance ((31)P-NMR) in porcine carotid arteries. pH(i) and intracellular phosphorus compounds were simultaneously monitored. Removal of extracellular divalent cations (Ca(2+) and Mg(2+)) in the absence of Na(+) caused a gradual decrease in [Mg(2+)](i) to approximately 60% of the control value after 125 min. On the other hand, the simultaneous removal of extracellular Ca(2+) and Na(+) in the presence of Mg(2+) gradually increased [Mg(2+)](i) in an extracellular Mg(2+)-dependent manner. 2-aminoethoxydiphenyl borate (2-APB) attenuated both [Mg(2+)](i) load and depletion caused under Na(+)- and Ca(2+)-free conditions. Neither [ATP](i) nor pH(i) correlated with changes in [Mg(2+)](i). RT-PCR detected transcripts of both TRPM6 and TRPM7, although TRPM7 was predominant. In conclusion, the results suggest the presence of Mg(2+)-permeable channels of TRPM family that contribute to Mg(2+) homeostasis in vascular smooth muscle cells. The low, basal [Mg(2+)](i) level in vascular smooth muscle cells is attributable to the relatively low activity of this Mg(2+) entry pathway.

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Polar body formation is an extremely unequal cell division. In order to understand the mechanism of polar body formation, morphological changes at the animal pole were investigated in living oocytes of the starfish, Asterina pectinifera, and the amounts of cortical actin filaments were quantitatively estimated after staining the maturing oocytes with fluorescently-labeled phallotoxins using a computer and image-processing software. Formation of a bulge, which is presumed to become a polar body, and the anaphase separation of chromosomes occurred simultaneously. When the bulge became large, one group of chromatids moved into the bulge. The dividing furrow then formed and finally a polar body formed. Just at the time of bulge formation, the intensity of the fluorescence produced by the actin filaments at the top of the animal pole began to decrease, and subsequently the intensity at the top fell to half of the original value. On the other hand, the fluorescence intensity at the base of the bulge increased gradually. This actin accumulation at the base created a dividing furrow around the top of the animal pole as the bulge grew. Even when the polar body formation was inhibited mechanically, a similar pattern of actin deficiency and accumulation in the cortex near the animal pole was observed. This indicates that such regulation of filamentous actin can take place without bulging. Therefore, polar body formation is initiated by the bulging of the cortex weakened by actin deficiency and followed by contraction of the base of the bulge reinforced by actin accumulation.

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Nuclear magnetic resonance (NMR) spectroscopy of the heart is normally carried out using whole heart preparations under coronary perfusion. In such preparations, either radical changes in ionic composition of the perfusate or applications of numerous drugs would affect coronary microcirculation. This report communicates the first (31)P NMR spectroscopy study using a heart slice preparation (left ventricular slices) superfused with extracellular medium. The ratio of phosphocreatine concentration to ATP concentration was approximately 2.1. Also, intracellular pH and Mg(2+) concentration ([Mg(2+)](i)), estimated from the chemical shifts of inorganic phosphate and ATP, were comparable with those under retrograde perfusion. [Mg(2+)](i) was significantly increased by the removal of extracellular Na(+), supporting the essential role of Na(+)-coupled Mg(2+) transport in Mg(2+) homeostasis of the heart. Heart slice preparation could also be used to evaluate the potency of cardiac drugs, regardless of their possible effects on coronary microcirculation.

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Aldosterone promotes vascular smooth muscle cell proliferation and endothelial dysfunction, suggesting the contribution to in-stent restenosis (ISR). This study evaluated any relation between plasma aldosterone levels and ISR 6 months after successful coronary stenting. We enrolled 156 consecutive patients with stable angina who underwent coronary bare metal stenting. Plasma aldosterone levels and other serum markers known to influence cardiovascular events were measured in all patients at baseline. Patients with restenosis were found to have significantly higher plasma aldosterone levels than their counterparts without restenosis (162 +/- 60 vs 122 +/- 60 pg/ml, p = 0.007). On logistic regression analysis, even after adjusting for clinical, angiographic, and other confounding variables, plasma aldosterone level per 10 pg/ml (odds ratio 1.34, 95% confidence interval 1.10 to 1.63, p = 0.006) proved to be the independent predictor of ISR. The area under the receiver-operating characteristic curve for plasma aldosterone level was 0.75, and the optimal cut-off value identified by receiver-operating characteristic analysis was 141.9 pg/ml, which had a predictive accuracy of 69%. In conclusion, the present findings indicate that plasma aldosterone levels at baseline are independent predictors of ISR and may constitute a potential therapeutic target.

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We determined the tension over the entire surface of the sea urchin eggs during cytokinesis, on the basis of the intracellular pressure and cell shape. This allowed us to determine the temporal changes in both the distribution of local forces and the total force produced in the whole cell cortex. A spike-like peak at anaphase and a broader peak at the onset of furrowing were observed in the time-course of the total force. Treatment of the eggs with cytochalasin D, blebbistatin, ML-9, or ML-7 significantly lowered the total force when they inhibited cytokinesis, suggesting that the tension results mainly from the interaction between intact actin filaments and activated myosin II. Myosin II would function as a motor, not only in the furrow region, but over a wide area of the cell surface, because the sum of the tensions outside the furrow region was larger than that inside the furrow region throughout cytokinesis. The distribution of the local force revealed that a global increase in the cortical force started well before the onset of furrowing, and that the force inside the furrow region continued to increase despite the decrease in the force outside the furrow region after the onset of furrowing. The spatial and temporal patterns of the force over the entire surface support the hypothesis that there are two separate but coordinated actomyosin activation mechanisms, one of which induces global activation of the cortex and the other of which then maintains the contractility only inside the furrow region.

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Kalihinol F, a naturally occurring diterpene from a marine sponge, Acanthella sp., inhibited chromosome separation in fertilized starfish (Asterina pectinifera) eggs but allows the first cleavage to occur, thereby forming unseparated metaphase chromosomes which were elongated between the two daughter cells. The chromosomes were eventually torn off in the embryonic cells. Most of the cells gradually lost the chromosomes during the cell cycle progression. The embryonic development halted at the morula stage just before the onset of blastulation. The mitotic failure occurred when kalihinol F was applied to a fertilized egg during the second meiotic process, but not after the completion of the second meiotic division. Kalihinol F inhibited topoisomerase I activity in vitro, but had no effects on activities of DNA polymerases alpha, beta, and gamma, and of topoisomerase II. These results suggest that the topoisomerase I plays an essential role in meiosis II in this species.

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The effects of the phosphatase inhibitors, okadaic acid (OA), adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and calyculin A (CL-A) on anaphase chromosome movement, cytokinesis, and cytoskeletal structures at cell division were examined by being microinjected into mitotic sand dollar eggs. When OA was injected, chromosome movement was inhibited and, moreover, chromosomes were ejected from the polar regions of the mitotic apparatus. By immunofluorescence, microtubules were observed to be severed in the OA-injected eggs, causing the smooth cell surface to be changed to an irregular surface. When ATPgammaS and CL-A were injected, the effect on cell shape was remarkable: In dividing eggs, furrowing stopped within several seconds after injection, small blebs appeared on the cell surface and became large, spherical or dumbbell cell shapes then changed to irregular forms, and subsequently cytoplasmic flow occurred. Microfilament detection revealed that actin accumulation in the cortex, which was not limited to the furrow cortex, occurred shortly after injection. Cortical accumulation of actin is thought to induce force generation and random cortical contraction, and accordingly to result in bleb extrusion from the cortex. Consequently, the phosphatase inhibitors inhibited the transition from mitosis to interphase by mediating cortical accumulation of actin filaments and/or fragmentation of microtubules.

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During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC.

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In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.

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Using the starfish oocyte and zygote, we investigated the abilities of the centrosome at maturation and cleavage divisions to form the aster and induce cytokinesis, in order to determine differences between these divisions. The transplanted centrosome originated from both maturation and cleavage, induced an additional furrow in cleavage in the recipient cells, but did not induce abnormal polar body formation at maturation. Although it organized an additional aster in the recipient cell in both divisions, a difference in size among asters formed was recognized. Therefore, mitotic asters were stabilized with hexylene glycol in order to measure their radius and clarify this difference. The mean radius (14.4 µm) of the first meiotic aster was significantly smaller than that (20.4 µm) of the aster at the first cleavage. The transplanted cleavage centrosome formed as small an aster as the recipient's own at maturation divisions. When zygotes were briefly treated with colcemid so that the zygotes could not perform cytokinesis but did perform karyokinesis, the size of aster became the same as that in meiosis. These results prove that although any centrosome functions as a microtubule organizing center independent of its origin, the size of the resultant aster decides whether or not cytokinesis would be induced.

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Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis. Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV-irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2-0.3 µm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 µm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule-dependent, perhaps sharing the same basic mechanisms.

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Inhibition of cleavage by colchicine was examined by microinjecting colchicine solution into one of the blastomeres of a sea urchin egg at the two-cell stage. Cleavage was inhibited if the microinjection was made before a critical point prior to the cleavage, whereas cleavage occurred in spite of the destruction of the mitotic apparatus if the microinjection was made after the critical point. The critical point was 10 min before the mid-stage of the cleavage in Clypeaster japonicus and 8 min before the mid-stage in Temnopleurus toreumaticus at 20 ± 1°C, corresponding to the beginning of anaphase. The threshold for the cleavage inhibition of colchicine was estimated to be 3 × 10-5 M to 3 × 10-6 M in final concentration in the cell.