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Biomedical subjects

E Nishida

Publications and source records attributed to E Nishida.

At least 235 records · Page 13Linked to original sources
PubMed1984

33K protein--an inhibitory factor of tubulin polymerization in porcine brain.

A factor (33K protein) that modulates tubulin polymerization in vitro has been purified to homogeneity from porcine brain by ammonium sulfate fractionation and Whatman DE52, Toyo-pearl HW65C and Bio-Gel A 0.5 m column chromatographies. The purified fraction was free of nucleic acids and sugars. The activity of the purified 33K protein is pronase E sensitive but apparently heat- and trypsin-resistant though it undergoes tryptic digestion. The 33K protein inhibits polymerization of brain microtubule proteins in a dose-dependent manner and partially depolymerizes preformed microtubules. It also inhibits polymerization of purified starfish tubulin and microtubule elongation involving fragellar outer doublet microtubules and purified porcine brain tubulin. This suggests that the target of the 33K protein is tubulin rather than microtubule-associated proteins. The 33K protein causes incomplete depolymerization of microtubules and a new steady state is quickly attained which is apparently independent of microtubule mass concentration. Divalent cations such as calcium and magnesium do not modulate the inhibitory activity of the 33K protein.

Animals↗
PubMed1984

An 18K protein from ascites hepatoma cell depolymerizes actin filaments rapidly.

Several actin binding proteins were isolated from ascites hepatoma cells AH7974 by DNase I affinity chromatography. Among them, a protein having a molecular weight of 18,000 was further purified by DEAE cellulose and hydroxyapatite column chromatographies and gel filtration on a Sephadex G-75 column. The 18K protein not only inhibits actin polymerization but also depolymerizes actin filaments. This conclusion was supported by viscosity and fluorescence intensity measurements and the DNase I inhibition assay. A chemical cross-linking experiment suggested that the 18K protein binds to monomeric actin and forms and 18K-actin 1:1 complex. The net depolymerization rate by the 18K protein measured by the DNase I inhibition assay was slower than the rapid reduction of the fluorescence intensity of pyrene-labeled F-actin upon addition of the 18K protein. This result suggests that the 18K protein not only binds to monomeric actin but also binds to actin filaments directly. The sedimentation assay showed that a part of the 18K protein was cosedimented with actin filaments. Electron microscopic observations demonstrated that the 18K protein decreased the amount of actin filaments and the remaining filaments appeared to be decorated and distorted by the 18K protein. The 18K protein had no Ca2+ ion sensitivity and exhibited the same effect on both this tumor actin and muscle actin.

Actins↗
PubMed1984

Spinal pathway of the milk-ejection reflex in the rat.

The effects of lesions of the spinal cord on the milk-ejection reflex evoked by suckling were studied in urethane-anesthetized lactating rats. All lesions were made between C6 and C7 vertebrae and milk ejection was monitored by recording intramammary pressure. In the first experiment on the rats with bilateral lesions, a 3-h suckling test with 5 pups on each side was performed. Eleven (84.6%) of 13 rats with the section of the dorsal funiculus (Group 2), and 12 (85.7%) of 14 rats with the combined section of the dorsal and ventral funiculi (Group 4) displayed regular milk ejection. The incidence of milk ejection in both groups was not significantly different from 81.8% (9 rats) of the 11 sham-operated rats (Group 1). In contrast, none of the 12 rats with bilateral section of the lateral funiculus (Group 3) displayed milk ejection and the incidence of milk ejection was significantly lower than that in Group 1. In the second experiment on the rats with unilateral section of the lateral funiculus, bilateral suckling with 10 pups (5 pups on each side) and unilateral suckling (both ipsilateral and contralateral to the lesion) with 5 pups were consecutively performed in the 10 rats. Milk ejection was induced in 50% by contralateral suckling and in 100% by bilateral suckling, and the incidence was significantly higher than that (0%) observed during ipsilateral suckling. A significant difference in the incidence of milk ejection was also observed between contralateral and bilateral sucklings.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗
PubMed1983

Kinetic analysis of actin polymerization.

The kinetics of actin polymerization were analyzed by measuring the changes in absorbance which accompany the G-F transformation of actin. In these studies, gel-filtered actin was polymerized in the absence of shearing stress under physiological ionic conditions. Self-polymerization was found to be characterized as a process having a lag phase followed by a pseudo-first-order decay process, suggesting that actin polymerization consists of distinct nucleation and elongation phases. The size of the nucleus was estimated to be two to four monomer units by analyzing the actin concentration-dependences of the rate constant for the pseudo-first-order process, the maximal rate of polymerization and the half-polymerization time. The elongation reaction was induced by mixing actin filament seeds with actin monomers under conditions where spontaneous nucleation is slow. This elongation was also found to be a pseudo-first-order process, indicating that spontaneous nucleation was negligible. Our data suggested that the initial rate of elongation was proportional to both the number concentration of actin filaments and the actin monomer concentration above the critical concentration, and that the depolymerization rate was proportional to the number concentration of actin filaments but independent of monomer concentration. The results of direct analysis of the depolymerization reaction were consistent with this suggestion. These studies strongly support the condensation polymerization mechanism as a model for actin polymerization.

Actins↗
PubMed1983

Kinetic and thermodynamic analyses of outer doublet tubulin polymerization.

The kinetics and thermodynamics of microtubule assembly were studied using outer doublet tubulin isolated from starfish sperm. Purified tubulin was gel-filtered just prior to the experiments. The self-polymerization process was found to be characterized by a lag followed by a growth phase that did not show a single first-order decay reaction. This suggested that the nucleation and elongation steps are not completely separate. The maximal rate of polymerization and the reciprocal of the duration of lag time increased approximately in proportion to the third and second powers of tubulin concentration above the critical concentration, respectively. Measurement of temperature dependence of the critical concentration gave a linear van't Hoff plot with delta H degree = 13.5 kcal/mol and delta S degree = 69 e.u.

Animals↗
PubMed1983

Pulsatile secretion of prolactin and oxytocin during nursing in the lactating rat.

The secretory profile of prolactin and oxytocin in response to suckling stimuli by litters was studied in unanesthetized and urethane-anesthetized lactating rats. Serum prolactin levels were determined by radioimmunoassay. Oxytocin released at milk-ejection reflex was monitored by the changes in the intramammary pressure and/or the characteristic pup's reaction associated with the milk ejection. Serum prolactin concentrations began to rise earlier than the first milk ejection in unanesthetized rats, but they were never elevated without the appearance of milk ejections in urethane-anesthetized rats. Pulsatile fluctuation in serum prolactin levels at 6-15 min intervals was observed in the nursing period when 10 pups were suckling continually. The intermittent milk-ejection reflex occurred not always but preponderantly (64-91%) when the serum prolactin levels were at the nadir of the fluctuation. Injection of an estimated dose of oxytocin released at each milk ejection (1 mU) did not change the serum prolactin levels. These results indicate that the mechanism for prolactin release may be more susceptible to the effects of anesthesia than that for oxytocin release in response to the suckling stimuli and that the release of both the hormones is pulsatile in nature and be influenced by a common biological clock during the nursing period.

Animals↗
PubMed1983

The regulation of actin polymerization by the 88K protein/actin complex and cytochalasin B.

The action of the 88K protein/actin complex (88K/A) and cytochalasin B on various aspects of actin polymerization kinetics was investigated, and the results were interpreted in terms of the condensation polymerization and treadmilling mechanism for actin polymerization. A substoichiometric concentration of 88K/A promotes actin nucleation under physiological salt conditions, especially in the presence of Ca2+. In addition, it reduces both the elongation rate and the depolymerization rate by up to 70% and inhibits annealing of the actin filaments. As a consequence, the average length of actin filaments polymerized with 88K/A becomes less than that of a control. These data indicate that 88K/A caps one end of actin filaments or actin oligomers where in the absence of 88K/A the rates for both association and dissociation of monomers are faster than at the other end. In a KCl/MgCl2 medium, 88K/A increases the steady state monomer concentration (the critical concentration) to a limited extent. This is explained by assuming that 88K/A caps the lengthening end (in treadmilling) of actin filaments, where the critical concentration is lower than at the other end (the shortening end). Moreover, cytochalasin B which has been shown to bind to the barbed end of actin filaments does not affect the 88K/A-nucleated actin polymerization. Therefore, it is strongly suggested that 88K/A caps the barbed end of actin filaments and that the barbed end is the lengthening end as well as the rapidly growing and rapidly depolymerizing end. The result obtained in the study on the action of cytochalasin B was consistent with this suggestion.

Actins↗
PubMed1982

Shear stress modulates the action of the 88 K protein-actin complex on actin polymerization.

An actin-regulatory protein, 88 K protein-actin complex (88 K-actin complex) isolated from porcine brain, increases the rate of actin polymerization as measured by viscometry which gives rise to shearing stresses. In contrast, when measured by the optical absorption method without solvent flow, the complex does not promote actin polymerization but inhibits both the initial rate and final extent of polymerization. These results suggest that shearing stresses are capable of modulating association of actin with the 88 K-actin complex.

Actins↗
PubMed1982

The interaction between calmodulin and microtubule proteins. IV. Quantitative analysis of the binding between calmodulin and tubulin dimer.

We reported previously that calmodulin binds to tubulin in a Ca2+-dependent manner, thereby inhibiting microtubule assembly. In this work, we quantitatively investigated the binding between calmodulin and tubulin by applying two analytical methods. One was the frontal analysis using affinity chromatography originally developed by Kasai and Ishii (J. Biochem. 84, 1061-1069, 1978). The use of tubulin-Sepharose columns gave a dissociation constant of 4.0 microM. The other was the equilibrium gel filtration developed by Hummel and Dreyer (Biochim. Biophys. Acta 63, 532-534, 1962). This method using a Sephadex G-100 column provided a dissociation constant of 3.5 microM under the same medium conditions as in the frontal analysis, and it was found that 2 mol calmodulin could bind to 1 mol tubulin. Furthermore, the frontal analysis method was convenient for studies on the effect of temperature and ionic strength on the binding. Upon elevating the temperature, the dissociation constant increased. Increase in the ionic strength also increased the dissociation constant.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

A new regulatory protein that affects the state of actin polymerization.

A new protein factor that regulates the state of actin polymerization was purified from porcine brain by DNase I-agarose chromatography. The purified protein factor consisted of a 1 : 1 complex of 88,000 dalton polypeptide and actin. When actin was polymerized by salt in the presence of the factor, the steady-state viscosity and the sedimentability were greatly reduced. The extent of the reductions was found to be greater in the presence of Ca2+ than in its absence.

Actins↗
PubMed1981

Phosphorylation of microtubule-associated proteins (MAPs) and pH of the medium control interaction between MAPs and actin filaments.

Interaction of microtubule-associated proteins (MAPs) with actin filaments at neutral pH is inhibited by phosphorylation of MAPs. Phosphorylated MAPs are less potent than unphosphorylated ones in increasing the low-shear viscosity of actin filaments in the neutral pH range. The ability of unphosphorylated MAPs to crosslink actin filaments falls off sharply above pH 7.5. Upon phosphorylation, the crosslinking ability of the MAPs peaks sharply between pH 6.2 and 6.3. Thus, the MAPs-actin interaction can be regulated by phosphorylation of MAPs and small changes in the physiological range of pH.

Actins↗
PubMed1981

Changes of serum concentrations of dehydroepiandrosterone, its sulfate, 11-deoxy-17-ketosteroids, cortisol, estradiol, testosterone, prolactin and gonadotropins in normal prepubertal and pubertal females.

The concentrations of serum dehydroepiandrosterone (DHA), DHA sulfate (DHA-S), 11-deoxy-17-ketosteroids (11-deoxy-17-KS), cortisol (F), estradiol (E2), testosterone (T), prolactin (PRL), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were measured by means of radioimmunoassays in 157 prepubertal and pubertal girls whose age ranged from 5 to 16. Among the hormones studied, DHA exhibited a first significant increment at age 8, ahead of all other hormones. The significant increases of 11-deoxy-17-KS and DHA-S occurred at age 9 and 10, respectively. After the occurrence of the significant increment, the concentrations of these androgens increased progressively with age. DHA/DHA-S ratio showed a decreasing trend with the advance of age. F did not show any significant variation with age. E2 increased significantly at age 11 and T at age 10. PRL increased significantly at age 12 but its concentration did not seem to vary very much with age after 9. FSH and LH started to increase after age 9, with their significant increases occurring at age 10 and 9, respectively. These results confirm that during the course of pubertal development in girls, adrenal androgens increase first, which is followed by pituitary gonadotropins and then gonadal estrogen.

17-Ketosteroids↗
PubMed1981

A new protein factor that modulates both microtubule assembly and actin polymerization.

A 94,000-dalton protein that has been shown to modulate microtubule assembly in a Mg2+- or Ca2+-dependent manner (Nishida & Sakai (1980) J. Biochem. 88, 1577-1586) was here shown to inhibit actin polymerization. The protein factor inhibited the rate and the extent of actin polymerization under nearly physiological conditions (for example, in 3 mM MgCl2 plus 90 mM KCl at pH 6.8). The inhibitory effect was dependent on divalent cation concentration; the lower the Mg2+ concentration was, the weaker the inhibitory effect. The inhibition was stoichiometric; addition of the protein factor caused a linear decrease in the extent of actin polymerization as measured by the viscosity increase, and under optimal conditions for inhibition about an equimolar amount of the protein factor was sufficient to inhibit the actin polymerization completely. Furthermore, inclusion of the protein factor increased the critical concentration of actin required for polymerization by a concentration nearly equivalent to that of the added factor. These results suggest the formation of a 1 : 1 complex between actin and the protein factor, which does not polymerize at all. Another assay for actin polymerization, pelleting of actin filaments by ultracentrifugation, confirmed the inhibitory effect of the protein factor. In addition to the inhibitory effect on polymerization, the protein factor had the ability to depolymerize actin filaments. We have temporarily called this protein factor PI factor. It may play an important role in cell structure and function through its interactions with actin and microtubules.

Actins↗
PubMed1980

Calcium sensitivity of sea urchin tubulin in in vitro assembly and the effects of calcium-dependent regulator (CDR) proteins isolated from sea urchin eggs and porcine brains.

Tubulin was purified from unfertilized eggs or embryos of the sea urchin. In vitro assembly of sea urchin tubulin into microtubules (MTs) was highly sensitive to Ca2+ ions. At low ionic strength, the self-assembly was inhibited by 5 x 10(-7) M free Ca2+, and MT elongation which was initiated by mixing sea urchin tubulin with Tetrahymena cilia outer fiber fragments was inhibited by 10(-6)--10(-5) M free Ca2+. Increase in the ionic strength of the assembly medium lowered the Ca2+ sensitivity, in contrast to brain MT assembly. Brain microtubule-associated proteins (MAPs), which stimulated the sea urchin MT assembly, lowered the Ca2+ sensitivity. Calcium-dependent regulator (CDR) protein was purified from unfertilized eggs of the sea urchin by the pruification procedures used for brain CDR. The sea urchin CDR associated with brain tubulin in a Ca2+-dependent manner indistinguishable from that of brain CDR when assayed by the ammonium sulfate fractionation method. Moreover, both CDRs had Ca2+-dependent inhibitory effects on brain MT assembly. However, neither procine brain CDR nor sea urchin CDR showed a Ca2+-dependent inhibitory effect on sea urchin MT assembly. Sea urchin tubulin assembly initiated by brain MAPs was not inhibited by these CDRs with Ca2+, either. These results suggest that CDRs are not species-specific, but that tubulins respond in a highly specific manner.

Animals↗
PubMed1980

Isolation of calmodulin from the protozoan, Tetrahymena pyriformis, by the use of a tubulin-Sepharose 4B affinity column.

We developed a simple new method for the isolation of calmodulin, a ubiquitous Ca2+-binding protein, by using tubulin-Sepharose 4B column chromatography, and succeeded in rapid isolation of calmodulin from Tetrahymena pyriformis. The procedure was also shown to be successfully applicable to the isolation of calmodulins from starfish ovary, porcine brain, and monkey brain and, therefore, may be of general use for the rapid isolation of calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗