Ken-ichi Nakamura

Sedative Effect on Humans of Inhalation of Essential Oil of Linalool: Sensory Evaluation and Physiological Measurements Using Optically Active Linalools

Abstract The sedative properties of linalool were examined using the optically active linalools, (R)-(−)-, (S)-(+)- and (RS)-(±)-forms. (R)-(−)-linalool with specific rotation of [α]D= −15.1° was isolated by repeated flash column chromatography from lavender oil, while (S)-(−)-linalool with [α]D= +17.4° and (RS)-(±)-linalool with [α]D=0° and content of (R)-form 50.9% and (S)-form 49.1% were obtained from coriander oil and commercial linalool, respectively, by using the same method. The effect of (RS)-(±)-linalool in our experiments, which was quite similar to that of linalool, with much more favorable impressions accompanied by a tendency to a greater decrease of the beta wave after hearing environmental sound after, rather than before work, appeared to be identical to that observed for (R)-(−)-linalool. The feature was just the reverse in the case of (S)-(+)-linalool.

Adenylate kinase is tightly bound to axonemes of Tetrahymena cilia.

Cilia of Tetrahymena thermophila possess adenylate kinase [ATP:AMP phosphotransferase, EC 2.7.4.3] activity. More than 95% of the total activity was recovered in the axonemal fraction when cilia were demembranated with 0.2% Nonidet P-40. There was no loss of the specific activity of adenylate kinase when axonemes were thoroughly washed with HMEK solution (10 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 M KCl, pH 7.4). These results suggest that adenylate kinase is tightly bound to axoneme. Solubilization of adenylate kinase was markedly increased when axonemes were incubated in HME buffer (10 mM HEPES, 1 mM MgCl2, 0.1 mM EDTA, pH 7.4) containing concentrations of NaCl (or KCl) exceeding 1 M. Therefore, routine isolation of adenylate kinase from axonemes involved pre-extracting axonemes with 0.5 M NaCl in HME buffer followed by extraction in HME buffer containing 1.5 M NaCl. Native-gel electrophoresis of the high salt extract revealed two protein bands (band I and band III). An active staining for adenylate kinase showed a single active band corresponding to the position of band III. Two-dimensional gel electrophoresis using native-gel electrophoresis in the first dimension and SDS-PAGE in the second dimension suggests that band III protein contains at least nine polypeptides ranging from 21 to 110 kDa.

Standard free energy maps for the hydrolysis of ATP as a function of pH and metal ion concentration: Comparison of metal ions

Abstract The observed equilibrium constant K obs for the hydrolysis of ATP to ADP and inorganic phosphate has been calculated as a function of pH and metal ion concentration pM (- log [M]) at 25 °C and μ = 0.2 with the use of literature values of the acid dissociation and complex dissociation constants for the phosphates. The resulting standard free energy changes ΔG °′ are presented by means of contour diagrams for the range pH 4–10 and pM 1–7. These maps summarize the results of some 1900 calculations per diagram, and clearly simulate a differential effect of the metal ions of interest, including Mg 2+ , Ca 2+ , Sr 2+ , Mn 2+ , Li + , Na + and K + , on the equilibrium hydrolysis of ATP.

Dynein of sperm flagella of oyster beloning to protostomia also has a two‐headed structure

Summary— An axonemal dynein was purified from the sperm of Crassostrea gigas, an oyster belonging to Protostomia. The molecular masses of component polypeptides were almost equivalent to those of other dyneins. Biochemical and biophysical properties were also quite similar. For example, UV‐cleavage, inhibition of ATPase by vanadate and induction of microtubule gliding were observed with the axonemal dynein. The oyster dynein had a two‐headed structure as had the outer arm dynein of Deuterostomia such as sea urchin, rainbow trout and bull spermatozoa. On the other hand, dyneins of Protozoa are three‐headed particles. From the evolutional point of view, it is likely that the number of heads of dynein molecule decreased when Metazoa evolved from Protozoa.

Analysis of tubulin isoforms by two-dimensional gel electrophoresis using SDS-polyacrylamide gel electrophoresis in the first dimension

A two-dimensional electrophoretic system using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the first dimension and isoelectric focusing (IEF) in the second dimension was devised. In spite of its simplicity, this method could show a markedly high resolution for tubulin isoforms and moreover could classify them into alpha- or beta-tubulin as a two-dimensional profile. With this method, seven alpha- and four beta-tubulin isoforms could be detected within axoneme from Tetrahymena cilia. Moreover this method could also resolve tubulin isoforms from the rabbit brain. These results indicate that the present two-dimensional gel electrophoresis is a useful tool for the electrophoretic analysis of tubulin isoforms from various sources.

Picosecond dynamics of magnetic polarons in Cd1−xMnxTe

Abstract We report the dynamical behavior of magnetic polarons (MPs) in Cd1−xMnxTe (x=0.05-0.6). Exciton Luminescence in samples of x=0.05−0.1 shows the dynamics of the acceptor-bound MP, where the formattion time of the bound MP is 1–2 ns. For x=0.2−0.4 the free MP is created and relaxes into deep localized states in the fluctuating band gap, forming the localized MP. For x⩾0.5 the lifetime of the MP decreases to less than 250 ps, which is caused by fast-energy transfer of the exciton energy to delectrons of the Mn ions. The spin-glass ordering of the Mn spins for x⩾0.2 at low temperature is found to affect the MP energies.

Application of stains-all staining to the analysis of axonemal tubulins: identification of β-tubulin and β-isotubulins

The cationic dye, Stains-all, is known to stain brain beta-tubulin blue and alpha-tubulin red (Serrano, L. et al. (1986) J. Biochem. Biophys. Methods 12, 281-287; Serrano, L. et al. (1989) Biochem. Int., 19, 235-246). The present experiments show that this stain can also be applied to detect beta-tubulin in axonemal tubulins from various sources such as cilia of protozoa, sperm flagella of echinoderm, and sperm flagella of mollusc. Furthermore, these experiments showed that it selectively stains isoforms of axonemal beta-tubulin blue following isoelectric focusing, whereas those of alpha-tubulin are stained red. These results indicate that Stains-all staining is a useful tool for electrophoretic analysis of axonemal tubulins.

Flagellar adenosine triphosphatases from annelid spermatozoa: Electrophoretic identification of dyneins

Abstract Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus . Dialysis of the axonemes against 1 m m Tris-HCl buffer (pH 8.3)-0.1 m m EDTA-0.1 m m dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas .

Comparison of axonemal proteins from two species of Tetrahymena. II. Difference in heat stability of microtubules

Abstract Tetrahymena thermophila , a heat resistant species, could swim at a speed of about 60% of that of the control even after being incubated at 40°C for 10 min, whereas Tetrahymena pyriformis did not show any motility after thermal treatment at 40°C for 5 min. We have found that more than 30% of tubulins are solubilized from the axonemes of T. pyriformis by heat treatment at 40°C for 30 min, while less than 15% of the tubulins are solubilized in T. thermophila . Electron microscopic observations revealed selective solubilization of tubulins from the B-microtubules of T. pyriformis by thermal treatment. Comparative analysis of axonemal tubulin isotypes shows highly similar profiles between the two Tetrahymena . This suggests that the heat stability of axonemal microtubules observed in T. thermophila is not due to the properties of the tubulin itself, but that other axonemal components which protect the microtubules against heat denaturation. In summary, combination of the present results and our previous findings on dynein (Takaya et al. Comp. Biochem. Physiol. 1995;112B:727–32) show that T. thermophila , a heat tolerant species, installs heat resistant microtubules together with heat-stable dynein in order to adapt to a high temperature environment.

Solubilization of dynein from Tetrahymena ssp. axonemes using phosphate analogues

One major protein was selectively solubilized when phosphate analogues, such as inorganic vanadate (Vi), beryllium fluoride (BeFx) or aluminum fluoride (AlFx), were added to ciliary axonemes of Tetrahymena ssp. (T. pyriformis or T. thermophila) in the presence of ATP. This protein contains three high molecular weight polypeptides, characteristic of an outer arm dynein. Electron microscopic observation of the axonemes after solubilization using ATP and Vi revealed axonemes partially lacking outer arm dyneins. These results suggest that the solubilized protein is an outer arm dynein and also that a dynein-ADP-phosphate complex decreases its affinity with the adjacent microtubules within axonemes. Limited digestion with chymotrypsin revealed that each solubilized dynein has a similar conformation, but it is markedly different from that of dynein in the absence of ATP or a phosphate analogue. The solubilized dynein obtained by the addition of Vi and ATP to axonemes was digested by UV irradiation to yield at least five new polypeptides (240, 230, 225, 180 and 160 kDa) but the dyneins solubilized by BeFx (or AlFx) in the presence of ATP did not produce any photocleavage products under the same conditions.