Biomedical subjects

K Mihashi

Publications and source records attributed to K Mihashi.

At least 19 recordsLinked to original sources

PubMed2001

Fluctuation of local points of F-actin sliding on the surface-fixed H-meromyosin molecules in the presence of ATP.

F-actin fragments fluorescently labeled with rhodamine-phalloidin were copolymerized with non-labeled F-actin fragments. F-actin copolymer consisted of several bright (fluorescent) and dark (non-fluorescent) stripes of approximately 1 microm in width. Local motion of individual speckled F-actin was investigated by measuring translocation fluctuation of several tracing points marked on the actin filament. The tracing points included the borders between neighboring bright and dark stripes, as well as the tip and tail of the filament. For speckled F-actin with an average sliding speed of 4.6 microm/s at 23 degrees C, the translocation distance of the tracing points (per 0.1 s) showed significant fluctuation, of the order of +/-0.12 microm/s, approximately 25% of the sliding speed. The fluctuation correlation of the translocation distance between two tracing points decreased as the distance between them increased. Statistical analysis of the correlation length of the translocation distance L(c) showed that L(c) increased with the sliding speed of the actin filament. The sliding speed, however, saturated as the correlation length became close to the persistence length of the bending elasticity of F-actin. On the contrary, the correlation length of change in the translocation direction was essentially equal to the persistence length of F-actin, independent of the sliding speed. These results suggest that elasticity of the actin filament underlies the sliding velocity of F-actin.

Actins↗

PubMed2001

An ellagic compound and iridoids from Cornus capitata root cultures.

An ellagic acid derivative, 3,3'-di-O-methylellagic acid 4-(5"-acetyl)-alpha-L-arabinofuranoside, and two iridoid glucosides, 6alpha-dihydrocornic acid and 6beta-dihydrocornic acid, were isolated from Cornus capitata adventitious roots cultured in Murashige-Skoog (Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-487) liquid medium containing 10 microM CuSO(4). Three known related metabolites, i.e. stenophyllin H1, dihydrocornin and cornin were also produced in the root cultures. The chemical structures were characterized by analysis of spectroscopic data.

Ellagic Acid↗

PubMed2001

Biodisposition of FITC-labeled aloemannan in mice.

Biodisposition of FITC-labeled aloemannan (F-AM) with the homogenate from some organs in mice was demonstrated. F-AM was metabolized only by the mucosa from the large intestine into smaller molecules that were effectively absorbed in mice. The homogenate from the other tissues did not affect the metabolism of F-AM. The degraded product (1) of F-AM after incubation with 10% feces homogenate for 24 h was chromatographed on a highly porous polymer and a Sephadex LH-20 column to provide an FITC-degraded fraction (2), which was shown to have a molecular weight of 800 D on Sephadex G-25 gel permeation. Metabolite 2 was examined by physicochemical methods and shown to be a mixture of FITC-hexose and -2 hexose on FAB-MS. An FITC-degraded fraction (3) with a molecular weight of 3 KD was obtained by 6-h incubation with 10% feces homogenate on Sephadex G-25 column chromatography and was shown to be a mixture of FITC-9 and 12 x hexose on TOF-MS.

Adjuvants, Immunologic↗

PubMed1999

Antiproliferative constituents from umbelliferae plants. V. A new furanocoumarin and falcarindiol furanocoumarin ethers from the root of Angelica japonica.

The CHCl3 extract of the root of Angelica japonica showed high inhibitory activity against human gastric adenocarcinoma (MK-1) cell growth. From this extract, a new furanocoumarin named japoangelone and four furanocoumarin ethers of falcarindiol, named japoangelols A-D, were isolated together with caffeic acid methyl ester, four polyacetylenic compounds (panaxynol, falcarindiol, 8-O-acetylfalcarindiol, and (9Z)-1,9-heptadecadiene-4,6-diyne-3,8,11-triol), eight coumarins (osthol, isoimperatorin, scopoletin, byakangelicin, xanthotoxin, bergapten, oxypeucedanin methanolate, and oxypeucedanin hydrate), and two chromones (3'-O-acetylhamaudol, and hamaudol). The structures of the new isolates were determined based on spectral evidence. The ED50 of isolates against MK-1, HeLa, and B16F10 cell lines are reported.

Animals↗

PubMed1998

Novel cytotoxic diterpenes from the stem of Dysoxylum kuskusense.

Three novel diterpenes, dysokusones A (1), B (2), and C (3), were isolated from the stem of Dysoxylum kuskusense as cytotoxic substances. The structures were established by spectroscopic examinations. Compounds 1, 2, and 3 were cytotoxic toward HL-60(TB) cells with EC50 values of 2.25, 6.35, and 2.37 microM, respectively. Compound 1 also displayed cytotoxicity against K-562 and NCI-H522 cells with EC50 values of 5.04 and 4.80 microM, respectively.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Novel antiproliferative falcarindiol furanocoumarin ethers from the root of Angelica japonica.

Four novel antiproliferative furanocoumarin ethers of falcarindiol, named japoangelols A (8.5), B (7.2), C (7.4), and D (8.4), were isolated from the root of Angelica japonica together with panaxynol (0.3), falcarindiol (3.2), (9Z)-1,9-heptadecadiene-4,6-diyne-3,8,11-triol (2.2), and 8-acetoxyfalcarinol (3.2). Structures were established from the spectroscopic evidence, and the inhibitory activities (ED50, microgram/ml, shown in the parentheses) were evaluated using the MTT assay.

Antineoplastic Agents, Phytogenic↗

PubMed1997

Antitumor agents. 180. Chemical Studies and cytotoxic evaluation of cumingianosides and cumindysoside A, antileukemic triterpene glucosides with a 14,18-cycloapotirucallane skeleton.

Treatment of cumingianosides and cumindysoside A, which possess a 14,18-cycloapotirucallane skeleton, with p-toluenesulfonic acid in CH2Cl2 yielded new triterpene glucosides. Cumingianoside A (1) gave 10 and 11, along with cumingianoside Q (5). The structures of 10 and 11 were determined on the basis of spectral examination and contained a dammar-13(17)-ene and a 17(R),23(R)-epoxydammarane skeleton, respectively. Cumingianoside C (2) afforded, together with cumingianoside P (6), products 12 and 13, which were similar to 10 and 11, respectively. With a short reaction time at room temperature, cumingianoside E (3) yielded cumingianoside D (4). In contrast, when 3 was treated with p-toluenesulfonic acid in CH2Cl2 overnight at 5 degrees C, it gave two products, 9 and 14. Extensive spectroscopic examination revealed that 9 possessed a dammar-12-ene skeleton, while 14 was a pentacyclic tetranortriterpene glucoside with a novel skeleton. Cumindysoside A (8) gave a product (15) similar to 14. The cytotoxicities of 9-15 were evaluated against a panel of 58 human tumor cell lines. Compounds 11-15 exhibited potent cytotoxicity with log GI50 values ranging from -7.11 to -4.94, especially against leukemia and colon-tumor cell lines.

Antineoplastic Agents, Phytogenic↗

PubMed1997

Antitumor agents. 169 Dysoxylum cumingianum. V. Cumingianosides P and Q, new cytotoxic triterpene glucosides with an apotirucallane-type skeleton from Dysoxylum cumingianum.

Detailed chemical studies on the cytotoxic fraction from the leaves of Dysoxylum cumingianum have resulted in the isolation of two new triterpene glucosides, cumingianosides P (18) and Q (19), with an apotirucallane-type skeleton. The structures of 18 and 19 were determined by spectral examinations, and by conversion of cumingianosides C (3) and A (1) into 18 and 19, respectively. The cytotoxicities of cumingianosides P and Q against over 50 human cancer cell lines were evaluated. Cumingianoside P exhibited significant (EC50 < 4 microM) cytotoxicity against 37 human cancer cell lines. Among them, the UO-31 (renal cancer) cell line was the most sensitive to this compound (EC50 0.267 microM). In contrast, cumingianoside Q showed selective cytotoxicity against NCI-H522 (non-small cell lung cancer) cells with an EC50 value of 1.67 microM, and exhibited no cytotoxicity (EC50 > 10 microM) against most of the remaining cancer cell lines.

Antineoplastic Agents, Phytogenic↗

PubMed1997

Antitumor agents. 168. Dysoxylum cumingianum. IV. The structures of cumingianosides G-O, new triterpene glucosides with a 14,18-cycloapotirucallane-type skeleton from Dysoxylum cumingianum, and their cytotoxicity against human cancer cell lines.

Nine new triterpene glucosides, named cumingianosides G-O (4-7, 9, 12-15), containing a 14,18-cycloapotirucallane-type skeleton were isolated from a cytotoxic fraction of the leaves of Dysoxylum cumingianum. The structures of the new compounds were established on the basis of chemical and spectral examinations. Evaluation of the cytotoxic activity of cumingianosides G-O showed that cumingianoside M exhibited significant (< 4 microM) cytotoxicity, especially against leukemia and melanoma cell lines.

Antineoplastic Agents, Phytogenic↗

PubMed1996

Mutual sensitization of ATP and GTP in driving F-actin on the surface-fixed H-meromyosin.

Using an in vitro motility assay on acto-H-meromyosin, we studied the sliding velocity of an actin filament driven by two kinds of H-meromyosin heads: H-meromyosin head with ATP bound (fast motor) and with GTP bound (slow motor). We found a significant increase in the sliding velocity owing to the coexistence of the fast motor and the slow motor. This phenomenon may give an important suggestion with regard to the integration over multiple interactions of H-meromyosin heads along the actin filament.

Actins↗

PubMed1996

Compressibility and specific volume of actin decrease upon G to F transformation.

We measured the densities as well as the sound velocities in solutions of G-actin, F-actin and the reconstituted thin filament. Using the data obtained, we determined their partial specific volumes and partial specific adiabatic compressibilities. The objectives were to investigate the volume change of actin upon polymerization and to detect the conformational change associated with the ca2+-binding to the reconstituted thin filament. The partial specific volume and the partial specific adiabatic compressibility of G-actin were 0.749 cm3/g and 9.3 x 10(-12) cm2/dyne, respectively. The results suggest that G-actin is a rather soft protein compared with other globular proteins. The partial specific volumes of F-actin were in a range of 0.63 -0.66 cm3/g depending on the solvent conditions. The partial specific adiabatic compressibilities of F-actin were negative (-(7-13) x 10(-12) cm3/dyne). These data indicate that the amount of hydration may increase by several times upon polymerization assuming that the size of the cavity remains constant. We detected little difference between the partial specific adiabatic compressibility of the reconstituted thin filament in a Ca2+-bound state and that in a Ca2+-unbound state. This suggests that the Ca2+ binding affected not the subunit itself but the inter-subunit junction.

Actin Cytoskeleton↗

PubMed1996

Effects of subtilisin cleavage of monomeric actin on its nucleotide binding.

The kinetics of ATP exchange on subtilisin-cleaved G-actin was investigated by measuring the fluorescence of 1,N6-ethenoadenosine 5'-triphosphate. The apparent dissociation rate of ATP (k-ATP) was 2.8-fold larger than that of intact G-actin in the presence of 300 microM free Ca2+. Analysis of the dependence of k-ATP on free Ca2+ showed that the dissociation rate constant of tightly bound Ca2+ was not significantly changed by subtilisin cleavage. On the other hand, an equilibrium binding study using 8-amino-2-[(2-amino-5-methylphenoxy)-methyl]-6-methoxyquinoline N,N,N',N'-tetraacetic acid (Quin 2) showed that the affinity of tightly bound Ca2+ for G-actin was reduced by about 13-fold after subtilisin treatment. Consequently, the stabilization by Ca2+ of ATP was weak in cleaved G-actin. Furthermore, the kinetic analysis of ATP exchange revealed that the binding equilibrium between ATP and divalent cation-free cleaved G-actin was much slower than that in the case of intact G-actin.

Actins↗

PubMed1995

Antitumor agents, 157. Absolute structures of cumingianosides A-F, antileukemic triterpene glucosides, and structures of the hydrolysates of cumingianoside A.

The stereostructures of cumingianosides A-F, a series of triterpene glucosides with a 14,18-cycloapoeuphane skeleton, have been established by X-ray crystallographic analysis on an aglycone [1c] the acid hydrolysate of cumingianoside A [1], which is a potent cytotoxic triterpene against MOLT-4 human leukemia cells with an EC50 value of < 0.00625 microM. The 14,18-cyclopropane ring in cumingianoside A [1] was opened under acidic conditions in two different directions to give compounds with an apoeuphane skeleton and a dammarane skeleton. Furthermore, it was found that subsequent hydrolysis yielded not only an aglycone with an apoeuphane skeleton [1c] but also an apo-rearrangement product [1d].

Antineoplastic Agents, Phytogenic↗

PubMed1994

Anti-AIDS agents. 15. Synthesis and anti-HIV activity of dihydroseselins and related analogs.

Forty-two dihydroseselins based on the structure of suksdorfin (1) were synthesized in order to evaluate their anti-HIV activity. These synthetic derivatives include 3',4'-di-O-acyl- and 3'- or 4'-O-acyl-cis-dihydroseselins (8-21) and 3',4'-trans-dihydroseselins with O-acyl and/or O-alkyl groups at the 3' and 4' positions (6, 22-43). Two 4'-azido (44, 45) and three 4'-alkylamido (46, 48, 49) derivatives were also prepared. By using optically pure reagents, three pairs of diastereoisomers were synthesized and separated as optically pure compounds (14, 15; 16, 17; 38, 39). Together with the above synthetic derivatives, seselin (3) and (+/-)-cis-(4), (+)-cis- (5), and (+/-)-trans-dihydroseselin-3',4'-diol (7) were also tested for their in vitro anti-HIV activity. An optically pure compound, 3',4'-di-O-(-)-camphanoyl-(+)-cis-khellactone (16), showed potent inhibitory activity and remarkable selectivity against HIV replication. The EC50 value and in vitro therapeutic index (TI) of 16 are 4 x 10(-4) microM and 136,719, respectively, which are better than those shown by AZT in the same assay. In addition, compound 16 is also active against HIV replication in a monocytic cell line and in peripheral blood mononuclear cells (PBMCs). Our in vitro assay indicated that, like compound 1, compound 16 is not an inhibitor of HIV-1 reverse transcriptase. Moreover, the anti-HIV activity of 16 is stereoselective as its three diastereoisomers (17, 38, 39) are at least 10,000 times less active. Since other synthetic dihydroseselin derivatives with different substituents or without any substituents are inactive or are active only at much higher concentration, the antiviral potency of 16 could be associated with the camphanoyl moieties of its structure. Therefore, compound 16 represents a unique coumarin structure with promising anti-HIV activity.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Adiabatic compressibility of myosin subfragment-1 and heavy meromyosin with or without nucleotide.

The partial specific adiabatic compressibilities of myosin subfragment-1 (S1) and heavy meromyosin (HMM) of skeletal muscle in solution were determined by measuring the density and the sound velocity of the solution. The partial specific volumes of S1 and HMM were 0.713 and 0.711 cm3/g, respectively. The partial specific adiabatic compressibilities of S1 and HMM were 4.2 x 10(-12) and 2.9 x 10(-12) cm2/dyn, respectively. These values are in the same range as the most of globular proteins so far studied. The result indicates that the flexibility of S1 region almost equals to that of HMM. After binding to ADP.orthovanadate, S1 and HMM became softer than their complexes with ADP. The bulk moduli of S1 and HMM were of the order of (4-6) x 10(10) dyn/cm2, which are very comparable with the bulk modulus of muscle fiber.

Adenine Nucleotides↗

PubMed1991

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

Actins↗

PubMed1991

High-affinity binding of cytochalasin B to the B-end of F-actin loses its inhibitory effect on subunit exchange when the bound nucleotide is ADP.

We investigated the mode of binding of cytochalasin B (CB) to F-actin in an ADP-solution with and without inorganic phosphate (Pi). In the presence of Pi (20 mM), a filament of F-actin had a single high-affinity CB binding site (Kd = 1.4 nM), just like in the case of an ATP-solution [Kd = 5.0 nM: Suzuki, N. & Mihashi, K. (1991) J. Biochem. 109, 19-23]. But in the absence of Pi, there were two low-affinity (Kd = 200 nM) CB binding sites as well as one high-affinity site (Kd = 1.6 nM). We determined the concentration of CB necessary for half-maximal inhibition of growth or shortening of F-actin (Ki) using of pyrene-labeled actin. We obtained Ki = 80 nM for growth and Ki = 800 nM for shortening in the presence of ATP. The addition of Pi to the ATP-solution reduced Ki for growth to 9 nM. We propose a model explaining these results. In the model, high-affinity CB binding to the terminal subunit dimer can inhibit subunit exchange at the B-end only when the terminal subunits bind ATP or ADP.Pi. When the terminal subunits bind ADP, additional low-affinity CB bindings to the terminal subunits are needed to inhibit the subunit exchange.

Actins↗

PubMed1990

Evidence for the existence of two equilibrium conformations of the ternary complex of myosin subfragment-1, ADP, and orthovanadate.

In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.

Adenosine Diphosphate↗