Biomedical subjects

A Kameyama

Publications and source records attributed to A Kameyama.

At least 19 recordsLinked to original sources

PubMed1999

A cytoplasmic factor, calpastatin and ATP together reverse run-down of Ca2+ channel activity in guinea-pig heart.

1. The cytoplasmic extract of bovine heart was separated into four fractions by gel filtration: H (molecular mass > 300 kDa), P (250-300 kDa), L1 (180-250 kDa) and L2 (< 180 kDa). The effects of these fractions on the run-down of L-type Ca2+ channel activity were investigated in guinea-pig ventricular myocytes. 2. After run-down induced by inside-out patch formation, Ca2+ channel activity was restored by P or H (+ 3 mM ATP) to 7.5 and 5.8 % of that in the cell-attached mode, respectively, but to as high as 86 % by P + H + ATP. 3. The reversal of run-down brought about by the P fraction was mimicked by calpastatin. 4. The restorative effect of calpastatin + ATP showed a biphasic time course: 38 % in the early transient phase and 11 % in the late phase. However, calpastatin + H + ATP showed a sustained effect: 66 % in the early transient phase, and 87 % in the late phase. 5. The effective component of the H fraction showed a protein-like nature: heat and trypsin sensitivity. 6. The activities of cAMP-dependent protein kinase, casein kinase I, casein kinase II, protein tyrosine kinase, protein serine/threonine or tyrosine phosphatases were measured. However, these kinases and phosphatases were not confirmed as the effective component of cytoplasm or the H fraction. 7. Run-down was not prevented by 2 microM phalloidin or 2 microM paclitaxel, suggesting that neither actin filaments nor microtubules are directly involved in the run-down. 8. Our results support the view that the basal activity of the Ca2+ channel is maintained by at least three factors: a protein-like factor in the H fraction, calpastatin, and ATP.

Adenosine Triphosphate↗

PubMed1999

Cloning and expression of the Ca2+ channel alpha1C and beta2a subunits from guinea pig heart.

Complimentary DNA clones encoding the alpha1C and beta2a subunits of guinea-pig cardiac L-type Ca2+ channels were isolated using the PCR method. The open reading frame encoded 2,169 amino acids for the alpha1C and 597 amino acids for the beta2a subunit. The proteins showed 94.2 and 94.8%, respectively, identity to the respective subunit of the rabbit protein. The message size of the guinea pig alpha1C and beta2a subunits was 8.0 and 3.5/4.0 kb, respectively. RT-PCR analysis revealed that the alpha1C subunit is expressed exclusively in the heart, while the beta2a subunit is expressed in the heart, cerebellum, whole brain, and stomach. The alpha1C and beta2a subunits are transiently expressed in BHK (baby hamster kidney) cells, and the channel currents were studied using the whole-cell patch clamp technique in medium containing 30 mM Ba2+. In cells expressing alpha1C alone, the Ba2+ current was activated at -30 mV and more positive potentials and peaked at about 10 mV. The co-expression of beta2a with alpha1C did not affect the voltage-dependence of the current, but increased the peak current and accelerated current decay. In cells transfected with guinea pig alpha1C and rabbit beta1+alpha2/delta, a Ba2+ current comparable to those in native myocytes was observed. The Ba2+ current can be blocked completely by nifedipine and is enhanced 3-fold by Bay K 8644. On the other hand, neither forskolin nor okadaic acid affects the Ba2+ current, suggesting that cAMP-mediated modulation is not easily reproduced in transfected cells, unlike that seen in native cardiac myocytes.

Amino Acid Sequence↗

PubMed1998

A Novel Synthesis of Poly(ester-alt-sulfide)s by the Ring-Opening Alternating Copolymerization of Oxiranes with gamma-Thiobutyrolactone Using Quaternary Onium Salts or Crown Ether Complexes as Catalysts.

Poly(ester-alt-sulfide) (polymer 1) was synthesized by the alternating copolymerization of glycidyl phenyl ether (GPE) with gamma-thiobutyrolactone (TBL) catalyzed by either quaternary onium salts or crown ether complexes. The copolymerization proceeded to produce polymer 1 with good yields in neat or in various organic solvents at 30-120 degreesC, in which quaternary onium salts having Cl- as a counteranion such as tetrabutylammonium chloride (TBAC) had higher activity than quaternary onium salts such as tetrabutylammonium bromide having Br- as a counteranion. It was also found that the alternate copolymer (polymer 1) of GPE with TBL was obtained selectively under different feed ratios of GPE and TBL, although ring-opening homopolymerizations of GPE and TBL did not proceed. Copolymerizations of various oxiranes such as butyl glycidyl ether, styrene oxide, and 1,2-hexene oxide with TBL catalyzed by TBAC also proceeded, and the corresponding poly(ester-alt-sulfide)s (polymers 2, 3, and 4) were obtained under the same conditions as for the synthesis of polymer 1.

Journal Article↗

PubMed1998

Run-down of L-type Ca2+ channels occurs on the alpha 1 subunit.

Run-down of L-type Ca2+ channels in CHO cells stably expressing alpha 1c, alpha 1c beta 1a, or alpha 1c beta 1a alpha 2 delta gamma subunits was studied using the patch-clamp technique (single channel recording). The channel activity (NPo) of alpha 1c channels was increased 4- and 8-fold by coexpression with beta 1a and beta 1a alpha 2 delta gamma, respectively. When membranes containing channels composed of different subunits were excised into basic internal solution, the channel activity exhibited run-down, the time-course of which was independent of the subunit composition. The run-down was restored by the application of calpastatin (or calpastatin contained in cytoplasmic P-fraction) + H-fraction (a high molecular mass fraction of bovine cardiac cytoplasm) + 3 mM ATP, which has been shown to reverse the run-down in native Ca2+ channels in the guinea-pig heart. The restoration level was 64.7, 63.5, and 66.4% for channels composed of alpha 1c, alpha 1c beta 1a, and alpha 1c beta 1a alpha 2 delta gamma, respectively, and was thus also independent of the subunit composition. We conclude that run-down of L-type Ca2+ channels occurs via the alpha 1 subunit and that the cytoplasmic factors maintaining Ca2+ channel activity act on the alpha 1 subunit.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Characterization and partial purification of the cytoplasmic factor that maintains cardiac Ca2+ channel activity.

Using the patch clamp method we attempted to characterize the cytoplasmic factor in guinea-pig cardiac myocytes which restores L-type Ca2+ channel activity after run-down. The factor was eluted from a diethylaminoethyl (DEAE) sepharose column by KCl at 100-360 mM. On gel filtration the factor had an apparent molecular mass (Mr) of 250-300 kDa. Two-dimensional electrophoresis of the partially purified factor showed at least nine spots, of which the major spot had a Mr of about 100 kDa and an isoelectric point of 4.8, suggesting that the physicochemical properties of the factor resemble those of calpastatin, an endogenous inhibitor of Ca2+-activated protease, calpain. Calpastatin activity was increased in the partially purified cytoplasm and an antibody raised against calpastatin recognized the major band. Reduction of calpastatin in the cytoplasm decreased the potency of Ca2+ channel activation. These results suggest that calpastatin might interact with the Ca2+ channel and maintain channel activity.

Adenosine Triphosphate↗

PubMed1998

Run-down of the cardiac L-type Ca2+ channel: partial restoration of channel activity in cell-free patches by calpastatin.

We have found previously that run-down of cardiac Ca2+ channels in cell-free patches is reversed by cytoplasm plus adenosine triphosphate (ATP). Characterization of the factor in cytoplasm revealed that it is likely to be calpastatin (CS), an endogenous inhibitor of calpain (Ca2+-activated neutral protease). We therefore investigated the possible restoring effect of CS obtained from various tissues (activity 1.3-23 U/ml) on Ca2+ channel activity after run-down in inside-out patches. Although CS from porcine erythrocytes (plus 3 mM ATP) had only a minimal effect in restoring channel activity (to 4% of the control level recorded before the run-down), CS from porcine heart restored channel activity to 19% of control. The product of recombinant complementary deoxyribonucleic acid (cDNA) of human heart CS, a membrane-bound CS partially purified from bovine heart and CS from rabbit skeletal muscle (Sigma) restored channel activity to 28%, 23% and 10% of control levels, respectively. These results suggest that tissue-type CS, but not erythrocyte-type (truncated) CS, seems to have an effect on the cardiac Ca2+ channel to maintain its activity. Purified CS had relatively small effects compared to that of crude cytoplasm, implying that some other factor(s) might contribute also to the regulation of Ca2+ channel activity.

Adenosine Triphosphate↗

PubMed1998

Steroidal saponins from the underground parts of Ruscus aculeatus and their cytostatic activity on HL-60 cells.

Phytochemical examination of the underground parts of Ruscus aculeatus has been undertaken as part of systematic study of plants of the Liliaceae. Six new spirostanol saponins and five new furostanol saponins were isolated, and their structures were assigned on the basis of spectroscopic analysis, including two-dimensional NMR techniques, and hydrolysis. Ruscogenin diglycoside with three acetyl groups attached to the inner galactosyl moiety and its corresponding 26-glucosyloxyfurostanol saponin showed cytostatic activity on leukemia HL-60 cells.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Steroidal saponins from the rhizomes of Hosta sieboldii and their cytostatic activity on HL-60 cells.

A total of eighteen steroidal saponins were isolated from the rhizomes of Hosta sieboldii, one of which appeared to be the first isolation from a plant source and six to be new compounds. The structures of the new saponins were determined by spectral data and a few chemical transformations to be (25R)-2 alpha, 3 beta-dihydroxy-5 alpha-spirostan-12-one (manogenin) 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-beta-D-glucopyranosyl -(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha,3 beta-dihydroxy-5 alpha-spirost-9-en-12-one (9,11-dehydromanogenin) 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, 9,11-dehydromanogenin 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[O-alpha-L- rhamnopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->3)]-O-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha,3 beta-dihydroxy-26-beta-D-glucopyranosyloxy-22-methoxy-5 alpha-furostan-12-one 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha, 3 beta-dihydroxy-26-beta-D-glucopyranosyloxy-22-methoxy-5 alpha-furost-9-en-12-one 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿ and (25R)-5 alpha-spirostan-2 alpha,3 beta,12 beta-triol 3-O-¿O-alpha-L- rhamnopyranosyl-(1-->2)-beta-D-galactopyranoside¿, respectively. Cytostatic activity of the isolated saponins on leukaemia HL-60 cells was examined.

Carbohydrate Conformation↗

PubMed1998

Aculeoside B, a new bisdesmosidic spirostanol saponin from the underground parts of Ruscus aculeatus.

From the underground parts of Ruscus aculeatus, a new bisdesmosidic spirostanol saponin named aculeosides B (2) was isolated, and its structure was determined on the basis of spectroscopic analysis, including 2D NMR techniques. Aculeoside A (1), which was previously isolated from the same plant source, exhibited inhibitory activity on cell growth of leukemia HL-60 cells with an IC50 value of 0.48 microgram mL(-1), while aculeoside B (2) was inactive.

Antineoplastic Agents, Phytogenic↗

PubMed1998

New steroidal constituents of the underground parts of Ruscus aculeatus and their cytostatic activity on HL-60 cells.

Phytochemical examination of the underground parts of Ruscus aculeatus has led to the isolation of a total of twelve steroidal saponins, including seven new ones. The structures of the new saponins were determined by spectroscopic analysis and chemical evidence. The furostanol saponin, having a diglycoside moiety modified with a (2S,3S)-2-hydroxy-3-methylpentanoic acid group and an acetic acid group, and its corresponding spirostanol saponin exhibited cytostatic activity on leukemia HL-60 cells.

Antineoplastic Agents, Phytogenic↗

PubMed1997

Run-down of the cardiac Ca2+ channel: characterization and restoration of channel activity by cytoplasmic factors.

Possible mechanisms for run-down in the Ca2+ channel, such as proteolysis or dephosphorylation of the channel, were examined in guinea-pig ventricular myocytes. The Ca2+ channel current, recorded in inside-out patches using a pipette solution containing 50 mM Ba2+ and 3 microM Bay K 8644, ran down with a mean survival time of 2.35 min. The survival time was not significantly affected by adenosine triphosphate (ATP) (3 mM), 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA) (2 mM), isoprenaline (l-5 microM), phosphate (l20 mM) and leupeptin (l0 microM). Stimulation of guanosine triphosphate (GTP)-binding proteins was also ineffective. The catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA, 0.5-2 microM) slightly and transiently increased channel activity, but had minimal effects on the channel when applied after complete run-down. On the other hand, cytoplasm from the heart, skeletal muscle, brain and liver, but not kidney, induced channel activity. There was a positive correlation between NPo (the product of the number of channels N and the open probability Po) value before run-down and that after the application of cytoplasm, suggesting that the activity of once-active channels was restored ba the exogenous cytoplasm. The potency of cytoplasm in tissues in inducing channel activity was not related to PKA activity nor to the number of dihydropyridine binding sites. These results suggest that the run-down of the cardiac Ca2+ channel is not mediated by dephosphorylation or proteolysis of the channel, but involves other factor(s), possibly interaction of the channel protein with a cytoplasmic regulatory protein.

Adenosine Triphosphate↗

PubMed1997

ATP regulates cardiac Ca2+ channel activity via a mechanism independent of protein phosphorylation.

The role of adenosine triphosphate (ATP) in the regulation of L-type Ca2+ channel activity was investigated in inside-out patches from guinea-pig ventricular cells, in which the Ca2+ channel activity had been reprimed by application of cytoplasm from bovine heart. Passing the cytoplasm through a diethylaminoethyl (DEAE)-sepharose column or heating at 60 degrees C for 20 min attenuated the induction Ca2+ channel activity to 6-13% of that in the preceding cell-attached patch. Addition of 10 mM MgATP to the cytoplasm greatly improved the potency of cytoplasm in restoring Ca2+ channel activity (to 83 +/- 22%, mean +/- SE). This effect of MgATP was also produced, although with lower potency, by K2ATP (61 +/- 20%) or 5'-adenylylimidodiphosphate (AMP-PNP, 39 +/- 7%), a non-hydrolyzable ATP analogue, suggesting that hydrolysis of ATP is not required for the stimulatory effect on channel activity. A non-specific protein kinase inhibitor H8 (50-100 microM) did not inhibit the effect of cytoplasm + MgATP on channel activity, suggesting the involvement of a pathway independent of phosphorylation. We conclude that ATP regulates Ca2+ channel activity in dual pathways: one with, and the other without, protein phosphorylation.

Adenosine Triphosphate↗

PubMed1997

Steroidal glycosides from the underground parts of Hosta plantaginea var. japonica and their cytostatic activity on leukaemia HL-60 cells.

A new C22-steroid glycoside was isolated from the underground parts of Hosta plantaginea var. japonica, together with a known furostanol saponin and three known spirostanol saponins. The structure of the new steroid glycoside was characterized by spectroscopic analysis and acid-catalysed hydrolysis as 2 alpha, 3 beta, 16 beta-trihydroxy-5 alpha-pregn-20(21)-ene-carboxylic acid gamma-lactone 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-beta-D-glucopyranosyl-(1--> 4)-beta-D-galactopyranoside¿. The isolated compounds were assayed for their cytostatic activity on leukaemia HL-60 cells. The spirostanol saponins showed cytostatic activity in a dose-dependent manner with the IC50 values ranging between 1 and 3 micrograms ml-1.

Antineoplastic Agents, Phytogenic↗

PubMed1996

Total synthesis of VIM-2 ganglioside isolated from human chronic myelogenous leukemia cells.

A total synthesis of the tumor-associated glycolipid antigen, VIM-2, is described [2]. Phenyl 2,3,4-tri-O-benzoyl-6-O-benzyl-beta-D-galactopyranosyl-(1-->4)-6-O-benzy l-2- deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (7), a key intermediate prepared by condensation of phenyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (6) and 2,3,4-tri-O-benzoyl-6-O-benzyl-alpha-D-galactopyranosyl bromide (5), was glycosylated with methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside (8) to give the trisaccharide donor 9, which, on coupling with 2-(trimethylsilyl)ethyl 2,4,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside (10), afforded the pentasaccharide 11. The regioselective glycosylation of 12 (derived by O-debenzoylation of 11) with 7 gave the heptasaccharide 13, which was converted by treatment with hydrazine monohydrate and subsequent N-acetylation into the hexasaccharide acceptor 14. The stereo- and regio-selective glycosylation of 14 with methyl (phenyl 5-acetamido-4,7,8,9-O-benzoyl-3,5-dideoxy-2-thio-D-glycero-beta-D-galact o-2- nonulopyranosid)onate (16) gave the desired octasaccharide 18. Hydrogenolytic removal of the benzyl groups in 18 and successive O-acetylation, removal of the 2-(trimethylsilyl)ethyl group, and treatment with trichloroacetonitrile gave the alpha-trichloro-acetimidate 21, which was then coupled with (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol (22) to give 23. Compound 23 was transformed, via selective reduction of the azido group, N-introduction of octadecanoic acid, O-desilylation, O-deacylation, and saponification of the methyl ester group, into the title VIM-2 ganglioside 26.

Carbohydrate Sequence↗

PubMed1996

Effects of calciseptine on unitary barium channel currents in guinea-pig portal vein.

Effects of synthesized calciseptine (CaS), found naturally in the venom of the black mamba, on voltage-dependent Ca2+ channels in smooth muscle cells of the guinea-pig portal vein were investigated. In the whole-cell voltage-clamp configuration, extracellular application of CaS (>/= 10 nM) inhibited the inward current in a concentration- and voltage-dependent manner at a holding potential of -90 mV. The Ca2+ current recorded at a high holding potential (-50 mV) was approximately 8 times more sensitive to CaS than that at a more negative holding potential (-90 mV). CaS (50 nM) shifted to the left the steady-state inactivation curve obtained by using single 8-s conditioning pulses of various amplitudes. When CaS (>/= 200 nM) was present in the pipette, the Ca2+ current remained for the duration of the experiments (more than 60 min) in the whole-cell configuration. Two different Ca2+ channel conductances are present in this tissue (25-pS and 12-pS channels). Both channels are blocked by dihydropyridine (DHP) derivatives, but have different sensitivities. In the cell-attached condition, CaS hardly changed the activity of either unitary Ca2+ channel current. To prevent the "run down" of the Ca2+ channels in cell-free conditions, we added cardiac cytosol, a supernatant from homogenized cardiac cells and an endogenous Ca2+ channel activating factor, in the pipette. The unitary Ca2+ channel currents were then recorded using the outside-out membrane patch configuration. Application of CaS (1 microM) in the bath completely blocked the open events of the 25-pS Ca2+ channel. CaS (10 nM) in the bath reduced the mean open time and channel availability, resulting in a decrease in the open probability of the 25-pS channel currents without affecting the amplitude of the single-channel conductance. CaS also reduced the open probability (though less potently) and channel availability of the 12-pS Ca2+ channel without a change in its amplitude. From these results, we conclude that CaS has inhibitory effects on the voltage-dependent Ca2+ current that are similar to those of DHP derivatives and that it acts from the outside of the membrane.

Animals↗

PubMed1996

Cyclic AMP-dependent protein kinase but not protein kinase C regulates the cardiac Ca2+ channel through phosphorylation of its alpha 1 subunit.

The voltage-dependent L-type Ca2+ channel in the heart is regulated by cAMP-dependent protein kinase (PKA) and possibly by protein kinase C (PKC). We have investigated the channel modulation through phosphorylation by these protein kinases, using liposomes into which Ca2+ channels from bovine heart were reconstituted. Phosphorylation of the proteoliposomes with PKA increased the dihydropyridine-sensitive Ca2+ efflux from them by about 70%. PKA rapidly phosphorylated membrane proteins of 210 and 170 kDa. A dihydropyridine-class Ca2+ channel blocker, [3H]azidopine, specifically photo-labeled a protein of 210 kDa, suggesting that the 210-kDa phosphoprotein might be the alpha 1 subunit of the Ca2+ channel. In contrast, phosphorylation of the proteoliposomes with PKC failed to modulate the Ca2+ efflux. Although PKC catalyzed the phosphorylation of membrane proteins of 150, 130, 95, 67, and 62 kDa, the 210- and 170-kDa proteins were not phosphorylated by this kinase. These results suggest that phosphorylation of the 210-kDa protein in the cardiac sarcolemma by PKA may be responsible for modulation of the channel function, whereas modulation of the channel by PKC, if it occurs, must be the result of an indirect mechanism, e.g. phosphorylation of a cytoplasmic protein or an associated channel polypeptide, that cannot function in the reconstituted system.

Animals↗

PubMed1996

A new cytotoxic cholestane bisdesmoside from Ornithogalum saundersiae bulbs.

Bioassay-guided fractionation of the MeOH extract of Ornithogalum saundersiae bulbs led to the isolation of a new cholestane bisdesmoside with potent cytotoxic activities toward leukemia HL-60 and MOLT-4 cells. The structure was deduced mainly from spectroscopic information.

Antineoplastic Agents, Phytogenic↗

PubMed1996

[Structure, function and expression of Ca2+ channels].

Structure, function and expression of voltage-dependent Ca2+ channels are reviewed. Ca2+ channels have been classified into one low-voltage activated subtype (T type) and at least 5 high-voltage activated subtypes (L, N, P, Q and R types), which are characterized by the sensitivity to specific blockers. Although L-type channel in skeletal muscle is shown to consist of alpha1, alpha2/delta, beta, gamma subunits, it is not clear whether other subtypes have similar subunit structures. Cloning and functional expression of cDNA encoding alpha1 subunits revealed existence of multiple genes and splicing variants. Thus, the diversity in the electrophysiological properties of Ca2+ channels in different tissues and developmental stages comes from, at least in part, the different molecular structure of the channels. Regulation of the expression of Ca2+ channels may be important for the elaborate control of cellular functions.

Animals↗