Biomedical subjects

Akito Tanaka

Publications and source records attributed to Akito Tanaka.

14 recordsLinked to original sources

PubMed2006

An easy preparation of 'monolithic type' hydrophilic solid phase: capability for affinity resin to isolate target proteins.

An easy preparation method of monolithic type hydrophilic solid phase was discussed. Newly invented functional monomer with a hydrophilic cross-linking agent was co-polymerized to realize well-controlled monolithic co-continuous structure by use of diethylene glycol as porogenic solvent. We were able to control the content of the functional monomer up to 40 vol% without loss of monolithic structure. Those prepared were utilized as affinity resins after immobilization of FK506, an immunosuppressive drug as a ligand. It was found that the affinity resins prepared were hydrophilic enough to eliminate non-specific adsorption of proteins, while two of the target proteins of FK506 tested were successfully captured.

Adsorption↗

PubMed2006

Isolating the whole complex of target proteins of FK506 using affinity resins from novel solid phases.

The development of novel solid phases enabled us to create affinity resins that could be used to isolate the whole complex of target proteins responsible for the immunosuppressive effects of FK506 from rat brain lysate, whereas the affinity resins from commercially available matrices could not achieve this isolation. The results illustrate the enhanced effectiveness of the affinity resin made from this novel material at identifying the target protein of the bioactive compound compared to resins made from the well-known materials Affigel or Toyopearl. This effectiveness arises because the novel material is hydrophilic enough to reduce nonspecific binding proteins and because it has a higher density of ligands that capture the nonubiquitous target protein.

Animals↗

PubMed2006

The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site.

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

Animals↗

PubMed2006

A versatile method of identifying specific binding proteins on affinity resins.

The isolation of both specific and nonspecific binding proteins on affinity matrices bearing bioactive compounds hinders the identification of drug cellular targets. Although solid-phase elution and competition methods conventionally are used to distinguish between specific and nonspecific receptor-ligand interactions, these approaches often are severely restricted by low ligand solubility and/or slow kinetic dissociation. This article describes an alternative and versatile method, termed serial affinity chromatography, to identify ligand receptors using affinity resins.

Affinity Labels↗

PubMed2006

Linoleic acid derivative DCP-LA improves learning impairment in SAMP8.

In the water-maze test, the linoleic acid derivative, 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (1 mg/kg, intraperitoneally), significantly shortened the prolonged latency for accelerated-senescence-prone mice 8 (SAMP8), reaching a level similar to the latency for accelerated-senescence-resistant mice 1 (SAMR1) as control. In the open-field test to assess motor activity, it was confirmed that the DCP-LA effect is not due to increased motor activity. In the passive avoidance test to assess fear memory, DCP-LA had no effect on the latency of acquisition and retention for SAMP8. The results of the present study, thus, suggest that DCP-LA could improve age-related learning impairment by enhancing cognitive functions.

Aging↗

PubMed2005

Reduction of nonspecific binding proteins to self-assembled monolayer on gold surface.

We developed a gold coated glass chip bearing a poly(ethyleneglycol) (PEG) type compound as hydrophilic spacer for surface plasmon resonance studies, which enabled adequate estimation of K(d) value between FK506 and FKBP12 not only using purified FKBP12 (K(d)=22 nM) but also using Escherichia coli lysate expressing FKBP12 (K(d)=15 nM). These results indicated effectiveness of the PEG spacer for reduction of nonspecific interactions. Chemical stability and simple surface-structure of the novel chip are also attractive.

Animals↗

PubMed2005

Development of chemically stable solid phases for the target isolation with reduced nonspecific binding proteins.

Poly(methacrylate) matrices for affinity resins were designed and synthesized based on our previous results that nonspecific protein absorption on affinity resins strongly depended on their hydrophobic property. The novel affinity resins bearing FK506 (6a, 6b) captured specific binding protein, FKBP12, with a small amount of nonspecific binding proteins. The amount of nonspecific binding proteins on 6a-6b was much reduced compared to that on commercially available poly(methacrylate) resins, Toyopearl (8), and was almost the same as that on one of the most popular resins, Affigel (9). Interestingly, 6a and 6b could isolate FKBP52 as a specific binding protein as well, although 8 and 9 could not.

Absorption↗

PubMed2005

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors.

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.

Animals↗

PubMed2005

Effects of cis-unsaturated free fatty acids on PKC-epsilon activation and nicotinic ACh receptor responses.

Linoleic acid (C18:2omega6) (10 microM) depressed ACh-evoked whole-cell membrane currents followed by potentiation of the currents in Xenopus oocytes expressing Torpedo ACh receptors. The potentiation was never obtained with linoleoylamide (10 microM), which contains CONH2 instead of COOH on linoleic acid. In the assay of protein kinase C (PKC) using a reversed phase high-performance liquid chromatography, linoleic acid activated PKC-epsilon but linoleoylamide otherwise had no effect on it. Of a variety of cis-unsaturated free fatty acids with 20 hydrocarbon chains (C20), 8-eicosenoic acid (C20:1omega12) potentiated ACh receptor channel currents without depression. 11,14-Eicosadienoic acid (C20:2omega6), 5,8,11-eicosatrienoic acid (C20:3omega9), 5,8,11,14-eicosatetraenoic acid (arachidonic acid) (C20:4omega6), and 5,8,11,14,17-eicosapentaenoic acid (C20:5omega3) depressed and potentiated the currents. In contrast, only a depression was obtained with 5-eicosenoic acid (C20:1omega15), 11-eicosenoic acid (C20:1omega9), and 8,11,14-eicosatrienoic acid (C20:3omega6). All the free fatty acids (C20) involved PKC-epsilon activation, with no significant difference in the potency among them. Taken together, it appears that carboxyl on cis-unsaturated free fatty acids plays a significant role in PKC-epsilon activation and potentiation of nicotinic ACh receptor responses. The role of the number and position of the cis-double bonds in the receptor responses, however, remains an open question.

Animals↗

PubMed2004

Design and synthesis of novel hydrophilic spacers for the reduction of nonspecific binding proteins on affinity resins.

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets. Reduction of nonspecific binding proteins is important for success in finding such targets. We herein disclose the design, synthesis, and effectiveness in reduction of nonspecific binding proteins, of novel hydrophilic spacers (2-5), which were introduced between matrices and a ligand. Among them, tartaric acid derivative (5) exhibited the most effective reduction of nonspecific binding proteins, whilst maintaining binding of the target protein. Introduction of 5 on TOYOPEARL reduced tubulin and actin by almost 65% and 90% compared to that without the hydrophilic spacer, respectively, with effective binding to the target protein, FKBP12.

Actins↗

PubMed2003

Stearic acid facilitates hippocampal neurotransmission by enhancing nicotinic ACh receptor responses via a PKC pathway.

Of a variety of saturated free fatty acids examined here, those with less than 20 hydrocarbon potentiated responses through Torpedo ACh receptors expressed in Xenopus oocytes, and the maximal effect was obtained with stearic acid (C18:0) at 10 microM (168+/-25% of basal levels 10 min after 10-min treatment). Stearic aid (10 microM) also potentiated alpha7 nicotinic ACh receptor responses, being evident 110 min after 10-min treatment (219+/-18% of basal levels), and the potentiation was inhibited by GF109203X, a selective inhibitor of protein kinase C (PKC). In the PKC assay using a reversed-phase HPLC, stearic acid (10 microM) enhanced PKC-epsilon activity approximately twice as much as the activity in the absence of stearic acid. Stearic acid (10 microM) induced a long-lasting facilitation of neurotransmission in the dentate gyrus of rat hippocampal slices, and the facilitation was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 receptors. The results presented here suggest that stearic acid facilitates hippocampal neurotransmission by enhancing alpha7 receptor responses via a PKC pathway.

Animals↗

PubMed2003

The newly synthesized linoleic acid derivative FR236924 induces a long-lasting facilitation of hippocampal neurotransmission by targeting nicotinic acetylcholine receptors.

The newly synthesized linoleic acid derivative, FR236924, induces a long-lasting facilitation of hippocampal neurotransmission based on a persistent enhancement in the activity of presynaptic nicotinic ACh receptors via a PKC pathway and the ensuing increase in glutamate release, not only in vitro but in vivo at a low dosage (2mg/kg, ip), which suggested the possibility of its use as a promising anti-dementia drug.

Alkanes↗

PubMed2002

FK228 (depsipeptide) as a natural prodrug that inhibits class I histone deacetylases.

FK228 is a histone deacetylase (HDAC) inhibitor, the molecular mechanism of inhibition of which has been unknown. Here we show that reduction of an intramolecular disulfide bond of FK228 greatly enhanced its inhibitory activity and that the disulfide bond was rapidly reduced in cells by cellular reducing activity involving glutathione. Computer modeling suggests that one of the sulfhydryl groups of the reduced form of FK228 (redFK) interacts with the active-site zinc, preventing the access of the substrate. HDAC1 and HDAC2 were more strongly inhibited by redFK than HDAC4 and HDAC6. redFK was less active than FK228 in inhibiting in vivo HDAC activity, due to rapid inactivation in medium and serum. Thus, FK228 serves as a stable prodrug to inhibit class I enzymes and is activated by reduction after uptake into the cells. The glutathione-mediated activation also implicates its clinical usefulness for counteracting glutathione-mediated drug resistance in chemotherapy.

Amino Acid Sequence↗

PubMedDate not supplied

A quantitative analysis and chemical approach for the reduction of nonspecific binding proteins on affinity resins.

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets of small molecules. Reduction of nonspecific binding proteins is important for the success of such biochemical approaches. To develop strategies to circumvent this problem, we quantitatively investigated the binding of tubulin and actin to a series of affinity resins bearing 15 variant ligands on 3 commercially available polymer supports. Nonspecific protein binding was proportional to the hydrophobicity of the affinity resins and could be quantitatively correlated to the CLOGP values of the ligands, which are a measure of compound hydrophobicity. When compounds had CLOGP values greater than 1.5, (amount of tubulin) = 0.73 x CLOGP - 1.1 (n = 7, r = 0.97), and (amount of actin) = 0.42 x CLOGP - 0.79 (n = 7, r = 0.99). On the basis of these studies, we designed a novel hydrophilic poly(ethylene glycol) (PEG) spacer (26) for the conjugation of ligands to chromatography resins. As predicted by our binding algorithm, introduction of this spacer reduced the amount of nonspecific protein binding in proportion to the number of ethylene glycol units.

Actins↗