Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [(14)C]p-aminohippurate and [(3)H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.     

Co-localization and interaction of human organic anion transporter 4 with caveolin-1 in primary cultured human placental trophoblasts

The human organic anion transporter 4 (hOAT4) has been identified as the fourth isoform of OAT family. hOAT4 contributes to move several negatively charged organic compounds between cells and their extracellular milieu. The functional characteristics and regulatory mechanisms of hOAT4 remain to be elucidated. It is well known that caveolin plays a role in modulating proteins having some biological functions. To address this issue, we investigated the co-localization and interaction between hOAT4 and caveolin-1. hOAT4 and caveolin-1 (mRNA and protein expression) were observed in cultured human placental trophoblasts isolated from placenta. The confocal microscopy of immuno-cytochemistry using primary cultured human trophoblasts showed hOAT4 and caveolin-1 were co-localized at the plasma membrane of the cell. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions and immune-precipitates from the trophoblasts. When synthesized cRNA of hOAT4 along with scrambled- or antisense-oligodeoxynucleotide (ODN) of Xenopus caveolin-1 were co-injected to Xenopus oocytes, the [3H]estrone sulfate uptake was significantly decreased by the co-injection of antisense ODN but not by scrambled ODN. These findings suggest that hOAT4 and caveolin-1 share a cellular expression in the plasma membrane and caveolin-1 up-regulates the organic anionic compound uptake by hOAT4 under the normal physiological condition.     

Identification of a novel high affinity copper binding site in the APP(145-155) fragment of amyloid precursor protein

The copper(II) binding features of the APP(145-155) and APP(145-157) fragments of the amyloid precursor protein, Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-NH2 and Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 were studied by NMR spectroscopy and NMR findings were supported by UV-vis, CD and EPR spectra. Potentiometric measurements were performed only for the more soluble Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-Glu-Thr-NH2 peptide fragment. The following was shown: (i) the imidazole rings of all the three His residues are involved in metal coordination; (ii) metal binding induces ionisation of Leu-148 and His-149 amide nitrogens that complete the donor set to copper(II) in the species dominant at neutral pH; (iii) the unusual coordination scheme of the His-Xxx-His-Xxx-His consensus sequence justifies the high specificity for Cu(II) when compared to SOD-like or albumin-like peptides or even in amyloid Abeta fragments. The present findings may represent the key for interpreting the observed requirement of His residues conservation for the redox cycling between Cu(II) and Cu(I) by soluble APP.     

Hydrostannation of 1-stannyl-1-alkynes,a reaction leading to novel vinyl anion precursors

Hydrostannation of 1-stannyl-1-alkynes leads to the formation of 1,1-distannyl- and 1,3-distannyl-1-alkenes while the reactions of 1-silyl-1-alkynes with a tin hydride are less regiospecific. The 1,1-distannyl-1-alkenes give α-stannylvinyl anions on treatment with methyllithium     

Band gap,sorption properties and fluorescence sensing behaviour of a novel 1D→2D cathenane‐like cobalt(II)–organic framework

A novel hydrolytic stable Co^II–organic framework, namely poly[[bis(2‐amino‐4‐sulfonatobenzoato‐κO¹)tetraaquatris{μ‐1,4‐bis[(imidazol‐1‐yl)methyl]benzene‐κ²N³:N^3′}dicobalt(II)] tetrahydrate], {[Co(C₇H₅NO₅S)(C₁₄H₁₄N₄)_1.5(H₂O)₂]·2H₂O}_n, ( 1 ), based on multifunctional 2‐amino‐5‐sulfobenzoic acid (H₂asba) and the auxiliary flexible ligand 1,4‐bis[(imidazol‐1‐yl)methyl]benzene (bix), was prepared using the solution evaporation method. The purity of ( 1 ) was confirmed by elemental analysis and powder X‐ray diffraction (PXRD) analysis. Complex ( 1 ) shows a novel 1D→2D interpenetrating network, which is further extended into a 3D supramolecular framework with channels occupied by the lattice water molecules. The 2‐amino‐4‐sulfonatobenzoate (asba^2?) ligand adopts a monodentate coordination mode. The bix ligands exhibit gauche–gauche (GG) and trans–trans (TT) conformations. A detailed analysis of the solid‐state diffuse‐reflectance UV–Vis spectrum reveals that an indirect band gap exists in the complex. The band structure, the total density of states (TDOS) and the partial density of states (PDOS) were calculated using the CASTEP program. The calculated band gap (E_g) matches well with the experimental one. The complex exhibits a reversible dehydration–rehydration behaviour. Interestingly, gas sorption experiments demonstrate that the new fully anhydrous compound obtained by activating complex ( 1 ) at 400 K shows selective adsorption of CO₂ over N₂. Complex ( 1 ) retains excellent framework stability in a variety of solvents and manifests distinct solvent‐dependent fluorescence properties. Moreover, the complex shows multiresponsive fluorescence sensing for some nitroaromatics in aqueous medium.     

Aurophilicity as a cofactor in crystal engineering. Dicyanoaurate(I) anion as a building block in a novel Co(II)-Au(I) bimetallic assembly

A 2D grid-shaped cyanide-bridged Co(II)-Au(I) bimetallic coordination polymer, [Co(DMF)2(Au(CN)2)2], has been prepared from the [Au(CN)2]- building block; sheets associate pair-wise by aurophilic interactions and the compound exhibits zeolite-like properties.     

A novel dendritic anion conductor: quaternary ammonium salt of poly(amidoamine) (PAMAM)

The quaternary ammonium salts of dendritic poly(amidoamine) generations 2 and 3 are prepared by methylation and quaternization with methyl iodide. The obtained dendritic anion conductors exhibit a conductivity of 10^–5–10^–6 S·cm^–1 at ambient temperature. The plots of conductivity vs. temperature are curves with the lowest point around 58°C, which appear to exhibit a substantially different conductive law compared with ordinary linear or comb-like polyelectrolytes.     

Spectral properties of a novel antimony(III)-phthalocyanine complex that behaves like J-aggregates in non-aqueous media

An unusual red-shift of phthalocyanine Q-band upon aggregation in non-aqueous media has been observed for antimony(III) derivative and has been studied by using optical absorption and magnetic circular dichroism spectroscopy.     

Identification of a novel protein regulating microtubule stability through a chemical approach

To identify novel proteins regulating the microtubule cytoskeleton, we screened a library of purine derivatives using mitotic spindle assembly in Xenopus egg extracts as an assay. Out of a collection of 1561 compounds, we identified 15 that destabilized microtubules without targeting tubulin directly, resulting in small spindles. Affinity chromatography with one compound, named diminutol, revealed a potential target as NQO1, an NADP-dependent oxidoreductase. A role for NQO1 in influencing microtubule polymerization was confirmed through inhibition studies using known inhibitors and immunodepletion. Therefore, this chemical approach has identified a novel factor required for microtubule morphogenesis and cell division.     

Configurational ordering of cationic chiral dyes using a novel C(2)-symmetric hexacoordinated phosphate anion

C(2)-Symmetry hexacoordinated phosphorus BINPHAT anion-of configuration controlled by a BINOL ligand-can be prepared readily in a one-pot process and behaves as an efficient NMR chiral shift agent and chiral inducer onto monomethinium dyes (CD, (1)H NMR).     

The novel chain‐like anion ∞1[NaMo8O26(mim)2]3− in (Hmim)3[NaMo8O26(mim)2] (mim is 1‐methylimidazole)

Tris(1‐methylimidazolium) bis(1‐methylimidazole)hexacosaoxidooctamolybdatesodium, (C₄H₇N₂)₃[NaMo₈O₂₆(C₄H₆N₂)₂], prepared from an aqueous solution containing Na₂MoO₄ and 1‐methylimidazole, contains the novel chain‐like anion _∞¹[NaMo₈O₂₆(mim)₂]^{3 −} (mim is 1‐methylimidazole). The [Mo₈O₂₆(mim)₂]⁴⁻ building unit, which lies across a center of inversion, is comprised of eight edge‐sharing MoO₆ and MoO₅(N_mim) octahedra. These molybdate units are interlinked by sodium, itself exhibiting a sixfold coordination with O atoms.     

Lead tetrakis(imidazolyl)borate solids: anion exchange, solvent intercalation, and self assembly of an organic anion

The coordination polymer Pb[B(Im)(4)](NO(3))(xH(2)O), constructed by using sodium tetrakis(imidazolyl)borate and lead(II) nitrate solutions, is a layered material with the metal centers facing the interlayer spacing. As in naturally occurring layered minerals, this compound can readily undergo anion exchange and reversible intercalation of solvent water in the solid state with retention of crystallinity. We observed changes in solvent intercalation by (207)Pb solid state NMR (SSNMR) and thermogravimetric analysis (TGA). Stoichiometric exchange of (15)N nitrate for nitrate and iodide for nitrate is monitored by (15)N and (207)Pb SSNMR, and single crystals of the iodide-exchanged material Pb[B(Im)(4)]I were isolated. While the iodide compound can be obtained through facile exchange from the nitrate parent compound, the organic anion benzoate is placed in the interlayer spacing for nitrate under self-assembly conditions and forms an alternating monolayer in Pb[B(Im)(4)](C(6)H(5)COO)(0.5H(2)O). The ion exchange versus self-assembly behavior correlates with the structural differences in the three compounds. In both Pb[B(Im)(4)]I and Pb[B(Im)(4)](C(6)H(5)COO)(0.5H(2)O), the lead sites act as Lewis acids for the iodide and benzoate, respectively.     

A novel method for the estimation of arsenic(V) in organic compounds

A novel method for the determination of arsenic(V) in organic compounds has been developed by reducing combined arsenic(V) to arsenic(III) in aqueous acetic acid medium with zinc dust. In some cases, addition of ethyl alcohol is necessary to dissolve the compound and to keep the arsenic(III) compound in solution. The arsenic(III) is titrated with iodine and the end-point is detected visually with starch as indicator or potentiometrically.     

Metal organic chemical vapour deposition (MOCVD) of bone mineral like carbonated hydroxyapatite coatings

For the first time, the MOCVD technique has been used to deposit carbonated hydroxyapatite onto Ti6AL4V substrates using volatile monomeric (liquid) complexes [Ca(beta-diketonate)(2)(L)] and P(OEt)(3).     

Development of a novel high-throughput assay for the investigation of GlyT-1b neurotransmitter transporter function

The glycine transporter (GlyT-1b) is a Na(+)/Cl(-)-dependent electrogenic transporter which mediates the rapid re-uptake of glycine from the synaptic cleft. Based on its tissue distribution, GlyT-1 has been suggested to co-localise with the NMDA receptor where it may modulate the concentration of glycine at its co-agonist binding site. This data has led to GlyT-1 inhibitors being proposed as targets for disorders such as schizophrenia and cognitive dysfunction. Radiolabelled uptake assays (e.g. [(3)H]glycine) have been traditionally used in compound screening to identify glycine transporter inhibitors. While such an assay format is useful for testing limited numbers of compounds, the identification of novel glycine uptake inhibitors requires a functional assay compatible with high-throughput screening (HTS) of large compound libraries. Here, the authors present the development of a novel homogenous cell-based assay using the FLIPR membrane potential blue dye (Molecular Devices) and FLEXstation. Pharmacological data for the GlyT-1 inhibitors Org 24598 and ALX 5407 obtained using this novel electrogenic assay correlated well with the conventional [(3)H]-glycine uptake assay format. Furthermore, the assay has been successfully miniaturised using FLIPR(3) and therefore has the potential to be used for high-throughput screening.