Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D Mg2+ imaging

The role of intracellular magnesium ions is of high interest in the fields of pharmacology and cellular biology. To accomplish the dynamic and three-dimensional imaging of intracellular Mg2+, there is a strong desire for the development of optimized Mg2+ fluorescent probes. In this paper we describe the design, synthesis, and cellular application of the three novel Mg2+ fluorescent probes KMG-101, -103, and -104. The compounds of this series feature a charged beta-diketone as a binding site specific for Mg2+ and a fluorescein residue as the fluorophore that can be excited with an Ar+ laser such as is widely used in confocal scanning microscopy. This molecular design leads to an intensive off-on-type fluorescent response toward Mg2+ ions. The two fluorescent probes KMG-103 and -104 showed suitable dissociation constants (Kd,Mg2+ = 2 mM) and nearly a 10-fold fluorescence enhancement over the intracellular magnesium ion concentration range (0.1-6 mM), allowing high-contrast, sensitive, and selective Mg2+ measurements. For intracellular applications, the membrane-permeable probe KMG-104AM was synthesized and successfully incorporated into PC12 cells. Upon application of the mitochondria uncoupler FCCP to the probe-incorporated cells, the resulting increase in the free magnesium ion concentration could be followed over time. By using a confocal microscope, the intracellular 3D magnesium ion concentration distributions were satisfactorily observed.     

Improved cross-axis synchronous flow-through coil planet centrifuge for performing counter-current chromatography. II. Studies on retention of stationary phase in short coils and preparative separations in multilayer coils

Performance of the apparatus was evaluated in terms of stationary phase retention, partition efficiency and sample loading capacity. Preliminary studies with short coils revealed high retention of the stationary phase under a proper combination of the head-tail elution and planetary motion. Preparative capability of the apparatus was successfully demonstrated on efficient multigram separations of 2,4-dinitrophenyl amino acids, indole auxins, and bacitracin in a pair of large multilayer coils with a total capacity of 1.5 l.     

Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

Identification of Shiga toxins in Shiga toxin-producing Escherichia coli using immunoprecipitation and high-performance liquid chromatography-electrospray ionization mass spectrometry

We developed a rapid and reliable identification method for Shiga toxins in Shiga toxin-producing Escherichia coli (STEC) using immunoprecipitation and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). Polyclonal antisera specific for Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) were raised in rabbits so as to be used for the immunoprecipitation. The immunoprecipitaion was carried out by mixing sample solutions with 50 microl each of the antisera to Stx1 and Stx2 followed by allowing the mixed solutions to stand for 30 min. The quantity required to obtain the immunoprecipitate was more than 0.5 microg of Shiga toxins. HPLC-ESI-MS analysis of the resulting immunoprecipitates provided accurate molecular weight information on Shiga toxins, leading to direct evidence for the presence of these toxins. It requires at most two days to perform our procedure from toxin extraction to measurement of HPLC-ESI-MS whereas the previous method using isolation procedures required about two weeks to complete. The usefulness of the present method has been demonstrated by identifying Stx1, Stx2 and a variant of Stx2 (Stx2e) in the immunoprecipitates prepared from STEC strains.     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Application of ion-exchange cartridge clean-up in food analysis IV. Confirmatory assay of benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin, in bovine tissues by liquid chromatography-electrospray ionization tandem mass spectrometry

A multiresidue analytical method was developed for the confirmation of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in bovine tissues using electrospray ionization liquid chromatography-tandem mass spectrometry (LC-ESI-MS-MS) with a product ion scan mode. All penicillins gave [M-H]-, [M-H-CO2]- and [M-H-141]- as the product ion, when [M-H]- was selected as the precursor ion. Combination of an ion-exchange cartridge clean-up and the LC-ESI-MS-MS method can reliably identify all of these penicillins fortified at a concentration of 0.05 mg/kg in bovine tissues, including liver, kidney and muscle. The limits of confirmation satisfy the maximum residue limits for each of the penicillins established by the World Health Organization, US Food and Drug Administration, European Union and Japan.     

An oxazaphospholidine approach for the stereocontrolled synthesis of oligonucleoside phosphorothioates

The stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates (PS-ODNs) using nucleoside 3'-O-oxazaphospholidine derivatives as monomer units is described. 2-Chloro-1,3,2-oxazaphospholidine derivatives were prepared from six kinds of enantiopure 1,2-amino alcohols and used for the phosphitylation reactions of 5'-O-protected nucleosides. A detailed study of these reactions revealed that the diastereoselectivity of the reaction depended on the structure of the enantiopure 1,2-amino alcohol, the reaction temperature, and the amine used as a scavenger of HCl. In addition, ab initio molecular orbital calculations for the 2-chlorooxazaphospholidine derivatives were carried out to elucidate the mechanism of these diastereoselective phosphitylation reactions. The LUMO of the 2-chloro-5-phenyloxazaphospholidine derivatives on the phosphorus atom was found to be almost orthogonal to the P-Cl bond. This LUMO may be involved in the phosphitylation reactions with predominant retention of the P-configuration. A series of dialkyl(cyanomethyl)ammonium salts were developed and used as activators for the condensation reactions of the diastereopure nucleoside 3'-O-oxazaphospholidines with 3'-O-protected nucleosides. In the presence of the new activators, the reactions proceeded rapidly to give the corresponding dinucleoside phosphite triesters. The diastereoselectivity of the condensation reaction did not depend on the counteranion but on the structure of the dialkyl(cyanomethyl)amine. In the presence of the activator, which consists of a relatively small dialkyl(cyanomethyl)amine, the condensation proceeded with excellent diastereoselectivity. After sulfurization and deprotection, diastereopure (R(p))- and (S(p))-dinucleoside phosphorothioates were obtained in excellent yields. The present methodology was also applied to the solid-phase synthesis of stereoregulated PS-ODNs. all-(R(p))-[T(PS)](3)T, all-(S(p))-[T(PS)](3)T, all-(R(p))-d[G(PS)A(PS)C(PS)]T, and all-(R(p))-[T(PS)](9)T were synthesized on a highly cross-linked polystyrene resin.     

Diastereocontrolled synthesis of dinucleoside phosphorothioates using a novel class of activators, dialkyl(cyanomethyl)ammonium tetrafluoroborates

A novel class of activators, dialkyl(cyanomethyl)ammonium tetrafluoroborates 1a-c, has been developed and applied to the condensations of diastereopure 5'-O-tert-butyldiphenylsilylthymidine 3'-cyclic phosphoramidites 3a-d with 3'-O-tert-butyldimethylsilylthymidine (4a). Among them, the condensation of 3a with 4a in the presence of 1a completed within 5 min and gave only one diastereoisomer of the corresponding phosphite 5a. After sulfurization and deprtection, almost diastereopure (Rp)-TpsT 7 was obtained (d.r. = 99:1). Next the 5'-O-(DMTr)nucleoside 3'-phosphoramidites 8a-d containing thymine, N6-benzoyladenine, N4-benzoylcytosine, and N2-phenylacetylguanine have been synthesized and allowed to condense with 3'-O-protected thymidine and 2'-deoxyadenosine. The 5'-O-DMTr group and the N-acyl groups of the nucleobases were compatible with the reaction conditions and the condensations completed quickly with excellent diastereoselectivity.     

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.