An automated fluorescence protein sequencer using 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS) as an Edman reagent

An automated fluorescence protein sequencer using 7-methylthio-5-(2,1,3-benzoxadiazolyl) isithiocyanate (MTBD-NCS), a fluorescent Edman reagent, is developed by the modification of a commercial protein sequencer. The generated MTBD-thiohydantoin amino acids fluoresced strongly, whereas the by-products such as MTBD-thiocarbamoyl amino acids and MTBD-carbamoly amino acids did not fluoresce. A few interfering peaks were observed in the chromatogram and amino acid sequence was easily determined. The coupling and cyclization/cleavage reaction conditions and extraction conditions of generated MTBD-thiazolinone amino acids were optimized using an autonalyzer. Finally, the sequence of a synthetic peptide (25 pmol), leucine-enkephalin-Thr-amide, was determined and up to six residues were successively analyzed.     

Isolation and structure of a GD3-Type ganglioside molecular species possessing neuritogenic activity from the starfish Luidia maculata

A GD3-type ganglioside molecular species, LMG-4 (1), has been obtained from the polar lipid fraction of the chloroform/methanol extract of the starfish Luidia maculata. The structure of this ganglioside has been determined on the basis of chemical and spectroscopic evidence to be 1-O-[(N-acetyl-alpha-D-neuraminosyl)-(2-->8)-(N-acetyl-alpha-D-neuraminosyl)-(2-->3)-beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranosyl]-ceramide. The ceramide moiety was composed of heterogeneous 2-hydroxy fatty acid and phytosphingosine units. This is the first report on the isolation and structure elucidation of GD3-type ganglioside from echinoderms. Moreover, 1 exhibited neuritogenic activity toward the rat pheochromocytoma PC12 cells in the presence of nerve growth factor.     

Constituents of Crinoidea. 3. Isolation and structure of a glycosyl inositolphosphoceramide-type ganglioside with neuritogenic activity from the feather star Comanthus japonica

A glycosyl inositolphosphoceramide-type ganglioside, CJP4, was obtained from the polar lipid fraction of the chloroform/methanol extract of the feather star Comanthus japonica. The structure of this ganglioside has been determined on the basis of chemical and spectroscopic evidence to be 9-O-methyl-(N-glycolyl-alpha-D-neuraminosyl)-(2-->11)-9-O-methyl-(N-glycolyl-alpha-D-neuraminosyl)-(2-->11)-9-O-methyl-(N-glycolyl-alpha-D-neuraminosyl)-(2-->3)-inositolphosphoceramide, which contains C(16)-sphingosine and C(22:0)-, C(24:0)-fatty acid. This is the first report on the isolation and structural elucidation of a trisialo-glycosyl inositolphosphoceramide-type ganglioside. Moreover, CJP4 exhibited neuritogenic activity toward the rat pheochromocytoma PC12 cells in the presence of nerve growth factor.     

Comparison of the migration behavior of nanoparticles based on polyethylene glycol and silica using micellar electrokinetic chromatography

Nanoparticles, spherical particles with diameters less than 100 nm, are promising theranostic devices for noninvasive diagnosis and therapy. In this study, nanoparticles composed of polyethylene glycol and silica were prepared, and their migration behavior was examined using capillary electrophoresis. The effects of the sodium dodecyl sulfate concentration in the electrolyte, the nanoparticle size, and the encapsulated molecule on the migration were examined. The addition of sodium dodecyl sulfate into the electrolyte had a significant effect on the electrophoretic mobility of polyethylene glycol nanoparticles, but a small effect on that of silica nanoparticles. As for the size effect, the mobility became a little faster for smaller nanoparticle sizes for both polyethylene glycol and silica nanoparticles. The encapsulated molecule affected the mobility of the nanoparticles through interactions between the encapsulated molecules and sodium dodecyl sulfate. We propose that the large effect of sodium dodecyl sulfate on the migration of the polyethylene glycol nanoparticles was due to the large spaces within the nanoparticles. These results indicate that nanoparticle migration is mainly determined by the nanoparticle components.     

Determination of the absolute configuration of sialic acids in gangliosides from the sea cucumber Cucumaria echinata

Enantiomeric pairs of sialic acid, D- and L-NeuAc (N-acetylneuraminic acid), were converted to D- and L-arabinose, respectively, by chemical degradation. Using this method, the absolute configuration of the sialic acid residues, NeuAc and NeuGc (N-glycolylneuraminic acid), in the gangliosides from the sea cucumber Cucumaria echinata was determined to be the D-form. Although naturally occurring sialic acids have been believed to be the D-form on the basis of biosynthetic evidence, this is the first report of the determination of the absolute configuration of the sialic acid residues in gangliosides using chemical methods.     

Development of a column-switching high-performance liquid chromatography for kynurenine enantiomers and its application to a pharmacokinetic study in rat plasma

Kynurenine (KYN), a tryptophan metabolite, is a precursor of kynurenic acid, which is an antagonist of N-methyl-d-aspartate receptor. In this study, an enantiomeric separation of d,l-KYN derivatized with the benzofurazan fluorescence reagent 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) (DBD-d,l-KYN) was first investigated by using a high-performance liquid chromatography (HPLC) with several chiral columns. As a consequence, DBD-d,l-KYN was enantiomerically separated on a cellulose-type chiral column (CHIRALCEL OJ-RH) with a mobile phase of H₂O/CH₃CN/MeOH (40/50/10) containing 0.1% acetic acid. Under this condition, the separation factor and resolution were 1.48 and 1.28, respectively. Next, a column-switching HPLC consisting of both octadecylsilica and chiral columns was developed and used to determine both d- and l-KYN enantiomers in 10 μL of rat plasma following the intraperitoneal administration of d,l-KYN to rats (10 mg kg⁻¹). The result revealed that the concentration of l-KYN was higher than that of d-KYN, suggesting that d-KYN was eliminated faster than l-KYN.     

Triterpenoid Saponins from the Cultivar “Green Elf” of Pittosporum tenuifolium

Four oleanane-type glycosides were isolated from a horticultural cultivar “Green Elf” of the endemic Pittosporum tenuifolium (Pittosporaceae) from New Zealand: three acylated barringtogenol C glycosides from the leaves, with two previously undescribed 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, 3-O-β-d-galactopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and the known 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C (Eryngioside L). From the roots, the known 3-O-β-d-glucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyloleanolic acid (Sandrosaponin X) was identified. Their structures were elucidated by spectroscopic methods including 1D- and 2D-NMR experiments and mass spectrometry (ESI-MS). According to their structural similarities with gymnemic acids, the inhibitory activities on the sweet taste TAS1R2/TAS1R3 receptor of an aqueous ethanolic extract of the leaves and roots, a crude saponin mixture, 3-O-β-d-glucopyranosyl-(1→2)-[α-l-arabinopyranosyl-(1→3)]-β-d-glucuronopyranosyl-21-O-angeloyl-28-O-acetylbarringtogenol C, and Eryngioside L were evaluated.     

Potentiality of IMAC as Sample Pretreatment Tool in Food Analysis for Veterinary Drugs

Immobilized metal ion affinity chromatography (IMAC) was evaluated as a sample pretreatment tool for residual veterinary drugs (VDs) in food. A test of six cations for the IMAC efficiency revealed that Cu²⁺ and Fe³⁺ were suitable in retaining tetracyclines, quinolones (QLs: fluoroquinolones, acid quinolones), macrolides, β-lactams, and aminoglycosides, but ineffective for sulfonamides and hormones. These results showed the IMAC had great potential for a multiclass multiresidual VDs analysis in a simple and selective way. This methodology was applied on milk analysis for eleven QLs, involving extraction with (1:1) acetonitrile/methanol and IMAC with Fe³⁺. Averaged recoveries ranged from 68 to 93% and limits of quantitation from 2 to 26 ng mL^?1. Ten commercial milk samples were found to be free from the QLs.