A novel matrix for high performance affinity chromatography and its application in the purification of antithrombin III

Viscose fiber, a regenerated cellulose, was evaluated for using as a novel matrix for high performance affinity chromatography. With a one-step activation with epichlorohydrin, heparin can be readily covalently attached to the matrix. This heparin-viscose fiber material was used for purifying antithrombin III (AT III) from human plasma. The purity of the AT III from this one-step purification is 93% as measured by SDS-PAGE and the protein recovery yield is about 90%. This column is highly specific as described by the dissociation constant of the complex of immobilized heparin and AT III, which was 2.83 x 10(-5)mol/L. And more important, this viscose fiber material demonstrated its excellent mechanical property that allows the flow rate to reach up to 900 cm/h or more.     

Determination of flavonoids from Orthosiphon stamineus in plasma using a simple HPLC method with ultraviolet detection

A simple liquid chromatographic method was developed for the simultaneous determination of flavonoids from Orthosiphon stamineus Benth, namely sinensitin, eupatorin and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, in plasma. Prior to analysis, the flavonoids and the internal standard (naproxen) were extracted from plasma samples using a 1:1 mixture of ethyl acetate and chloroform. The detection and quantification limits for the three flavonoids were similar being 3 and 5 ng/ml, respectively. The within-day and between-day accuracy values, expressed as percentage of true values, for the three flavonoids were between 95 and 107%, while the corresponding precision, expressed as coefficients of variation, for the three flavonoids were less than 14%. In addition, the mean recovery values of the extraction procedure for all the flavonoids were between 92 and 114%. The calibration curves were linear over a concentration range of 5-4000 ng/ml. The present method was applied to analyse plasma samples obtained from a pilot study using rats in which the mean absolute oral bioavailability values for sinensitin, eupatorin and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone was 9.4, 1.0 and 1.5%, respectively.     

Simultaneous determination of six phenolic constituents of danshen in human serum using liquid chromatography/tandem mass spectrometry

The six phenolic constituents are water-soluble components extracted from the Chinese medical herb danshen, the dried roots of Salvia miltiorrhiza Bunge (Labiatae). An liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method has been developed for the simultaneous quantification of six phenolic constituents of danshen (magnesium lithospermate B (MLB), rosmarinic acid (RA) and lithospermic acid (LA), caffeic acid (CAA), protocatechuic aldehyde (3,4-dihydroxybenzaldehyde, Pal), 3,4-dihydroxyphenyllactic acid (danshensu)) in human serum with chloramphenicol as internal standard. The serum samples were treated by special liquid-liquid extraction, and the analytes were determined using electrospray negative ionization mass spectrometry in the multiple reaction monitoring (MRM) mode, with sufficient sensitivity to allow analysis of human serum samples generated following administration of a clinically relevant dose. Good linearity over the range 8-2048 ng/mL for six phenolic constituents was observed. The intra- and inter-day precisions (CV) of analysis were <13%, and the accuracy ranged from 88 to 116%. This quantitation method was successfully applied to a pharmacokinetic study of i.v. drip infusion of Danshen injection fluid in human.     

ReAS: Recovery of ancestral sequences for transposable elements from the unassembled reads of a whole genome shotgun

We describe an algorithm, ReAS, to recover ancestral sequences for transposable elements (TEs) from the unassembled reads of a whole genome shotgun. The main assumptions are that these TEs must exist at high copy numbers across the genome and must not be so old that they are no longer recognizable in comparison to their ancestral sequences. Tested on the japonica rice genome, ReAS was able to reconstruct all of the high copy sequences in the Repbase repository of known TEs, and increase the effectiveness of RepeatMasker in identifying TEs from genome sequences.     

Peroxisomal proteomics, a new tool for risk assessment of peroxisome proliferating pollutants in the marine environment

In an attempt to improve the detection of peroxisome proliferation as a biomarker in environmental pollution assessment, we have applied a novel approach based on peroxisomal proteomics. Peroxisomal proteins from digestive glands of mussels Mytilus galloprovincialis were analyzed using 2-DE and MS. We have generated a reference 2-DE map from samples obtained in a well-studied reference area and compared this with peroxisomal proteomes from other sequenced genomes. In addition, by comparing 2-DE maps from control samples with samples obtained in a polluted area, we have characterized the peroxisome proliferation expression pattern associated with exposure to a polluted environment. Over 100 spots were reproducibly resolved per 2-DE map; 55 differentially expressed spots were quantitatively detected and analyzed, and 14 of these showed an increase in protein expression of more than fourfold. Epoxide hydrolase, peroxisomal antioxidant enzyme, and sarcosine oxidase (SOX) have been identified by ESI MS/MS, and acyl-CoA oxidase, multifunctional protein, and Cu,Zn-superoxide dismutase were immunolocalized by Western blotting. Our results indicate that a peroxisomal protein pattern associated to marine pollutant exposure can be generated, and this approach may have a greater potential as biomarker than traditional, single-protein markers.     

"In vitro" protection of DNA from Fenton reaction by plant polyphenol verbascoside

The protection effect of verbascoside (Ver) against Fenton reaction on plasmid pBR322 DNA was studied using agarose gel electrophoresis and UV-visible spectroscopy. The pBR322 plasmid DNA is damaged by hydroxyl radical (OH*) generated from the Fenton reaction with H2O2 and Fe(II) or Fe(III). This DNA damage is characterized by the diminution of supercoiled DNA forms or by the increase of relaxed or linear DNA forms after oxidative attack. The UV spectrum study showed that verbascoside can form complexes with Fe(II) or Fe(III), and the complexation can be reversed by the addition of EDTA. The formation constants of verbascoside-Fe complexes were estimated as 10(21.03) and 10(31.94) M(-2) for Fe(II) and Fe(III) respectively. The inhibition of Fenton reaction by verbascoside could be partially explained by the sequestration of Fe ions.     

Structural investigation of a novel rhamnoglucogalactan isolated from the fruiting bodies of the fungus Hericium erinaceus

A new heteropolysaccharide (HEP-1) was isolated from the fruiting bodies of Hericium erinaceus. It was estimated to have a molecular weight of 1.8x10(4) da and showed [alpha](D)(20) +129 (c 0.295, H(2)O). HEP-1 is composed of rhamnose, galactose, and glucose in the ratio of 1.19:3.81:1.00. Its structural features were investigated using composition analysis, methylation analysis, partial hydrolysis, and IR and NMR spectroscopy. The results showed that HEP-1 has a (1-->6)-linked alpha-d-galactopyranosyl backbone with branches that are composed of rhamnose and glucose attached to O-2.     

Axial patterning of C. elegans male sensilla identities by selector genes

The fan and rays of the C. elegans male tail constitute a compound sensory organ essential for mating. Within this organ, the individual sensilla, known as rays, have unique identities. We show that ray identities are patterned by a selector gene mechanism in a manner similar to other serially homologous axial structures. One selector gene that promotes the identities of a subset of the rays is the Hox gene egl-5. Within EGL-5-expressing rays, further patterning is provided by a Pax-6 homolog and a signal of the TGFβ family. These genes and pathway coordinately specify multiple ray properties affecting all three terminal ray cell types. These properties include complex patterns of FMRFamide-like (FaRP) neuropeptides, serotonin (5HT) and dopamine expression, and ray morphology. Differences in these differentiated characteristics give each sensillum a unique identity and potentially endow the compound ray organ with a higher-order information gathering capacity.     

Synthesis, characterization and supramolecular structures of two Ni(II) complexes with potentially heptadentate ligand N,N-bis(2-hydroxybenzyl)-1,3-bis[(2-aminoethyl)amino]-2-propanol

A potentially heptadentate ligand H₃L (N,N^′-bis(2-hydroxybenzyl)-1,3-bis[(2-aminoethyl)amino]-2-propanol) and its two Ni(II) complexes, [Ni(H₂L)H₂O](H₂O)₃ClO₄ (1) and [Ni(H₂L)(H₂O)](H₂O)Cl (2) were prepared and characterized. X-ray structural analyses indicate that complex 1 has a distorted octahedral coordination geometry, with four amine N atoms of H₂L⁻ defining the equatorial plane, one aqua O atom and one phenoxo O atom of the ligand occupying two axial positions, respectively. The Ni(II) center of 2 has coordination geometry similar to that of 1. IR and electronic spectra of 1 and 2 are in agreement with their crystal structural features. Approximately along the ab plane, 2D supramolecular structure of 1 is assembled through multiple hydrogen bonds between hydroxy groups of the ligands, coordinated and crystal lattice H₂O and π-π stacking interactions between adjacent phenyl rings of the ligands, while for that of 2, probably along the a axis, 1D chain structure is also formed by multiple hydrogen bonds, but lack of π-π stacking interactions.