Identification and quantitative determination of benproperine metabolites in human plasma and urine by liquid chromatography-tandem mass spectrometry

Two novel metabolites of benproperine (BPP), 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-3-piperidinol (3-OH-BPP) and 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-4-piperidinol (4-OH-BPP), were confirmed by comparison of retention times and mass spectra with those of synthetic standards using liquid chromatography-tandem mass spectrometry. Selective and sensitive procedures were developed for the simultaneous determination of BPP, 3-OH-BPP and 4-OH-BPP in human plasma and urine. The analytes were extracted from plasma sample and enzymatically hydrolyzed urine samples by liquid-liquid extraction, separated through a Diamonsil C(18) column (150 mm x 4.6 mm i.d.) and determined by tandem mass spectrometry with an electrospray ionization interface in selected reaction monitoring mode. Dextromethorphan was used as internal standard. The mobile phase consisted of acetonitrile-water-formic acid (34:66:1, v/v/v), and flow-rate was 0.5 ml min(-1). This method has a lower limit of quantification (LLOQ) of 60, 4.0 and 4.0 nmol l(-1)for BPP, 3-OH-BPP and 4-OH-BPP in plasma, 4.9, 4.7 and 2.4 nmol l(-1) in urine, respectively. The intra- and inter-run precision were measured to be below 9.2%, and the accuracy was within +/-4.3% for the analytes. The method was successfully used to determine BPP, 3-OH-BPP and 4-OH-BPP in plasma and urine for pharmacokinetic investigation. The results indicated residue of 3-OH-BPP in the body at least 192 h after an oral dose of BPP.     

Chemical fingerprint analysis of rhizomes of Gymnadenia conopsea by HPLC-DAD-MSn

A high-performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS(n)) method has been firstly developed for chemical fingerprint analysis of rhizomes of Gymnadenia conopsea R. Br. and rapid identification of major compounds in the fingerprints. Comparing the UV and MS spectra with those of reference compounds, seven main peaks in the fingerprints were identified as adenosine (1), 4-hydroxybenzyl alcohol (2), 4-hydroxybenzyl aldehyde (3), dactylorhin B (4), loroglossin (5), dactylorhin A (6) and militarine (7). Compounds 4-7 were succinate derivative esters and firstly discovered from this species. The Computer Aided Similarity Evaluation System (CASES) for chromatographic fingerprint of traditional Chinese medicine was employed to evaluate the similarities of 10 samples of the rhizomes of G. conopsea collected from Sichuan, Qinghai and Hebei provinces, Tibet autonomous region of China, and Nepal. These samples from different sources had similar chemical fingerprints. This method is specific and may serve for quality identification and comprehensive evaluation of this traditional Tibetan remedy.     

Plasma phospholipid metabolic profiling and biomarkers of mouse IgA nephropathy

IgA nephropathy is the most common form of glomerulonephritis (GN) and it could progress to end-stage renal failure within 10 years. Participating in biological processes in various pathways, phospholipids as a class of important constituents in the biomembranes have been paid increasing attention in many fields. However, phospholipids metabolism in glomerular disease was not clear, especially in IgA nephropathy. In this paper, the plasma phospholipid metabolic profile in mouse IgA nephropathy was investigated to discover the potential biomarkers on the progression of this disease by using high performance liquid chromatography/mass spectrometry (HPLC/MS) and the principal components analysis (PCA) as well as partial least squares-discriminant analysis (PLS-DA). The experimental mouse models of IgA nephropathy were established by oral immune and BSA injection. It was found that expression of intercellular adhesion molecule-1 (ICAM-1) in the glomeruli had a significant correlation with proteinuria in mouse IgA nephropathy. The association between plasma phospholipids and expression of ICAM-1 in the glomeruli of IgA nephropathy suggested C18:0/C18:0 PS (phosphatidylserine), C18:0/C22:5 PS (phosphatidylserine) and C18:0/C20:4 PI (phosphatidylinositol) were possible biomarkers of IgA nephropathy. The results show that the plasma phospholipid metabolic profiles from HPLC/MS combining with PCA and PLS-DA can be used not only to differentiate the IgA nephropathy from the controls, but also to discover and identify the potential biomarkers.     

Atomic force microscopy-based cell nanostructure for ligand-conjugated quantum dot endocytosis

While it has been well demonstrated that quantum dots (QDs) play an important role inbiological labeling both in vitro and in vivo,there is no report describing the cellular nanostructure basis ofreceptor-mediated endocytosis.Here,nanostructure evolution responses to the endocytosis of transferrin(Tf)-conjugated QDs were characterized by atomic force microscopy (AFM).AFM-based nanostructureanalysis demonstrated that the Tf-conjugated QDs were specifically and tightly bound to the cell receptorsand the nanostructure evolution is highly correlated with the cell membrane receptor-mediated transduction.Consistently,confocal microscopic and flow cytometry results have demonstrated the specificity anddynamic property of Tf-QD binding and internalization.We found that the internalization of Tf-QD is linearlyrelated to time.Moreover,while the nanoparticles on the cell membrane increased,the endocytosis was stillvery active,suggesting that QD nanoparticles did not interfere sterically with the binding and function ofreceptors.Therefore,ligand-conjugated QDs are potentially useful in biological labeling of cells at a nanometerscale.     

A comparison of the genetic diversity in Dipteronia sinensis Oliv. and Dipteronia dyeriana Henry

Dipteronia is an endemic genus to China and includes only two species, Dipteronia sinensis and D. dyeriana. Based on random amplified polymorphic DNA (RAPD) markers, a comparative study of the genetic diversity and genetic structure of Dipteronia was performed. In total, 128 and 103 loci were detected in 17 D. sinensis populations and 4 D. dyeriana populations, respectively, using 18 random primers. These results showed that the proportions of polymorphic loci for the two species were 92.97% and 81.55%, respectively, indicating that the genetic diversity of D. sinensis was higher than that of D. dyeriana. Analysis, based on similarity coefficients, Shannon diversity index and Nei gene diversity index, also confirmed this result. AMOVA analysis demonstrated that the genetic variation of D. sinensis within and among populations accounted for 56.89% and 43.11% of the total variation, respectively, and that of D. dyeriana was 57.86% and 42.14%, respectively. The Shannon diversity index and Nei gene diversity index showed similar results. The abovementioned characteristics indicated that the genetic diversity levels of these two species were extremely similar and that the interpopulational genetic differentiation within both species was relatively high. Analysis of the genetic distance among populations also supported this conclusion. Low levels of interpopulational gene flow within both species were believed to be among the leading causes for the above-mentioned phenomenon. The correlation analysis between genetic and geographical distances showed the existence of a remarkably significant correlation between the genetic distance and the longitudinal difference among populations of D. sinensis (p < 0.01), while no significant correlation was found between genetic and geographical distances among populations of D. dyeriana. This indicated that genetic distance was correlated with geographical distances on a large scale rather than on a small scale. This result may be related to differences in the selection pressure on species by their habitats with different distribution ranges. We suggest that in situ conservation efforts should focus on establishing more sites to protect the natural populations and their habitats. Ex situ conservation efforts should focus on enhancing the exchange of seeds and seedlings among populations to facilitate gene exchange and recombination, and to help conserve genetic diversity. __________ Translated from Acta Phytoecologica Sinica, 2005, 29(5): 785–792 [译自: 植物生态学报, 2005, 29(5): 785–792]     

Species composition and correlation of understory woody plants in Chinese fir plantation in the lower subtropical area

There are 71 species in the shrub layer of the Cunninghamia lanceolata plantation after natural succession. The species richness and diversity have increased with slight anthropogenic disturbance. The status and function of understory woody species were judged by the analysis of the important value (IV). x ² statistics and r test were used for testing the significance of interspecific association and correlation among 25 main understory woody plants selected from the woody population. The results clearly showed their interspecific relationships and their differences in resource utilization. Species-pairs of positive association were in the majority. Most species were accommodated in the shady habitat. There was a positive correlation between the IV of the species and the interspecific association. The higher the IV of the species, the closer and more significant was the interspecific association. Based on analytical results of interspecific association and correlation, 25 woody plants in the shrub layer could be divided into four ecological species groups: I. Ficus hispida + Antidesma bunius + Mallotus barbatus + Ficus cunia + Saurauia tristyla + Mallotus philippinensis + Maesa japonica + Ficus hirta + Alchornea rugosa + Ficus fulva + Mallotus apelta; II. Cudrania tricuspidata + Schefflera octophylla; III. Cunninghamia lanceolata + Clerodendron cytophyllum + Millettia semicastrata + Randia spinosa + Litsea cubeba + Litsea pungens; IV. Ardisia japonica + Psychotria rubra + Vitex quinata + Cephalanthus occidentalis + Pithecellobium lucidum + Mycetia sinensis. If species group III or II is the advantaged species in the shrub layer, the community would change from a coniferous forest to a sparse evergreen broad-leaved forest. For group IV, the community would be relatively stable. For group I, the coniferous forest would be mixed with coniferous-broad leaved forest. The classification of ecological species groups would provide a theoretical basis on judging its ecological function, adjusting the stand structure of the plantation and directing the suitable natural vegetation type through the close-natural restoration process. __________ Translated from Acta Ecologica Sinica, 2005, 25(9): 2173–2179 [译自: 生态学报, 2005, 25(9): 2173–2179]     

Predicting protein-protein interactions from sequences in a hybridization space

To understand the networks in living cells, it is indispensably important to identify protein-protein interactions on a genomic scale. Unfortunately, it is both time-consuming and expensive to do so solely based on experiments due to the nature of the problem whose complexity is obviously overwhelming, just like the fact that "life is complicated". Therefore, developing computational techniques for predicting protein-protein interactions would be of significant value in this regard. By fusing the approach based on the gene ontology and the approach of pseudo-amino acid composition, a predictor called "GO-PseAA" predictor was established to deal with this problem. As a showcase, prediction was performed on 6323 protein pairs from yeast. To avoid redundancy and homology bias, none of the protein pairs investigated has > or = 40% sequence identity with any other. The overall success rate obtained by jackknife cross-validation was 81.6%, indicating the GO-PseAA predictor is very promising for predicting protein-protein interactions from protein sequences, and might become a useful vehicle for studying the network biology in the postgenomic era.     

The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts

This study presents an analysis of the stromal proteome in its oligomeric state extracted from highly purified chloroplasts of Arabidopsis thaliana. 241 proteins (88% with predicted cTP), mostly assembled in oligomeric complexes, were identified by mass spectrometry with emphasis on distinguishing between paralogues. This is critical because different paralogues in a gene family often have different subcellular localizations and/or different expression patterns and functions. The native protein masses were determined for all identified proteins. Comparison with the few well characterized stromal complexes from A. thaliana confirmed the accuracy of the native mass determination, and by extension, the usefulness of the native mass data for future in-depth protein interaction studies. Resolved protein interactions are discussed and compared with an extensive collection of native mass data of orthologues in other plants and bacteria. Relative protein expression levels were estimated from spot intensities and also provided estimates of relative concentrations of individual proteins. No such quantification has been reported so far. Surprisingly proteins dedicated to chloroplast protein synthesis, biogenesis, and fate represented nearly 10% of the total stroma protein mass. Oxidative pentose phosphate pathway, glycolysis, and Calvin cycle represented together about 75%, nitrogen assimilation represented 5-7%, and all other pathways such as biosynthesis of e.g. fatty acids, amino acids, nucleotides, tetrapyrroles, and vitamins B(1) and B(2) each represented less than 1% of total protein mass. Several proteins with diverse functions outside primary carbon metabolism, such as the isomerase ROC4, lipoxygenase 2 involved in jasmonic acid biosynthesis, and a carbonic anhydrase (CA1), were surprisingly abundant in the range of 0.75-1.5% of the total stromal mass. Native images with associated information are available via the Plastid Proteome Database.     

Assessment of the pre-clinical immunogenicity of a new VEGF receptor Fc-fusion protein FP3 with ELISA and BIACORE

Purpose  

A new VEGF receptor fusion protein FP3 was shown to have promising antitumor potency better than Bevacizumab. Characterization of its immune response is essential to the safe and effective administration in clinical trials. In this study, both BIACORE and ELISA assays were employed to assess pre-clinical immunogenicity of FP3 in monkeys.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.