Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs

The substrates of most protein kinases remain unknown because of the difficulty tracing signaling pathways and identifying sites of protein phosphorylation. Here we describe a method useful in detecting subclasses of protein kinase substrates. Although the method is broadly applicable to any protein kinase for which a substrate consensus motif has been identified, we illustrate here the use of antibodies broadly reactive against phosphorylated Ser/Thr-motifs typical of AGC kinase substrates. Phosphopeptide libraries with fixed residues corresponding to consensus motifs RXRXXT*/S* (Akt motif) and S*XR (protein kinase C motif) were used as antigens to generate antibodies that recognize many different phosphoproteins containing the fixed motif. Because most AGC kinase members are phosphorylated and activated by phosphoinositide-dependent protein kinase-1 (PDK1), we used PDK1-/- ES cells to profile potential AGC kinase substrates downstream of PDK1. To identify phosphoproteins detected using the Akt substrate antibody, we characterized the antibody binding specificity to generate a specificity matrix useful in predicting antibody reactivity. Using this approach we predicted and then identified a 30-kDa phosphoprotein detected by both Akt and protein kinase C substrate antibodies as S6 ribosomal protein. Phosphospecific motif antibodies offer a new approach to protein kinase substrate identification that combines immunoreactivity data with protein data base searches based upon antibody specificity.     

Human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes

Recent epidemiologic studies show increasing human immunodeficiency virus type 1 (HIV-1) transmission through oral-genital contact. This paper examines the possibility that normal human oral keratinocytes (NHOKs) might be directly infected by HIV or might convey infectious HIV virions to adjacent leukocytes. PCR analysis of proviral DNA constructs showed that NHOKs can be infected by CXCR4-tropic (NL4-3 and ELI) and dualtropic (89.6) strains of HIV-1 to generate a weak but productive infection. CCR5-tropic strain Ba-L sustained minimal viral replication. Antibody inhibition studies showed that infection by CXCR4-tropic viral strains is mediated by the galactosylceramide receptor and the CXCR4 chemokine coreceptor. Coculture studies showed that infectious HIV-1 virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection.     

Single-step recovery of ephedrine hydrochloride from raw materials using expanded bed adsorption

Expanded bed adsorption, using a cation resin 001 x 7 Styrene-DVB, was used to recover and purify ephedrine hydrochloride from a powdered herb. The axial liquid-phase dispersion coefficient was about 10(-5) m(2) s(-1) and the recovery yield and purification reached 86% and 22, respectively. Compared with using conventional extraction with dimethylbenzene, this method is safer and also more efficient.     

Mining single nucleotide polymorphisms from EST data of silkworm, Bombyx mori, inbred strain Dazao

We made use of 81,635 expressed sequence tags (ESTs) derived from 12 different cDNA libraries of the silkworm, Bombyx mori, inbred strain Dazao (P50), to identify high-quality candidate single nucleotide polymorphisms (SNPs). By PHRAP assembling, 12,980 contigs containing 11,537 contigs assembled by more than one read were obtained, and 101 candidate SNPs and 27 single base insertions/deletions were identified from 117 contigs assembled from 1576 high-quality reads base-called with PHRED and screened on the basis of the neighborhood quality standard (NQS). Simultaneously, we also predicted 40 SNPs in coding regions (cSNPs), of which 26 were predicted to lead to amino acid non-synonymous variations and 14 synonymous substitutions. Also, the 1.66:1 ratio of transition/transversion is different from that of other insects. As the first SNP analysis of a Lepidoptera, B. mori, the single nucleotide polymorphic density is estimated to be 1.3 x 10(-3) by sequence diversity. This analysis shows that expressed sequences from multiple libraries may provide an abundant source of comparative reads to mine for cSNPs from the silkworm genome.     

Fabrication of gelatin nanofibrous scaffolds using ethanol/phosphate buffer saline as a benign solvent

Electrospinning of natural polymer nanofibers useful for biomedical applications often requires the use of cytotoxic organic solvents. In this study, gelatin nanofibers are electrospun from phosphate buffer saline/ethanol binary mixtures as a benign solvent at ambient temperature. The influences of ionic strength, ethanol concentration, and gelatin concentration on the electrospinnability of gelatin solutions and the fiber microarchitectures are analyzed. The electrospun scaffolds retain their morphologies during vapor‐phase crosslinking with glutaraldehyde in ethanol and the subsequent removal of salts contained in the nanofibers via water rinsing. When fully hydrated, the mechanically preconditioned scaffolds display a Young's modulus of 25.5 ± 5.3 kPa, tensile strength of 55.5 ± 13.9 kPa, deformability of 160 ± 15%, and resilience of 89.9 ± 1.8%. When cultured on the gelatin scaffolds, 3T3 fibroblasts displayed spindle‐like morphology, similar to the cell's normal morphology in a 3D extracellular matrix. © 2012 Wiley Periodicals, Inc. Biopolymers 97:1026–1036, 2012.     

Plasma metabonomics study of rheumatoid arthritis and its Chinese medicine subtypes by using liquid chromatography and gas chromatography coupled with mass spectrometry

Rheumatoid arthritis (RA) is the most severe type of chronic inflammatory disease and has always been a research hotspot in different fields. In this study, a non-targeted metabonomics approach was carried out to profile metabolic characteristics of RA and its Chinese medicine subtypes by using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). Plasma samples of 57 RA patients and 23 healthy controls were collected. On the basis of the traditional Chinese medicine (TCM), RA patients were classified into two main patterns, the cold pattern and the heat pattern. By using univariate and multivariate data analysis, we found that the RA patients presented diverse dysfunctions in inositol phosphate metabolism, lipid metabolism, amino acid metabolism, glucose metabolism, ascorbate metabolism, glyoxylate and dicarboxylate metabolism. The metabolic phenotypes were different between the RA cold pattern and the RA heat pattern. Compared with the RA cold pattern, the RA heat pattern showed elevated plasma concentrations of glycochenodeoxycholate, proline, saturated and mono-unsaturated phosphatidylcholine (PC) but decreased levels of urea, free fatty acid (FFA) and polyunsaturated PC. Our data show that metabonomics is a valuable tool in disease and TCM subtype research.     

Diterpenoids and a Diarylheptanoid from Hedychium coronarium with Significant Anti‐Angiogenic and Cytotoxic Activities

Two new labdane diterpenoids, namely hedycoronals A and B ( 1 and 2 , resp.), were isolated from the rhizomes of Hedychium coronarium, together with eight known diterpenoids, 4 – 11 , and a known diarylheptanoid, 3 . The structures of 1 and 2 were established by detailed interpretation of their 1D‐ and 2D‐NMR spectra and HR‐ESI‐MS data. Inhibitory activities against human umbilical vein endothelial cells (HUMECs) proliferation and cytotoxic activities against four cancer cell lines were assessed for all the isolates. Most of these metabolites showed moderate or potent cytotoxic activities against four cancer cell lines. Moreover, compounds 3 and 8 exhibited promising inhibitory activities against HUMECs with the IC₅₀ values of 6.4 to 3.3 μM .     

BVT.2733, a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor, attenuates obesity and inflammation in diet-induced obese mice

Background

Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is being pursued as a new therapeutic approach for the treatment of obesity and metabolic syndrome. Therefore, there is an urgent need to determine the effect of 11β-HSD1 inhibitor, which suppresses glucocorticoid action, on adipose tissue inflammation. The purpose of the present study was to examine the effect of BVT.2733, a selective 11β-HSD1 inhibitor, on expression of pro-inflammatory mediators and macrophage infiltration in adipose tissue in C57BL/6J mice.

Methodology/Principal Findings

C57BL/6J mice were fed with a normal chow diet (NC) or high fat diet (HFD). HFD treated mice were then administrated with BVT.2733 (HFD+BVT) or vehicle (HFD) for four weeks. Mice receiving BVT.2733 treatment exhibited decreased body weight and enhanced glucose tolerance and insulin sensitivity compared to control mice. BVT.2733 also down-regulated the expression of inflammation-related genes including monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α) and the number of infiltrated macrophages within the adipose tissue in vivo. Pharmacological inhibition of 11β-HSD1 and RNA interference against 11β-HSD1 reduced the mRNA levels of MCP-1 and interleukin-6 (IL-6) in cultured J774A.1 macrophages and 3T3-L1 preadipocyte in vitro.

Conclusions/Significance

These results suggest that BVT.2733 treatment could not only decrease body weight and improve metabolic homeostasis, but also suppress the inflammation of adipose tissue in diet-induced obese mice. 11β-HSD1 may be a very promising therapeutic target for obesity and associated disease.