Design and synthesis of a novel class of furan-based molecules as potential 20S proteasome inhibitors

A novel class of furan-based compounds as potential 20S proteasome inhibitors have been designed and synthesized, among which nine compounds are peptide derivatives and six molecules are statine peptidomimetics. The C-terminal furanyl moiety was introduced to target molecules as furan-based amino acids. All the compounds were obtained steadily with moderate to high yield. Compound 12 was a selective moderate potent proteasome peptidomimetic inhibitor. It inhibited HepG2 and HL-60 proliferation effectively.     

Bromodeoxyuridine labelling and fluorescence‐activated cell sorting of polyamine‐transforming bacterioplankton in coastal seawater

Polyamines (PAs) are a group of nitrogen‐rich dissolved organic nitrogen (DON) compounds that are ubiquitously distributed in marine environments. To identify bacteria that are involved in PA transformations, coastal bacterioplankton microcosms were amended with a single PA model compound, i.e. putrescine (PUT) or spermidine (SPD), or with no addition as controls (CTRs). Bromodeoxyuridine (BrdU) was added to all the microcosms to label newly synthesized DNAs. Fluorescence‐activated cell sorting (FACS) analysis indicated significant increases in numbers of total cells and cells with both high and low levels of BrdU incorporation in the PUT and SPD microcosms, but not in the CTRs. 16S rDNA pyrotag sequencing of FACS‐sorted cells indicated that PUT‐ and SPD‐transforming bacteria were composed similarly of a diverse group of taxa affiliated with Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria (especially Roseobacter of its alpha lineage). Broad taxonomic distribution of PA‐transforming bacteria was also indicated by the abundance and distribution of PA transporter gene homologues in a survey of sequenced marine bacterial genomes. Our results suggest that PAs may be common DON substrates for marine bacterioplankton, in line with the hypothesis that bacterially mediated PA transformation accounts for an important proportion of marine DON flux.     

Analysis of fatty acids in A. szechenyianum Gay. by microwave‐assisted extraction and gas chromatography‐mass spectrometry

Introduction – Aconitum szechenyianum Gay. is a traditional Chinese medicinal herb with the detumescent and styptic effects and antitumor activity. There have been only a few researches on its chemical components, but no detailed report has appeared on its fatty acids. Objective – To develop a simple and effective method for the extraction of fatty acids from A. zechenyianum Gay. and then to investigate the fatty acid components. Methodology – Microwave‐assisted extraction (MAE) was optimized with response surface methodology, and the fatty acid compositions of extract were determined by GC–MS with previous derivatisation to fatty acid methyl esters (FAMEs). The results were compared with that obtained by classical Soxhlet extraction (SE). Results – Compared with SE, MAE showed significantly higher fatty acid yields, shorter extraction time, and lower energy and solvent consumption. The major fatty acids in A. szechenyianum Gay. are linoleic acid, palmitic acid, linolenic acid, oleic acid and stearic acid, and the unsaturated fatty acids occupy 66.4% of the total fatty acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Affinity purification of a cholesterol oxidase expressed in Escherichia coli

A cholesterol oxidase (COD) gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), an affinity protocol was developed for the preparation, and industrial application of this method was of great potential. Riboflavin was chosen as the affinity ligand, and it was coupled with Sepharose 4B through some spacers. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture the target protein. The purified cholesterol oxidase was 99.5% pure analyzed on HPLC Vydac C4 column, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 9.8% of crude extracted proteins; the recovery of typical cholesterol oxidase activity was 90.1%, higher than that of other reported traditional protocols. Reducing SDS-PAGE analysis showed that the enzyme was a single polypeptide with the mass of ～50 kDa. The desorption constant K(d) and the theoretical maximum absorption Q(max) on the affinity medium were 1.0 μg/g medium and 74.5 mg/g medium in absorption analysis. K(m) and V(max) of cholesterol oxidase activity for the purified enzyme were 25.5 μM and 16.4 μmol/(min mg), respectively.     

Genes "waiting" for recruitment by the adaptive immune system: the insights from amphioxus

In seeking evidence of the existence of adaptive immune system (AIS) in ancient chordate, cDNA clones of six libraries from a protochordate, the Chinese amphioxus, were sequenced. Although the key molecules such as TCR, MHC, Ig, and RAG in AIS have not been identified from our database, we demonstrated in this study the extensive molecular evidence for the presence of genes homologous to many genes that are involved in AIS directly or indirectly, including some of which may represent the putative precursors of vertebrate AIS-related genes. The comparative analyses of these genes in different model organisms revealed the different fates of these genes during evolution. Their gene expression pattern suggested that the primitive digestive system is the pivotal place of the origin and evolution of the AIS. Our studies support the general statement that AIS appears after the jawless/jawed vertebrate split. However our study further reveals the fact that AIS is in its twilight in amphioxus and the evolution of the molecules in amphioxus are waiting for recruitment by the emergence of AIS.     

Anti-inflammatory effects of tocopherol metabolites

Our objective was to assess the anti-inflammatory effects of alpha-tocopherol, gamma-tocopherol, and their metabolites 2,5,7,8-tetramethyl-2-(beta-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC) in defined cell culture systems. Rat aortic endothelial cells and mouse microglial cultures were treated with tumor necrosis factor TNFalpha or bacterial lipopolysaccharide (LPS) and nitrite and prostaglandin E(2) (PGE(2)) were measured. alpha-CEHC suppressed TNFalpha-stimulated nitrite production in both cell types, whereas both CEHC derivatives inhibited LPS-stimulated microglial nitrite efflux. Both alpha-CEHC and gamma-CEHC inhibited microglial PGE(2) production, but neither alpha- nor gamma-tocopherol was effective at inhibiting cytokine-stimulated inflammatory processes. These results show that the anti-inflammatory effects of tocopherols are highly cell type-, stimulus-, and endpoint-dependent.     

The arachidonic acid 5-lipoxygenase inhibitor nordihydroguaiaretic acid inhibits tumor necrosis factor alpha activation of microglia and extends survival of G93A-SOD1 transgenic mice

Familial forms of amyotrophic lateral sclerosis (ALS) can be caused by mutations in copper, zinc-superoxide dismutase (SOD1). Mice expressing SOD1 mutants demonstrate a robust neuroinflammatory reaction characterized, in part, by up-regulation of tumor necrosis factor alpha (TNFalpha) and its primary receptor TNF-RI. In an effort to identify small molecule inhibitors of neuroinflammation useful in treatment of ALS, a microglial culture system was established to identify TNFalpha antagonists. Walker EOC-20 microglia cells were stimulated with recombinant TNFalpha, with or without inhibitors, and the cell response was indexed by NO2- output. Three hundred and fifty-five rationally selected compounds were included in this bioassay. The arachidonic acid 5-lipoxygenase (5LOX) and tyrosine kinase inhibitor nordihydroguaiaretic acid (NDGA), a natural dicatechol, was one of the most potent non-cytotoxic antagonists tested (IC50 8 +/- 3 microm). Investigation of the G93A-SOD1 mouse model for ALS revealed increased message and protein levels of 5LOX at 120 days of age. Oral NDGA (2500 p.p.m.) significantly extended lifespan and slowed motor dysfunction in this mouse, when administration was begun relatively late in life (90 days). NDGA extended median total lifespan of G93A-SOD1 mice by 10%, and life expectancy following start of treatment was extended by 32%. Disease-associated gliosis and cleaved microtubule-associated tau protein, an indicator of axon damage, were likewise reduced by NDGA. Thus, TNFalpha antagonists and especially 5LOX inhibitors might offer new opportunities for treatment of ALS.