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Central neuromedin U 2 receptor (NMU2R) plays important roles in the regulation of food intake and body weight. Identification of NMU2R agonists may lead to the development of pharmaceutical agents to treat obesity. Based on the structure of rutin, a typical flavonoid and one of the NMU2R agonists we previously identified from an in-house made natural product library, 30 flavonoid derivatives have been synthesized and screened on a cell-based reporter gene assay. A number of compounds were found to be selective and highly potent to NMU2R. For example, the EC50 value of compound NRA 4 is very close to that of NMU, the endogenous peptide ligand of NMU2R. Structure-activity relationship analysis revealed that a 3-hydroxyl group in ring C and a 2'-fluoride group in ring B were essential for this class of compounds to be active against NMU2R.

Three new (1-3) and several known (4-6) steroids were isolated from the leaves of Chinese Melia azedarach. The structures of the new compounds were elucidated by means of spectroscopic methods including 2D NMR techniques and mass spectrometry to be (20S)-5,24(28)-ergostadiene-3beta,7alpha,16beta,20-tetrol (1), (20S)-5-ergostene-3beta,7alpha,16beta,20-tetrol (2), and 2alpha,3beta-dihydro-5-pregnen-16-one (3). The cytotoxicities of the isolated compounds against three human cancer cell lines (A549, H460, U251) were evaluated; only compounds 1, 2, and (20S)-5-stigmastene-3beta,7alpha,20-triol (4) were found to show significant cyctotoxic effects with IC(50)s from 12.0 to 30.1 microg/mL.

In addition to twenty-nine known compounds, two new guaiane sesquiterpenes and two new furanocoumarins were isolated from the chloroform extract of the rhizomes of Notopterygium incisum. The new structures were elucidated by means of spectroscopic methods including 2 D NMR techniques and mass spectrometry to be 8 beta-acetoxy-4 alpha,6 alpha-dihydroxy-1 alpha,5 alpha( H)-guai-9-ene (incisumdiol A, 1), 4 alpha,6 alpha-dihydroxy-1 alpha,5 alpha( H)-guai-9-ene (incisumdiol B, 2), 5-[(2 E,5 Z)-7-hydroxy-3,7-dimethyl-2,5-octadienoxy]psoralene ( 3) and 5-[(2,5)-epoxy-3-hydroxy-3,7-dimethyl-6-octenoxy]psoralene ( 4).

Interleukin (IL)-2 is a pharmacologically important cytokine secreted by T-lymphocytes. Recombinant IL-2 (rIL-2) has been modified and produced in many systems. Mass production of rIL-2 is the prerequisite for its wide application. Using a site-directed mutagenesis strategy, we first generated a gene coding for a new type of mutant of human IL-2 (MhIL-2), in which we replaced the cysteine-125 in human IL-2 with alanine, the leucine-18 with methionine, and the leucine-19 with serine. Then we investigated the possibility of its production of MhIL-2 in a Pichia pastoris system. High-level secreted expression of MhIL-2 was achieved by methanol induction. When purified with ultrafiltration, cation-exchange chromatography, and Sephadex G100 gel filtration, about 100 mg of MhIL-2 with high purity was obtained from 1 L of ferment supernatant. Biologic activity assay revealed that the purified recombinant protein displayed increased activity on proliferation of IL-2-dependent CTLL-2 cells. These results suggest that MhIL-2 is an improved IL-2 mutant that might hold great promise for clinical use, and that P. pastoris is an excellent system for the mass production of biologically active hIL-2.

Aim: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library. Methods: Synthetic responsive elements 6timescAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. Results: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC 50 values of SBG492 were 342.7 mug/mL for the D1 receptor and 31.7 mug/mL for the D5 receptor. Conclusion: We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

A new dimeric psoralen-type furanocoumarin, notopterol-(18-O-20′)-notopol (1), was isolated from Notopterygium incisum for the first time and its structure was elucidated by spectroscopic methods.

Nine fructose-derived carbohydrates were obtained from the methanol extract from the rhizome of Alisma orientalis. On the basis of spectroscopic analysis, their structures were determined to be alpha-D-fructofuranose (1), beta-D-fructofuranose (2), ethyl alpha-D-fructofuranoside (3), ethyl beta-D-fructofuranoside (4), 5-hydroxymethyl-furaldehyde (5), sucrose (6), raffinose (7), stachyose (8) and verbascose (9), along with two oligosaccharides of manninotriose (10) and verbascotetraose (11). Compounds 3, 4 and 7-11 were isolated from this plant for the first time. A hypothetical biosynthesis pathway among these isolated carbohydrates (1-11) was briefly introduced.

Background The G-protein-coupled receptor (GPCR) superfamily represents the largest protein family in the human genome. These proteins have a variety of physiological functions that give them well recognized roles in clinical medicine. In Xenopus tropicalis, a widely used animal model for physiology research, the repertoire of GPCRs may help link the GPCR evolutionary history in vertebrates from teleost fish to mammals. Results We have identified 1452 GPCRs in the X. tropicalis genome. Phylogenetic analyses classified these receptors into the following seven families: Glutamate, Rhodopsin, Adhesion, Frizzled, Secretin, Taste 2 and Vomeronasal 1. Nearly 70% of X. tropicalis GPCRs are represented by the following three types of receptors thought to receive chemosensory information from the outside world: olfactory, vomeronasal 1 and vomeronasal 2 receptors. Conclusion X. tropicalis shares a more similar repertoire of GPCRs with mammals than it does with fish. An examination of the three major groups of receptors related to olfactory/pheromone detection shows that in X. tropicalis, these groups have undergone lineage specific expansion. A comparison of GPCRs in X. tropicalis, teleost fish and mammals reveals the GPCR evolutionary history in vertebrates.

We have investigated the effects of lysophosphatidylcholine (LPC), a product of lipid peroxidation, on Abeta(1-42)-induced SH-SY5Y cell apoptosis.The viability of cultured SH-SY5Y cells was measured using a CCK-8 kit. Apoptosis was determined by Chip-based flow cytometric assay. The mRNA transcription of Bcl-2, Bax, and caspase-3 were detected by using reverse transcription and real-time quantitative PCR and the protein levels of Bax and caspase-3 were analyzed by Western blotting. The cytosolic calcium concentration of SH-SY5Y cells was tested by calcium influx assay. G2A expression in SH-SY5Y cells was silenced by small interfering RNA.Long-term exposure of SH-SY5Y cells to LPC augmented the neurotoxicity of Abeta(1-42). Furthermore, after LPC treatment, the Bax/Bcl-x(L) ratio and the expression levels, as well as the activity of caspase-3 were, elevated, whereas the expression level of TRAF1 was reduced. Because LPC was reported to be a specific ligand for the orphan G-protein coupled receptor, G2A, we investigated LPC-mediated changes in calcium levels in SH-SY5Y cells. Our results demonstrated that LPC can enhance the Abeta(1-42)-induced elevation of intracellular calcium. Interestingly, Abeta(1-42) significantly increased the expression of G2A in SH-SY5Y cells, whereas knockdown of G2A using siRNA reduced the effects of LPC on Abeta(1-42)-induced neurotoxicity.The effects of LPC on Abeta(1-42)-induced apoptosis may occur through the signal pathways of the orphan G-protein coupled receptor.

To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library.Synthetic responsive elements 6 cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings.A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC(50) values of SBG492 were 342.7 microg/mL for the D1 receptor and 31.7 microg/mL for the D5 receptor.We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

Interleukin-2 (IL-2) was initially isolated as a T cell growth factor and had been shown to direct the expansion and differentiation of several hematopoietic cell types. Clinical studies using IL-2 in the treatment of AIDS have been encouraging, due to its critical role as a proliferative signal for activated T-lymphocytes. IL-2 has also undergone trials in the treatment of several types of cancer, based on its stimulation of cytotoxic, antitumor cells. Today, human IL-2 is produced completely by genetically engineered method, and it has been proved that genetically engineered recombinant human IL-2 has almost the same function and clinical effect as wild IL-2. In the former study, recombinant human IL-2 usually comes from E. coli, in this paper the mutant IL-2 was successfully expressed and purified in Pichia pastoris for the first time. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression level. Expression conditions of human mutant interleukin-2(the codon for cysteine-125 of human IL-2 with alanine; the codon for leucine-18 with methionine; the codon for leucine-19 with serine) in the recombinant Pichia pastoris strain were optimized via test of some factors such as the rate of aeration, the inductive duration, the initial pH and the concentration of methanol. The results from tests showed that the most important parameter for efficient expression of interleukin-2 in recombinant Pichia pastoris strain is adequate aeration during methanol induction, and the optimum inductive condition for interleukin-2 expression was: more than 80% aeration, 2 days for induction, the initial pH of 6.0, the final methanol concentration of 1.0%. With this condition, the expressed IL-2 was secreted into fermentation broth and reached a yield of 30%, approximately 200 mg/L. Expressed interleutin-2 (MvIL-2) was isolated and purified by centrifugation, millipore filtration to concentration, Econo-PacS strongly acidic cation exchanger cartridge and molecular sieve chromatography and the yield of MvIL-2 was 27%. MvIL-2 was purified to electrophoretic purity by SDS-PAGE and only one peak being loaded on HPLC. Purified MvIL-2 protein had stimulating activity similar to the wild type of IL-2 as assayed by IL-2-dependent CTLL-2 cells. However, the stability of MvIL-2 was superior than that of IL-2 at different temperatures. The activity of obtained MvIL-2 was 4 - 5 times of the wild type of IL-2, So MvIL-2 had an advantage over wild type of rhIL-2 in storage stability and activity.