Purification and characterization of UDPG:ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng

Heterogeneity of ginsenosides is an interesting and important issue because those structure-similar secondary metabolites have different or even totally opposite pharmacological activities. In this work, a new enzyme UDP-glucose:ginsenoside Rd glucosyltransferase (UGRdGT), which catalyzes the formation of ginsenoside Rb₁ from ginsenoside Rd [Biotechnol. Bioeng. 89: 444–52, 2005], was purified approximately 145-fold from suspended cells of Panax notoginseng with an overall yield of 0.2%. Purification to apparent homogeneity, as judged by SDS-PAGE, was successfully achieved by using sequential ammonium sulphate precipitation, anion-exchange chromatography and native PAGE. The enzyme had a molecular mass of 36 kDa, and its activity was optimal at pH 8.5 and 35 °C. The enzyme activity was enhanced by Mn²⁺, Ca²⁺ and Mg²⁺, but strongly inhibited by Zn²⁺, Hg²⁺, Co²⁺, Fe²⁺ and Cu²⁺. The apparent K_m value for UDP-glucose and ginsenoside Rd was 0.32 and 0.14 mM, respectively. The biotransformation yield from ginsenoside Rd to Rb₁ by UGRdGT in 50 mM Tris–HCl buffer at pH 8.5 and 35 °C was over 80%. This work provides a basis for further molecular study on the ginsenoside Rb₁ biosynthesis by P. notoginseng cells and it is also useful for potential application to in vitro biotransformation from ginsenoside Rd to Rb₁.     

乳蛋白生物活性肽的序列及其功能

乳蛋白经消化产生的肽类除了具有营养作用外,还具有多种生物活性,包括阿片肽和阿片拮抗肽活性及免疫调节,抗高血压、抗血栓、抗菌抗病毒,促进矿质元素吸收,防止腹泻等作用。本文对近几年发现的乳蛋白生物活性肽的序列结构及功能做了综述。     

Cytokine-induced cell death in immortalized Schwann cells: roles of nitric oxide and cyclic AMP

Tumor necrosis factor-alpha and interferon-gamma are pleiotropic cytokines that regulate Schwann cell responses during injury and inflammatory demyelination. We have previously shown that cyclic AMP (cAMP)-elevating agents decrease the demyelination and Wallerian degeneration in experimental allergic neuritis. In this study, we examined the role of cAMP in cytokine-mediated signaling in a spontaneously immortal Schwann cell clone (iSC). We found that tumor necrosis factor-alpha and interferon-gamma exert synergistic inhibitory action on Schwann cell viability via the production of nitric oxide (NO) and ceramide (cer). Furthermore, we found that: (i) NO synthase inhibitors attenuate the cytokine-induced cer accumulation and cell death indicating that NO acts upstream of cer; and (ii) cytokine-induced cell death is decreased in iSCs pretreated continuously for 48-72 h with forskolin, an activator of adenylate cyclase. Although forskolin modulates the phosphorylation of ERKs and Akt, it decreases the susceptibility of iSC to cytokines via a separate mechanism operating after NO induction and before cer accumulation. We propose that the protective effect of cAMP-elevating agents in experimental allergic neuritis may be mediated in part via modulation of Schwann cell responses to cytokines.     

Recent encounters with atropisomerism in drug discovery

Atropisomerism is stereochemistry arising from restricted bond rotation that creates a chiral axis. Atropisomers are subject to time-dependent inversion of chirality via bond rotation, a property which in drug molecules introduces complexity and challenges for drug discovery and development processes. Greater recognition of the occurrence of atropisomerism and improved characterization techniques have helped medicinal chemists successfully advance atropisomeric drug molecules. This review provides recent examples of atropisomerism encountered in medicinal chemistry efforts and the strategies used to address the accompanying challenges.     

MDR1 P-glycoprotein transports endogenous opioid peptides

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic μ-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a K_m of 15 and 12 μM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.     

The Role of Ceramide Synthases in the Pathogenicity of Cryptococcus neoformans

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