Preparation of cyclodextrin chiral stationary phases by organic soluble catalytic 'click' chemistry

We describe an effective and simple protocol that uses click chemistry to attach native β-cyclodextrin (β-CD) to silica particles, resulting in a chiral stationary phase (CCNCSP) that can be used for the enantioseparation of chiral drugs by high-performance liquid chromatography (HPLC). Starting from β-CD, the CCNCSP is prepared in several steps: (i) reaction of β-CD with 1-(p-toluenesulfonyl)-imidazole to afford mono-6-toluenesulfonyl-β-CD; (ii) azidolysis of mono-6-toluenesulfonyl-β-CD in dimethylformamide to give mono-6-azido-β-CD (N(3)-CD); (iii) reaction of cuprous iodide with triphenylphosphine to form an organic soluble catalyst CuI(PPh(3)); (iv) preparation of alkynyl-modified silica particles; and (v) click chemistry immobilization of N(3)-CD onto alkynyl-modified silica to afford the desired chiral stationary phase. Synthesis of the stationary phase and column packing takes ～1 week.     

Electro-acupuncture potentiates the disulphide-reducing activities of thioredoxin system by increasing thioredoxin expression in ischemia-reperfused rat brains

Reactive oxygen species can directly affect the conformation and activity of sulfhydryl-containing proteins by oxidation of their thiol moiety. During the process of ischemia-reperfusion, the thioredoxin (Trx) system (consisting of thioredoxin reductase (TR), Trx and NADPH) prevents susceptible proteins from this oxidative modification. Oxidative damage is one of the most damaging stress in ischemia. If oxidative stress could be minimized, the damage occurred will be minimized accordingly. We therefore investigated whether electroacupuncture (EA) treatment at Fengchi (GB20) or Zusanli (ST36) acupoints in post-ischemic rats could increase TR-related activities and Trx expression which would translate into maintaining the intact thiol moiety of susceptible proteins in the surrounding. Our results indicated that EA treatment at either acupoint increased the Trx expression in ischemic-reperfused brain tissues. Induced Trx expressed levels gradually increased from post-ischemia day 1 to day 4. Statistical analysis revealed that there was no observable difference in the effect of EA treatment at GB20 and ST36. Sham EA treatment did not induce any Trx expression. EA at either acupoint did not alter TR activities in both non-ischemic and ischemic-reperfused rat brains. Taken overall, our finding suggests that EA treatment at GB20 or ST36 could increase Trx expression which could minimize oxidative modifications of thiol groups of surrounding proteins.     

Expression of recombinant MDA-BF-1 with a kinase recognition site and a 7-histidine tag for receptor binding and purification

Prostate cancer metastasizes predominantly to bone, where it induces osteoblastic lesions. Paracrine factors secreted by the metastatic cancer cells are thought to mediate these events. We previously isolated a novel bone metastasis-related factor (MDA-BF-1) from bone marrow aspirate samples from patients with prostate cancer and bone metastasis, and found that this factor stimulated osteoblast differentiation, possibly by interacting with a receptor on the osteoblasts. Identifying this putative MDA-BF-1 receptor biochemically requires the expression of MDA-BF-1 for receptor binding assays and for the preparation of a ligand-affinity column. We tagged MDA-BF-1 with a peptide containing a protein kinase A phosphorylation site plus a 7-histidine sequence to facilitate the labeling of MDA-BF-1 for receptor binding assay and the binding of MDA-BF-1 to an immobilized metal affinity column. The recombinant MDA-BF-1 protein (MDA-BF1-kinase-his) was expressed in Sf9 cells using a baculovirus expression system. About 0.8 mg of purified MDA-BF1-kinase-his protein was obtained from 4 x 10(8) Sf9 cells. MDA-BF1-kinase-his can be phosphorylated by PKA with a specific activity around 10(5)cpm/mug protein. Receptor binding assays using this (32)P-labeled MDA-BF-1 showed that MDA-BF-1 bound to membranes prepared from Saos-2, an osteosarcoma cell line, and C2C12, a mouse pluripotent mesenchymal precursor cell line that can be induced to become osteoblast by BMP-2. In contrast, MDA-BF-1 did not bind to membranes from PC-3 human prostate cancer cells or HEK293 human embryonic kidney cells. These observations suggest that the MDA-BF-1 receptor is expressed in cells of osteoblastic lineage. In addition to its use as a ligand for receptor binding assays, a ligand affinity column can be prepared by binding MDA-BF1-kinase-his to an IMAC for the purification of MDA-BF-1 receptor.     

Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values

To facilitate the identification of candidate molecular biomarkers that are linked to the pathogenesis of hepatocellular carcinoma (HCC), we investigated protein-expression profiles of 146 tissue specimens including 67 pairs of tumors and adjacent non-tumors resected from HCC patients as well as 12 normal livers by 2-DE. Among the 1800 spots displayed in the liver proteome, a total of 90 protein species were found to be significantly different between the three groups (P < 0.05). Three of the top candidate markers up-regulated in HCC, with high receiver operating characteristic (ROC) curves, were identified by MS/MS analysis and belonged to the chaperone members: heat-shock protein (Hsp)27, Hsp70 and glucose-regulated protein (GRP)78. Over-expression of these chaperone proteins in HCC tissues was confirmed by Western blotting and immunohistochemistry. In correlation with clinico-pathological parameters, expression of Hsp27 was linked to alpha-fetoprotein level (P = 0.007) whereas up-regulation of GRP78 was associated with tumor venous infiltration (P = 0.035). No significant association of Hsp70 with any pathologic features was observed. The present HCC proteome analysis revealed that in response to the stressful cancerous microenvironment, tumor cells strived to increase the expression of chaperone proteins for cyto-protective function and to enhance tumor growth and metastasis.     

A data-mining approach for multiple structural alignment of proteins

Comparing the 3D structures of proteins is an important but computationally hard problem in bioinformatics. In this paper, we propose studying the problem when much less information or assumptions are available. We model the structural alignment of proteins as a combinatorial problem. In the problem, each protein is simply a set of points in the 3D space, without sequence order information, and the objective is to discover all large enough alignments for any subset of the input. We propose a data-mining approach for this problem. We first perform geometric hashing of the structures such that points with similar locations in the 3D space are hashed into the same bin in the hash table. The novelty is that we consider each bin as a coincidence group and mine for frequent patterns, which is a well-studied technique in data mining. We observe that these frequent patterns are already potentially large alignments. Then a simple heuristic is used to extend the alignments if possible. We implemented the algorithm and tested it using real protein structures. The results were compared with existing tools. They showed that the algorithm is capable of finding conserved substructures that do not preserve sequence order, especially those existing in protein interfaces. The algorithm can also identify conserved substructures of functionally similar structures within a mixture with dissimilar ones. The running time of the program was smaller or comparable to that of the existing tools.     

CDK5RAP2 stimulates microtubule nucleation by the gamma-tubulin ring complex

CDK5RAP2 is a human microcephaly protein that contains a γ-tubulin complex (γ-TuC)-binding domain conserved in Drosophila melanogaster centrosomin and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC-tethering proteins. In this study, we show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC-mediated nucleation activator (γ-TuNA). γ-TuNA but not its γ-TuC-binding-deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2-6. RNA interference-mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC-mediated microtubule nucleation.     

Senkyunolides reduce hydrogen peroxide-induced oxidative damage in human liver HepG2 cells via induction of heme oxygenase-1

Rhizoma Chuanxiong is widely used as folk medicine to treat the diseases caused by oxidative stress and inflammation. To delineate the underlying molecular mechanisms, we recently found that Rhizoma Chuanxiong extract significantly induced heme oxygenase-1 (HO-1), an enzyme that degrades intracellular heme into three bioactive products: biliverdin, carbon monoxide and free iron. The anti-inflammatory, antiapoptotic and antiproliferative actions of these products highlight HO-1 as a key endogenous antioxidant and cytoprotective gene. This study was designed to further characterize HO-1 induction of Rhizoma Chuanxiong through bioactivity-guided fractionation. All isolated fractions were assayed for HO-1 induction in human HepG2 cell line at mRNA and protein levels. Based on chromatographic profiling, nuclear magnetic resonance (NMR) and mass spectrometric analysis, the active compounds were identified as senkyunolide-H and its stereoisomer senkyunolide-I. Both senkyunolide isomers inhibited the formation of reactive oxygen species and lipid peroxidation and enhanced the cellular resistance to hydrogen peroxide-induced oxidative damage. Notably, heme oxygenase inhibitor tin protoporphyrin IX (SnPP) significantly suppressed the antioxidant activity of senkyunolide stereoisomers. Thus, this study demonstrated that senkyunolide-H and -I attenuated oxidative damage via activation of HO-1 pathway.