Construction of recombinant plasmids containing rat thyroglobulin mRNA sequences

Two plasmids containing rat thyroglobulin cDNA sequences have been constructed and characterized. A plasmid with a 500-bp insert (pRT6) was isolated and identified as thyroglobulin-specific on the basis of the tissue specificity of the inserted sequence and of its ability to retain thyroglobulin mRNA on a nitrocellulose filter. The cDNA insert in pRT6 was subsequently used to screen a rat thyroid cDNA library constructed with large cDNA. A plasmid was found containing a 1700-bp insert. The polarity and the fidelity of the insert is demonstrated by S1 mapping.     

A critical role of protein-bound water in the catalytic cycle of cytochrome P-450 camphor.

The rates of NADH oxidation during the hydroxylation of camphor by cytochrome P-450cam were followed in the presence of co-solvents used to increase the osmotic pressure surrounding the protein-bound water. As a result, the measured Vmax decreases independently of the perturbant tested. Roughly 28 molecules of water, involved during the catalytic cycle, are deduced from the variation of Vmax as a function of osmotic pressure. These molecules, in part, could be those present in the cytochrome P-450cam-putidaredoxin interface.     

Triptolide inhibits colon cancer cell proliferation and induces cleavage and translocation of 14‐3‐3 epsilon

Triptolide is a diterpenoid triepoxide derived from the traditional Chinese medical herb Tripterygium wilfordii. In the present study, we demonstrated that this phytochemical attenuated colon cancer growth in vitro and in vivo. Using a proteomic approach, we found that 14‐3‐3 epsilon, a cell cycle‐ and apoptosis‐related protein, was altered in colon cancer cells treated with triptolide. In this regard, triptolide induced cleavage and perinuclear translocation of 14‐3‐3 epsilon. Taken together, our findings suggest that triptolide may merit investigation as a potential therapeutic agent for colon cancer, and its anticancer action may be associated with alteration of 14‐3‐3 epsilon. Copyright © 2012 John Wiley & Sons, Ltd.     

Molecular mechanisms of <Emphasis Type="Italic">Bombyx batryticatus</Emphasis> ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.     

Role of Drosophila gene dunc-115 in nervous system

Axonal guidance signals are transduced through growth cone surface receptors to the interior leading to changes of actin dynamics and actin binding proteins, which are critical in determining the outcome of actin cytoskeleton reorganization. We report here the characterization of the Drosophila actin binding protein abLIM/Unc-115 homolog Dunc-115 and its role in the nervous system. Three Dunc-115 isoforms are identified as Dunc-115L, M and S, respectively. While Dunc-115L is a canonical homolog of Unc-115 with four LIM domains and one villin headpiece domain, Dunc-115M and S are novel isoforms without counterparts in other species. Our molecular modeling shows Dunc-115L is likely to bind to actin. Mutant analysis reveals that Dunc-115 is involved in axonal projection in both the visual and central nervous system.