Monte Carlo simulation of trabecular bone remodelling and absorbed dose coefficients for tritium and 14C.

A Monte Carlo simulation of multiple trabecular bone cavities in adult bone was developed and the absorbed radiation dose factors evaluated for 3H and 14C. The model was developed to assess the dose from radionuclide uptake in quiescent bone, but also the effects of temporal changes in bone turnover by incorporating bone-modelling units (BMU). Absorbed dose fractions were calculated for target regions that include the endosteal layer where radiation-sensitive stem cells in bone marrow are considered to reside preferentially. There were large differences in the absorbed fractions for two types of bone surface, quiescent and forming. Tritium in quiescent bone results in a dose to the endosteum about 20 times that for the same activity in forming bone surface irradiating osteoblasts. When the quiescent bone surface source was extended from an infinitely thin layer to a more realistic 1 microm thick, the tritium absorbed fractions for endosteum and red marrow targets fell by more than 2-fold.

Anti-HIV agent trichosanthin enhances the capabilities of chemokines to stimulate chemotaxis and G protein activation, and this is mediated through interaction of trichosanthin and chemokine receptors.

Trichosanthin (TCS), an active protein component isolated from a traditional Chinese medicinal herb Trichosanthes kirilowii, has been shown to inhibit HIV infection and has been applied in clinical treatment of AIDS. The recent development that chemokines and chemokine receptors play important roles in HIV infection led us to investigate the possible functional interaction of TCS with chemokines and their receptors. This study demonstrated that TCS greatly enhanced both RANTES (regulated upon activation, normal T cell expressed and secreted)- and stromal cell-derived factor (SDF)-1 alpha-stimulated chemotaxis (EC50 approximately equal to 1 nM) in leukocytes (THP-1, Jurkat, and peripheral blood lymphocyte cells) and activation of pertussis toxin-sensitive G proteins (EC50 approximately equal to 20 nM). TCS also significantly augmented chemokine-stimulated activation of chemokine receptors CCR5 and CXCR4 as well as CCR1, CCR2B, CCR3, and CCR4 transiently expressed in HEK293 cells. A mutant TCS with 4,000-fold lower ribosome-inactivating activity showed similar augmentation activity as wild-type TCS. Moreover, flow cytometry demonstrated that the specific association of TCS to the cell membranes required the presence of chemokine receptors, and laser confocal microscopy reveals that TCS was colocalized with chemokine receptors on the membranes. The results from TCS-Sepharose pull-down and TCS and chemokine receptor coimmunoprecipitation and cross-linking experiments demonstrated association of TCS with CCR5. Thus, our data clearly demonstrated that TCS synergizes activities of chemokines to stimulate chemotaxis and G protein activation, and the effects of TCS are likely to be mediated through its interaction with chemokine receptors.

[Activation of G protein on the membrane of TCS-sensitive cells].

Trichosanthin (TCS), a Type I Ribosome Inactivating Protein isolated from the root tuber of Trichosanthes Kirilowii M. has various biological activities including abortion induction, anti-tumor and anti-HIV. The mechanism of TCS specifically killing sensitive cells has not been studied clearly. In this study, we initially found that there exists TCS-affinity molecule on Syncytiotrophoblast cells and Jar cells. Furthermore, by [35S]GTP gamma S Binding Assay, we find that TCS can activate G protein on the membrane of TCS-sensitive cells. These results indicate that on the membrane of TCS-sensitive cells exists TCS-specific receptor.

[Study on Trichosanthin induced apoptosis of leukemia K562 cells].

Trichosanthin (TCS), an eukaryotic ribosome-inactivating protein isolated from the root tuber of Trichosanthes plant, has various biological activities including abortion induction, antitumor, and anti-HIV. In this study, cultured human leukemia K562 cells treated with trichosanthin were examined. Analysis of the cells by single laser flow cytometry showed the sub-G1 peak. DNA extracted from these cells formed a characteristic "ladder" on agarose gel electrophoresis. Under electromicroscope, typical morphological changes of apoptosis were also observed. From all of these findings, we concluded that trichosanthin was able to induce apoptosis in K562 cells.

Structure-function relationship of trichosanthin.

Trichosanthin (TCS), a 27 kDa ribosome inactivating protein extracted from the root tuber of Trichosanthes plant, was subjected to limited chymotrypsin digestion and three peptide fragments designated TCS-F1 72-247, TCS-F2 100-247, and TCS-F3 133-247 were generated. The RNA-N-glycosidase and cytotoxic activities of the TCS fragments were compared with that of inact TCS. TCS-F1 and TCS-F2 were biologically active, while TCS-F3 was completely inactive. Dose-dependent studies showed that TCS-F1 and TCS-F2 were less potent in their trophoblast cytotoxicity than intact TCS, however, full biological effect could still be obtained with a higher dosage. Based on the known three-dimensional structure of TCS, we postulate that the putative active site of TCS is located at amino acid residues 110 to 174.

Synthesis of the 5'-half molecule of yeast alanine tRNA.

In this paper, we report the synthesis of the 5'-half molecule of yeast alanine tRNA (tRNAAlay) by ligating three oligonucleotide fragments corresponding to the nucleotide sequences 1-13, 14-22 and 23-35 respectively under the catalysis of T4 RNA ligase (Fig. 1). Because of the high purity of the oligonucleotide fragments and the excellent quality of T4RNA ligase and polynucleotide kinase we prepared, the isolation steps were simplified and the overall yields were much higher. The ligating yield of the docosamer (IV) was 75%, that of the pentatriacontamer (V), 90%, and the isolated yield of the final product was 21% calculated on the basis of the tridecamer (III) used in the first reaction. Under the action of T4 RNA ligase the synthetic 5'-half molecule was joined with the natural 3'-half molecule forming a semi-synthetic tRNAAlay, which possessed the biological activities of both accepting (3H)-alanine and incorporating it into proteins. The correctness of the structure of the synthetic 5'-half molecule was verified by both chemical analysis and biological activity assay.