Ribosome Inactivating Proteins from Plants Inhibiting Viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

Isolation of a genomic DNA for Jatropha curcas ribosome inactivating protein and its tobacco transformation

Genomic DNA for Jatropha curcas ribosome inactivating protein （JRIP） was cloned from total DNA of its leaves by polymerase chain reaction （PCR）. The no intron character was confirmed. The plant expression vector pBI121-JRIP was constructed by inserting the JRIP gene into pBI121 plasmid. The recombinant Agrobacterium EHA105 strain harboring pBI121-JRIP was constructed by conducting pBI121-JRIP to strain EHA 105. PCR and Southern blotting were carried out, and the results proved that the JRIP gene was integrated into tobacco genome. It might provide a new material for disease resistance tobacco species breeding.

The Study of Anti-tumor Activity of Trichosanthin by Cyclic Voltammogram

The anti-tumor activity of Trichosanthin (TCS) has been frequently reported in recent years. In our experiments, electrochemical methods were applied to detect the effects of TCS on human leukemia cells U937. 50 mu g/ml TCS treatment for 40 hours can cause irreversible negative effects on the viability of U937 cells. This effect largely depends on the concentration of TCS and the time period of treatment.

Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C-treated Tobacco Leaves

A new ribosome-inactivating protein （RIP） with a molecular weight of 31 kDa induced by Cinchonaglycoside C （1） designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that it belongs to the RIP family and it was distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence. CIP31 can directly impair synthesis of coat protein （CP） of tobacco mosaic virus （TMV）, which resulted in inhibition of TMV long distance movement and multiplication in tobacco plants at concentrations of ng/mL. Furthermore, no toxicity was shown to the growth and fertility of the plants. CIP31 was synthesized only in the presence of Cinchonaglycoside C （1） and was independent of the salicylic acid （SA） signal pathway. We provided evidence for the SA-independent biological induction of resistance.

Molecular Replacement Studies of Cucurmosin from Cucurbita Moschata:Structure Homology with Trichosanthin

High diffraction quality crystals of cucurmosin, a type I ribosome inactivating protein isolated from the sarcocarp of Cucurbita moschata (pumpkin), have been grown under newly optimised conditions. With in-house rotating anode X-ray source, these crystals diffract to 1.65 ?resolution which is much higher than that of the previously reported crystals that diffracted only to 3 ?resolution. The crystals belong to space group P212121 with cell parameters a = 41.5, b = 58.4 and c = 99.3 . Molecular replacement studies indicate that the cucurmosin structure is homologous to trichosanthin. The initial structural model has been obtained and the model fitting/ refinement is in progress.

Resistance identification of bivalent fungi-resistant genes transformed soybean to Phytophthora sojae

Soybean is one of the most important sources of edible oil and proteins in the world. However, it suffers from many kinds of fungal diseases which is a major limiting factor in soybean production. The fungal disease can be effectively controlled by breeding plant cultivars with genetic transformation. In this study, the resistance to Phytophthora sojae of five bivalent transgenic soybean lines was identified using the hypocotyls inoculation technique. The lines were the T2 of the transgenic soybean which were transformed with kidney bean chitinase gene and barley ribosome inactivating protein gene, and were positive by Southern Blot analysis. The resistance difference was studied through comparing the death percentage of transgenic soybean with the control. The results showed that four lines were more resistant to P sojae, whereas other one had no significant difference in comparison with the control. These transgenic soybean lines with enhanced resistance to P sojae will be useful in soybean resistance breeding.

Activation of c-Jun N-terminal Kinases by Ribotoxic Stresses

The c-Jun N-terminal kinases （JNKs） are classic stress-activated protein kinases. Many cellular stresses have been shown to stimulate JNK activation. In this review, we focus on ribotoxic stresses based on their multiple biological potencies including anti-HIV-1 activity. Some of the functions of ribotoxins and the signaling transduction pathway that mediated are mentioned. Different from other stimulators, ribotoxic stresses act on special motifs of 28S rRNA in translationally active mammal ribosomes. Binding and damaging on the motif leads to JNK activation and subsequently biological response to the signal initiator, which is named ribotoxic stress response. Cellular ＆ Molecular Immunology. 2005;2（6）：419-425.

Chemical synthesis of a structural gene coding for trichosanthin

A structural gene (750 bp), which codes for a type I ribosome inactivating protein, trichosanthin, has been designed according to the codon usage of highly expressed gene in E. coli and chemically synthesized. In the synthesized gene, twenty-seven unique restriction sites were evenly dispersed with an average distance between two adjacent sites less than 50 bp to facilitate a systematic investigation on structure-functional relationship of this protein by site-directed mutagenesis. To synthesize it, the whole gene was divided into three large fragments (EP, PN and NH) which were assembled from several chemical synthetic oligonucleotides by enzymatic method. The assembly of both the fragment EP from six oligonucleotides (A-F) and the fragment PN from four oligomers (G-J) was catalyzed by T-4 DNA ligase in using the single stranded DNA method [Chen, H.-B. et al., Nucl. Acids Res., 18, 871(1990)]. And fragment NH was formed from three duplexes K, L and M by the classical double stranded DNA method. Finally, each fragment was cloned into vector pUC18 in succession to form the plasmid, pC0TCS, to complete the whole gene synthesis, The sequencing data for the synthetic gene coincides with the designed one.