Heterogeneity of the ribosome-inactivating protein trichosanthin in Trichosanthes kirilowii tubers

The ribosome-inactivating protein trichosanthin isolated from the tubers of Trichosanthes kirilowii, the Chinese drug Tianhuafen, has a molecular mass of approximately 26 kDa. We show here that T. kirilowii tubers also contain ribosome-inactivating proteins with a small extent of structural variation from and a larger molecular mass than trichosanthin.     

Expression of a gene encoding trichosanthin in transgenic rice plants enhances resistance to fungus blast disease

. The gene encoding mature trichosanthin, a type I ribosome-inactivating protein isolated from the tuber of Trichosanthes kirilowii Maximowicz, was transformed into calli of rice (Oryza sativa L.) by bombardment. Transgenic rice plants were obtained and confirmed by Southern and Western blot analysis. When transgenic rice plants expressing trichosanthin were inoculated with the spores of Pyricularia oryzae, a major rice fungus blast pathogen, the lesions on leaves were much less severe, and the seedling survival rate and whole plant weight were higher than those of control plants with the gus gene. The presented data demonstrate a novel, potential role of trichosanthin in antifungal protection in transgenic plants.     

Biosynthesis and bioactivity of trichosanthin in cultured crown gall tissues of Trichosanthes kirilowii Maximowicz

Trichosanthin (TCS) from Trichosanthes kirilowii Maximowicz (T. kirilowii) can be used to treat choriocarcinoma. In this work, we established a novel system to produce TCS in crown gall tissues of T. kirilowii infected by Agrobacterium tumefaciens C58 (A. tumefaciens). In the crown gall tissues, a nopaline synthase (NOS) gene of A. tumefaciens was identified by polymerase chain reaction (PCR), and nopaline accumulation was confirmed by a high-voltage filter paper electrophoresis. Furthermore, we optimized conditions to culture the crown gall tissues able to grow fast and produce TCS in an auxin-free medium, and found that a fungal elicitor of Armillaria mellea was capable of stimulation of TCS secretion into the medium. Moreover, we identified that the TCS purified from the crown gall tissues could induce gastric cancer cell death. These data underscore the usefulness of our system as an inexpensive and virtually unlimited source of TCS.     

天花粉收白诱发白血病细胞K562凋亡的研究

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.     

Two novel proteins bind specifically to trichosanthin on choriocarcinoma cell membrane

Trichosanthin is the active protein component in the Chinese herb Trichosanthes kirilowi, which has distinct pharmacological properties. The cytotoxicity of trichosanthin was demonstrated by its selective inhibition of various choriocarcinoma cells. When Jar cells were treated with trichosanthin, the influx of calcium into the cells was observed by confocal laser scanning microscopy. When the distribution of trichosanthin-binding proteins on Jar cells was studied, two classes of binding sites for trichosanthin were shown by radioligand binding assay. Furthermore, the cytoplasmic membrane of Jar cells was biotinylated and the trichosanthin-binding proteins were isolated with trichosanthin-coupled Sepharose beads. Two protein bands with molecular masses of about 50 kDa and 60 kDa were revealed, further characterization of which should shed light on the mechanism of the selective cytotoxicity of trichosanthin to Jar cells.     

Purification and characterization of trichosanthin. Homology to the ricin A chain and implications as to mechanism of abortifacient activity

Trichosanthin, a protein from the Chinese medicinal herb Trichosanthes kirilowii, was purified in two essentially quantitative steps involving CM-Sephadex chromatography and reverse-phase high performance liquid chromatography. The protein was found to have a molecular mass of 25-26 kDa, to contain no cysteine, and to contain no glycosidic linkages. Pure trichosanthin was found to have potent abortifacient activity in pregnant mice. In order to understand the molecular basis of this unique biological activity, we have examined the amino acid sequence of the protein. As purified, trichosanthin was found to contain two amino-terminal sequences which differed only in the absence or presence of a tyrosine at residue 1. Sequence analysis of trichosanthin has allowed for determination of the NH2-terminal 38-amino acid residues. Comparison of this sequence to those present in a data base revealed homology with the ricin A-chain. Consistent with this structural homology, we have found that trichosanthin is a potent inhibitor of protein synthesis in a reticulocyte lysate system.     

The mechanism of action of trichosanthin on eukaryotic ribosomes--RNA N-glycosidase activity of the cytotoxin.

Trichosanthin is a ribosome-inactivating protein from root tubers of Trichosanthes kirilowii Maxim. In this paper, the mechanism of action of trichosanthin on eukaryotic ribosomes was studied. A fragment of about 450 nucleotides was released from 28S ribosomal RNA after treatment of rat liver ribosome with trichosanthin and its isolated ribosomal RNAs were treated with aniline. Analysis of nucleotide sequence of 5' terminus of this fragment revealed that the aniline-sensitive site of the phosphodiester bond was between positions A4324 and G4325 in the 28S rRNA. Adenine was recovered by ion-exchange column chromatography from the 50% ethanol soluble fraction of the reaction mixture in which rat liver ribosomes were treated with trichosanthin. Thin-layer chromatographic analysis indicated that 1 mol of adenine was released from 1 mol of ribosomes. When the ribosomes were incubated with trichosanthin in the presence of inorganic [32P]phosphate, little incorporation of radioactivity into 28S rRNA was observed, indicating that the release of adenine was not mediated by phosphorolysis. These results demonstrate that trichosanthin inactivates the ribosomes by cleaving the N-C glycosidic bond of adenylic acid at 4324 of 28S rRNA in a hydrolytic fashion.     

Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.

     

Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro.

Trichosanthin, an abortifacient, immunosuppressive and anti-tumor protein purified from the traditional Chinese herb medicine Tian Hua Fen, is a potent inhibitor against HIV-1 replication. Under normal enzymatic digestion conditions, trichosanthin cleaves the supercoiled double-stranded DNA to produce nicked circular and linear DNA. Trichosanthin has no effect on linear double-stranded DNA. Neither does it convert relaxed circular duplex DNA into a supercoiled form in the presence of ATP. Thus trichosanthin is not a DNA gyrase. However, trichosanthin can cleave the relaxed circular DNA into a linear form, indicating that both the circular as well as the supercoiled forms are essential for trichosanthin recognition. In addition, trichosanthin contains one calcium metal ion per protein molecule, which presumably is related to its endonucleolytic activity.     

A chemotaxonomic study of flavonoids in the leaves of six Trichosanthes species

The 7-, 3′- and 4′-glucosides of luteolin, the 7-glucoside and 6,8-di-C-glucoside of apigenin were isolated from Trichosanthes kirilowii var. japonica. Kaempferol 3,7-di-rhamnoside and 3-glucoside-7-rhamnoside were identified from T. cucumeroides, kaempferol 3-galactoside and 3-sophoroside were also identified from T. anguina. Quercetin-3-rutinoside was detected from T. multiloba and T. rostrata. T. bracteata afforded luteolin 3′-glucoside and kaempferol 3-rutinoside, and T. kirilowii afforded luteolin 7-, 3′- and 4′-glucosides and apigenin 7-glucoside.     

Production of expansin from light/dark growing Trichosanthes kirilowii var. Japonicum root cultures

Summary Actively growing Trichosanthes kirilowii var. Japonicum root culture grown under light produces two proteins that are similar to the S1 and S2 proteins (expansin) extracted from Cucumis sativus cv Burpee Pickler stems. These proteins play a role in the expansion (swelling) of cell walls. Since the root cultures can be easily handled and scaled up, root cultures may be an alternative source for the production of expansin.     

Beta-trichosanthin: a new abortifacient protein from the Chinese drug, wangua, Trichosanthes cucumeroides

beta-Trichosanthin was a new abortifacient protein purified from the Chinese drug, Wangua, root tubers of Trichosanthes cucumeroides (Cucurbitaceae). The purification procedure involved acetone fractionation, ammonium sulfate precipitation, ion-exchange chromatography on CM-Sepharose and preparative agarose electrophoresis. Homogeneity of beta-trichosanthin was demonstrated in immunoelectrophoresis, agarose electrophoresis and SDS-polyacrylamide gel electrophoresis. It had a molecular weight of 28,000 and no cysteine in its molecule. It differed from trichosanthin, a known abortifacient protein isolated from a related Chinese drug, Tianhuafen, root tubers of Trichosanthes kirilowii (Cucurbitaceae), in molecular weight, carbohydrate content, charge and amino acid composition. beta-Trichosanthin was, however, immunochemically identical to trichosanthin and was about twice as potent as trichosanthin in inducing mid-term abortion in mice.     

Improved isolation and further characterization of beta-trichosanthin,a ribosome-inactivating and abortifacient protein from tubers of trichosanthes cucumeroides (cucurbitaceae)

• 1.1. Beta-trichosanthin was isolated from root tubers of Trichosanthes cucumeroides with a procedure involving acetone fractionation, ion exchange chromatography on CM-Sepharose and DEAE-Sepharose and gel filtration on Sephadex G-50.
• 2.2. The protein was homogeneous by SDS-polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. It possessed a molecular weight of 28,000 and was a strongly basic glycoprotein.
• 3.3. It was immunochemically identical to trichosanthin but different from alpha- and beta-momorcharins.
• 4.4. It possessed potent abortifacient and ribosome-inactivating activities. In the latter type of activity it was more potent than trichosanthin.

     

Ribosomal protein L10a, a bridge between trichosanthin and the ribosome

Trichosanthin is a type I ribosome-inactivating protein with many pharmacological activities. The trichosanthin-coupled Sepharose affinity purification revealed a protein, which was identified by mass spectrometry as the ribosomal protein L10a. The interaction between trichosanthin and recombinant L10a was further confirmed by in vitro binding assay. Kinetic analysis by surface plasmon resonance technology revealed that L10a had a high affinity to trichosanthin with a K(D) of 7.78nM. The study with mutated forms of trichosanthin demonstrated that this specific association correlates with the ribosome-inactivating activity of trichosanthin. This finding might provide insight into the mechanisms by which trichosanthin inactivates ribosome and that underlies its pharmacological effect.     

Properties of bromodextran-trichosanthin: a comparison with trichosanthin, an anti-AIDS protein.

Trichosanthin was coupled with bromodextran and the reaction mixture chromatographed on Sephadex G-75 fine to yield two peaks. The first peak was judged to be bromodextran-trichosanthin by SDS-polyacrylamide gel electrophoresis. The yield was optimal when 4% trichosanthin and 8% bromodextran T20 were reacted for 210 hours. Bromodextran-trichosanthin exhibited an ultraviolet absorption spectrum similar to that of free trichosanthin and the complex reacted positively with the trichosanthin antiserum. It had lower abortifacient and protein synthesis inhibiting activities but it also possessed lower allergenicity, suggesting that it may be useful as a substitute of trichosanthin, especially when the problem of hypersensitivity caused by prolonged administration of trichosanthin is serious.     

Sterine in Trichosanthes kirilowii

Four Δ⁵-sterols and six Δ⁷-sterols were isolated from the seed oil of Trichosanthes kirilowii and identified as campesterol, sitosterol, stigmasterol, Δ⁷-campesterol, Δ⁷-stigmasterol, Δ^7,22-stigmastadienol, 24-ethylcholesta-5,25-diene-3β-ol, 24-ethylcholesta-7,24(25)-diene-3β-ol, 24-ethylcholesta-7,25-diene-3β-ol, and 24-ethylcholesta-7,22,25-trine-3β-ol.     

Identification of a stable complex of trichosanthin with nicotinamide adenine dinucleotide phosphate

Trichosanthin, a type I ribosome-inactivating protein with RNAN-glycosidase activity, forms a stable complex with nicotinamide adenine dinucleotide phosphate, a substrate analog. Difference UV spectroscopy, fluorescence spectroscopy, and³¹P NMR are used to identify the formation of the complex, followed by a crystal structure analysis carried out to elucidate the active-site structure of trichosanthin. The determination of germinal vesicle breakdown indicates that the complex does not, at least for abortion-inducing activity, result in competitive inhibition to the protein.     

Fusion with maltose-binding protein (MBP) affects neither RNA N-glycosidase activity nor immunogenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

A genomic clone encoding mature karasurin-A (KRNA), a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica, was efficiently expressed in E. coli using an expression cassette vector pMAL-c2. The resultant recombinant KRNA fused with maltose-binding protein (MBP) was recovered from the soluble fraction of the bacterial cells and purified to near homogeneity after one round of the affinity chromatography. Neither the karasurin precursor retaining both N- and C-terminal peptides, nor the protein with the N-terminal peptide was successufully produced even as a MBP-fusion. The protein with its C-terminal peptide was over-produced but was recovered in an insoluble fraction. Both the recombinant MBP-KRNA fusion protein and recombinant KRNA with MBP removed were as active as the native KRNA from root tubers. The immunogenicity of the recombinant KRNA was also unaffected by fusion with MBP.     

利用栝楼发根生产天花粉蛋白的研究

利用发根农杆菌 (Agrobacterium rhizogenes)菌株 R1 60 1 ,成功地从药用植物栝楼 (Trichosantheskirilowii Maxim.)中诱导出发根并建立了栝楼发根的离体培养系统。通过蛋白质印迹法对栝楼发根的天花粉蛋白进行了鉴定。利用 SDS- PAGE和 TLC扫描 ,测定出每克鲜重栝楼发根中天花粉蛋白的含量可达 8.1 6mg。     

Differential response of leaf gas exchange to enhanced ultraviolet-B (UV-B) radiation in three species of herbaceous climbingplants

We measured diurnal changes in photosynthetic rate, transpiration rate, stomatal conductance and water use efficiency in three species of herbaceous climbing plants (Luffa cylindrica, Trichosanthes kirilowii and Dioscorea opposita) exposed to two intensities of UV-B radiation: 3.0 μw cm^?2 (R1) and 8.0 μw cm^?2 UV-B (R2) radiation under ambient growth conditions. Responses differed per species and per treatment. In Luffa all values increased compared to the Control in both treatments, except for stomatal conductance in R2. In Trichosanthes photosynthetic rates and water use efficiency increased, while the transpiration rates decreased under both treatments, and stomatal conductance was lower in R1. In Dioscorea photosynthetic rates and water use efficiency decreased under both treatments, while the transpiration rates and stomatal conductance increased. The results suggested that to some extent increased UV-B radiation was beneficial to the growth of L. cylindrica and T. kirilowii, but detrimental to D. opposita.