Induction of nitric oxide synthase by Oldenlandia diffusa in mouse peritoneal macrophages.
(1/21)
Oldenlandia diffusa (OD) has been used to treat malignant tumors. In this study using mouse peritoneal macrophages, we have examined the mechanism by which OD regulates nitric oxide (NO) production. When OD (1 mg/ml) was used in combination with 10 U/ml of recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production (36.13+/-7.12 microM) by the Griess method (nitrite). Treatment of macrophages with rIFN-gamma plus OD (1 mg/ml) caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production (4.49+/-1.43 ng/ml) by enzyme-linked immunosorbent assay. The increased production of NO and TNF-alpha from rIFN-gamma-plus OD-stimulated cells was almost completely inhibited by pretreatment with 100 microM of pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). PDTC also inhibited phosphorylation of IkappaB in rIFN-gamma-plus OD-stimulated cells. These findings demonstrate that OD increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of OD. (+info)
Disulfide folding pathways of cystine knot proteins. Tying the knot within the circular backbone of the cyclotides.
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The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (Le-Nguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway. (+info)
Disulfide mapping of the cyclotide kalata B1. Chemical proof of the cystic cystine knot motif.
(3/21)
The cyclotides are a recently discovered family of plant proteins that have the fascinating structural feature of a continuous cyclic backbone and, putatively, a knotted arrangement of their three conserved disulfide bonds. We here show definite chemical proof of the I-IV, II-V, III-VI knotted disulfide connectivity of the prototypic cyclotide kalata B1. This has been achieved by a new approach for disulfide analysis, involving partial reduction and stepwise alkylation including introduction of charges and enzymatic cleavage sites by aminoethylation of cysteines. The approach overcomes the intrinsic difficulties for disulfide mapping of cyclotides, i.e. the cyclic amide backbone, lack of cleavage sites between cysteines, and a low or clustered content of basic amino acids, and allowed a direct determination of the disulfide bonds in kalata B1 using analysis by mass spectrometry. The established disulfide connectivity is unequivocally shown to be cystine knotted by a topological analysis. This is the first direct chemical determination of disulfides in native cyclotides and unambiguously confirms the unique cyclic cystine knot motif. (+info)
Clinical observation in 86 cases of acne vulgaris treated with Compound Oldenlandis Mixture.
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86 cases of acne vulgaris were treated with Fu Fang She She Cao He Ji ([symbol: see text] Compound Oldenlandis Mixture), with the other 34 cases treated with Dang Gui Ku Shen Wan ([symbol: see text] Pills Prepared from Chinese Angelica and Flavescent Sophora Root) as the controls, to observe the therapeutic effect of the former. The results showed that the cure plus markedly effective rate was 73.26% in the treatment group, and 47.06% in the control group, with a significant difference in the cure plus markedly effective rate between the two groups (P < 0.01), and also in the total effective rate between the two groups significant (P < 0.05). It may be concluded that the Compound Oldenlandis Mixture is a better agent for the illness. (+info)
Processing of a 22 kDa precursor protein to produce the circular protein tricyclon A.
(5/21)
Cyclotides are a family of plant proteins that have the unusual combination of head-to-tail backbone cyclization and a cystine knot motif. They are exceptionally stable and show resistance to most chemical, physical, and enzymatic treatments. The structure of tricyclon A, a previously unreported cyclotide, is described here. In this structure, a loop that is disordered in other cyclotides forms a beta sheet that protrudes from the globular core. This study indicates that the cyclotide fold is amenable to the introduction of a range of structural elements without affecting the cystine knot core of the protein, which is essential for the stability of the cyclotides. Tricyclon A does not possess a hydrophobic patch, typical of other cyclotides, and has minimal hemolytic activity, making it suitable for pharmaceutical applications. The 22 kDa precursor protein of tricyclon A was identified and provides clues to the processing of these fascinating miniproteins. (+info)
Kalata B8, a novel antiviral circular protein, exhibits conformational flexibility in the cystine knot motif.
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The cyclotides are a family of circular proteins with a range of biological activities and potential pharmaceutical and agricultural applications. The biosynthetic mechanism of cyclization is unknown and the discovery of novel sequences may assist in achieving this goal. In the present study, we have isolated a new cyclotide from Oldenlandia affinis, kalata B8, which appears to be a hybrid of the two major subfamilies (Mobius and bracelet) of currently known cyclotides. We have determined the three-dimensional structure of kalata B8 and observed broadening of resonances directly involved in the cystine knot motif, suggesting flexibility in this region despite it being the core structural element of the cyclotides. The cystine knot motif is widespread throughout Nature and inherently stable, making this apparent flexibility a surprising result. Furthermore, there appears to be isomerization of the peptide backbone at an Asp-Gly sequence in the region involved in the cyclization process. Interestingly, such isomerization has been previously characterized in related cyclic knottins from Momordica cochinchinensis that have no sequence similarity to kalata B8 apart from the six conserved cysteine residues and may result from a common mechanism of cyclization. Kalata B8 also provides insight into the structure-activity relationships of cyclotides as it displays anti-HIV activity but lacks haemolytic activity. The 'uncoupling' of these two activities has not previously been observed for the cyclotides and may be related to the unusual hydrophilic nature of the peptide. (+info)
Determination of iridoid glucosides for quality assessment of Herba Oldenlandiae by high-performance liquid chromatography.
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Herba Oldenlandiae, the dried herb of Oldenlandia diffusa (WILLD.) ROXB. (Family Rubiaceae), is officially listed in the Chinese Pharmacopoeia. In the herbal market, two substitutes originated from O. corymbosa (L.) LAM and O. tenelliflora BL. are commonly used. In light of this, the target in setting up a method for quality assessment of Herba Oldenlandiae is urgently needed. In this article, a simple and reliable high-performance liquid chromatographic (HPLC) method was developed for quantifying asperuloside (1), E-6-O-p-coumaroyl scandoside methyl ester (2) and E-6-O-p-coumaroyl scandoside methyl ester-10-methyl ether (3) in Herba Oldenlandiae derived from O. diffusa. Among them, compound 3 is a new compound isolated from O. diffusa. All these unique compounds were used as markers for the first time in the quality assessment of Herba Oldenlandiae. The results showed that the contents of compounds 1-3 were significantly varied among different samples whilst those of compounds 2 and 3 were found to be lower in contents in the two substitutes of O. diffusa. The analytical method is suitable for quality control of Herba Oldenlandiae and useful in differentiation from its confusable species. The method has been fully validated with satisfactory linearity, accuracy, precision and stability. (+info)
Evidence for Oldenlandia diffusa-evoked cancer cell apoptosis through superoxide burst and caspase activation.
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BACKGROUND & OBJECTIVE: Oldenlandia diffusa (Bai Hua She She Cao) is one of the herbs most commonly used in traditional Chinese medicine for treating cancer. Various studies using the herb alone or in combination with other therapy plans have evidenced the effectiveness of the herb in the management of cancers of different tissue origin. However, the mechanisms underlying its anti-cancer activity are unknown. In the present study, we attempted to investigate the apoptotic activity of crude extracts of the herb as well as the possible molecular pathways. METHODS: We incubated human promyelocytic leukemia cell line HL60 cells with ethanol or aqueous extracts of the herb, and determined the levels of intracellular superoxide at 2 and 4 hours as well as caspase activity at 3, 6 and 8 hours using photospectrometry. Cancer cell survival and apoptosis were quantified at 24 hours by using MTT and flow cytometry analyses respectively. RESULTS: We found that it dose-dependently inhibited the cancer cell growth in MTT assay. Flow cytometry analysis revealed that it elicited significant production of sub-G(1) population of the cells, indicating the extract-evoked cell apoptotic death. The LD(50) of the ethanol extract was estimated to be approximately 320 microg/ml. Moreover, treatment of the cancer cells with the ethanol component markedly increased the production of superoxide within few hours. Significant elevation in the protease activities of caspases-2 and -3 were detected at as early as 3 and 6 hours respectively. CONCLUSION: Our results show that the ethanol extract of the herb effectively evokes cancer cell apoptosis, possibly through burst-mediated caspase activation. (+info)