Gypsophila Paniculata Research Articles

The Endocytic Uptake Pathways of Targeted Toxins Are Influenced by Synergistically Acting Gypsophila Saponins

The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better.

English

Gypsophila L. is the third biggest genus of Caryophyllaceae family in Turkey. The genus has 55 species in the country. 33 species are endemics and total number of the taxa is 58. Ankyropetalum Fenzl is a small genus with 3 species and 1 of them is endemic. Saponaria L. has 20 taxa of 18 species and 10 taxa of them are endemics to Turkey. Turkey is the gene center of all the genera. All of the genera are known as coven, female coven and halva root and developed roots or rhizomes are economically very important. Extract produced from the roots are known as fire extinguisher, gold polishing, cleaner and softener of delicate fabrics and crispness giving to halva. The extracts are often used for making liqueur, herbal cheese, ice cream and some foods. Some taxa are Boron (B) hyperacumulators, so they can be planted and used for destroyed agriculture to control erosion. Gypsophila paniculata L. is very important in horticulture. Gypsophila bicolor, Gypsophila arrostii, Gypsophila perfoliata, Ankyropetalum gypsophiloides and Saponaria officinalis L. are very important because of their saponin contents. In this paper, economic importance of the plants in the light of our observations and literatures was given. Key words: Gypsophila, Ankyropetalum, Saponaria, economy, saponin.

In vitro Flowering of Shoots Regenerated from Cultured Nodal Explants

A protocol for the regeneration of Gypsophila paniculata L. using nodal explants from 2-month-old field grown plants was established. The induction of multiple shoots was best obtained on Murashige and Skoog (MS) medium supplemented with 13.3 μM BA. Callus growth was observed on MS medium containing 44.3 μM BA. Calluses were transferred to MS medium supplemented with 2, 4-D (4.5, 13.5, 22.6 μM), NAA (5.3, 16.1, 26.8 μM) or BA (4.4, 13.3, 22.1 μM) for 2 months to induce shoot formation. After 6 weeks of initial culture, multiple shoots were regenerated from calluses cultured on MS medium supplemented with 13.3 μM BA. All regenerated shoots produced roots on 16.1 μM NAA containing MS medium within 4 weeks. Rooted plantlets were hardened and established in pots at 100% survival. For induction of in vitro flowering, regenerated shoots could be induced to flower efficiently when cultured on MS medium containing 13.3 μM BA and 50 g/l sucrose.

Abstract 767: Gypsophila saponins significantly augment the cytotoxicity of saporin-based anti-CD19, -CD22, -CD38 and -CD71 immunotoxins in human leukemia and lymphoma

Abstract Expanding the therapeutic window of immunotoxins (IT) is an important goal that would open up greater therapeutic possibilities for this class of drug. Saponins from Gypsophila paniculata have been shown to augment the cytotoxicity of the ribosome-inactivating protein (RIP) saporin and saporin containing cytotoxins that are directed against EGFR in cell culture and mouse models of human cancer. The mechanism through which saponins exert this effect is not clearly understood but is probably mediated through membrane disruption of endosomes that contain internalised IT thus allowing more efficient escape of IT to the cytosol and thence direct access of the RIP to target ribosomes. Here we show that Gypsophila saponins (SA) dramatically and significantly augment the cytotoxicity of saporin-based ITs directed against CD19, CD22, CD38 and CD71 expressed on the membrane surface of five different human leukaemia and lymphoma cell lines. We observed a high degree of variability in the augmentive effect exerted by SA, not only between different cell lines but also between different target molecules expressed on the same or on different cell lines. For instance SA augmented the anti-CD19 IT BU12-Saporin by only 10-fold in NALM-6 cells (representing a reduction in IT IC50 from 3 × 10−10M to 3 × 10−11M by SA) but by a massive 9.43 million-fold in Daudi cells (representing a reduction in IT IC50 from 5 × 10−9M to 5.3 × 10−16M by SA), despite there being a similar expression level of CD19 by each cell line. Through antibody blocking experiments and the use of cells not expressing the antigen targeted by the IT under study we revealed that SA does bring about a modest reduction in the immunospecificity of IT activity. However, by scheduling pulse exposure of target cells to IT first followed by washout and then exposure to SA it was possible to retain full immunospecificity. We speculate that the differences in SA augmentive effect on IT cytotoxicity between different cell lines and different target molecules on the same cell line probably represents individual variations in lipid/cholesterol membrane composition, particularly within lipid microdomains (lipid rafts) to which target molecules such as CD19 and CD38 migrate prior to receptor-mediated endocytosis following cross linking by the antibody component of IT. The variations in augmentive effect of SA on IT cytotoxicity observed for the different cell lines in the present study makes understanding the mechanism behind this phenomenon very important if saponins are to be sucessfully developed as a treatment modality to be used in conjunction with saporin-based ITs or cytotoxins for individual patients with haematological and other types of malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 767. doi:10.1158/1538-7445.AM2011-767

Flowering and expression of flowering-related genes under long-day conditions with light-emitting diodes

The effects of light quality on flowering time were investigated in Gypsophila paniculata, which is a long-day cut flower, and with Arabidopsis under long-day conditions with light-emitting diodes (LEDs). Gypsophila paniculata plants were grown under natural daylight and flowering was controlled by long-day treatment with a weak LED light of a single color in the night. Flowering was promoted not by blue light, but by far-red light in G. paniculata, while flowering was promoted by both light colors in Arabidopsis. FT homologs of G. paniculata GpFT1 and GpFT2 were differentially expressed under long-day conditions with white light, suggesting that they play roles in flowering at different stages of reproductive development. GpFTs and FT gene expression was not induced by far-red light in G. paniculata or Arabidopsis. Instead, the expression of the SOC1 homolog of G. paniculata GpSOC1 and SOC1 was induced by far-red light in G. paniculata and Arabidopsis. Flowering was promoted by induction of FT and SOC1 expression with blue light in Arabidopsis, whereas GpFTs and GpSOC1 expression was low with blue light induction in G. paniculata. The relationship between flowering and the expression of FT and SOC1 in Arabidopsis was confirmed with ft and soc1 mutants. These results suggest that long-day conditions with far-red light promote flowering through SOC1 and its homologs, while the conditions with blue light do not promote flowering in G. paniculata, because of low expression of GpFTs and GpSOC1 in contrast to that in Arabidopsis.

Pollinator Visits to Threatened Species Are Restored Following Invasive Plant Removal

An indirect consequence of plant invasions is the disruption of native plant-pollinator interactions. We examined effects of invasive baby’s breath (Gypsophila paniculata) and spotted knapweed (Centaurea maculosa) on floral visitors to federally threatened Pitcher’s thistle (Cirsium pitcheri) in Lake Michigan dunes. In sweep net surveys, abundances of pollinator taxa were five times higher in invaded than in naturally invader-free sites. However, plot-level G. paniculata removal treatments increased pollinator visits to C. pitcheri relative to invaded plots and restored visitation to levels found in naturally uninvaded plots. Invader removal also increased native plant species richness, which was positively correlated with pollinator visitation to C. pitcheri, suggesting an indirect effect on pollinators mediated through invader-altered plant composition. In temporary floral arrays, the rate of pollinator visitation to C. pitcheri was not affected by neighbor plant species identity. However, compared with native Monarda punctata, invasive C. maculosa attracted more total pollinators to the array but reduced the proportion of total visits that were to C. pitcheri and increased pollinator movements between plant species. While both G. paniculata and C. maculosa appear to act as magnet species by attracting more pollinators at the plot level, these invaders have the potential to reduce reproduction of C. pitcheri by decreasing pollinator visits and increasing interspecific pollen transfer.

First Report of Crown Rot on Gypsophila (Gypsophila paniculata) Caused by Fusarium proliferatum in Korea.

Gypsophilas commonly cultivated are Gypsophila elegans B. and G. paniculata L. In September of 2009 and 2010, a severe wilt symptom due to crown rot was observed on G. paniculata (cv. Bristol Fairy) in greenhouses in Yeosu, South Korea. The area of cultivation (~8 ha) in Yeosu covers 90% of production in the Jeonnam Province. Disease outbreak was 20 to 30% in affected greenhouses. Early symptoms included brown discoloration surrounding basal stems and slight wilting. Late symptoms included a sunken stem rot next to the roots, root rot, severe wilting, and dying plants. The causal fungus appeared to invade plants through the basal stem, causing a crown rot that prevented the plant from taking up water and nutrients. Crown rot occurred on young and mature plants. Ten fungal isolates were recovered from basal stems and roots of wilted plants. Microconidia were abundantly produced on potato dextrose agar (PDA), V8 juice agar (VA), carnation leaf agar (CLA), and oatmeal agar (OA). Microconidia were single celled, variable, oval-ellipsoid cylindrical, straight to curved, club-to-kidney shaped or spindle shaped on OA, more slender on VA. Macroconidia were not found on any media used. Microconidia on PDA were 5.9 to 15.1 (9.9) × 2.7 to 4.3 (3.5) μm. Germinated conidia (or false conidia) were often formed on CLA. Conidiophores as phialides were singly formed but often branched. Length of conidiophores was up to 31.1 μm on CLA. Small-sized chlamydospores were rarely found. Fusarium isolates (EML-GYP1, 2, and 3) were selected and identified. From extracted genomic DNA, the internal transcribed spacer (ITS) region including 5.8S rDNA was amplified using ITS1F (5'-CTTGGTCATTTAGAGGAAGT-3') and LR5F (5'-GCTATCCTGAGGGAAAC-3') primers. Sequence analyses by BLAST indicated that the isolates (GenBank HM560019, HM560020, and HM560021) were most similar to F. proliferatum (EF4534150) with sequence identity values of 99.3, 99.4, and 99.1%, respectively. The causal fungus was determined to be F. proliferatum based on morphological data and ITS rDNA sequences. Pathogenicity tests with the three isolates were performed on 10 plants of G. paniculata using the dipping method. Healthy roots and basal stems were soaked in a conidial suspension adjusted to ~1.2 × 106 conidia/ml (distilled water) for 15 min. Plants were potted in sterile soil, kept in a humid chamber for 72 h, and moved to a greenhouse. The experiment was carried out in duplicate and repeated two times. Similar symptoms to those observed in the greenhouses were seen 7 days after inoculation. The causal fungus was reisolated from the artificially inoculated basal stems, fulfilling Koch's postulates. Control plants whose basal stems and roots were dipped in sterile water showed no crown rot and wilt symptoms. EML-GYP2 was determined to be the most pathogenic. Ten records of disease caused by three Fusarium species (Fusarium sp., F. oxysporum, and F. udum) have been found on gypsophilas (1), but only F. oxysporum has been reported to cause wilt on G. elgans in Korea (2). To our knowledge, this is the first report of crown rot on gypsophila caused by F. proliferatum in Korea as well as the world.

Targeted toxins and saponins – a powerful cooperation

Saponins from Gypsophila species are known to enhance the cytotoxicity of targeted toxins. In general targeted toxins consist of two components: a toxin moiety with enzymatic activity and a ligand moiety, which mediates specific binding to cancer cells. It was shown that saponins from Gypsophila paniculata L. enhanced the toxicity of a targeted toxin, composed of the N-glycosidase saporin from Saponaria officinalis L. and human epidermal growth factor up to 2,500 000-fold in primary breast cancer cells. Meanwhile the efficiency of the combined application of saponins and targeted toxins was shown in a mouse tumour model. Compared to the single application of the targeted toxin (SA2E), the combination with saponins lead to a tremendous reduction (at least 50-fold) of the SA2E dosage. By this dose reduction side effects were only moderate. This represents an enormous step forward in the targeted anti tumour therapy with recombinant fusion toxins. However, in all these studies a mixture of saponins was used. For the further successful development of a combinatorial anti cancer therapy with targeted toxins and saponins pure saponins are mandatory. This study was intended to isolate pure saponins from Gypsophila paniculata L. in order to scruntinize the toxicity enhancing properties of isolated Gypsophila saponins on targeted toxins. One of the four isolated saponins showed strong, one moderate and two no cytotoxicity enhancing properties. On the basis of these results we determined the structural prerequisites for the saponin-mediated toxicity enhancement of targeted toxins.

Epidermal growth factor receptor expression affects the efficacy of the combined application of saponin and a targeted toxin on human cervical carcinoma cells

Cervical cancer is the second most common cancer in women worldwide. Targeting the epidermal growth factor receptor (EGFR) is a very promising approach since it is overexpressed in about 90% of cervical tumors. Here, we quantified the toxic effect of SE, a targeted toxin consisting of epidermal growth factor (EGF) as targeting moiety and the plant toxin saporin-3, on 3 common human cervical carcinoma cell lines (HeLa, CaSki and SiHa) and recently established lines (PHCC1 and PHCC2) from 2 different individuals. A human melanocytic and a mouse cell line served as negative control. Additionally, we combined SE with saponinum album, a saponin composite from Gypsophila paniculata, which exhibited synergistic properties in previous studies. The cell lines, except for SiHa cells, revealed high sensitivity to SE with 50% cell survival in the range of 5-24.5 nM. The combination with saponin resulted in a remarkable enhancement of cytotoxicity with enhancement factors ranging from 9,000-fold to 2,500,000-fold. The cytotoxicity of SE was clearly target receptor specific since free EGF blocks the effect and saporin-3 alone was considerably less toxic. For all cervical carcinoma cell lines, we evinced a clear correlation between EGFR expression and SE sensitivity. Our data indicate a potential use of targeted toxins for the treatment of cervical cancer. In particular, the combination with saponins is a promising approach since efficacy is drastically improved.

Root in vitro cultures of six Gypsophila species and their saponin contents

A simple and rapid method of excised root cultures from six Gypsophila species was performed allowing continuous growth without phytohormones. Established on MH3 medium from solid-grown seedlings these roots were subcultured for 1 year on a solid medium before being transferred in a liquid medium to obtain substantial biomass for saponin content analysis. Morphologically, the different root lines presented different growth behaviors and different physical aspects: some have linear growth by the tip of the main axial root from the original seedling; others have additional lateral root initiations producing a hairy root-system more or less dense. A marked increase of biomass was observed in the light by comparison with dark conditions. Significant growth for Gypsophila glomerata was achieved within 3 weeks on liquid medium; biomass grew up to 50-fold in batch cultures reaching 10 g DW. The fingerprints of the saponin HPLC profiles of the six Gypsophila species were drastically different with at least up to 30 different saponins detected for some of them. The roots of Gypsophila elegans accumulated saponins up to 65 mg/g DW. These amounts were higher than in Gypsophila paniculata roots classically found as the best producing ones. On the contrary the root lines of G. glomerata showed a smaller quantitative amount of saponins (between 1.3 and 7.10 mg/g DW) than those of G. elegans but nearly the same HPLC profiles as for root extracts of G. paniculata plants grown directly in the fields.

Abstract 5624: Saponins from Gypsophila paniculata L significantly potentiate the immunospecific cytotoxic activity of anti-CD19 and CD38 saporin-based immunotoxins for a human lymphoma cell line

Abstract Saponins are a highly diverse group of glycosides of plant origin containing either a steroidal or triterpenoid aglycone to which one or more sugar chains are attached. They have been variously described as enhancing the cytotoxic activity of ribosome-inactivating proteins (RIPs) and ligand-directing conjugates constructed with these for eukaryotic cells. Saponins are thought to facilitate the entry of RIPs into the cytosol across the membranes of intracellular vesicular structures. This characteristic of saponins therefore makes them of interest as molecules that could be useful in improving the therapeutic index of immunotoxins (IT) in a clinical setting. In the current study we used a protein synthesis inhibition assay based on leucine incorporation together with cell proliferation studies in culture to demonstrate that saponins from Gypsophila paniculata L. potentiate the cytotoxicity of the anti-CD19 and CD38 saporin-based immunotoxins (BU12-SAPORIN and OKT10-SAPORIN, respectively) by several thousand-fold for the human lymphoma cell line Daudi. Various experiments revealed that the enhancing effect of saponins on immunotoxin activity did not appear to be dependent on extra cellular pH in the assay system we used. Kinetic studies further revealed that saponin at a predetermined optimal concentration of 2 μg/ml reduced the t10 (time taken to reduce protein synthesis to 10% of the level seen in control Daudi cells) for OKT10-SAPORIN at 0.1μg/ml from 50 hours without saponin to 12.5 hours with saponin. In blocking experiments using OKT10-SAPORIN IT at the IC50 concentration in combination with saponin together with a gross excess of native OKT10 antibody we established that the immunospecific cytotoxic activity of OKT10-SAPORIN was fully retained when leucine incorporation was used as the surrogate measure of cytotoxicity but only partially so when thymidine incorporation was used. This observation of a partial uncoupling of protein and DNA synthesis as a measure of cytotoxicity was further supported by cell proliferation experiments in culture which were undertaken in parallel. If saponins are to be of any clinical value as candidate molecules for improving the therapeutic index of immunotoxins it is imperative that the immunospecificity of IT activity is fully retained. Whilst this study clearly demonstrates that saponins from G. paniculata L. do indeed significantly potentiate IT activity it seems that this effect may only be partially immunospecific. Nevertheless, the dramatic improvements that saponins confer on IT activity argue in favor of in vivo experiments in transgenic animal models of human lymphoma combined with additional studies to explore other strategies that might be employed to improve the immunospecific effect achievable. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5624.

안개초(Gypsophila paniculata L.)로부터 dihydroflavonol 4-reductase 유전자의 분리 및 분석

Dihydroflavonol 4-reductase (DFR) is a key enzyme of the flavonoid biosynthesis pathway which catalyses the NADPH-dependent reduction of 2R,3R-trans-dihydroflavonols to leucoanthocyanidins. In this study we describe cloning and expression of the genes encoding the flavonoid-bio- synthetic enzyme DFR in Gypsophila paniculata L. Inspec- tion of the 1279 bp long sequence revealed an open reading frame 1063 bp, including a 36 bp 5' leader region and 181 bp 3' untranslated region. Comparison of the coding region of this DFR cDNA sequence including the sequences of Arabidopsis thaliana, Citrus sinensis, Dianthus caryophyllus, Ipomoea batatas, Matthiola incana, Nierembergia sp, Pe- tunia hybrida, Solanum tuberosum, Vitis vinifera reveals an identity higher than 69% at the nucleotide level. The func- tion of this nucleotide sequences was verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRNA from wild type and mutant plants, by in vitro expression yielding and enzymatically active reductase, as indicated by the small leucopelargonidin peak. Genomic southern blot analysis showed the presence of only one gene for DFR in Gypsophila paniculata. 서 론

New Triterpenoid Saponins from the Roots of Gypsophila paniculata L.

AbstractThe seven new triterpenoid saponins 1–7 were isolated from the roots of Gypsophila paniculata L. Their structures were established by 1D‐ and 2D‐NMR techniques, HR‐MS, and acid hydrolysis. The isolated compounds include 3,28‐O‐bidesmosides with or without a 4‐methoxycinnamoyl group (see 1 vs. 2 and 3), and 3‐O‐monoglucosides 4–7. All isolated saponins 1–7 and their aglycones were evaluated for their α‐glucosidase inhibition activity. Compound 1 showed inhibitory activity against yeast α‐glucosidase with an IC50 value of 100.9±3.3 μM, whereas compounds 2–7 were inactive.

A convenient method for saponin isolation in tumour therapy

Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy.

The distribution of saponins in vivo affects their synergy with chimeric toxins against tumours expressing human epidermal growth factor receptors in mice

Certain saponins synergize with antitumour drugs to enhance their efficacy, but the mechanisms underlying this synergy in vivo are not well studied. Here, we describe the distribution of Saponinum album (Spn) from Gypsophila paniculata L. in mice after subcutaneous injection. The [(3)H]-labelled Spn used for in vivo experiments was biologically active, as it still increased the cytotoxicity of a chimeric toxin in vitro. Distribution of [(3)H]-Spn was measured in BALB/c mice, with or without subcutaneous tumours in the flank. Labelled Spn was subcutaneously injected in the neck, and samples of organs, blood, urine and tumour tissue were analysed for radioactivity, 5-240 min after the injection. The majority of [(3)H]-Spn distributed within 10 min throughout the entire animal, with high levels of radioactivity in the urine by 30 min. No preferential accumulation in tumour tissue or other organs was observed. In tumour-bearing mice, using a sequential combination of Spn (given first) and a chimeric toxin against the epidermal growth factor receptor, ErbB1, we tested two different pretreatment times for Spn. There was high antitumour efficacy (66% inhibition of tumour growth) after 60 min pre treatment with Spn, but no significant inhibition after 10 min pre treatment with Spn. [(3)H]-Spn was rapidly cleared from the mice after s.c. injection, and antitumour synergy with chimeric toxins was correlated with the removal of excess Spn from tissues. Disposition of Spn in vivo may critically determine antitumour synergy with chimeric toxins.

Phytotoxicity of biosolids and screening of selected plant species with potential for mercury phytoextraction

Mercury contaminated stockpiles of biosolids (3.5-8.4 mg kg(-1) Hg) from Melbourne Water's Western Treatment Plant (MW-WTP) were investigated to evaluate the possibility for their phytoremediation. Nine plant species (Atriplex codonocarpa, Atriplex semibaccata, Austrodanthonia caespitosa, Brassica juncea, Brassica napus, Gypsophila paniculata, Sorghum bicolor, Themeda triandra and Trifolium subterraneum) were screened for phytoextraction potential in Hg-contaminated biosolids from MW-WTP. In addition, the same plant species were germinated and grown in two other substrates (i.e. potting mix and potting mix spiked with mercury(II)). Growth measurements and the mercury uptake for all three substrates were compared. Some plant species grown in potting mix spiked with mercury(II) grew more vigorously than in the other two substrates and showed higher levels of sulphur in their tissues. These results suggested that the mercury stress activated defence mechanisms and it was hypothesised that this was the likely reason for the enhanced production of sulphur compounds in the plant species studied which stimulated their growth. Some species did not grow in biosolids because of the combined effect of high mercury toxicity and high salt content. Atriplex conodocarpa and Australodanthonia caespitose proved to be the most suitable candidates for mercury phytoextraction because of their ability to translocate mercury from roots to the above-ground tissues.

A BRIEF COMMUNICATION

Saponins are amphiphilic substances consisting of a hydrophobic backbone with one or two hydrophilic sugar units. Recently it was shown that saponinum album (SA) from Gypsophila paniculata L. enhanced cytotoxicity of a saporin-based chimeric toxin (up to 385,000-fold) as well as the toxicity of saporin (up to 100,000-fold) with N-glycosidase activity. Previously we have shown that toxicity of other N-glycosidases such as ricin A-chain, nigrin b, and toxins such as diphtheria toxin or microcystin-LR was not enhanced by SA. This points to a specific SA-dependent mechanism of toxicity enhancement on saporin and saporin-based toxins. Although the cytotoxicity enhancing effect was observed in up to 10 different cell lines, nothing is known about the kinetic of SA under cell culture conditions. Therefore SA was titrated, and the uptake respective liberation profile of SA was investigated in ECV-304 cells. Treatment of cells with [3H]-SA leads to an immediate uptake of saponin molecules. After cells were saturated with [3H]-SA, a first equilibrium (first eq.) was reached. The first eq. was disturbed by washing until a second equilibrium was reached between the activity observed within cells and that seen in the supernatant. After a further extensive washing, a small portion of saponin molecules remained durable associated with the cell. This portion was sufficient to induce a drastic toxicity enhancement on saporin indicating a long-lasting sensitization of cells against the toxin.

Enhancement of saporin cytotoxicity by Gypsophila saponins—More than stimulation of endocytosis

Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that saporin exerts no cytotoxicity on seven human cell lines. However, the combination of saporin with a special mixture of Gypsophila saponins ( Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein. In this study we investigated whether the enhancement of the saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged saporin ( hissaporin) in ECV-304 cells. For this purpose hissaporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of hissaporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic hissaporin was significantly higher in saponin-treated cells than in cells, which were only incubated with hissaporin. This indicates a saponin mediated endosomal escape of saporin.

A Simple Method for Isolation ofGypsophilaSaponins for the Combined Application of Targeted Toxins and Saponins in Tumor Therapy

Saponinum album (SAP) is a complex mixture of triterpene saponins from Gypsophila paniculata L. Although most of the saponins from SAP are characterized, the separation of pure saponins remains time consuming and costly, involving different chromatographic techniques. Recently it was shown that SAP drastically enhanced the cytotoxicity of a chimeric toxin consisting of the N-glycosidase saporin and human epidermal growth factor (Sap-EGF) in cell culture experiments. In view of a potential therapeutic use of the coadministration of SAP and Sap-EGF in tumor therapy, an economic and time-saving method for the isolation of pure saponins from the crude SAP mixture in high amounts is required. In this study we isolated a single saponin by a simple chromatographic method. The isolated saponin was characterized by mass spectrometry and was shown to enhance the cytotoxicity of Sap-EGF on HER14 cells.

Carbon addition interacts with water availability to reduce invasive forb establishment in a semi-arid grassland

Increases in nitrogen (N) availability can favor fast-growing invasive species over slow- growing native species. One way to reduce N availability is to add labile carbon (C) to the soil, which can lead to microbial immobilization of plant available N. This method has been used, with widely varying degrees of success, to both study and control plant invasions. One reason that C addition might not work as expected is that N is not always the limiting resource for plant growth. For example, if plant growth is limited by water, changes in N availability might have little effect on invasion. Here I ask whether effects of C addition on N availability, resident plant biomass, and invasion depend on water availability in semi-arid mixedgrass prairie. Six invasive species were seeded into plots treated with a factorial combination of water (ambient or added) and N (?C, control or ?N). Carbon addition reduced capture of mineral N by resin probes (by an average of 73%), and reduced biomass of resident species (from 336 g m -2 to 203 g m -2 ), both with and without added water. In contrast, because there was little invasion in ambient-water plots, C addition reduced invasion only in added-water plots. Given added water, C addition reduced biomass of Centau- rea diffusa by 95%, and prevented invasion by Gypsophila paniculata and Linaria dalmatica. Mech- anisms by which C addition reduced invasion varied by species, with added C reducing the growth of individual C. diffusa plants, but reducing numbers of G. paniculata and L. dalmatica individuals.