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The present study was carried out under plastic house conditions at the nursery of Zohria garden, Hort. Res. Inst., ARC, Cairo, Egypt throughout the two successive seasons of 2015 and 2016 to reveal the effect of planting dates (winter, spring, summer and autumn), some rooting promoters at different concentrations i.e. IBA, NAA, 2,4-D, catechol, cinnamic acid and tryptophan (alone or in combination) and their interactions on rooting of hard-to-root ornamental tree Phytolacca dioica.Results revealed thatplanting in spring increased rooting percentage (%) and root length (cm) in both seasons to the highest values. Treating the cuttings with IBA at 4000 ppm + NAA at 4000 ppm formulation resulted in the highest rooting percentage (%) in both seasons. Although there was no clear trend that could be observed from the obtained results, planting cuttings in either spring or autumn in addition to treating with IBA at 4000 ppm + NAA at 4000 ppm formulation increased rooting percentage (%) to the highest values.In conclusion, to achieve a successful vegetative propagation and to induce rooting on cuttings it is recommended to plant the cuttings in either spring or autumn in addition to treating with IBA at 4000 ppm + NAA at 4000 ppm formulation.

This work was carried out in the Tissue Culture Laboratory, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt from the period elapsed from 2014 to 2016 to study the effect of sterilization on propagation of Phytolacca dioica, L.. Results of this study could be briefed in the following outlines: Chlorox concentrations at 30 or 35 % gave the highest survival percentage of Phytolacca dioica, L.. In addition, soaking explants in the sterilant solution for 20 or 25 minutes resulted in the highest survival percentage. Similarly, the interaction treatment demonstrated that using chlorox at 35% for 25 minutes resulted in the highest survival%. In the multiplication stage it was found that using BAP at 10 ppm resulted in the highest number of shoots and leaves. BAP at 6 or 8 ppm induced the highest content of total chlorophyll. The longest shoots were a result of applying BAP at 3 ppm. Inoculating explants on half strength medium resulted in the highest values of number of shoots and leaves, as well as highest contents of total chlorophyll and carotenoids. MS medium at 3 quarters strength shared in the first position of number of leaves. The longest shoots were a result of using MS medium at full strength. The interaction between BAP levels and MS medium strength, indicated that using MS medium at half strength supplemented with BAP at 10 ppm produced highest number of shoots. The same medium strength with BAP at 8 or 6 ppm gave the highest contents of total chlorophyll and carotenoids. MS medium at 3 quarters strength combined with PAP at 10 ppm gave highest number of shoots and leaves. MS medium at full strength plus BAP at either 3 or 10 ppm resulted in the highest shoot length and number of leaves.

Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis.

The ribosome inactivating protein PD-L4 from Phytolacca dioica is a N-β-glycosidase, probably involved in plant defence. The crystal structures of wild type PD-L4 and of the S211A PD-L4 mutant with significantly decreased catalytic activity were determined at atomic resolution. To determine the structural determinants for the reduced activity of S211A PD-L4, both forms have also been co-crystallized with adenine, the major product of PD-L4 catalytic reaction. In the structure of the S211A mutant, the cavity formed by the lack of the Ser hydroxyl group is filled by a water molecule; the insertion of this non-isosteric group leads to small albeit concerted changes in the tightly packed active site of the enzyme. These changes have been correlated to the different activity of the mutant enzyme. This work highlights the importance of atomic resolution studies for the deep understanding of enzymatic properties. Proteins 2008. © 2007 Wiley-Liss, Inc.

We investigated the antimicrobial activity of PD-L4, a type 1 RIP from Phytolacca dioica. We found that this protein is active on different bacterial strains both in a native and denatured/alkylated form and that this biological activity is related to a cryptic peptide, named PDL440-65, identified by chemical fragmentation. This peptide showed the same antimicrobial activity of full-length protein and possessed, similarly to several antimicrobial peptides, an immunomodulatory effect on human cells. It assumes an alpha-helical conformation when interact with mimic membrane agents as TFE and likely bacterial membranes are a target of this peptide. To date PDL440-65 is the first antimicrobial peptide identified in a type 1 RIP. We investigated the antimicrobial activity of PD-L4, a type 1 RIP from Phytolacca dioica. We found that this protein is active on different bacterial strains both in a native and denatured/alkylated form and that this biological activity is related to a cryptic peptide, named PDL4(40-65), identified by chemical fragmentation. This peptide showed the same antimicrobial activity of full-length protein and possessed, similarly to several antimicrobial peptides, an immunomodulatory effect on human cells. It assumes an alpha-helical conformation when interact with mimic membrane agents as TFE and likely bacterial membranes are a target of this peptide. To date PDL4(40-65) is the first antimicrobial peptide identified in a type 1 RIP. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ribosome-inactivating proteins isolated fromPhytolacca dioicaL. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adultP.dioicaplants, produces both free 3′-OH and 5′-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [α-32P]dCTP and [γ-32P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity withE.coliHB101 competent cells in comparison to untreated control plasmid DNA.

Four triterpene saponins were isolated from Arenaria filicaulis Boiss., Phytolacca dioica L. and Koelreuteria paniculata Laxm. Their structures and conformational behaviour were investigated by one- and two-dimensional 1H and 13C NMR spectroscopy. Complete 1H and 13C assignments were achieved. Copyright © 2001 John Wiley & Sons, Ltd.

Discs of SBR containing Phytolacca dioica L. plant can be used to release saponin of the plant which has an efficient toxic effect on Bimpholaria alexandrine snails, the intermediate hosts of Schistosomiasis. Such a snail control system is used to protect the human beings from schistosomiasis. Schistosomiasis is a major health problem of increasing severity, especially in the developing countries. High abrasion furnace black (HAF) and hydrated silica (HISIL) are some inert fillers which improve the properties of rubber, and they play an important role in controlling the porosity of controlled release styrene butadiene rubber formulations containing Phytolacca dioica L. plant. Also, the leaching rate of saponin depends on the concentration of filler, type of water, temperature of the environment as well as surface to volume ratio of the discs. The scanning electron micrographs illustrate the cracks and pore structure which are formed after release of saponin. The leaching rate of saponin from SBR/HAF filled formulation is greater than the leaching rate from SBR/HISIL filled formulation.

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and 50.0°C, respectively and seasonal variation. The Michaelis-Menten constant (K(M)) for H(2)O(2) was 5.27 mM, and the velocity maximum (V(max)) 1.31 nmol min(-1), while the enzyme turnover was 0.148 s(-1). Finally, the presence of Ca(2+) and Mg(2+) enhanced the PD-cP activity, with Mg(2+) 1.4-fold more effective than Ca(2+)

Evolution of Plant Ribosome-Inactivating Proteins.- RNA N-Glycosidase Activity of Ribosome-Inactivating Proteins.- Enzymatic Activities of Ribosome-Inactivating Proteins.- Type I Ribosome-Inactivating Proteins from Saponaria officinalis.- Type 1 Ribosome-Inactivating Proteins from the Ombu Tree (Phytolacca dioica L.).- Sambucus Ribosome-Inactivating Proteins and Lectins.- Ribosome-Inactivating Proteins from Abrus pulchellus.- Ribosome-Inactivating Proteins in Cereals.- Ribosome Inactivating Proteins and Apoptosis.- The Synthesis of Ricinus communis Lectins.- How Ricin Reaches its Target in the Cytosol of Mammalian Cells.- Ribosome-Inactivating Protein-Containing Conjugates for Therapeutic Use.

The toxicity of plant proteins, later identified as ribosome-inactivating proteins (RIPs), was described more than a century ago and their enzymatic activity was established more than 30 years ago. However, their physiological role and related biological activities are still uncertain. Therefore, despite the body of literature, research on RIPs is ongoing. This review deals with new RIPs being purified, sequenced, characterized, and cloned, and an increasing number of 3D-structures that are determined at high resolution. This is the case of the five type 1 RIPs (PD-S1-3, PD-L1/2, PD-L3/4, dioicin 1, and dioicin 2) from seeds and leaves of the ombú tree (Phytolacca dioica L.), native of the grassy pampas of Argentina. The data collected so far will contribute to our understanding of important issues of RIP research: (1) identifying structural determinants responsible for new enzymatic activities such as the DNA cleaving activity; (2) glycosylation and its influence on the catalytic and biological activities; (3) cellular localization of endogenous RIPs and their physiological role(s). © Springer-Verlag Berlin Heidelberg 2010.

Leaves from Phytolacca heterotepala H. Walter (Mexican pokeweed) contain at least 10 type 1 RIP isoforms, named heterotepalins. Their Mr values are included in the range 28,000-36,000, as shown by SDS-PAGE performed under reduced conditions and the pI values in the pH range 8.50-9.50. Some heterotepalins are glycosylated. ESI-QTOF mass spectrometry provides the accurate Mr of heterotepalin 4 (29,326.00) and heterotepalin 5b (30,477.00), two isoforms purified to homogeneity by conventional chromatographic techniques. The N-terminal sequences up to residue 35, show that heterotepalins exhibit an high percentage identity with other type 1 RIPs isolated from Phytolaccaceae. Some heterotepalins cross-react with antisera raised against RIPs isolated from Phytolacca dioica leaves. The complete amino acid sequence of heterotepalin 4 matches that of Phytolacca heterotepala anti-viral protein PAP (RIP1), deduced from the cDNA sequence of PhRIP1 gene (AC: AY327475), with one exception concerning residue 245 which, in the native protein, is Ile instead of Met. This substitution, found by mass spectrometry mapping, has been directly confirmed by Edman degradation sequencing of the C-terminal tryptic peptide 242-262. The results show the high potential of mass spectrometry and Edman degradation to verify and to uncover possible amino acid substitutions between native proteins and their cDNA deduced sequences. © 2006 Elsevier Ltd. All rights reserved.

Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29,407 +/- 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf chitinase (PLC-B) by only one amino acid (R17I). The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50% inhibition at the picomolar level, and produced the beta-fragment, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical to PD-L2 [designated the isoleucine (Ile) form from the N-terminal residue] and PD-L3 is identical to PD-L4 [designated the valine (Val) form from the N-terminal residue] and that there are 35 identical residues between the two forms. Furthermore, the Val form presents the same number of identical residues as PD-S2, an RIP isolated from the seeds of the same plant. With the exception of PD-L4, the purified RIPs gave a positive reaction when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass spectrometry, had M(r) values of 32,715 +/- 1 (PD-L1), 31,542 +/- 1 (PD-L2), 30,356 +/- 1 (PD-L3) and 29,185 +/- 1 Da (PD-L4). The 1171 kDa difference in M(r), within the same RIP form, could be due to glycosylation. Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases. This property was less pronounced in PD-L1 and PD-L3 than in PD-L2 and PD-L4, respectively. The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivity with antisera to PAP-R saporin-S6, momordin 1 and even PD-S2, an RIP isolated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%.

Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405–412) were purified from the seeds of Phytolacca dioica . These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M r approx. 30 000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908–5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana . PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30+ Hodgkin's lymphoma-derived L540 cell line.

A saponin-rich extract of Phytolacca dioica L. berries, its acid hydrolysate, and its major aglycone, phytolaccagenin, were assayed for antifungal activity against ATCC standard cultures of Candida albicans and Cryptococcus neoformans, and against clinical isolates of these fungi. The activity of the extract was either low or negligible, but the hydrolysate, containing the sapogenins, including phytolaccagenin, and also pure phytolaccagenin, showed promising antifungal potency. Hydrolysis of a natural product extract is shown to be a useful modification leading to improved bioactivity.