Biochemical characterization of Euphorbia resinifera floral cyathia.

Euphorbia resinifera O. Berg is a plant endemic to the Northern and Central regions of Morocco known since the ancient Roman and Greek times for secreting a poisonous latex containing resiniferatoxin. However, E. resinifera pseudo-inflorescences called cyathia are devoid of laticifers and, therefore, do not secrete latex. Instead, they exudate nectar that local honey bees collect and craft into honey. Honey and cyathium water extracts find a broad range of applications in the traditional medicine of Northern Africa as ointments and water decoctions. Moreover, E. resinifera monofloral honey has received the Protected Geographic Indication certification for its outstanding qualities. Given the relevance of E. resinifera cyathia for bee nutrition, honey production, and the health benefit of cyathium-derived products, this study aimed to screen metabolites synthesized and accumulated in its pseudo-inflorescences. Our analyses revealed that E. resinifera cyathia accumulate primary metabolites in considerable abundance, including hexoses, amino acids and vitamins that honey bees may collect from nectar and craft into honey. Cyathia also synthesize volatile organic compounds of the class of benzenoids and terpenes, which are emitted by flowers pollinated by honey bees and bumblebees. Many specialized metabolites, including carotenoids, flavonoids, and polyamines, were also detected, which, while protecting the reproductive organs against abiotic stresses, also confer antioxidant properties to water decoctions. In conclusion, our analyses revealed that E. resinifera cyathia are a great source of antioxidant molecules and a good food source for the local foraging honeybees, revealing the central role of the flowers from this species in mediating interactions with local pollinators and the conferral of medicinal properties to plant extracts.

Investigate the anaerobic degradation of high-acetone latex wastewater with magnetite supplement.

This study evaluated the effects of acetone on the anaerobic degradation of synthetic latex wastewater, which was simulated from the wastewater of the deproteinized natural rubber production process, including latex, acetate, propionate, and acetone as the main carbon sources, at a batch scale in 5 cycles of a total of 60 days. Fe3O4 was applied to accelerate the treatment performance from cycle 3. Acetone was added in concentration ranges of 0%, 0.05%, 0.1%, 0.15%-included latex, and 0.15%-free latex (w/v). In the Fe3O4-free cycles, for latex-added vials, soluble chemical oxygen demand (sCOD) was removed at 43.20%, 43.20%, and 12.65%, corresponding to the input acetone concentrations varying from 0.05% to 0.15%, indicating the interference of acetone for COD reduction. After adding Fe3O4, all flasks reported a significant increase in COD removal efficiency, especially for acetone-only and latex-only vials, from 36.9% to 14.30%-42.95% and 83.20%, respectively. Other highlighted results of COD balance showed that Fe3O4 involvement improved the degradation process of acetate, propionate, acetone, and the other COD parts, including the intermediate products of latex reduction. Besides, during the whole batch process, the order of reduction priority of the carbon sources in the synthetic wastewater was acetate, propionate and acetone. We also found that the acetate concentration appeared to be strongly related to reducing other carbon sources in natural rubber wastewater. Microbial community analysis revealed that protein-degrading bacteria Bacteroidetes vadinHA17 and Proteinniphilum and methylotrophic methanogens might play key roles in treating simulated deproteinized-natural-rubber wastewater.

Insight into the collagen-degrading activity of a serine protease in the latex of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.

Latex Proteins from Plumeria pudica with Therapeutic Potential on Acetaminophen-Induced Liver Injury.

Liver disease is global health problem. Paracetamol (APAP) is used as an analgesic drug and is considered safe at therapeutic doses, but at higher doses, it causes acute liver injury. N-acetyl-p- Benzoquinone Imine (NAPQI) is a reactive toxic metabolite produced by biotransformation of APAP. NAPQI damages the liver by oxidative stress and the formation of protein adducts. The glutathione precursor N-acetylcysteine (NAC) is the only approved antidote against APAP hepatotoxicity, but it has limited hepatoprotective effects. The search for new drugs and novel therapeutic intervention strategies increasingly includes testing plant extracts and other natural products. Plumeria pudica (Jacq., 1760) is a plant that produces latex containing molecules with therapeutic potential. Proteins obtained from this latex (LPPp), a well-defined mixture of chitinases, proteinases proteinase inhibitors have shown anti-inflammatory, antinociceptive, antidiarrheal effects as well as a protective effect against ulcerative colitis. These studies have demonstrated that LPPp acts on parameters such as Glutathione (GSH) and Malondialdehyde (MDA) concentration, Superoxide Dismutase (SOD) activity, Myeloperoxidase (MPO) activity, and TNF- α IL1-ß levels. Since oxidative stress and inflammation have been reported to affect the initiation and progression of liver injury caused by APAP, it is suggested that LPPp can act on aspects related to paracetamol hepatoxicity. This article brings new insights into the potential of the laticifer proteins extracted from the latex of P. pudica and opens new perspectives for the treatment of this type of liver disease with LPPp.

Comparative study on cellular entry of incinerated ancient gold particles (Swarna Bhasma) and chemically synthesized gold particles.

Gold nanoparticles (AuNPs) are used for a number of imaging and therapeutic applications in east and western part of the world. For thousands of years, the traditional Indian Ayurvedic approach to healing involves the use of incinerated gold ash, prepared with a variety of plant extracts and minerals depending on the region. Here, we describe the characterization of incinerated gold particles (IAuPs) in HeLa (human cells derived from cervical cancer) and HFF-1 (human foreskin fibroblast cells) in comparison to synthesized citrate-capped gold nanoparticles (AuNPs). We found that while individual IAuP crystallites are around 60 nm in size, they form large aggregates with a mean diameter of 4711.7 nm, some of which can enter cells. Fewer cells appeared to have IAuPs compared to AuNPs, although neither type of particle was toxic to cells. Imaging studies revealed that IAuPs were in vesicles, cytosol, or in the nucleus. We found that their nuclear accumulation likely occurred after nuclear envelope breakdown during cell division. We also found that larger IAuPs entered cells via macropinocytosis, while smaller particles entered via clathrin-dependent receptor-mediated endocytosis.

Latex proteins from Calotropis procera: Toxicity and immunological tolerance revisited.

Many thousands of plants are disseminated worldwide in traditional and folk medicines based on the belief that their leaves, roots, seeds, bark or secretions, when adequately handled, can treat, alleviate or ameliorate numerous disease symptoms. Calotropis procera (Apocynaceae) is a popular medicinal plant and the claims of this shrub's phytomedicinal properties have been scientifically validated. In this study, further prospects towards the in vivo toxicity and oral immunological tolerance of phytomodulatory proteins isolated from the latex of C. procera are reported. Acute toxicity was determined in mice by oral and intraperitoneal administration of latex proteins (LP) and was followed behavioral, hematological and histological analyses. Oral immunological tolerance to LP was assessed by intraperitoneal immunization in mice that had received LP orally before. Animals given 5000 mg/kg orally exhibited only discrete behavioral alterations and augmentation of monocytes. Death was not notified 14 days after exposure. However, all animals receiving LP 150 mg/kg by i.p. died in 1 h. Death (20%) was documented when LP (75 mg/kg) was given in the peritoneum and signs of harmful effects were observed in the survivors (80%). Oral immunological tolerance was observed in animals previously given LP orally, when they were further immunized/challenged with peritoneal exposure to different doses of LP. This was confirmed by the lowering of IgE and IgG in the serum, IL-4 and IFN-γ in spleen homogenates and the absence of anaphylaxis signs. It is therefore concluded that LP exhibited quite discrete adverse effects when orally administrated at higher concentrations and this route of administration did not stimulate adverse immunological reactions. Instead it was observed immunological tolerance. The present study contributes very important information concerning the safe use of C. procera as a phytotherapeutic agent.

Proteomic comparison of Chelidonium majus L. latex in different phases of plant development.

Chelidonium majus L. (Papaveraceae) latex is used in traditinonal folk medicine to treat papillae, warts, condylomas, which are visible effects of human papilloma virus (HPV) infections. The aim of this work was to provide new insights into the biology and medicinal use of C. majus milky sap in the flowering and fruit ripening period of the plant by comparing the protein content between samples collected on respective developmental stages using LC-MS-based label-free proteome approach. For quantification, the multiplexed LC-MS data were processed using comparative chemometric approach. Progenesis LC-MS results showed that in green fruit phase (stage IV), comparing to flowering phase (stage III) of plant development, a range of proteins with higher abundance were identified as stress- and defense-related. On the other hand at stage III very intense protein synthesis, processes of transcription, protein folding and active transport of molecules (ABC transporters) are well represented. 2-DE protein maps showed an abundant set of spots with similar MWs (about 30-35 kDa) and pIs (ca. 5.5-6.5), which were identified as major latex proteins (MLPs). Therefore we suggest that biological activity of C. majus latex could be related to its protein content, which shifts during plant development from intense biosynthetic processes (biosynthesis and transport of small molecules, like alkaloids) to plant defense mechanisms against pathogens. Further studies will help to elucidate if these defense-related and pathogenesis-related proteins, like MLP, together with small-molecule compounds, could inhibit viral infection, what could be a step to fully understand the medicinal activity of C. majus latex.

Combination of transcriptomic and proteomic approaches helps to unravel the protein composition of Chelidonium majus L. milky sap.

MAIN CONCLUSION: A novel annotated Chelidonium majus L. transcriptome database composed of 23,004 unique coding sequences allowed to significantly improve the sensitivity of proteomic C. majus assessments, which showed novel defense-related proteins characteristic to its latex. To date, the composition of Chelidonium majus L. milky sap and biosynthesis of its components are poorly characterized. We, therefore, performed de novo sequencing and assembly of C. majus transcriptome using Illumina technology. Approximately, 119 Mb of raw sequence data was obtained. Assembly resulted in 107,088 contigs, with N50 of 1913 bp and N90 of 450 bp. Among 34,965 unique coding sequences (CDS), 23,004 obtained CDS database served as a basis for further proteomic analyses. The database was then used for the identification of proteins from C. majus milky sap, and whole plant extracts analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach. Of about 334 different putative proteins were identified in C. majus milky sap and 1155 in C. majus whole plant extract. The quantitative comparative analysis confirmed that C. majus latex contains proteins connected with response to stress conditions and generation of precursor metabolites and energy. Notable proteins characteristic to latex include major latex protein (MLP, presumably belonging to Bet v1-like superfamily), polyphenol oxidase (PPO, which could be responsible for browning of the sap after exposure to air), and enzymes responsible for anthocyanidin, phenylpropanoid, and alkaloid biosynthesis.

Proteomic analysis and purification of an unusual germin-like protein with proteolytic activity in the latex of Thevetia peruviana.

MAIN CONCLUSION: The latex from Thevetia peruviana is rich in plant defense proteins, including a 120 kDa cysteine peptidase with structural characteristics similar to germin-like proteins. More than 20,000 plant species produce latex, including Apocynaceae, Sapotaceae, Papaveraceae and Euphorbiaceae. To better understand the physiological role played by latex fluids, a proteomic analysis of Thevetia peruviana (Pers.) Schum latex was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86 %) were identified, including storage proteins, a peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. An unusual cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme (glycoprotein) was inhibited by E-64 and iodoacetamide and exhibited high specific activity towards azocasein (K m 17.6 µM), with an optimal pH and temperature of 5.0-6.0 and 25-37 °C, respectively. Gel filtration chromatography, two-dimensional gel electrophoresis, and mass spectrometry revealed that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits (20 kDa). A unique N-terminal amino acid sequence was obtained to oligomer and monomers of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36). High-resolution images from atomic force microscopy showed the homohexameric structure of peruvianin-I may be organized as a trimer of dimers that form a central channel similar to germin-like proteins. Peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects. Peruvianin-I represents the first germin-like protein (GLP) with cysteine peptidase activity, an activity unknown in the GLP family so far.

Improved sanguinarine production via biotic and abiotic elicitations and precursor feeding in cell suspensions of latex-less variety of Papaver somniferum with their gene expression studies and upscaling in bioreactor.

Elicitors play an important role in challenging the plant defense system through plant-environment interaction and thus altering the secondary metabolite production. Culture filtrates of four endophytic fungi, namely, Chaetomium globosum, Aspergillus niveoglaucus, Paecilomyces lilacinus, and Trichoderma harzianum were tested on embryogenic cell suspensions of latex-less Papaver somniferum in dose-dependent kinetics. Besides this, abiotic elicitors salicylic acid, hydrogen peroxide, and carbon dioxide were also applied for improved sanguinarine production. Maximum biomass accumulation (growth index (GI) = 293.50 ± 14.82) and sanguinarine production (0.090 ± 0.008 % dry wt.) were registered by addition of 3.3 % v/v T. harzanium culture filtrate. Interestingly, it was further enhanced (GI = 323.40 ± 25.30; 0.105 ± 0.008 % dry wt.) when T. harzanium culture filtrate was employed along with 50 µM shikimate. This was also supported by real-time (RT) (qPCR), where 8-9-fold increase in cheilanthifoline synthase (CFS), stylopine synthase (STS), tetrahydroprotoberberine cis-N-methyltransferase (TNMT), and protopine 6-hydroxylase (P6H) transcripts was observed. Among abiotic elicitors, while hydrogen peroxide and carbon dioxide registered low level of sanguinarine accumulation, maximum sanguinarine content was detected by 250 µM salicylic acid (0.058 ± 0.003 % dry wt.; GI = 172.75 ± 13.40). RT (qPCR) also confirms the downregulation of sanguinarine pathway on CO2 supplementation. Various parameters ranging from agitation speed (70 rpm), impeller type (marine), media volume (2 l), inoculum weight (100 g), and culture duration (9 days) were optimized during upscaling in 5-l stirred tank bioreactor to obtain maximum sanguinarine production (GI = 434.00; 0.119 ± 0.070 % dry wt.). Addition of 3.3 % v/v T. harzanium culture filtrate and 50-µM shikimate was done on the 6th day of bioreactor run.

Novel route for rapid biosynthesis of copper nanoparticles using aqueous extract of Calotropis procera L. latex and their cytotoxicity on tumor cells.

This paper accounts for novel, low-cost, eco-friendly route for rapid biosynthesis of copper nanoparticles. Cysteine proteases present in the latex of Calotropis procera L. were used to fabricate copper nanoparticles from copper acetate. Copper nanoparticles were initially characterized by transmission electron microscopy (TEM) and X-ray diffraction technique (XRD). Transmission electron microscopy (TEM) was used to estimate the size and shape of nanoparticles. The average size of copper nanoparticles was found to be 15 ± 1.7 nm. Energy dispersive analysis of X-rays (EDAX) showed distinct peaks of copper. Fourier transform infrared spectroscopy (FTIR) was performed to confirm capping behavior of the latex proteins that contributed to long term stability of copper nanoparticles (6 months) in aqueous medium. Copper nanoparticles synthesized by above method were monodisperse type. Cytotoxicity studies of latex stabilized copper nanoparticles were carried out on HeLa, A549 and BHK21 cell lines by MTT dye conversion assay. HeLa, A549 and BHK21 cells showed excellent viability even at 120 µM concentration of copper nanoparticles. This shows that copper nanoparticles synthesized by above method hold excellent biocompatibility.

Green synthesis of colloidal silver nanoparticles using natural rubber latex extracted from Hevea brasiliensis.

Colloidal silver nanoparticles were synthesized by an easy green method using thermal treatment of aqueous solutions of silver nitrate and natural rubber latex (NRL) extracted from Hevea brasiliensis. The UV-Vis spectra detected the characteristic surface plasmonic absorption band around 435 nm. Both NRL and AgNO(3) contents in the reaction medium have influence in the Ag nanoparticles formation. Lower AgNO(3) concentration led to decreased particle size. The silver nanoparticles presented diameters ranging from 2 nm to 100 nm and had spherical shape. The selected area electron diffraction (SAED) patterns indicated that the silver nanoparticles have face centered cubic (fcc) crystalline structure. FTIR spectra suggest that reduction of the silver ions are facilitated by their interaction with the amine groups from ammonia, which is used for conservation of the NRL, whereas the stability of the particles results from cis-isoprene binding onto the surface of nanoparticles. Therefore natural rubber latex extracted from H. brasiliensis can be employed in the preparation of stable aqueous dispersions of silver nanoparticles acting as a dispersing and/or capping agent. Moreover, this work provides a new method for the synthesis of silver nanoparticles that is simple, easy to perform, pollutant free and inexpensive.

SDS induced molten globule state of heynein; a new thiol protease: Evidence of domains and their sequential unfolding.

The molten globule state can be an intermediate in the protein-folding pathway and its detailed description can help understand the protein folding and an insight into the molecular structure of a protein. Sodium dodecyl sulfate (SDS), an anionic surfactant is known to induce molten globule sate in some proteins. SDS-induced changes in heynein were monitored by CD, fluorescence, 8-anilino-1-napthalenesulfonic acid (ANS) binding and proteolytic activity measurements. An enhancement in the α-helicity of protein with increasing concentration of SDS along with exposure of tryptophans is seen. At a concentration of SDS (∼2mM) heynein loses activity and rigid tertiary structure but possesses considerable amount of secondary structure along with strong ANS binding, indicating the presence of an intermediate state, which is like molten globule state seen in the case heynein. Chemical and temperature induced unfolding of SDS-induced state of heynein is non-cooperative contrary to the protein in the absence of detergent. Further, the cooperative unfolding transition curve of heynein in the absence of SDS intersects at a point where the second transition of SDS-induced state starts suggesting that the molecule of heynein consist of at least two structural domains which are stabilized differentially and unfolds sequentially. Enhancement of α-helicity of heynein in the presence of SDS suggests the α-rich domain of the protein was stabilized and unfold later as compared with ß-rich domain during temperature and chemical induced denaturation. Equilibrium unfolding pathway of heynein in SDS-induced state provides knowledge of the structure and stability of this plant cysteine protease at domain level.

Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes.

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.

Benghalensin, a highly stable serine protease from the latex of medicinal plant Ficus benghalensis.

A serine protease was purified to homogeneity from the latex of medicinal plant Ficus benghalensis by a single step procedure using anion exchange chromatography. The enzyme, named benghalensin, has a molecular mass of 47 kDa (MALDI-TOF and SDS-PAGE). The purified protein is a glycoprotein, and the enzymatic activity is solely inhibited by PMSF and chymostatin, indicating that the enzyme belongs to the serine protease class. The isoelectric point of the enzyme is pH 4.4 with optimum pH and temperature of pH 8.0 and 55 degrees C respectively. The extinction coefficient (epsilon(1%)(280)) of the enzyme is 29.25, and the molecular structure consists of 17 tryptophan, 31 tyrosine and 09 cysteine residues. Peptide mass fingerprinting and de novo sequencing of tryptic-digested fragments of the protein did not find any putative conserved domains in BLAST analysis. The enzyme is stable and retains full activity over a broad range of pH and temperature or prolonged storage at 4 degrees C. Simple purification, high yield and stability enable exploration of the protein for structure-function relationship studies as well as other applications.

Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases.

AIM OF THE STUDY: To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. MATERIALS AND METHODS: A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. RESULTS: The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing A alpha and B beta subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen-agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). CONCLUSION: Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.

Catalase and antiquitin from Euphorbia characias: two proteins involved in plant defense?

Here we report the cDNA nucleotide sequences of a calmodulin-binding catalase and an antiquitin from the latex of the Mediterranean shrub Euphorbia characias. Present findings suggest that catalase and antiquitin might represent additional nodes in the Euphorbia defense systems, and a multi-enzymatic interaction contributing to plant's protection against biotic and abiotic stresses is proposed to occur in E. characias laticifers.

A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies [correction of interspecie] cross-reactivity.

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy.

Opium poppy produces a diverse array of pharmaceutical alkaloids, including the narcotic analgesics morphine and codeine. The benzylisoquinoline alkaloids of opium poppy accumulate in the cytoplasm, or latex, of specialized laticifers that accompany vascular tissues throughout the plant. However, immunofluorescence labeling using affinity-purified antibodies showed that three key enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), involved in the biosynthesis of morphine and the related antimicrobial alkaloid sanguinarine, are restricted to the parietal region of sieve elements adjacent or proximal to laticifers. The localization of laticifers was demonstrated using antibodies specific to the major latex protein (MLP), which is characteristic of the cell type. In situ hybridization showed that CYP80B1, BBE, and COR gene transcripts were found in the companion cell paired with each sieve element, whereas MLP transcripts were restricted to laticifers. The biosynthesis and accumulation of alkaloids in opium poppy involves cell types not implicated previously in plant secondary metabolism and dramatically extends the function of sieve elements beyond the transport of solutes and information macromolecules in plants.

Expression and activity of cell-wall-degrading enzymes in the latex of opium poppy, Papaver somniferum L.

The alkaloid-rich latex of the opium poppy, Papaver somniferum L., is valued as a source of pharmaceuticals including thebaine, codeine, and morphine, but is also harvested for heroin production. The poppy laticifer system develops through the gradual disappearance of the common walls between differentiating laticifer elements throughout the plant. Gene homologues for cell-wall-degrading enzymes were found during random sequencing of an opium poppy latex cDNA library. RNA gel blot analysis of cellulase, polygalacturonase beta-subunit, 1,3-beta-glucanase, and xyloglucan endotransglycosylase homologues showed their expression was not limited to laticifers. In contrast, poppy gene homologues to pectin methylesterase (PME), pectin acetylesterase (PAE) and pectate lyase (PL) where all highly expressed and latex-specific. Enzyme assays confirmed the presence of PME, PAE, and PL activities in latex serum. The abundance of transcripts encoding pectin-degrading enzymes in latex suggests that these enzymes may play an important role in laticifer development.