Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds

A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l^–1 TDZ followed by three cycles of selection on medium with 0.5 mg l^–1 BA and increasing concentrations of hygromycin (20–40–60 mg l^–1). Selected shoot clusters were transferred to medium with 0.5 mg l^–1 BA for proliferation and 0.2 mg l^–1 BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l^–1 NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor.     

Ricin A Chain from <Emphasis Type="Italic">Ricinus sanguineus</Emphasis>: DNA Sequence,Structure and Toxicity

The gene coding for the ricin A-chain from Ricinus sanguineus (R_sTA) was isolated and sequenced (GB: DQ661048). Comparison of R_sTA with the ricin A-chain from Ricinus communis (RTA) revealed the presence of five differences in the gene sequence. At the protein level only two differences were noticed, the replacement of Asn136 by Ser (N136S) and Ile173 by Val (I174 V). From the R_sTA structure model (PMDB: PM0074652), the N136S mutation was predicted to have no effect on R_sTA structure. The I174V mutation is believed to have no effect on the R_sTA structure or on toxicity since this replacement was found in the Ricinus agglutinin’s A-chain and the latter has a comparable toxicity to RTA. The Ser221 of the putative lipase active site was found. Toxicity experiments showed that R_sTA and RTA have similar toxicities. This finding proves that the N136S and I174 V mutations have no effect on R_sTA toxicity.     

Methyl jasmonate changes the levels of rubisco and other leaf proteins in Ricinus communis

The effect of methyl jasmonate (MJ) on the water-soluble protein pattern of Ricinus communis leaves was analyzed. Several dynamic changes occurred after a period of 24 and 48h including six proteins (M_r 13,000, 15,000, 16,000, 27,000, 29,000 and 60,000) whose levels increased by 48h and seven others (M_r 11,000, 18,000, 20,000 30,000, 37,000, 40,000 and 58,000) whose levels decreased. Four proteins (M_r 24,000, 34,000, 64,000 and 66,000) were induced after 24h of treatment, but returned to control levels by 48h. On the other hand, the levels of three proteins (M_r 74,000, 84,000 and 88,000) decreased after 24h, but returned to control levels after 48h. One of the proteins that accumulated after 48 h had the 13 first residues sequenced. This polypeptide named MJRC-15, was identical to the C-terminal sequence of Rubisco-large chain polypeptide (position 337–350) from tobacco chloroplast. Western-blot analysis using polyclonal antibodies against Rubisco supports the hypothesis that MJRC-15 is a degradation product of Rubisco.     

The phytoremediation potential of bioenergy crop Ricinus communis for DDTs and cadmium co-contaminated soil

Cadmium (Cd) and dichlorodiphenyltrichloroethane (DDT) or its metabolite residues are frequently detected in agricultural soils and food, posing a threat to human health. The objective of this study was to compare the ability of 23 genotypes of Ricinus communis in mobilizing and uptake of Cd and DDTs (p,p′-DDT, o,p′-DDT, p,p′-DDD and p,p′-DDE) in the co-contaminated soil. The plant genotypes varied largely in the uptake and accumulation of DDTs and Cd, with mean concentrations of 0.37, 0.43 and 70.51 for DDTs, and 1.22, 2.27 and 37.63 mg kg⁻¹ dw for Cd in leaf, stem and root, respectively. The total uptake of DDTs and Cd varied from 83.1 to 267.8 and 66.0 to 155.1 μg per pot, respectively. These results indicate that R. communis has great potential for removing DDTs and Cd from contaminated soils attributed to its fast growth, high biomass, strong absorption and accumulation for both DDTs and Cd.     

Specificities of Ricinus communis agglutinin 120 interaction with sulfated galactose

Lectins are used extensively as research tools to detect and target specific oligosaccharide sequences. Ricinus communis agglutinin I (RCA₁₂₀) recognizes non-reducing terminal β-d-galactose (Galβ) and its specificities of interactions with neutral and sialylated oligosaccharides have been well documented. Here we use carbohydrate arrays of sulfated Galβ-containing oligosaccharide probes, prepared from marine-derived galactans, to investigate their interactions with RCA₁₂₀. Our results showed that RCA₁₂₀ binding to Galβ1–4 was enhanced by 2-O- or 6-O-sulfation but abolished by 4-O-sulfation. The results were corroborated with competition experiments. Erythrina cristagalli lectin is also a Galβ-binding protein but it cannot accommodate any sulfation on Galβ.     

Effects of ricinoleic acid esters from castor oil of Ricinus communis on the vitellogenesis of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks

This study examines the effects of ricinoleic acid esters from Ricinus communis castor oil on the vitellogenesis of Rhipicephalus sanguineus ticks attached to hosts that were fed with commercial rabbit food containing these esters. The oocytes of ticks from the treatment group (TG) showed cytoplasmic changes that inhibited the development of oocytes I and II to the advanced stages (IV and V) in addition to preventing the maturation of oocytes V, resulting in small ones. In addition, sperm was not observed in ampoules. Our findings confirm the acaricide potential of ricinoleic acid esters.     

Analysis of castor bean ribosome-inactivating proteins and their gene expression during seed development

Ribosome-inactivating proteins (RIPs) are enzymes that inhibit protein synthesis after depurination of a specific adenine in rRNA. The RIP family members are classified as type I RIPs that contain an RNA-N-glycosidase domain and type II RIPs that contain a lectin domain (B chain) in addition to the glycosidase domain (A chain). In this work, we identified 30 new plant RIPs and characterized 18 Ricinus communis RIPs. Phylogenetic and functional divergence analyses indicated that the emergence of type I and II RIPs probably occurred before the monocot/eudicot split. We also report the expression profiles of 18 castor bean genes, including those for ricin and agglutinin, in five seed stages as assessed by quantitative PCR. Ricin and agglutinin were the most expressed RIPs in developing seeds although eight other RIPs were also expressed. All of the RIP genes were most highly expressed in the stages in which the endosperm was fully expanded. Although the reason for the large expansion of RIP genes in castor beans remains to be established, the differential expression patterns of the type I and type II members reinforce the existence of biological functions other than defense against predators and herbivory.     

Effects of Ricinus communis oil esters on salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

This study showed the interference of esters extracted from Ricinus communis in the secretory cycle of salivary glands of Rhipicephalus sanguineus ticks, which consequently caused collateral effects on their feeding process. Ticks attached on hosts which were fed with commercial feed containing different concentrations of R. communis oil esters suffered damages such as cytoplasmic changes in their salivary glands, notably in the acinar cells, impairing the functioning of the acini and accelerating the organs degeneration as a whole. It was found that esters interfered with the activity of cellular secretion by changing the glycoprotein of salivar composition especially in acini II cells. It was also shown that the damages caused by esters in the salivary glands cells of these ectoparasites increased in higher concentrations of the product and degenerative glandular changes were more pronounced.     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Homologous very-long-chain 1,3-alkanediols and 3-hydroxyaldehydes in leaf cuticular waxes of Ricinus communis L

Surface extracts from primary leaves of Castor bean were found to contain 1.8 microg cm(-2) of cuticular waxes. The mixture comprised alkanes (C(26)-C(29)), primary alcohols (C(22)-C(38)), aldehydes (C(26) and C(28)), fatty acids (C(20)-C(34)) and triterpenoids (lupeol, beta- and alpha-amyrin). Besides, a series of n-alkane-1,3-diols was detected, with chain lengths ranging from C(22) to C(28), a strong predominance of even-numbered homologs, and a maximum for hexacosane-1,3-diol. Seven other compounds were assigned to a novel class of wax constituents and identified as homologous unbranched 3-hydroxyaldehydes ranging from C(22) to C(28). As the chain length distribution of this series closely paralleled the homolog pattern of 1,3-diols, it seems likely that both compound classes are biosynthetically related.     

Carbohydrate-binding activity of the type-2 ribosome-inactivating protein SNA-I from elderberry (Sambucus nigra) is a determining factor for its insecticidal activity

In recent years, different classes of proteins have been reported to promote toxic effects when ingested. Type-2 ribosome-inactivating proteins (RIPs) are a group of chimeric proteins built up of an A-chain with RNA N-glycosidase activity and a B-chain with lectin activity. These proteins are thought to play a role in plant protection. Sambucus nigra agglutinin I (SNA-I) is a type-2 RIP, isolated from the bark of elderberry (S. nigra L.). This study demonstrated the insecticidal potency of SNA-I on two Hemipteran insect species using two different methods. An artificial diet supplemented with different concentrations of the purified RIP reduced survival and fecundity of pea aphids Acyrthosiphon pisum. In addition, feeding of tobacco aphids, Myzus nicotianae, on leaves from transfected plants constitutively expressing SNA-I, resulted in a delayed development and reduced adult survival and also the fertility parameters of the surviving aphids were reduced, suggesting that a population of aphids would build up significantly slower on plants expressing SNA-I. Finally, a series of experiments with transgenic lines in which a mutant RIP was expressed, revealed that the carbohydrate-binding activity of SNA-I is necessary for its insecticidal activity. In a first set of mutants, the B-chain was mutated at one position (Asp231ΔGlu), and in the second set both carbohydrate-binding sites were mutated (Asn48ΔSer and Asp231ΔGlu). Mutation of one carbohydrate-binding site strongly reduced the insecticidal activity of SNA-I, whereas mutation of both lectin sites (almost) completely abolished the SNA-I effect on tobacco aphids.     

Induction of apoptosis by the mistletoe lectins: A review on the mechanisms of cytotoxicity mediated by Viscum album L.

This review focuses on the cytotoxic properties of Viscum album L. (VAL). Apart from well-established results of protein synthesis inhibition by the mistletoe lectins (MLs), namely their catalytic A chain, there is now convincing evidence that the VAL-mediated cytotoxicity is mainly due to an induction of apoptosis. Among the more than 1,000 proteins detected in VAL, the MLs and the viscotoxins (VTs) are the predominant toxic proteins. Using purified components, such as the D-galactose-specific ML I, the N-acetyl-D-galactosamine-specific ML II and ML III, crude VTs and oligosaccharides, only the MLs induced apoptosis. The in vitro studies suggest that interaction of lectin B chains with appropriate receptors on the cell surface activates distinct signalling pathways that ultimately leads to apoptosis in a large fraction of cells, while others survive, however, with a conservation of their DNA. Inhibition of protein synthesis by the A chain of the hololectin probably accelerates the B chain-induced course of events.     

Configuration of polyisoprenoids affects the permeability and thermotropic properties of phospholipid/polyisoprenoid model membranes

The influence of α-cis- and α-trans-polyprenols on the structure and properties of model membranes was analyzed. The interaction of Ficaprenol-12 (α-cis-Prenol-12, α-Z-Prenol-12) and Alloprenol-12 (α-trans-Prenol-12, α-E-Prenol-12) with model membranes was compared using high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC) and fluorescent methods. l-α-Phosphatidylcholine from egg yolk (EYPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as the main lipid components of unilamellar (SUVs) and multilamellar (MLVs) vesicles were used. The two-step extraction procedure (n-pentane and hexane, respectively) allowed to separately analyze the fractions of polyprenol as non-incorporated (Prenol_NonInc) and incorporated (Prenol_Inc) into liposomes. Consequently, distribution coefficients, P′, describing the equilibrium of prenol content between phospholipid (EYPC) membrane and the aqueous phase gave different log P′ for α-cis- and α-trans-Prenol-12, indicating that the configuration of the α-terminal residue significantly alters the hydrophobicity of the polyisoprenoid molecule and consequently the affinity of polyprenols for EYPC membrane. In fluorescence experiments α-trans-Pren-12 increased up to 1.7-fold the permeability of EYPC bilayer for glucose while the effect of α-cis-Pren-12 was almost negligible. Considerable changes of thermotropic behavior of DPPC membranes in the presence of both prenol isomers were observed. α-trans-Pren-12 completely abolished the pretransition while in the case of α-cis-Pren-12 it was noticeably reduced. Furthermore, for both prenol isomers, the temperature of the main phase transition (T_m) was shifted by about 1 °C to lower values and the height of the peak was significantly reduced. The DSC analysis profiles also showed a new peak at 38.7 °C, which may suggest the concomitant presence of more that one phase within the membrane.Results of these experiments and the concomitant occurrence of alloprenols and ficaprenols in plant tissues suggest that cis/trans isomerization of the α-residue of polyisoprenoid molecule might comprise a putative mechanism responsible for modulation of the permeability of cellular membranes.     

Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp

Hyaluronidase (HAase) activity was detected in the culture supernatants of Penicillium purpurogenum and Penicillium funiculosum. The HAase from Penicillium spp. (HAase-P) was a hyaluronate 4-glycanohydrolase, which catalyzed the endolytic hydrolysis of the β-1,4 glycosidic linkage, as do vertebrate HAases. The gene encoding HAase-P was cloned and expressed in Escherichia coli. According to homology analyses of the deduced amino acid sequences, HAase-P is not classified into any of the known HAase groups, but belongs to glycoside hydrolase family 16, which includes endo-β-1,3(4)-glucanase. Regarding the substrate specificities, no chondroitinase and glucanase activities were detected. Judging from homology analyses and enzymatic properties, HAase-P seems to be a new type of HAase.     

Lectin activity of the nucleocytoplasmic EUL protein from Arabidopsis thaliana

The Euonymus lectin (EUL) domain was recognized as the structural motif for a novel class of putative carbohydrate binding proteins. Confocal microscopy demonstrated that the lectin from Euonymus europaeus (EEA) as well as the EUL protein from Arabidopsis thaliana (ArathEULS3) are located in the nucleocytoplasmic compartment of the plant cell. ArathEULS3 as well as its EUL domain were successfully expressed in Pichia pastoris and purified. The EUL domain from Arabidopsis interacts with glycan structures containing Lewis Y, Lewis X and lactosamine, indicating that it can be considered a true lectin domain. Despite the high sequence identity between the EUL domains in EEA and ArathEULS3, both domains recognize different carbohydrate structures.     

Sucrose uptake by cotyledons of Ricinus communis L.: Characteristics,mechanism, and regulation

Cotyledons of Ricinus communis take up externally supplied sucrose at a rate of up to 150 mol/h/g fresh weight, which is very high when compared with other sugar transport systems of higher plants. The uptake of sucrose is catalysed with a K _m of 25 mmol l^–1; at high sucrose concentrations a linear (diffusion) component becomes obvious. Other mono-, di-, or trisaccharides do not compete for sucrose uptake. Sucrose is accumulated by the cotyledons up to 100-fold, whereby most of the transported, externally supplied sucrose mixes with sucrose present in the tissue. At low sucrose concentrations, however; a small unexchangeable internal pool of sucrose becomes evident. Poisons of energy metabolism such as FCCP inhibit uptake and accumulation of sucrose. The transport of sucrose induces an increase of respiration, from which an energy requirement of 1.4 ATP/sucrose taken up can be calculated. Sucrose is taken up together with protons at an apparent stoichiometry of 0.3 protons/sucrose. Other sugars do not cause proton uptake. The K _m for sucrose induced proton uptake is 5 mmol l^–1; the discrepancy to the K _m for sucrose uptake as well as the low proton: sucrose stoichiometry might possibly be caused by a large contribution of diffusion barriers. The estimated proton-motive potential difference would by sufficient to explain an electrogenic sucrose accumulation. The rate of uptake of sucrose is subject to feedback inhibition by internal sucrose. It is also regulated during growth of the seedlings since it develops rapidly during the first days of germination and declines again after the 4th day of germination, though no substantial increase of passive permeability resistance was observed.Abbreviations DMO dimethyloxazolidinedione - FCCP trifluoromethoxy (carbonyl-cyanide) phenylhydrazon - fr. wt. fresh weight     

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA.     

Control of growth cone motility and neurite outgrowth by SPIN90

SPIN90 is an F-actin binding protein thought to play important roles in regulating cytoskeletal dynamics. It is known that SPIN90 is expressed during the early stages of neuronal development, but details of its localization and function in growth cones have not been fully investigated. Our immunocytochemical data show that SPIN90 is enriched throughout growth cones and neuronal shafts in young hippocampal neurons. We also found that its localization correlates with and depends upon the presence of F-actin. Detailed observation of primary cultures of hippocampal neurons revealed that SPIN90 knockout reduces both growth cone areas and in the numbers of filopodia, as compared to wild-type neurons. In addition, total neurite length, the combined lengths of the longest (axonal) and shorter (dendritic) neurites, was smaller in SPIN90 knockout neurons than wild-type neurons. Finally, Cdc42 activity was down-regulated in SPIN90 knockout neurons. Taken together, our findings suggest that SPIN90 plays critical roles in controlling growth cone dynamics and neurite outgrowth.