Genetic Engineering Methods to Overcome Toxicity Challenges in Castor Industry

The toxicity of castor, in the form of ricin, is one of the key challenges that is slowing the adoption of castor as a crop in some countries, especially in the developed parts.

Toxicity challenges in castor industry can be overcome through genetic engineering.

An example of this is the Genetic Engineering of Lesquerella fendleri for castor oil production.

  • Seed oil of Lesquerella fendleri (Lf) contains a valuable hydroxy fatty acid (HFA), lesquerolic acid (20:1OH).
  • Lf does not have biological toxic compounds, thus its oil represents an alternative source of HFA. Lesquerolic acid is derived by a 2 carbon elongation of 18:1OH.
  • By suppressing the elongation step through genetic engineering, it is possible to generate a new Lf crop that could produce 18:1OH.

Novo Synthetix, USA has followed the genetic engineering technique in castor such that it no longer produces ricin. No new DNA is introduced, mainly tweaking of genes.

Anti-cancer activity from new-world ricins

Biological systems have a complex chemistry which tend to exhibit functions under conditions of demand and proteins are one such prime classes of bio-molecules, persisting in organisms with a tremendous degree of diversity in their constituent composition and functions.Characterization of distinct candidates of proteins for functional vitality is a consensus process, independent of organism and has been followed for Castor here in the context of functional evaluation of strange variants of toxin,ricin and agglutinin in bio-medicine.

Ricin protein is complex protein with well defined lethal functional activity in human cells but its variation inside the castor plant and populations has not been elucidated extensively either on materialistic or functional basis which triggered researchers from Taiwan to initiate freak identification of ricin protein and related toxins from castor seed,richest source of castor toxins.To address this question,they adopted a protein fractionation chromatographic and electrophoresis techniques to resolve  multiple variants of ricin and evaluating the activity of purified, anomalous forms.The results indicated three principal isoforms of ricin(A,B,C) and an agglutinin, in which toxic activity was seen to a maximum level from ricin A upon oral feeding of it in mice and further observation from them revealed that the concerned protein isolate turned out to a novel member.The scientists subsequently subjected it to display its impact against tumour cells and noticed cell division inhibitory activity which was also selective and differential in mode of action to diseased cells.

It is an exciting study to see as the protein chemists followed an elegant and lucid experimental design but ended up with a potent toxin coupled to anti-tumor activity.However the thing that is to be monitored is the contrasting functional behaviour of this new toxin between an intact organism and a cancer cell line which obviously demands for a proper bio-safety evaluation to scale up the promotion of this toxin (declared as a lectin as per authour’s interpretations) for any disease management actions in humans.

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Return of Castor in North America

Castor, an oil seed crop is currently confined to oriental countries to a vast extent in terms of cultivation and oil production but the demand is constantly mounting from the western countries so as to produce precious chemical derivatives from it.It is a crop relatively suitable for cultivation in West as well but  has been strongly ignored on account of its toxic properties including allergens and toxins.Research efforts have been continuously made to overcome the defined, negatives of castor and yielded appreciable results in recent years particularly in USA.

Such committed actions in castor biology are not restricted to research levels but tend to proceed further when a collaborative agreement has been made between a Canadian bio-products company Lubbock-Castagra and Texas AgriLife Research to produce castor seed with less toxin and high, sustainable yield. Reports from concerned authourities said that  encouraging the crop improvement in castor would restore the abandoned castor cultivation with desired modifications in cropping patterns of the Texas state and directly reduce the burdens of import of this plant oil commodity from countries including India.CEO of Canadian Castagra-Lubbock, Mr:Peter Roosen further added that the oil extracted from this quantitatively toxin compromised, castor would be exploited for the production of renewable bioplastics and hence carrying a tremendous potential for the generation of employment in green technology sector.Presently,the castor crop with reduced levels of toxin is at the trial stage in Texas and evaluation studies are being undertaken for a bulk cultivation, expected in 2012.

It is inspiring to watch such an activity adopted by the legal bodies in developed nations to complement the defects what they are experiencing in their environment with respect to castor. Similar sort of co-ordinated plans are mandatory to maximize the revenues from castor in India.This is well punched with reference to the promotion of end derivatives of castor oil in which India is placed way behind the Westerners in the production tally.It not alone ensures economic exchanges of profits but depicts India an industrially competitive nation at the global level.

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Functional significance for castor allergens

Castor seed has abundant level of proteins which are evolved spontaneously to serve the survival demands of the organism in response to the selection pressures. Among multiplicity in proteins, scientists have started focussing on three candidate proteins 2S namely albumins,Ric c1 and Ric c3 which have been earlier characterized as allergens for their biological significance.In general, bio-molecular functional definition can be arrived from analysis of its constituents and its spatial configuration that stood as bases for the functional implications in chosen castor proteins.The allergen proteins under study are found to contain a domain for digestive enzyme inhibition which made researchers to design an insect feeding bio-assay in insects Callosobruchus maculatus, Zabrotes subfasciatus, and Tenebrio molitor over their most optimal diet with parameters of insect larvae mortality and body mass gain from diet containing castor protein allergens .The results confirmed that a loss of body weight by one-fifth fraction compared to control lines which enabled researchers to arrive at the enzyme inhibitory activity from concerned plant proteins due to test domain in them.Further scientists claimed that the castor proteins are potential targets in the design of inhibitors with importance in crop protection and biomedical field.

The results seem to be promising for the feeding deterrent activity from castor proteins  but it would be convincing if an in vitro enzyme assay with gut enzymes against castor allergens and histopathological analysis of insect tissues for molecular complexes would have been performed for confirmed functional implication.

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Profiling of castor toxin by genetic amplification

Ricin toxin is a threatening agent with significance in global warfare. Scientists have been working in the development of several distinct analytical methods for the detection of toxin with improved sensitivity and specificity  but they still tend to suffer from critical limitations especially under circumstances when direct immuno probe based protein toxin profiling was made in complex biochemical samples including food stuffs that resulted in ambiguities.It stood as one of the fundamental reasons which has diverted researchers to concentrate on genetic profiling of toxin in samples for precise interpretation.

US scientists have now developed a technique called real time PCR which targets the genetic material in samples.They applied a genetic probe specific for the toxin in DNA samples isolated from milk and egg for generating a fingerprint.Experimental results showed that their probe functioned relatively well and was able to detect toxin at concentrations of 5ng per ml of milk or liquid egg.It was also reported that negative interferences from the samples were obvious but researchers were quite content with the level of detection of sub-lethal doses of toxin in biological fluids.

It is interesting to watch technique such as this since the analysts were targetting the core biomolecule,DNA which permitted them to reduce the chemical complexity of samples by fractionation for convenient profiling. Nevertheless it is vital to note that advanced PCR systems are expensive and prone for producing artifacts, also demanding for skills in the preparation of quality samples and optimization of probes in the detection of multiple variants of ricin DNA from the genomic pools of biological sources standing at different loci.

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Biosensor development for ricin toxin

Ricin detection in  biological samples is of great significance as the toxin is reported to be a candidate substance for intoxication in bio-terrorism, suicide etc.By classical methods people have exploited antibody based immuno probes for detection of test toxin in materials under observation.However it suffers from certain drawbacks including sensitivity,cross-reactivity,time and technical preparation.It prompted researchers around the world to surf for alternate probes in bio-sensor developments which can effectively circumvent these problems.

Scientists from Japan have made use of natural molecular interaction behaviour of toxin and designed a novel probe to profile the toxin.By applying knowledge in ligand-receptor chemistry of ricin in biological systems,they have designed a synthetic analogue that acts as a ligand to toxin.It was immobilized onto a solid matrix to serve in stationary phase and ricin incubation was performed.The toxin bound to the test probe was assessed by a special spectroscopic technique Surface Plasmon Resonance(SPR) which indicated ricin at concentrations of 5 pg/ml in 5 minutes which the authours claimed as a benchmark in biosensor designing.The results obtained from this new probe were further validated by alternate methods for specificity.

The experimental research work was exciting as a analysts have completed a broad study and generated promising results with cutting edge technologies in the qualitative and quantitative fingerprinting of castor toxin.A technique that is pointed in nature proportionately requires professional perfection for absolute analysis.

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Candidate catalyst for ricin toxin activation

Ricin toxin from castor is a dimeric toxin and initially synthesized as a toxoid but found to be processed in castor beans during the course of seed development.As the protein is localized in the cellular sub components, protein bodies of seed endosperm, researchers from UK have made their experimental investigation on concerned cell organelles of storage function for the detection of molecular factor with catalytic potential to transform the inactive toxin to its lethally active phase.Protein profiling was adopted after isolation and fractionation of crude mixtures from protein bodies to identify the specific target and the results confirmed the materialistic presence of an acid endopeptidase enzyme.The functional relevance for this enzyme over toxin transformation was evaluated by in vitro assays where active toxin release was noticed  from its precursors.

Scientists from this study were able to detect a key candidate acting as an accelerator of ricin toxicity and offered us a solution to target the molecular components in castor with flexibility for producing non toxic seeds instead of a restricted direct focus over the toxin.However it is vital to understand the specificity in this toxin-catalyst association for precise targeting to improve the application potential of castor crop.

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Vaccine developments to ricin toxin

Ricin is a potent lethal cytotoxin derived from castor seeds and combatting its toxicity is a tedious task.Currently no effective antidote has ever been formulated so far and makes therapy difficult.It also places the victims survival purely as a matter of chance.As chemotherapy andd other mehods fail to produce productive results,scientists have started to employ vaccines as a possible tool to tackle the toxin.

US researchers from Army Laboratory in Frederick have found a vaccine containing an inactivated toxin that selectively retains an activity to boost the immune system without inducing lethal toxicity to mount systemic defence response in the body by protein engineering princples and found to be effective on animal models.The US Army has reported that currently trials are underway in 30 human volunteers and plans to conduct clinical trials as well at Fort Detrick.

Vaccines are powerful tools to produce solutions in a timely consistent and biologically safe way.Historical credits to vaccines in global health are something extraordinary  with reference to pathogenic diseases and attempts such as this to focus on vaccine vehicles and expanding their horizon to toxins neutralization would most probably yield a fruitful result in future to address the problems of global threat from castor toxin.

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Catalytic transformation of castor cake by Penicillium fungus

Castor cake is a by product from castor seed but remains as a substance with restricted application potential due to its allergenic and toxic properties.People traditionally have treated the castor cake by wild fermentation for the catalytic transformation of anti-metabolites into their corresponding non-toxic forms for improving its utility.

This age-old practice was scientifically approached by the Brazilian researchers where they subjected castor cake to solid state fermentation.Penicillium simplicissimum fungus was chosen as the catalytic factor because of its potency to manipulate in castor cake substrate and generate lipid metabolizing lipase enzymes.Solid state fermentation(dessicated fermentation) was performed for the cultivation of fungus and the products, enzyme and fermented castor cake were partitioned by solid-liquid phase differential extraction.Enzyme lipase magnitude in crude isolated fraction was determined from its activity against synthetic substrate while the debris cake fermented was evaluated for its toxic protein contents and functional activities by immunoassays and immuno sensitive cell culture assays respectively.The results proved to be promising as the toxin titre and allergens induced cell degranulation were seen to be minimum and the microbial enzyme production was noticed to a significant extent as well from specific activity studies.

Results turn out to be promising but the experiment would have been better if an utility for the fermented castor cake in biology was defined.The solid state technology by fungus seems to be a viable one on commodities like castor cake to generate high value products when time factor is not signified to a greater extent.

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Medium formulation in ricin vaccine

Ricin is a noxious cytotoxin present in castor bean.People have long been pursuing research to understand its functional activities,physical structure and quantitative magnitude in beans etc.In spite of such extensive studies, no effective solution has been formulated to ricin intoxication. However researchers have found vaccine therapy as a promising route to combat the toxin.In ricin vaccinology, the wild toxin has been modified in such a way so that it tends to retain its antigenicity to boost the host defence system but not the toxic activity, called it as “ricin toxoid”.Genetic engineering  is a standardized technique to precisely produce variants in ricin and the candidate with expected property are subsequently chosen.

US scientists have identified an elite ricin toxoid from earlier studies and made an evaluation on vaccine formulation to achieve enhanced antigenicity and stability.Initially they treated the protein in phosphate buffer solution and found structural and functional tolerance in protein after cyclic exposures to extreme temperature ranges.To further improve its adjuvant adherence and related antigenicity, toxoid was treated with aluminium hydroxide adjuvant in succinate buffer and evaluated by differential centrifugation and immuno assays.The results indicated that aluminium hydroxide showed a good compatibility with toxoid by co-sedimentation of protein with gel matrix and also elicited a strong immune response, confirmed by antibody titre studies.Functional confirmation did not seem to be significantly affected by conjugation with adjuvant and Fourier transform infrared spectroscopy was applied to understand it.

Scientists in this study have made a complete profile in ricin vaccine formulation and its influence in conferring active immunity to primitive model systems but the confirmation is to be arrived from human clinical trials as extrapolation of data from mice to humans is difficult .

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