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PubMed1997

Trypsin isolated from the midgut of the tobacco hornworm, Manduca sexta, is inhibited by synthetic pro-peptides in vitro.

The seven residue synthetic peptides VPAYPQR and VPANPQR, identical to the pro-regions of midgut trypsins from Manduca sexta, inhibit the peptidase activity of the mature enzymes. The inhibitory potencies of these peptides towards purified insect trypsin were determined and were found to be more potent at pH 7.0 than at pH 10.0. These peptides did not inhibit porcine trypsin, nor were they degraded rapidly by purified insect trypsin. We suggest that these particular peptides, and insect proregions in general, may be suitable for use in genetically engineered plants as insect specific inhibitors of digestive proteases.

Amino Acid Sequence↗
PubMed1990

Evolving strategies for the genetic engineering of herbicide resistance in plants.

Developments in plant genetic engineering technology will shortly permit the commercial introduction of transgenic crop varieties resistant to a number of non-selective herbicides. High levels of tolerance have been achieved both by overexpression of a target protein and by modification of that target to an insensitive form. However the results of preliminary trials suggest that in some instances the yield penalty for such genetic alterations will be prohibitive. An alternative strategy, based on the transfer and expression of a gene encoding a herbicide-detoxifying enzyme, appears to offer high resistance levels at low metabolic cost and is expected to assume increasing importance, although it may not prove suitable for all herbicides.

Journal Article↗
PubMed2006

Histaminase PEGylation: preparation and characterization of a new bioconjugate for therapeutic application.

Copper amine oxidase catalyses the oxidative deamination of primary amino groups of several biogenic amines, one of which is histamine, the principal chemical mediator of the first phase of allergic reactions. Looking forward to a possible future therapeutic application of this enzyme in the field of histamine-mediated afflictions, we developed a simple method for the purification of a histaminase from grass pea shoots, a source particularly enriched with the enzyme. Furthermore, in order to improve its therapeutic potential, in particular to reduce the high impurity due to its heterologous source, we conjugated the protein with poly(ethylene glycol) and tested the molecular, immunogenic and pharmacokinetic properties of the native and modified forms. The PEGylated enzyme showed molecular and enzymatic properties similar to those of the unmodified one, but the PEGylation extended the permanence of the injected drug in the body and eliminated its high immunogenic behaviour. The considerable ease of native histaminase production as well as the improved properties after PEGylation, make this engineered plant enzyme a suitable drug candidate for alternative treatment of histamine-mediated affections.

Algorithms↗
PubMed2005

Recent advances using rodent models for predicting human allergenicity.

The potential allergenicity of newly introduced proteins in genetically engineered foods has become an important safety evaluation issue. However, to evaluate the potential allergenicity and the potency of new proteins in our food, there are still no widely accepted and reliable test systems. The best-known allergy assessment proposal for foods derived from genetically engineered plants was the careful stepwise process presented in the so-called ILSI/IFBC decision tree. A revision of this decision tree strategy was proposed by a FAO/WHO expert consultation. As prediction of the sensitizing potential of the novel introduced protein based on animal testing was considered to be very important, animal models were introduced as one of the new test items, despite the fact that non of the currently studied models has been widely accepted and validated yet. In this paper, recent results are summarized of promising models developed in rat and mouse.

Administration, Oral↗
PubMed2002

The sequestration switch: removing industrial CO2 by direct ocean absorption.

This review paper considers direct injection of industrial CO2 emissions into the mid-water oceanic column below 500 m depth. Such a process is a potential candidate for switching atmospheric carbon emissions directly to long term sequestration, thereby relieving the intermediate atmospheric burden. Given sufficient research justification, the argument is that harmful impact in both the Atmosphere and the biologically rich upper marine layer could be reduced. The paper aims to estimate the role that active intervention, through direct ocean CO2 storage, could play and to outline further research and assessment for the strategy to be a viable option for climate change mitigation. The attractiveness of direct ocean injection lies in its bypassing of the Atmosphere and upper marine region, its relative permanence, its practicability using existing technologies and its quantification. The difficulties relate to the uncertainty of some fundamental scientific issues, such as plume dynamics, lowered pH of the exposed waters and associated ecological impact, the significant energy penalty associated with the necessary engineering plant and the uncertain costs. Moreover, there are considerable uncertainties regarding related international marine law. Development of the process would require acceptance of the evidence for climate change, strict requirements for large industrial consumers of fossil fuel to reduce CO2 emissions into the Atmosphere and scientific evidence for the overall beneficial impact of ocean sequestration.

Absorption↗
PubMed1998

Perspective: edible vaccines--a concept coming of age.

In addition to their traditional role as a source of natural medicines, it is now possible to genetically engineer plants to produce pharmaceuticals. Transgenic plants expressing antigens from pathogenic microorganisms offer many advantages as low-cost production systems and effective delivery systems for vaccines. This new technology might contribute to global vaccine programs and might have a dramatic impact on health care in developing countries.

Administration, Oral↗
PubMed2006

Native or raw starch digestion: a key step in energy efficient biorefining of grain.

Improved molecular disassembly and depolymerization of grain starch to glucose are key to reducing energy use in the bioconversion of glucose to chemicals, ingredients, and fuels. In fuel ethanol production, these biorefining steps use 10-20% of the energy content of the fuel ethanol. The need to minimize energy use and to raise the net yield of energy can be met by replacing high-temperature, liquid-phase, enzymatic digestion with low temperature, solid-phase, enzymatic digestion. Also called cold hydrolysis, the approach is a step toward a "green" method for the production of fuel ethanol. There has been substantial prior and increased recent interest in this approach that is presented in this first review of the subject. We include incentives, developmental research, fundamental factors of raw starch digestion, and novel approaches in enzymology and processing. The discussion draws on resources found in enzymology, engineering, plant physiology, cereal chemistry, and kinetics.

Amylases↗
PubMed2006

Bacterial pathways for degradation of nitroaromatics.

The last one hundred years have seen a massive expansion in the chemicals industry; however, with this progress came the concomitant pollution of the environment with a significant range of xenobiotics.Nitroaromatic compounds form one such category of novel environmental contaminants and are produced through a large number of industrial processes, most notably the pesticides, dyes and explosives industries. Whilst singly nitrated aromatic compounds are usually mineralised in the environment, multiply nitrated aromatics, such as the explosive 2,4,6-trinitrotoluene (TNT), are recalcitrant and highly toxic. The predominant route of biological transformation of aromatic compounds is oxidation; however, the presence of three electron-withdrawing nitro-groups around the ring prevents oxidation, rendering such compounds resistant to biodegradation. The subsequent accumulation of these contaminants has stimulated much research leading to the isolation of bacteria that possess, to varying extents, the ability to remediate explosives and other nitroaromatic pollutants.The extreme environments created by these toxic substances accelerate the evolutionary process and examples of bacteria that have conscripted metabolic enzymes for novel remediatory pathways are included. This Highlight ends with a discussion of the future of nitroaromatic bioremediation including engineering plants to express bacterial enzymes for use in bioremediation programs.

Bacteria↗
PubMed2006

Midgut bacteria required for Bacillus thuringiensis insecticidal activity.

Bacillus thuringiensis is the most widely applied biological insecticide and is used to manage insects that affect forestry and agriculture and transmit human and animal pathogens. This ubiquitous spore-forming bacterium kills insect larvae largely through the action of insecticidal crystal proteins and is commonly deployed as a direct bacterial spray. Moreover, plants engineered with the cry genes encoding the B. thuringiensis crystal proteins are the most widely cultivated transgenic crops. For decades, the mechanism of insect killing has been assumed to be toxin-mediated lysis of the gut epithelial cells, which leads to starvation, or B. thuringiensis septicemia. Here, we report that B. thuringiensis does not kill larvae of the gypsy moth in the absence of indigenous midgut bacteria. Elimination of the gut microbial community by oral administration of antibiotics abolished B. thuringiensis insecticidal activity, and reestablishment of an Enterobacter sp. that normally resides in the midgut microbial community restored B. thuringiensis-mediated killing. Escherichia coli engineered to produce the B. thuringiensis insecticidal toxin killed gypsy moth larvae irrespective of the presence of other bacteria in the midgut. However, when the engineered E. coli was heat-killed and then fed to the larvae, the larvae did not die in the absence of the indigenous midgut bacteria. E. coli and the Enterobacter sp. achieved high populations in hemolymph, in contrast to B. thuringiensis, which appeared to die in hemolymph. Our results demonstrate that B. thuringiensis-induced mortality depends on enteric bacteria.

Animals↗
PubMed2002

Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis.

In view of their perceived chemopreventive activities against hormone-dependent cancers, cardiovascular disease, and postmenopausal ailments, there is considerable interest in engineering plants to contain isoflavone phytoestrogens. However, attempts to date have only resulted in low levels of isoflavone accumulation in non-legumes. Introducing soybean isoflavone synthase (IFS) into Arabidopsis thaliana leads to accumulation of low levels of genistein glycosides. Leaves of wild-type A. thaliana contain high levels of similar conjugates of the flavonols quercetin and kaempferol, which could be increased by threefold on introduction of an alfalfa chalcone isomerase transgene. Levels of genistein were not increased by expressing both IFS and alfalfa chalcone isomerase, but levels of flavonol conjugates were reduced to a greater extent than could be accounted for by flux into isoflavone. Introduction of IFS into the tt6/tt3 double mutant blocked in flavonol, and anthocyanin synthesis resulted in high levels of genistein. The bottleneck for constitutive isoflavone production in Arabidopsis is, therefore, competition for flavanone between IFS and endogenous flavonol synthesis, and the flavonol pathway is reciprocally but disproportionately affected by IFS.

Alcohol Oxidoreductases↗
PubMed1987

T-DNA organization in tumor cultures and transgenic plants of the monocotyledon Asparagus officinalis.

Asparagus officinalis was the first monocotyledonous plant from which hormone-independent and opine-producing crown gall tissue could be isolated. We confirm by DNA hybridization that tumor lines obtained after infection of this plant by Agrobacterium strains harboring wild-type nopaline and octopine tumor-inducing (Ti) plasmids are stably transformed and contain transferred DNA (T-DNA) segments identical to the T-DNA found in dicotyledonous plants. We have also infected Asparagus with a nononcogenic T-DNA vector that carries a chimeric aminoglycoside phosphotransferase [NOS-APH(3')II] gene and selected transformed tissues on kanamycin-containing medium. The transformed status of these tissues was then confirmed by DNA hybridization. From these calli we regenerated kanamycin-resistant shoots that were subsequently rooted. Thus we report the isolation of transgenic monocotyledonous plants engineered via the Agrobacterium vector system.

Journal Article↗
PubMed1995

Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.

Plants possess multiple resistance mechanisms that guard against pathogen attack. Among these are inducible systems such as systemic acquired resistance (SAR). SAR is activated by pathogen exposure and leads to an increase in salicylic acid (SA), high-level expression of SAR-related genes, and resistance to a spectrum of pathogens. To identify components of the signal transduction pathways regulating SAR, a mutant screen was developed that uses 2,6-dichloroisonicotinic acid as an activator of SAR gene expression and pathogen resistance, followed by assays for resistance to the fungal pathogen Peronospora parasitica. Mutants from this screen were subsequently examined to assess their defense responses. We describe here a recessive mutation that causes a phenotype of insensitivity to chemical and biological inducers of SAR genes and resistance. These data indicate the existence of a common signaling pathway that couples these diverse stimuli to induction of SAR genes and resistance. Because of its non-inducible immunity phenotype, we call this mutant nim1. Although nim1 plants fail to respond to SA, they retain the ability to accumulate wild-type levels of SA, a probable endogenous signal for SAR. Further, the ability of nim1 plants to support growth of normally incompatible races of a fungal pathogen indicates a role for this pathway in expression of genetically determined resistance, consistent with earlier findings for transgenic plants engineered to break down SA. These results suggest that the wild-type NIM1 gene product functions in a pathway regulating acquired resistance, at a position downstream of SA accumulation and upstream of SAR gene induction and expression of resistance.

Journal Article↗
PubMed1996

Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.

Administration, Oral↗
PubMed2004

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae).

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.

Animals↗
PubMed1997

Regulation of levels of proline as an osmolyte in plants under water stress.

Compatible osmolytes are potent osmoprotectants that play a role in counteracting the effects of osmotic stress. Proline (Pro) is one of the most common compatible osmolytes in water-stressed plants. The accumulation of Pro in dehydrated plants is caused both by activation of the biosynthesis of Pro and by inactivation of the degradation of Pro. In plants, L-Pro is synthesized from L-glutamic acid (L-Glu) via delta(1)-pyrroline-5-carboxylate (P5C) by two enzymes, P5C synthetase (P5CS) and P5C reductase (P5CR). L-Pro is metabolized to L-Glu via P5C by two enzymes, proline dehydrogenase (oxidase) (ProDH; EC 1.5.99.8) and P5C dehydrogenase (P5CDH; EC 1.5.1.12). Such metabolism of Pro is inhibited when Pro accumulates during dehydration and it is activated when rehydration occurs. Under dehydration conditions, when expression of the gene for P5CS is strongly induced, expression of the gene for ProDH is inhibited. By contrast, under rehydration conditions, when the expression of the gene for ProDH is strongly induced, the expression of the gene for P5CS is inhibited. Thus, P5CS, which acts during the biosynthesis of Pro, and ProDH, which acts during the metabolism of Pro, appear to be the rate-limiting factors under water stress. Therefore, it is suggested that levels of Pro are regulated at the level of transcriptional the genes of these two enzymes during dehydration and rehydration. Moreover, it has been demonstrated that Pro acts as an osmoprotectant and that overproduction of Pro results in increased tolerance to osmotic stress of transgenic tobacco plants. Genetically engineered crop plants that overproduce Pro might, thus, acquire osmotolerance, namely, the ability to tolerate environmental stresses such as drought and high salinity.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed2005

The value of short amino acid sequence matches for prediction of protein allergenicity.

Typically, genetically engineered crops contain traits encoded by one or a few newly expressed proteins. The allergenicity assessment of newly expressed proteins is an important component in the safety evaluation of genetically engineered plants. One aspect of this assessment involves sequence searches that compare the amino acid sequence of the protein to all known allergens. Analyses are performed to determine the potential for immunologically based cross-reactivity where IgE directed against a known allergen could bind to the protein and elicit a clinical reaction in sensitized individuals. Bioinformatic searches are designed to detect global sequence similarity and short contiguous amino acid sequence identity. It has been suggested that potential allergen cross-reactivity may be predicted by identifying matches as short as six to eight contiguous amino acids between the protein of interest and a known allergen. A series of analyses were performed, and match probabilities were calculated for different size peptides to determine if there was a scientifically justified search window size that identified allergen sequence characteristics. Four probability modeling methods were tested: (1) a mock protein and a mock allergen database, (2) a mock protein and genuine allergen database, (3) a genuine allergen and genuine protein database, and (4) a genuine allergen and genuine protein database combined with a correction for repeating peptides. These analyses indicated that searches for short amino acid sequence matches of eight amino acids or fewer to identify proteins as potential cross-reactive allergens is a product of chance and adds little value to allergy assessments for newly expressed proteins.

Allergens↗
PubMed1986

General occupational health history and examination.

This paper presents a method for developing occupational medical examinations using the preplacement examination (PPE) and the periodic monitoring examination (PME). The steps for developing an appropriate PPE include goal definition, analysis of job requirements and potential exposures, an occupational and medical history, and a targeted physical examination derived from the preceding steps. The PPE should result in the proper placement of the worker. It also serves as a baseline for comparison to future testing or health-related developments. The PME checks the effectiveness of more primary preventive measures such as plant engineering controls and personal protection. The contents and frequency of the PME depend on the pathophysiology and toxicology of the hazardous exposures, the interval exposure and medical history, and legal requirements. Individual and group results should be assessed periodically for trends suggesting work-related problems that would need further evaluation.

Humans↗
PubMed1993

Interference with brome mosaic virus replication by targeting the minus strand promoter.

Sense and antisense strategies for interfering with the replication of brome mosaic virus (BMV) were examined. The effects of 200 nucleotide-long sense and antisense transcripts, corresponding to the viral 3' end (-) strand promoter, on the accumulation of progeny viral RNAs were studied by co-inoculation with wildtype BMV RNAs. Progeny accumulation in barley protoplasts transfected with either sense or antisense transcripts of the (-) strand promoter and BMV RNAs-1 and -2 was decreased by more than 90%, and by 60 to 80% when RNA-3 was also present. This trans interference was concentration-dependent, and reduced both (+) and (-) strand progeny accumulation to a similar extent. The appearance of complementary (-) strands indicated that sense interfering transcripts could serve as templates for (-) strand synthesis, and the use of deletion mutants revealed that the observed interference was in part mediated by this template activity. The reproducibility of the protoplast assay used here allows rapid evaluation of interference strategies and comparisons to be made of alternative approaches to engineered resistance. The results presented here suggest that targeting viral (-) strand promoters with sense and antisense transcripts may be an effective method for engineering plant resistance to viral infection.

Base Sequence↗