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Biomedical subjects

P Christou

Publications and source records attributed to P Christou.

At least 19 recordsLinked to original sources
PubMed2006

Transgenic plants for insect pest control: a forward looking scientific perspective.

One of the first successes of plant biotechnology has been the creation and commercialisation of transgenic crops exhibiting resistance to major insect pests. First generation products encompassed plants with single insecticidal Bt genes with resistance against major pests of corn and cotton. Modelling studies predicted that usefulness of these resistant plants would be short-lived, as a result of the ability of insects to develop resistance against single insecticidal gene products. However, despite such dire predictions no such collapse has taken place and the acreage of transgenic insect resistance crops has been increasing at a steady rate over the 9 years since the deployment of the first transgenic insect resistant plant. However, in order to assure durability and sustainability of resistance, novel strategies have been contemplated and are being developed. This perspective addresses a number of potentially useful strategies to assure the longevity of second and third generation insect resistant plants.

Animals↗
PubMed2005

Recent progress in plantibody technology.

Antibodies are an important class of proteins that can be used for the prevention, treatment and diagnosis of many diseases. Consequently, there is an intense and growing demand for recombinant antibodies, placing immense pressure on current production capacity which is based largely on microbial cultures and mammalian cells. Alternative systems for cost effective antibody production would be very welcome, and plants are now gaining widespread acceptance as green bioreactors with advantages in terms of cost, scalability and safety. Several plant-produced antibodies (plantibodies) are undergoing clinical trials and the first commercial approval could be only a few years away. The performance of the first generation of products has been very encouraging so far. In terms of product authenticity, differences in glycosylation between plantibodies and their mammalian counterparts have been defined, and the scientific evaluation of any possible consequences is underway. Ongoing studies are addressing the remaining biochemical constraints, and aim to further improve product yields, homogeneity and authenticity, particularly where the antibody is intended for injection into human patients. A remaining practical challenge is the implementation of large-scale production and processing under good manufacturing practice conditions that are yet to be endorsed by regulatory bodies. The current regulatory uncertainty and the associated costs represent an entry barrier for the pharmaceutical industry. However, the favourable properties of plants are likely to make the plant systems a useful alternative for small, medium and large scale production throughout the development of new antibody-based pharmaceuticals.

Animals↗
PubMed2004

Producing transglutaminases by molecular farming in plants: minireview article.

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities, both in terms of biology and biotechnology, is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases in research, medicine and industry has increased the demand for an inexpensive, efficient and safe source of recombinant enzymes. We describe initial results concerning the production of a mammalian transglutaminase in transgenic rice plants as a first step towards the large-scale molecular farming of this enzyme.

Animals↗
PubMed2004

Transgenic rice as a vehicle for the production of the industrial enzyme transglutaminase.

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. We explored the use of plant-based systems for the production of this important industrial enzyme. Transgenic rice plants engineered with a rat prostate transglutaminase (rTGp), driven by the strong constitutive maize-1 ubiquitin promoter and its first intron, were shown to express the recombinant enzyme at the mRNA and protein levels. The Ca2+ dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterisation of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.

Animals↗
PubMed2004

Long-term vertical changes of the anterior maxillary teeth adjacent to single implants in young and mature adults. A retrospective study.

AIM: To evaluate the effects of the tooth eruption process on the position of teeth adjacent to implant-borne restorations in adult patients compared to patients in their late adolescence. SUBJECT AND METHODS: The sample included 28 patients divided into two groups. A "young adult" group consisting of 14 patients, aged from 15.5 to 21 years, and a "mature adult" group consisting of 14 patients, aged from 40 to 55 years. All patients presented missing anterior teeth, requiring insertion of 40 implant fixtures (16 central incisors, 12 lateral incisors, 12 canines). The implants were of the Straumann Dental Implant System, clinically and radiologically re-evaluated 1 year or more after the surgical procedure (mean interval=4.2 years). Assessment of the eruption of the adjacent teeth was performed using the implant as a stable point of reference: measurements of the different reference points were compared after implant placement and at follow-up examination. RESULTS: In the "young adult" group, all patients showed infra-occlusion of the implant-supported crowns: the vertical step measured on radiographs varied between 0.1 and 1.65 mm. In the "mature adult" group, all patients showed a vertical difference between the teeth adjacent to the implant-supported crown and the implant: the measured step ranged from 0.12 to 1.86 mm. No difference was found in the amount of vertical eruption between male and female patients, nor according to localization of the implant. CONCLUSION: Mature adults can exhibit major vertical steps after anterior restorations with osseointegrated fixtures to the same extent as adolescents or "young adult" individuals with residuous growth potential.

Adolescent↗
PubMed2001

Expression of legumin and vicilin genes in pea mutants and the production of legumin in transgenic plants.

Pea seeds contain two major storage proteins, legumin and vicilin, in proportions that are genetically and environmentally determined. They are synthesized from at least 40 genes and at least 10 different genetic loci. Mutant alleles at loci involved in starch synthesis, which result in perturbations in starch accumulation, also affect the expression of legumin genes, thereby influencing the legumin: vicilin ratio within the total seed protein. Examples of such alleles include r (starch-branching enzyme) and rb (ADP-glucose pyrophosphorylase), both of which result in a reduction in legumin synthesis; double mutants (rrb) show a particularly severe reduction in the amount of legumin. The effects of such mutations are specific to legumins. The amounts of vicilin are unaffected by mutations at r or rb. One of the consequences of the production of legumin from many genes is structural heterogeneity that is believed to preclude the purification of homogeneous legumin for crystallization and 3D-structure determination. Expression of cloned legumin cDNA in E. coli can result in sequence homogeneity, but E. coli is unable to carry out the normal proteolytic processing of legumin precursors and consequently such material is different from that produced in pea seeds. This paper describes the high-level synthesis, processing and assembly of pea legumin in transgenic wheat seeds, leading to the spontaneous in vitro formation of paracrystalline arrays of legumin, which may be attributed to the fact that the legumin consists of a single type of subunit. Such material might be used as a source of single-sequence, processed and assembled pea legumin for structural investigation.

Gene Expression Regulation, Plant↗
PubMed2001

Tagged Transcriptome Display (TTD) in indica rice using Ac transposition.

We have developed a population of transgenic indica rice lines containing the autonomous Activator transposon (Ac) from maize. A transposon excision assay using as the reporter the green fluorescent protein (GFP) gene driven by the ubiquitin promoter was used to monitor Ac excision in various tissues. Our results, based on Ac excision and re-insertion events in 289 independent rice transformants, provide an insight into transposon biology in this heterologous model cereal system. Twenty percent of the transformed calli displayed uniform GFP activity, indicating very early Ac excision, while later excision in another 40% of calli was revealed by mosaic GFP activity. Both phenotypes were confirmed by molecular analysis. Progeny analysis revealed active transposition, with some lines displaying transposition to unlinked positions. Amplification of the Ac copy number was observed in approximately 30% of the lines, thus tagging multiple sites. We developed a "transposon insertion display" procedure, involving the use of CpG methylation-sensitive enzymes and a macroarray-based approach with cDNA as a complex hybridization probe, to selectively detect transposon insertions in transcribed sequences. Sequencing of tagged sites identified by hybridization to leaf cDNA revealed that all the hybridizing tags were homologous to genes or ESTs present in databases. Extrapolation from these data suggests that 12% of all amplified tags display homology to genes or ESTs - five times more than would be expected on the basis of random transpositional insertion. This Tagged Transcriptome Display (TTD) technique, using a population of Ac insertion-tagged lines, therefore represents a convenient approach for the identification of tagged genes involved in specific processes, as revealed by their expression patterns.

Base Sequence↗
PubMed2001

Over-expression of a cDNA for human ornithine decarboxylase in transgenic rice plants alters the polyamine pool in a tissue-specific manner.

We investigated how over-expression of a cDNA for human ornithine decarboxylase (odc) affects the polyamine pools in transgenic rice. We further investigated tissue-specific expression patterns and product accumulation levels of the transgene driven by either constitutive or seed-specific promoters. Our results indicate that: (1) whereas the expression of a heterologous arginine decarboxylase (adc) cDNA in rice resulted in increased putrescine and spermine levels only in seeds, plants engineered to express odc cDNA exhibited significant changes in the levels of all three major polyamines in seeds and also in vegetative tissues (leaves and roots); (2) there was no linear correlation between odc mRNA levels, ODC enzyme activity and polyamine accumulation, suggesting that control of the polyamine pathway in plants is more complex than in mammalian systems; (3) ODC activity and polyamine changes varied in different tissues, indicating that the pathway is regulated in a tissue-specific manner. Our results suggest that ODC rather than ADC is responsible for the regulation of putrescine synthesis in plants.

Carboxy-Lyases↗
PubMed2001

Transgenic rice plants expressing the ferredoxin-like protein (AP1) from sweet pepper show enhanced resistance to Xanthomonas oryzae pv. oryzae.

We used particle bombardment to cotransform mature seed-derived rice callus (Oryza sativa L., ssp. japonica, cv. Eyi 105) with plasmids containing the linked marker genes gusA and hpt, and the ap1 gene encoding an amphipathic protein previously shown to delay the hypersensitive response induced in non-host plants by the pathogen Pseudomonas syringae pv. syringae (Pss). Thirty-two independent lines of transgenic rice plants were regenerated, and 27 of these lines carried all three transgenes as shown by molecular analysis. A bacterial blight inoculation test was carried out on ten lines. In each case, plants carrying the ap1 gene showed enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo) race 6 at various levels. This suggests the ap1 gene could be a useful candidate for genetic engineering strategies in rice to provide bacterial blight resistance.

Journal Article↗
PubMed2001

Pea legumin overexpressed in wheat endosperm assembles into an ordered paracrystalline matrix.

Legumin, a major component of pea seed storage vacuoles, is synthesized by a number of paralogous genes. The polypeptides are cleaved posttranslationally and can form mixed hexamers. This heterogeneity hampers structural studies, based on the production of hexamer crystals in vitro. To study a single type of homogenous legumin we produced pea legumin A in transgenic wheat (Triticum aestivum) endosperm where prolamins are predominant and only small amounts of globulins accumulate in separate inclusions. We demonstrated that the legumin precursor was cleaved posttranslationally and we confirmed assembly into 11S hexamers. Legumin was deposited within specific regions of the inclusion bodies. Angular legumin crystals extended from the inclusion bodies into the vacuole, correlating with the high legumin content. This suggests that the high-level production of a single type of legumin polypeptide resulted in the spontaneous formation of crystals in vivo. The use of a heterologous cereal system such as wheat endosperm to produce, isolate, and recrystallize homogenous 11S legume globulins offers exciting possibilities for structural analysis and characterization of these important seed storage proteins.

DNA, Complementary↗
PubMed2001

Expression of Arabidopsis GAI in transgenic rice represses multiple gibberellin responses.

Bioactive gibberellins (GAs) are essential endogenous regulators of plant growth. GA signaling is mediated via GAI, a nuclear member of the GRAS family of plant transcription factors. Previous experiments have suggested that GAI is a GA-derepressible repressor of plant growth. Here we test this hypothesis by examining the effects of the expression of Arabidopsis GAI in transgenic Basmati rice. High-level expression of GAI caused dwarfism and reduced GA responses, and the strength of this effect was correlated with the level of transgene expression. In particular, the expression of GAI abolished the GA-mediated induction of rice aleurone alpha-amylase activity, thus implicating GAI orthologs in the well-characterized cereal aleurone GA response. The GA derepressible repressor model predicts that high-level expression of GAI should confer dwarfism, and these observations are consistent with this prediction.

Arabidopsis↗
PubMed2001

Native and artificial reticuloplasmins co-accumulate in distinct domains of the endoplasmic reticulum and in post-endoplasmic reticulum compartments.

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.

Calcium-Binding Proteins↗
PubMed2000

The maize streak virus coat protein transcription unit exhibits tissue-specific expression in transgenic rice.

Maize streak geminivirus (MSV) is a single-stranded DNA virus that infects cereals and other grasses. A promoter region incorporating the MSV large intergenic region and movement protein gene sequence was ligated to the gus (beta-glucuronidase) reporter gene which replaced the virus coat protein (CP) gene. The CP promoter activity was analysed in transgenic rice plants (Oryza sativa L.) and was compared with that obtained in plants transformed with the gus gene downstream of the cauliflower mosaic virus (CaMV) 35S promoter. The MSV CP promoter activity varied in the five plant lines tested, but was always less than that of the CaMV promoter. Histochemistry showed that the MSV CP promoter was active in cells of regenerating callus but in regenerated plants it provided an expression pattern restricted to the vascular tissues of the root, stem, leaf and floral organs. Expression was highest in phloem-associated tissues of the vegetative organs and was absent from the tip and elongation region of seedling roots. Thus, the MSV CP promoter shows a degree of developmental regulation and can be used to confer tissue-specific expression in transgenic rice plants.

Journal Article↗
PubMed2000

Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene, single-copy transgenic locus in rice.

We investigated transgene silencing in a line of rice plants that carries a single-copy 6.6-kb transgenic locus comprising three heterologous transgenes: bar, hpt and gusA. We identified at least three distinct types of silencing effects associated with different methylation patterns, including a novel form of transcriptional silencing involving methylation of cytosine residues only at non-conventional acceptor sites in the coding region. Silencing arose de novo in individual R1, R2 and R3 plants despite the stability of the transgenic locus, although the basic structure of the locus, transgene dosage and position effects remained constant within the line. We found that different silencing effects could occur concurrently in adjacent heterologous transgenes in the same plant, with no evidence for spreading of silenced states or methylation patterns from one transgene to another.

Base Sequence↗
PubMed2000

Simultaneous reduction of the activity of two related enzymes, involved in early steps of the polyamine biosynthetic pathway, by a single antisense cDNA in transgenic rice.

Transgenic rice cell lines transformed with a heterologous cDNA derived from the arginine decarboxylase gene of oat, in an antisense orientation, exhibited significant (P < 0.05) down-regulation of the activity of the endogenous arginine and ornithine decarboxylases, compared to wild type and controls transformed only with the selectable marker (hpt). Changes in enzyme activity were reflected in a marked decrease in the level of putrescine (P < 0.001) and spermidine (P < 0.01) but not spermine (P > 0.05) in the majority of cell lines analyzed. In agreement with previous results, we confirmed that cell lines with low levels of polyamines exhibited normal morphogenic responses. In vegetative tissue at the whole plant level no significant variation (P > 0.05) in polyamine levels was observed. However, we measured significant reductions (P < 0.001) in putrescine levels in seeds derived from three out of five plants analyzed in detail. Thus, simultaneous reduction of the activity of the two alternative enzymes in the early steps of the polyamine pathway results in significant reduction in end-product accumulation in the seeds of transgenic plants.

Avena↗
PubMed2000

Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA).

Transgenic rice plants expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA) were screened for resistance to green leafhopper (Nephotettix virescens; GLH), a major homopteran pest of rice. Survival was reduced by 29% and 53% (P<0.05) respectively, on plants where GNA expression was tissue-specific (phloem and epidermal layer) or constitutive. Similar levels of resistance in GNA-expressing transgenic rice were previously reported for rice brown planthopper (Nilaparvata lugens; BPH). GNA binding to glycoproteins in gut tissues showed that BPH contained more "receptors" than GLH, and that the binding affinity was stronger, particularly in the midgut. Subsequent toxicity of GNA is thus unlikely to be directly related to the amount of lectin bound. GNA was not detected in the honeydew of either insect species when they were fed on GNA-expressing plants, in contrast to results from artificial diet studies. This result suggests that GNA is not being delivered to the insect efficiently. When offered a free choice vs control plants, BPH nymphs tended to avoid plants expressing GNA; avoidance was less pronounced and took longer to develop on plants where GNA expression was tissue-specific, In contrast to BPH, GLH nymphs were attracted to plants expressing GNA, whether constitutively or in a tissue-specific manner.

Journal Article↗
PubMed2000

Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies.

This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 microg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

Animals↗