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At least 271 records · Page 15Linked to original sources
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↗
PubMed1997

Plant cell suspension cultures: some engineering considerations.

Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings and fragrances. For some of these compounds, plant cell culture can provide a potential production alternative to traditional cultivation methods or chemical synthesis routes. Many systems have been patented and the last 20 years have seen considerable industrial and academic interest in the development of large scale cultures to produce pharmaceutically active, high value substances. However, the industrial application of plant cell suspension cultures has, to date, been limited. Commercialisation has essentially been impeded by economic feasibility, arising from both biological and engineering considerations. This paper reviews the commercial development of the technology to date and focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cell suspension cultures. Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to the physical characteristics of the suspended cells. Recent studies indicate that shear sensitivity may not be as important, in some cases, as initially anticipated.

Bioreactors↗
PubMed2000

Degradation of Aux/IAA proteins is essential for normal auxin signalling.

The growth substance auxin mediates many cellular processes, including division, elongation and differentiation. PSIAA6 is a member of the Aux/IAA family of short-lived putative transcriptional regulators that share four conserved domains and whose mRNAs are rapidly induced in the presence of auxin. Here PSIAA6 was shown to serve as a dominant transferable degradation signal when present as a translational fusion with firefly luciferase (LUC), with an in vivo half-life of 13.5 min in transgenic Arabidopsis seedlings. In a transient assay system in tobacco protoplasts using steady-state differences as an indirect measure of protein half-life, LUC fusions with full-length PSIAA6 and IAA1, an Aux/IAA protein from Arabidopsis, resulted in protein accumulations that were 3.5 and 1. 0%, respectively, of that with LUC alone. An N-terminal region spanning conserved domain II of PSIAA6 containing amino acids 18-73 was shown to contain the necessary cis-acting element to confer low protein accumulation onto LUC, while a fusion protein with PSIAA6 amino acids 71-179 had only a slight effect. Single amino acid substitutions of PSIAA6 in conserved domain II, equivalent to those found in two alleles of axr3, a gene that encodes Aux/IAA protein IAA17, resulted in a greater than 50-fold increase in protein accumulation. Thus, the same mutations resulting in an altered auxin response phenotype increase Aux/IAA protein accumulation, providing a direct link between these two processes. In support of this model, transgenic plants engineered to over-express IAA17 have an axr3-like phenotype. Together, these data suggest that rapid degradation of Aux/IAA proteins is necessary for a normal auxin response.

Arabidopsis↗
PubMed2025

mamp-ml: A deep learning approach to epitope immunogenicity in plants.

Eukaryotes detect biomolecules through surface-localized receptors, key signaling components. A subset of receptors survey for pathogens, induce immunity, and restrict pathogen growth. Comparative genomics of both hosts and pathogens has unveiled vast sequence variation in receptors and potential ligands, creating an experimental bottleneck. We have developed mamp-ml, a machine learning framework for predicting plant receptor-ligand interactions. We leveraged existing functional data from over two decades of foundational research, together with the large protein language model ESM-2, to build a pipeline and model that predicts immunogenic outcomes using a combination of receptor-ligand features. Our model achieves 73% prediction accuracy on a held-out test set, even when an experimental structure is lacking. Our approach enables high-throughput screening of LRR receptor-ligand combinations and provides a computational framework for engineering plant immune systems.

Journal Article↗
PubMed1989

[Volatile ashes and their biological effect. 2. Fibrogenic effect of volatile ashes].

In experiments on white Wistar rats fibrogenic effects of 6 samples of fly-ashes collected from electric precipitators in power engineering plants have been evaluated. The coal came from different national deposits. All the ashes have been found to contain: quartz and mullite, 3 ashes contained additionally orthoclase, whereas 1, apart from quartz and mullite, contained kaolinite; naturally radioactive elements (Ra226, K40, Th228) and trace elements (As, Ba, Be, Cd, Ce, Cu, Fe, Pa, Mo, Ni, Pb, Se, U Zu). Experimental pneumoconiosis was induced through intratracheal administration of single doses of 50 mg of dust; the experiment was carried out at 3 time intervals of 3, 6 and 9 months. The fibrogenic activity was evaluated both qualitatively (histopathological methods) and quantitatively (lung weight, hydroxyproline content in lungs, dust elimination from lungs); control groups consisted of animals which obtained NaCl solution and quartz sands. Fly-ashes were found to exhibit different fibrogenic effects, yet, their fibrogenic activity was weaker, compared to quartz sands. No clear correlation was found between fibrogenic effects of ashes and test physico-chemical properties, such as the content of SiO2, trace elements or naturally radioactive elements. Analysis of occupational diseases (for the period section): (1979-1983) demonstrated occupational diseases of dust-related aetiology among power engineering workers, pneumoconioses, constituting 7.8% of 127 cases of occupational diseases.

Animals↗
PubMed2006

Gradual soil water depletion results in reversible changes of gene expression, protein profiles, ecophysiology, and growth performance in Populus euphratica, a poplar growing in arid regions.

The responses of Populus euphratica Oliv. plants to soil water deficit were assessed by analyzing gene expression, protein profiles, and several plant performance criteria to understand the acclimation of plants to soil water deficit. Young, vegetatively propagated plants originating from an arid, saline field site were submitted to a gradually increasing water deficit for 4 weeks in a greenhouse and were allowed to recover for 10 d after full reirrigation. Time-dependent changes and intensity of the perturbations induced in shoot and root growth, xylem anatomy, gas exchange, and water status were recorded. The expression profiles of approximately 6,340 genes and of proteins and metabolites (pigments, soluble carbohydrates, and oxidative compounds) were also recorded in mature leaves and in roots (gene expression only) at four stress levels and after recovery. Drought successively induced shoot growth cessation, stomatal closure, moderate increases in oxidative stress-related compounds, loss of CO2 assimilation, and root growth reduction. These effects were almost fully reversible, indicating that acclimation was dominant over injury. The physiological responses were paralleled by fully reversible transcriptional changes, including only 1.5% of the genes on the array. Protein profiles displayed greater changes than transcript levels. Among the identified proteins for which expressed sequence tags were present on the array, no correlation was found between transcript and protein abundance. Acclimation to water deficit involves the regulation of different networks of genes in roots and shoots. Such diverse requirements for protecting and maintaining the function of different plant organs may render plant engineering or breeding toward improved drought tolerance more complex than previously anticipated.

Climate↗
PubMed1995

In vitro toxicological investigations of isoxazolinone amino acids of Lathyrus sativus.

Two non-protein amino acids of Lathyrus sativus, beta-(isoxazoline-5-on-2-yl)-alanine (BIA) and its higher homologue alpha-amino-gamma-(isoxazoline-5-on-2-yl)-alanine (ACI) were tested for excitotoxic potential. BIA (0.5-2.0 mM) but not ACI (2.0 mM) produced a concentration-dependent neurodegeneration in mouse cortical explants. The neuronal damage was prevented by the prior and simultaneous application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), indicating that it was mediated by non-N-methyl-D-aspartate type receptors. BIA (0.5-2.0 mM) activated CNQX-sensitive currents which were significantly smaller than those activated by 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the majority of neurons. In a small number of cells, BIA (2 mM) produced currents which were similar in amplitude to those activated by beta-ODAP (50 microM). These results suggest that Lathyrus sativus plants engineered to block the synthesis of beta-ODAP may accumulate a neurotoxic precursor and therefore must be tested for the presence of both BIA and beta-ODAP.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships.

Cinnamoyl CoA:NADP oxidoreductase (CCR, EC 1.2.1.44) catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, i.e. the first specific step in the synthesis of the lignin monomers. The cloning of a cDNA encoding CCR in Eucalyptus gunnii (EUCCR) is reported here. The identity of the EUCCR cDNA was demonstrated by comparison with peptide sequence data from purified CCR and functional expression of the recombinant enzyme in Escherichia coli. Sequence analysis revealed remarkable homologies with dihydroflavonol-4-reductase (DFR), the first enzyme of the anthocyanin biosynthetic pathway. Moreover, significant similarities were found with mammalian 3 beta-hydroxysteroid dehydrogenase and bacterial UDP-galactose-4-epimerase, suggesting that CCR shared a common ancestor with these enzymes and can therefore be considered as a new member of the mammalian 3 beta-hydroxysteroid dehydrogenase/ plant dihydroflavonol reductase superfamily. In Eucalyptus gunnii, CCR is encoded by one gene containing four introns whose positions are similar to those of introns I, II, III and V in DFR genes from dicots. In agreement with the involvement of CCR in lignification, the CCR transcript was shown to be expressed in lignified organs, i.e. root and stem tissues, and was localized mainly in young differentiating xylem. On the other hand, its abundance in Eucalyptus leaves suggests that monolignols may be precursors of end products other than lignins. This first characterization of a gene corresponding to CCR opens new possibilities to genetically engineer plants with lower lignin content. This is particularly important for woody plants such as Eucalyptus which are used for pulp making.

Aldehyde Oxidoreductases↗
PubMed1994

Perception and response in plant disease resistance.

Plants express sophisticated mechanisms for recognizing pathogens. The functionally defined repertoire of non-self perception is large; the number and nature of subsequent molecular events required for resistance is unknown. Recent cloning of disease resistance genes, and genetic identification of loci required for their function, allows dissection of the structure, evolution, and deployment within populations of pathogen-perception mechanisms. Roles for reactive oxygen species and programmed cell death in resistance have also been suggested recently. New results document a role for salicylic acid as a lynchpin in the establishment and maintenance of the 'effector functions' of disease resistance, and strategies for engineered plant protection are moving closer to reality.

Apoptosis↗
PubMed1994

Early events in virus-plant interactions.

1. Plant viruses can only enter their host through a wounded plant cell. Once in the cytoplasm, the virion must be disassembled, and for certain viruses with a "+" RNA genome, cotranslational disassembly of virus particles has been described. 2. Subsequent to viral protein synthesis which requires the host translational machinery, the "+" RNA genome is replicated in the cytoplasm. Viral genome amplification requires at least one viral-coded non-structural protein in conjunction with one or more host factors. 3. Early events in virus infection can be studied in systems that hinder these events. This is the case of natural hosts that are resistant to viruses: mutant viruses which overcome such resistance have been described. It is also the case of genetically engineered plants that are protected from virus infection. Both types of systems should help in determining the mode of interaction involved, and possibly also the host factor(s) involved in the various steps of virus infection.

Capsid↗
PubMed2002

Over-expression of a seed specific hevein-like antimicrobial peptide from Pharbitis nil enhances resistance to a fungal pathogen in transgenic tobacco plants.

Two hevein-like peptides from the seed of Pharbitis nil, designated Pharbitis nil antimicrobial peptide 1 (Pn-AMP1) and Pn-AMP2, had been purified previously. Both exhibit potent in vitro antifungal activity against a broad spectrum of phytopathogenic fungi. We now report the isolation of two cDNA clones, designated pnAMP-h1 and pnAMP-h2, and the corresponding genomic clones encoding these proteins from mature seeds of P. nil. Comparison of the deduced amino acid sequence to that of the mature protein suggests that the peptides are produced as a prepropeptide consisting of an N-terminal signal peptide, the mature protein and C-terminal domains. The transcripts of the two genes are accumulated seed--specifically, and the maximum transcripts are observed in the mid-to-late stage of seed development. Constitutive over-expression of the pnAMP-h2 cDNA in transgenic tobacco under the control of the cauliflower mosaic virus 35S promoter conferred enhanced resistance against the oomycete Phytophthora parasitica, the causal agent of black shank disease. Thus the Pn-AMPs may play a role in the protection of seeds and may be useful as a novel gene source to engineer plants resistant to fungal pathogens.

Amino Acid Sequence↗
PubMedDate not supplied

[Relation of actual nutritional characteristics to the health status of retirees and persons of preretirement age employed in mechanical engineering production].

Actual nutrition and health state were investigated in 406 males of pre-pension and pension age (40-70 years) engaged in physical work (III category of difficulty) at an engineering plant. The chemical composition of their food was evaluated with respect to 74 chemical elements. The state of the workers' health was evaluated by the data obtained from the registers kept at the Plant medical unit. A relationship has been established between the character of the actual nutrition and the presence of an age-dependent disease, as well as the role of certain nutrients in the development of this or that disease in different age periods. A conclusion has been made on the necessity of the development of differential physiological requirements in the nutrients and energy for elder subjects engaged in the social industry, and inclusion of the rational nutrition into the complex of measures for prevention of age-dependent diseases.

Adult↗
PubMed2005

Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants.

Genetically engineered tobacco (Nicotiana tabacum) with the ability to synthesis glycinebetaine was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea). The genetic engineering enabled the plants to accumulate glycinebetaine mainly in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO2 assimilation of transgenic plants was significantly more tolerant to high temperatures than that of wild-type plants. The analyses of chlorophyll fluorescence and the activation of Rubisco indicated that the enhancement of photosynthesis to high temperatures was not related to the function of photosystem II but to the Rubisco activase-mediated activation of Rubisco. Western-blotting analyses showed that high temperature stress led to the association of Rubisco activase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than in transgenic plants. The results in this study suggest that under high temperature stress, glycinebetaine maintains the activation of Rubisco by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fractions and thus enhances the tolerance of CO2 assimilation to high temperature stress. The results seem to suggest that engineering of the biosynthesis of glycinebetaine by transformation with the BADH gene might be an effective method for enhancing high temperature tolerance of plants.

Betaine↗
PubMed2006

An in vivo internal deletion in the N-terminus region of Arabidopsis cystathionine gamma-synthase results in CGS expression that is insensitive to methionine.

Cystathionine gamma-synthase (CGS), the first enzyme of methionine biosynthesis in higher plants, plays an important role in the biosynthesis pathway and in regulating methionine metabolism in plant cells. In response to methionine, the expression of this enzyme is regulated via amino acid sequences located in its N-terminal. Here, using reverse transcription PCR and ribonuclease protection analysis, we demonstrate that, in addition to the full-length CGS transcript, a deleted form exists in Arabidopsis. The deleted transcript of CGS that lacks 90 or 87 nt located internally in the regulatory N-terminal region of CGS maintains the reading frame of the protein. Its association with polyribosomes indicates that this deleted form of CGS is translated. In order to study the function of this deleted form of CGS, we overexpressed it in transgenic tobacco plants. We found that the transgenic plants engineered to express only the deleted form of CGS accumulated methionine to a much higher level than those that expressed the full-length CGS. Furthermore, in vitro feeding experiments revealed that the deleted form of CGS is not subject to feedback regulation by methionine, as reported for the full-length transcript. Therefore, although most likely produced from the full-length CGS, the transcript of the deleted form is insensitive to methionine application and its expression may be important for maintaining methionine metabolism even in the presence of a high level of methionine.

Amino Acid Sequence↗
PubMed2003

Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation.

The initial step in the synthesis of isoleucine (Ile) is the conversion of threonine to alpha-ketobutyrate. This reaction is carried out by threonine deaminase (TD), which is feedback-regulated by Ile. Mutations in TD that manifest insensitivity to Ile feedback inhibition result in intracellular accumulation of Ile. Previous reports have shown that in planta expression of the wild-type Escherichia coli TD, ilvA, or an Ile-insensitive mutant designated ilvA-466, increased cellular concentrations of Ile. A structural analog of Ile, l-O-methylthreonine (OMT), is able to compete effectively with Ile during translation and induce cell death. It has been postulated that OMT could therefore be utilized as an effective selective agent in plant engineering studies. To test this concept, we designed two binary plasmids that harbored an nptII cassette and either the wild-type ilvA or mutant ilvA-466. The ilvA coding sequences were fused to a plastid transit peptide down stream of a modified 35S CaMV promoter. Tobacco transformations were set up implementing a selection protocol based on either kanamycin or OMT. The ilvA gene was effectively utilized as a selectable marker gene to identify tobacco transformants when coupled with OMT as the selection agent. However, the transformation efficiency was substantially lower than that observed with nptII using kanamycin as the selection agent. Moreover, in a subset of the ilvA transformants and in a majority of the ilvA-466 transgenic lines, a severe off-type was observed under greenhouse conditions that correlated with increased levels of expression of the ilvA transgene.

Apoptosis↗
PubMed1997

A single amino acid substitution in the antimicrobial defense protein cecropin B is associated with diminished degradation by leaf intercellular fluid.

Degradation is one of several factors that may affect the level of accumulation of transgene products in plants. In plants engineered to secrete antimicrobial proteins to the intercellular compartment of leaves, the degenerative activity of proteases residing in leaf intercellular fluid (IF) could be critical to achieving the expected transgene function. We synthesized a structural analogue (MB39) of the antibacterial protein cecropin B and compared the susceptibility of both proteins to degradation in vitro by IF extracted from leaves of various crops. The half-life of the two proteins in the various IF extracts ranged from 3 min to 25.5 h, with the analogue MB39 displaying the longer half-life in IF from nine of 10 species. Overall, the half-life of MB39 averaged 2.9 times greater than that of cecropin B. Analysis of the peptides produced by endopeptidase activity in potato iF indicated that the 5.7-fold lower degradation rate of MB39 was associated with the substitution of valine for methionine at residue 11 of cecropin B. These findings point to the possibility of tailoring antimicrobial protein genes to reduce the rate of protein degradation in a particular target crop.

Amino Acid Sequence↗
PubMed2000

Radiotracer and computer modeling evidence that phospho-base methylation is the main route of choline synthesis in tobacco.

Among flowering plants, the synthesis of choline (Cho) from ethanolamine (EA) can potentially occur via three parallel, interconnected pathways involving methylation of free bases, phospho-bases, or phosphatidyl-bases. We investigated which pathways operate in tobacco (Nicotiana tabacum L.) because previous work has shown that the endogenous Cho supply limits accumulation of glycine betaine in transgenic tobacco plants engineered to convert Cho to glycine betaine. The kinetics of metabolite labeling were monitored in leaf discs supplied with [(33)P]phospho-EA, [(33)P]phospho-monomethylethanolamine, or [(14)C]formate, and the data were subjected to computer modeling. Because partial hydrolysis of phospho-bases occurred in the apoplast, modeling of phospho-base metabolism required consideration of the re-entry of [(33)P]phosphate into the network. Modeling of [(14)C]formate metabolism required consideration of the labeling of the EA and methyl moieties of Cho. Results supported the following conclusions: (a) The first methylation step occurs solely at the phospho-base level; (b) the second and third methylations occur mainly (83%-92% and 65%-85%, respectively) at the phospho-base level, with the remainder occurring at the phosphatidyl-base level; and (c) free Cho originates predominantly from phosphatidylcholine rather than from phospho-Cho. This study illustrates how computer modeling of radiotracer data, in conjunction with information on chemical pool sizes, can provide a coherent, quantitative picture of fluxes within a complex metabolic network.

Choline↗
PubMedDate not supplied

Segmented regression with errors in predictors: semi-parametric and parametric methods.

We consider the estimation of parameters in a particular segmented generalized linear model with additive measurement error in predictors, with a focus on linear and logistic regression. In epidemiologic studies segmented regression models often occur as threshold models, where it is assumed that the exposure has no influence on the response up to a possibly unknown threshold. Furthermore, in occupational and environmental studies the exposure typically cannot be measured exactly. Ignoring this measurement error leads to asymptotically biased estimators of the threshold. It is shown that this asymptotic bias is different from that observed for estimating standard generalized linear model parameters in the presence of measurement error, being both larger and in different directions than expected. In most cases considered the threshold is asymptotically underestimated. Two standard general methods for correcting for this bias are considered; regression calibration and simulation extrapolation (simex). In ordinary logistic and linear regression these procedures behave similarly, but in the threshold segmented regression model they operate quite differently. The regression calibration estimator usually has more bias but less variance than the simex estimator. Regression calibration and simex are typically thought of as functional methods, also known as semi-parametric methods, because they make no assumptions about the distribution of the unobservable covariate X. The contrasting structural, parametric maximum likelihood estimate assumes a parametric distributional form for X. In ordinary linear regression there is typically little difference between structural and functional methods. One of the major, surprising findings of our study is that in threshold regression, the functional and structural methods differ substantially in their performance. In one of our simulations, approximately consistent functional estimates can be as much as 25 times more variable than the maximum likelihood estimate for a properly specified parametric model. Structural (parametric) modelling ought not be a neglected tool in measurement error models. An example involving dust concentration and bronchitis in a mechanical engineering plant in Munich is used to illustrate the results.

Bias↗