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Plants and insects have been coexisting for more than 350 million years. During this time, both have evolved many strategies to successfully exploit or respond to reciprocal adaptation and defense reactions. Plants tend to minimize the damage caused by pest feeding, while pests tend to manipulate plant response by suppressing plant defense mechanisms or developing strategies to overcome plant defense systems. Plants recognize insect pests by the wounding that they cause and elicitors present in pest oral secretions (saliva and/or regurgitant). These elicitors or insect-associated microorganisms can modulate plant response to the benefit of their insect hosts. In this article, we have undertaken an analysis of gene expression in serine and cysteine proteinase inhibitors (SerPI and CysPI, respectively) in wheat (Triticum aestivum) plants exposed to cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) larvae feeding, and the impact of microbes associated with CLB on the expression levels of these genes. Using three wheat varieties and antibiotic-treated and untreated CLB larvae, we found that SerPI plays a more important role than CysPIs in plant defense against CLB larvae. Additionally, higher levels of SerPI gene expression were observed in systemic leaves in comparison to the wounded leaves (local response). Each of the tested wheat varieties reacted in a specific way to the particular treatment. Moreover, the presence of microbial components associated with insects influenced plant response to CLB larvae feeding. [ABSTRACT FROM AUTHOR]

Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α-proteinase inhibitor (rα-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH)SO precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα-PI with 54 % recovery. The plant-purified rα-PI showed molecular mass and structural conformation comparable to native serum α-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα-PI protein. This work demonstrates a simple and efficient one-step purification of rα-PI from transgenic plants, which is an essential prerequisite for further therapeutic development. [ABSTRACT FROM AUTHOR]

Expression pattern of a sugar beet serine proteinase inhibitor gene, BvSTI, was characterized in response to mechanical and fall armyworm (FAW; Spodoptera frugiperda J.E. Smith) induced wounding. BvSTI expression was analyzed in three breeding lines moderately resistant to sugar beet root maggot (Tetanops myopaeformis Roder) and in a susceptible line, F1010. Increased mechanical wound induced levels of BvSTI expression were observed in all resistant lines as compared to F1010 during the first 24 h. The most intensive response to wounding was observed in one of the resistant lines, F1016, with a maximum 5- and 2.5-fold increase of BvSTI transcript levels over non-wounded roots and leaves, respectively. In contrast, slight increase of BvSTI transcript levels in leaves and even an initial decrease in roots were observed in F1010. BvSTI transcript accumulation in F1016 and F1010 tissues wounded by FAW showed a similar gene expression pattern, but it was delayed and less intense than the response incited by abiotic wounding. On the protein level, BvSTI specific antibody confirmed increased accumulation of the 30 kDa BvSTI protein in wounded leaves but not in roots of F1016 and F1010. Using trypsin inhibition assays, the activity of BvSTI was confirmed in F1016 roots and leaves and F1010 leaves. In F1010 roots BvSTI activity was completely lacking. We conclude that BvSTI gene expression was wound induced in the insect resistant germplasm suggesting that BvSTI can be used in biotechnological approaches or in breeding programs for improving insect resistance. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors play a significant role in plant defense against insect pests and phytopathogens by inhibiting their proteases. A thermotolerant monomeric trypsin inhibitor with molecular weight ∼18kD was purified from Cocculus hirsutus (ChTI) using trypsin sepharose affinity column. Western blot analysis using ChTI IgY revealed its presence in vegetative parts and seeds. The second and third instar larvae of H. armigera fed with ChTI (5000TIU/ml) resulted in 84.59 and 58.71% reduction in mean larval weight respectively. An increase in the larval growth period was observed in ChTI fed larvae at all instars and inhibitor fed larvae could not complete their life cycle. ChTI caused 74 and 59.53% inhibition of bovine trypsin and Helicoverpa gut proteases respectively. ChTI exhibited strain specificity and inhibited growth and development of plant fungal pathogens. Bioassay studies on yeast strains indicated that ΔYNK and MNN1 are more sensitive to ChTI. The results suggest that phosphodiester linkage in cell wall components is likely to be the key determinants for binding of ChTI. Taken together, these studies indicate that ChTI is a potential candidate for development of transgenic plants against foliar diseases and insect pests. [ABSTRACT FROM AUTHOR]

Two new serine proteinase inhibitors (RmIn I and RmIn II) from the tropical sea anemone Radianthus macrodactylus have been isolated and characterized. The purification procedure includes polychrome-1 hydrophobic chromatography, Superdex™ Peptide 10/30 FPLC, and Nucleosil C18 reverse-phase HPLC. The molecular masses of RmIn I, RmIn II, and the complexes RmIn II/trypsin and RmIn I,II/α-chymotrypsin have been determined. The K i values of RmIn I and RmIn II for trypsin and α-chymotrypsin have been determined. The polypeptides RmIn I and RmIn II are shown to be nontoxic and to exhibit antihistamine activity. The N-terminal amino acid sequences of RmIn I (GICSEPIVVGPCKAG-) and RmIn II (GSTCLEPKVVGPCKA-) have been determined. A high homology of the amino acid sequences is demonstrated for the proteinase inhibitors produced by such evolutionarily distant species as coelenterates, reptiles, and mammals. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Kallikrein-binding protein (KBP) is a serine proteinase inhibitor (serpin). It specifically binds to tissue kallikrein and inhibits kallikrein activity. Our study was designed to test its effects on retinal neovascularization and vascular permeability. Methods. Endothelial cell proliferation was determined by [3H] thymidine incorporation assay and apoptosis quantified by Annexin V staining and flow cytometry. Effect on retinal neovascularization was determined by fluorescein angiography and count of pre-retinal vascular cells in an oxygen-induced retinopathy (OIR) model. Vascular permeability was assayed by the Evans blue method. Vascular endothelial growth factor (VEGF) was measured by Western blot analysis and ELISA. Results. Kallikrein-binding protein specifically inhibited proliferation and induced apoptosis in retinal capillary endothelial cells. Intravitreal injection of KBP inhibited retinal neovascularization in an OIR model. Moreover, KBP decreased vascular leakage in the retina, iris and choroid in rats with OIR. Blockade of kinin receptors by specific antagonists showed significantly weaker inhibition of endothelial cells, when compared to that of KBP, suggesting that the anti-angiogenic activity of KBP is not through inhibiting kallikrein activity or kinin production. KBP competed with 125I-VEGF for binding to endothelial cells and down-regulated VEGF production in endothelial cells and in the retina of the OIR rat model. Conclusion/interpretation. Kallikrein-binding protein is a multi-functional serpin, and its vascular activities are independent of its interactions with the kallikrein-kinin system. Inhibition of VEGF binding to its receptors and down-regulation of VEGF expression could represent a mechanism for the vascular activities of KBP. [ABSTRACT FROM AUTHOR]

Trypsin inhibitor SFTI-1 isolated from sunflower seeds (comprising 14 amino acid residues and two cycles: head-to-tailcyclisation and disulfide bridge) is the smallest naturally occurring plant serine proteinase inhibitor. In our recent paperwe have shown that the elimination head-to-tail cyclisation did not change trypsin inhibitory activity as judged by measuredby association equilibrium constants K. The removal of disulfide bridge produced 2.4-fold lower activity. In the present paper we described chymotrypsin inhibitory activity. SFTI-1 inhibits significantly lower bovine α-chymortypsin(K = (5.20±1.56) × 10 M). The activity of the analogue with disulfide bridge only was practically the same, whereas the K value determined forhomodetic peptide was almost 3-fold lower. Considering the results obtained and the recent literature data we postulate thelower inhibitory activity against both enzyme of the analogue with head-to-tail cyclisation only reflect its lower proteolytic stability. [ABSTRACT FROM AUTHOR]

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against α1-proteinase inhibitor (human and sheep), inter-α-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, α2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus pulposus and annulus fibrosus of the ovine intervertebral disc. [ABSTRACT FROM AUTHOR]

The sequence specific assignment of theH NMR spectrum of barley serine proteinase inhibitor 2 is described. The sequential assignment procedure has been applied to the residues 22 to 83. The 21 residues in the N-terminal part of the structure were shown to be a random coil for which sequential assignment was not immediately possible. For the C-terminal part of the protein starting at Thr-22 and ending with the C-terminal residue Gly-83, sequence specific assignment of all the H, H, H, H resonances was achieved and complete sequence specific assignment of the non labile protons of the amino acid residue types of glycine, alanine, threonine, serine, valine, isoleucine, leucine, asparagine, aspartic acid, glutamine, glutamic acid, phenylalanine, tyrosine and tryptophan is reported. [ABSTRACT FROM AUTHOR]