Deployment of gene specific marker in development of kunitz trypsin inhibitor free soybean genotypes.

Genetic elimination of kunitz trypsin inhibitor in soybean seed would obviate the need for boiling required to inactivate the antinutritional factor and therefore economize the soy processing. PI542044, the source of null variant of kunitz trypsin inhibitor gene is being used in the development of kunitz trypsin inhibitor free soybean genotypes in India. Gene specific marker can expedite the genetic elimination of this undesirable trait from popular soybean genotypes. In the present study, we tested the DNA amplification of soybean genotype PI542044 and kunitz trypsin inhibitor null lines derived from this genotype with a gene specific primer developed from the null variant of PI157740. The amplicons so obtained corresponded to the absence of kunitz trypsin inhibitor protein band on 10% polyacrylamide gel. The gene specific marker also amplified the null allele of template DNA of F1, BC1F1 and BC2F1 plants developed during marker assisted introgression of null allele of kunitz trypsin inhibitor into elite soybean cultivar JS97-52. The results presented show the utility of this gene specific marker developed from null allele of kunitz trypsin inhibitor for identification of kunitz trypsin inhibitor free genotypes developed from PI542044, the only source of null variant available in India.

Antigenicity of native and modified Kunitz soybean trypsin inhibitors.

Food provides a continuous antigenic stimulus to the immune system and the antigenicity of processed food proteins should be considered in toxicological evaluations. The antigenicity of the Kunitz trypsin inhibitor was studied using antibodies prepared by inoculating rabbits with native, heat-denatured, and N-acetylcysteine-treated Kunitz soybean trypsin inhibitors. Immunochemical studies using a competitive solid-phase enzyme immunoassay and two groups of sera revealed two patterns of antigenicity. Antibodies elicited with the denatured inhibitor were specific for the denatured conformation of the protein. In contrast, native inhibitor elicited antibodies that selectively recognized determinants in both native and heat-treated protein, but that did not bind trypsin inhibitors treated with N-acetylcysteine. These results imply that: the disulfide bonds must be intact to maintain the native antigenic conformation and the cysteine treatment may suppress allergic manifestations of soybean trypsin inhibitors and possibly other food proteins. These studies were extended by analyzing a panel of monoclonal antibodies prepared against native Kunitz trypsin inhibitor. The inhibitor has at least two distinct antigenic sites (epitopes), one of which is retained under denaturing conditions. The measurement of native Kunitz trypsin inhibitor in food samples by immunoassay appears practical. The relevance of these findings to food processing, food safety, and health is also discussed.

New properties of the soybean trypsin inhibitor: Inhibition of human neutrophil elastase and its effect on acute pulmonary injury.

Seeds from legumes including the Gilcine max are known to be a rich source of protease inhibitors. The soybean Kunitz trypsin inhibitors (SKTIs) have been well characterised and have been found to exhibit many biological activities. However their effects on inflammatory diseases have not been studied to date. In this study, SKTI was purified using anion exchange chromatography using a Resource Q column. The purified protein was able to inhibit human neutrophil elastase (HNE) and bovine trypsin. Purified SKTI inhibited HNE with an IC(50) value of 8mug or 0.3nM. At this concentration SKTI showed neither cytotoxic nor haemolytic effects on human blood cell populations. SKTI showed no deleterious effects on organs, blood cells or the hepatic enzymes ALT and AST in the mouse model of acute systemic toxicity. Human neutrophils incubated with SKTI released less HNE than control neutrophils when stimulated with PAF or fMLP (83.1% and 70% respectively). These results showed that SKTI affected both pathways of elastase release by PAF and fMLP stimuli, suggesting that SKTI is an antagonist of fMLP/PAF receptors. In an in vivo mouse model of LPS acute lung injury, SKTI significantly suppressed the inflammatory effects caused by elastase in a dose-dependent manner. Histological sections stained by hematoxylin/eosin confirmed this decrease in inflammation. These results showed that SKTI could be used as a pharmacological agent for the therapy of many inflammatory diseases.

Impact of transgenic oilseed rape expressing oryzacystatin-1 (OC-1) and of insecticidal proteins on longevity and digestive enzymes of the solitary bee Osmia bicornis.

The risk that insect-resistant transgenic plants may pose for solitary bees was assessed by determining longevity of adult Osmia bicornis (O. rufa) chronically exposed to transgenic oilseed rape expressing oryzacystatin-1 (OC-1) or to the purified insecticidal proteins recombinant rOC-1, Kunitz soybean trypsin inhibitor (SBTI), Galanthus nivalis agglutinin (GNA), or Bacillus thuringiensis toxin Cry1Ab dissolved in sugar solution (at 0.01 and 0.1%, w:v, Cry1Ab only at 0.01%). Compared to control bees, longevity was significantly reduced by SBTI and GNA at both concentrations and by rOC-1 at 0.1%, but not by Cry1Ab or rOC-1 at 0.01%. Longevity on the OC-1 oilseed rape was not significantly different from the control plants. The effects of SBTI and rOC-1 on longevity were investigated through characterization of the digestive proteinases of O. bicornis and analysis of the response in proteinase profiles to ingestion of these proteinase inhibitors. A relatively complex profile of at least four types of soluble proteolytic enzymes was identified. Serine proteinases were found to be predominant, with metallo and especially cysteine proteinases making a smaller albeit significant contribution. The compensatory response to in vivo enzyme inhibition was similar for SBTI and rOC-1 although less pronounced for rOC-1. It consisted of a non-specific overproduction of native proteinases, both sensitive and insensitive, and the induction of a novel aspartic proteinase.

Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.

Calcicludine (CaC) is a 60-amino acid polypeptide from the venom of Dendroaspis angusticeps. It is structurally homologous to the Kunitz-type protease inhibitor, to dendrotoxins, which block K+ channels, and to the protease inhibitor domain of the amyloid beta protein that accumulates in Alzheimer disease. Voltage-clamp experiments on a variety of excitable cells have shown that CaC specifically blocks most of the high-threshold Ca2+ channels (L-, N-, or P-type) in the 10-100 nM range. Particularly high densities of specific 125I-labeled CaC binding sites were found in the olfactory bulb, in the molecular layer of the dentate gyrus and the stratum oriens of CA3 field in the hippocampal formation, and in the granular layer of the cerebellum. 125I-labeled CaC binds with a high affinity (Kd = 15 pM) to a single class of noninteracting sites in rat olfactory bulb microsomes. The distribution of CaC binding sites in cerebella of three mutant mice (Weaver, Reeler, and Purkinje cell degeneration) clearly shows that the specific high-affinity labeling is associated with granule cells. Electrophysiological experiments on rat cerebellar granule neurons in primary culture have shown that CaC potently blocks the L-type component of the Ca2+ current (K0.5 = 0.2 nM). Then CaC, in the nanomolar range, appears to be a highly potent blocker of an L-subtype of neuronal Ca2+ channels.

Hypertrophy and hyperplasia of the rat pancreas produced by short-term dietary administration of soya-derived protein and soybean trypsin inhibitor.

Feeding soy protein concentrate to weanling rats over a one-week period produced a dose-related increase in pancreatic weight due to an increase in acinar cell size. Hyperplastic changes occur simultaneously, as evidenced by an increase in mitotic activity after two days on the test diet. Similar changes were also obtained by feeding soybean Kunitz trypsin inhibitor over the same time period. The results suggest that this approach may be useful as a model to investigate the effect of plant-derived material on the pancreas, in particular proliferative lesions.