Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R₁ progeny. SaPIN2a mRNA was detected in both the R₀ and R₁ transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.Abbreviations CaMV cauliflower mosaic virus - cDNA complementary DNA - NOS nopaline synthase - PAGE polyacrylamide gel electrophoresis - PI proteinase inhibitor - SaPIN2a Solanum americanum proteinase inhibitor IIa - SDS sodium dodecyl sulphate - T-DNA transferred DNA     

Daffodil flowers delay senescence in cut Iris flowers

Visible symptoms of tepal senescence in cut Iris x hollandica (cv. Blue Magic) flowers were delayed by placing one cut daffodil flower (Narcissus pseudonarcissus, cv. Carlton) in the same vase. Addition of mucilage, exuded by daffodil stems, to the vase water had the same effect as the flowering daffodil stem. The active compound in the mucilage was identified as narciclasine (using LC/MS, GC/MS, 1H and 13C-NMR, and comparison with an authentic sample of narciclasine). The delay of senescence, either by mucilage or purified narciclasine, was correlated with a delayed increase in protease activity, and with a considerable reduction of maximum protease activity. Narciclasine did not affect in vitro protease activity, but is known to inhibit protein synthesis at the ribosomal level. Its effects on senescence and protease activity were similar to those of cycloheximide (CHX), another inhibitor of protein synthesis, but the effective narciclasine concentration was about 100 times lower than that of CHX. It is concluded that the delay of Iris tepal senescence by daffodil stems is due to narciclasine in daffodil mucilage, which apparently inhibits the synthesis of proteins involved in senescence.     

Growth-inhibition effects of pacifastin-like peptides on a pest insect: the desert locust, Schistocerca gregaria

The main reason for the varying degrees of success of peptidase inhibitors (PI) as biological insecticides is the existence of a poorly understood mechanism, which allows pest insects to compensate for PI present in their diet. To challenge this highly flexible physiological mechanism and to prolong the inhibitory effect of PI on insect growth, a number of measures were taken into account before and during experiments with a notorious pest insect, the desert locust, Schistocerca gregaria: (i) non-plant PI (pacifastin-related inhibitors) were used to reduce the risk of a specific co-evolutionary adaptation of the pest insect, (ii) based on the main types of digestive enzymes present in the midgut, mixtures of multiple PI with different enzyme specificity were selected, allowing for a maximal inhibition of the proteolytic activity and (iii) digestive peptidase samples were taken during oral administration experiments to study compensatory mechanisms. Contrary to larvae fed on a diet containing plant-derived PI, a significant growth impediment was observed in larvae that were fed a mixture of different pacifastin-like PI. Nevertheless, the growth inhibition effect of this PI mixture attenuated after a few days, Moreover, a comprehensive study of the observed responses after oral administration of PI revealed that S. gregaria larvae can adjust their secreted digestive enzyme activities in two distinct ways depending on the composition/concentration of the PI-mixture.     

地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。     

Isolation,cDNA Cloning,and Structure-based Functional Characterization of Oryctin,a Hemolymph Protein from the Coconut Rhinoceros Beetle,Oryctes rhinoceros,as a Novel Serine Protease Inhibitor

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant ¹³C,¹⁵N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K_i values of 3.9 × 10⁻¹⁰ m, 6.2 × 10⁻¹⁰ m, 1.4 × 10⁻⁹ m, and 1.2 × 10⁻⁸ m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.     

外来入侵植物的氮代谢及其土壤氮特征

研究了4种外来入侵植物(五爪金龙、南美蟛蜞菊、金腰箭和马缨丹)和1种本地植物鸡矢藤(对照)的氮代谢及其土壤氮特征.结果表明:外来人侵植物的组织硝酸还原酶活性、根际土壤NH4-N、NO3-N含量、蛋白酶活性和脲酶活性均较高,分别为鸡矢藤的1.65～4.34、1.56～2.15、1.72～3.11、1.43～3.23和1.41～3.33倍,而植物组织硝态氮含量则较低,仅为鸡矢藤的17.5%～50.6%.相关分析表明:植物组织硝酸还原酶活性与根际土壤总氮、NH4-N、NO3-N含量呈显著正相关(P<0.05),与蛋白酶活性和脲酶活性呈极显著正相关(P<0.01).这说明,外来植物入侵使土壤氮代谢加快,氮的生物有效性增强,氮同化能力提高,并且较好地将植物体氮素代谢与土壤氮素代谢协调起来.因此,较强的氮素同化能力与加速土壤氮素的转化可能是植物成功入侵的重要机制之一.     

Detrimental role for human high temperature requirement serine protease A1 (HTRA1) in the pathogenesis of intervertebral disc (IVD) degeneration

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.