大豆胰蛋白酶抑制剂对棉铃虫幼虫消化生理和生长发育的影响

本研究根据棉铃虫Helicotverpa ormigera(Hubner)幼虫中肠蛋白酶在离体条件下对蛋白酶抑制剂的反应,选择具有较强抑制作用的大豆胰蛋白酶抑制剂,以0.21-4.2%(干重)的浓度配入幼虫人工饲料,测定了幼虫短期和长期取食这些饲料引起的中肠类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力的变化和生长抑制效应。短期取食抑制剂的幼虫,中肠弱碱性类胰蛋白酶活力显著增高,在4.2%。浓度下比对照高出21%;强碱性类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力显著降低,生长发育受到明显抑制。长期取食低浓度(0.84%)抑制剂的幼虫,弱碱性类胰蛋白酶和类胰凝乳蛋白酶活力显著增高,强碱性类胰蛋白酶活力显著降低。总蛋白酶活力变化不显著;长期取食高浓度(4.2%)抑制剂的幼虫,强碱性类胰蛋白酶和总蛋白酶活力显著降低,其它酶活力变化不显著。抑制剂随浓度的增高对幼虫生长的抑制作用加强,但浓度高于0.84%后,抑制强度的变化减小。据此作者认为,蛋白酶抑制剂对昆虫抗营养效应在于它对蛋白酶的激活和抑制作用,从而导致各种蛋白酶间的协调性破坏,昆虫消化过程受阻,影响生长发育。     

Gene expression patterns of Helicoverpa armigera gut proteases

Relative quantification of reported gut proteinase cDNAs from Helicoverpa armigera larvae fed on various host plants (cotton, chickpea, pigeonpea, tomato and okra), non-host plant PIs (winged bean, bitter gourd, ground nut, and capsicum) and during larval development has been carried out using semi-quantitative RT-PCR. Five trypsin-like and three chymotrypsin-like proteinases were categorized as insensitive or sensitive to most of the proteinase inhibitors (PIs) and insensitive/sensitive to specific PIs based on their expression analysis. These results were supported by amino acid sequence analysis, where diverged amino acids were observed in the regions, which are reported to be involved in typical trypsin-trypsin inhibitor interactions and critical for proteinase inhibitor resistance. Among exopeptidases (five aminopeptidase and three carboxypeptidase), HaAmi4 and HaAmi5 of aminopeptidase and HaCar1 of carboxypeptidase exhibited considerable differential expression. Elastase and cathepsin B-like proteinases were expressed at negligible levels. The proteases identified in the study would be ideal candidates for further interactions studies with protease inhibitors to understand the structural reasons of protease inhibitor insensitivity.     

Expression patterns of anterior Hox genes in the polychaete Chaetopterus: correlation with morphological boundaries

Expression patterns for five Hox genes were examined by whole-mount in situ hybridization in larvae of Chaetopterus, a polychaete annelid with a tagmatized axial body plan. Phylogenetic analysis demonstrates that these genes are orthologs of the Drosophila genes labial, proboscipedia, zen, Deformed, and Sex combs reduced and are termed CH-Hox1, CH-Hox2, CH-Hox3, CH-Hox4, and CH-Hox5, respectively. Expression studies reveal a biphasic expression pattern. In early larval stages, well before any indications of segmental organization exist, a novel pattern of expression in bilateral posterior proliferating cell populations, corresponding to the teloblasts, was detected for each of the genes, with CH-Hox1 and CH-Hox2 expressed before the remaining three. In middle larval stages, all five genes are expressed in bilateral strips along the ventral midline, corresponding with the developing ventral nerve cord. In addition, CH-Hox1 and CH-Hox2 show strong expression at the foregut-midgut boundary. By late larval stages the expression is generally confined to the ventral CNS and ectoderm of the anterior parapodia. Anterior boundaries of expression are "colinear," at later larval stages, with CH-Hox2 expressed most rostrally, in the first segment, and anterior expression boundaries for CH-Hox1, CH-Hox3, CH-Hox4, and CH-Hox5 in segments 2, 3, 4, and 5, respectively. Like vertebrates and spiders, but unlike insects, CH-Hox3 participates in this colinear axial expression pattern. CH-Hox1 and CH-Hox2 have distinct posterior boundaries of expression in the ninth segment, which corresponds to a major morphological boundary, and may reflect a reorganization of Hox gene regulation related to the evolutionary reorganization of the Chaetopterus body plan.     

3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.     

Bioinsecticidal activity of Archidendron ellipticum trypsin inhibitor on growth and serine digestive enzymes during larval development of Spodoptera litura

The roles of serine proteases involved in the digestion mechanism of the cutworm Spodoptera litura (Lepidoptera: Noctuidae) were examined (in vitro and in vivo) following feeding of plant protease inhibitors. A trypsin inhibitor from Archidendron ellipticum (AeTI) was purified by ammonium sulfate fractionation, ion-exchange chromatography and size-exclusion chromatography (HPLC) and its bioinsecticidal properties against S. litura were compared with Soybean Kunitz trypsin inhibitor (SBTI). AeTI inhibited the trypsin-like activities of the midgut proteases of fifth instar larvae of S. litura by over 70%. Dixon plot analysis revealed competitive inhibition of larval midgut trypsin and chymotrypsin by AeTI, with an inhibition constant (K(i)) of 3.5x10(-9) M and 1.5x10(-9) M, respectively. However, inhibitor kinetics using double reciprocal plots for both trypsin and chymotrypsin inhibitions demonstrated a mixed inhibition pattern. Feeding experiments conducted on different (neonate to ultimate) instars suggested a dose-dependent decrease for both the larval body weight as well as % survival of larva fed on diet containing 50, 100 and 150 microM AeTI. Influence of AeTI on the larval gut physiology indicated a 7-fold decrease of trypsin-like protease activity and a 5-fold increase of chymotrypsin-like protease activity, after being fed with a diet supplemented with 150 microM AeTI. This study suggests that although the early (1st to 3rd) larval instars of S. litura are susceptible to the trypsin inhibitory action of AeTI, the later instars may facilitate the development of new serine proteases, insensitive to the inhibitor.     

Digestive proteolytic activity in larvae of tomato moth,Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo

Three distinct digestive protease activities, with strongly alkaline pH optima, were identified in the gut of tomato moth (Lacanobia oleracea) larvae, and characterised using specific synthetic substrates and inhibitors. These were; a trypsin-like activity, a chymotrypsin-like activity specific for substrates and inhibitors containing more than one amino acid residue, and an elastase-like activity, accounting for 40%, 30% and 20% of overall proteolysis respectively. The protease activities differed in their sensitivities to inhibition by different plant protein protease inhibitors (PIs), as estimated by I(50) values. Soya bean Kunitz trypsin inhibitor (SKTI) was the only plant PI tested to inhibit all three digestive protease activities at concentrations <40 &mgr;g/ml (approx. 5x10(-6)M). Incorporation of SKTI into a potato leaf-based artificial diet at 2% of total protein, decreased larval survival and growth (by approx. 33% and 40% respectively after 21 days) and retarded development (by approx. 2 days). However, when SKTI was expressed in transgenic potato plants at approx. 0.5% of total protein, only marginal effects on L. oleracea larvae were observed, which decreased with time. Whilst the presence of SKTI in artificial diet increased endogenous larval trypsin-like activity by up to four-fold, no effects on this activity were observed in larvae feeding on transgenic plants.     

杠柳新苷P和E对东方粘虫和小地老虎幼虫中肠蛋白酶活性的影响

从杀虫植物杠柳Periploca sepium Bunge根皮中分离得到的杠柳新苷P具有很高的杀虫活性, 为了探索其杀虫机理, 采用经典的昆虫蛋白酶活性测定方法, 比较研究了杠柳新苷P和无杀虫活性的杠柳新苷E对东方粘虫Mythimna separata与小地老虎Agrotis ypsilon 6龄幼虫中肠类胰蛋白酶和类胰凝乳蛋白酶活性的影响。结果表明： 对东方粘虫弱碱性类胰蛋白酶, 杠柳新苷P表现出强激活作用（酶活性为对照的3.43倍）, 激活时间可长达8 h, 而杠柳新苷E则无明显激活作用。杠柳新苷P和E对东方粘虫弱碱性类胰蛋白酶活性的影响二者差异显著（P=0.01）, 杠柳新苷P药后2, 4和8 h, 东方粘虫中肠弱碱性类胰蛋白酶的活性分别是杠柳新苷E药后的15.4, 106.8和242.7倍。酶活性测定结果还表明, 与东方粘虫相比, 小地老虎中肠类胰蛋白酶活性相对较低, 且杠柳新苷P的激活作用也较弱, 这可能是杠柳新苷P对东方粘虫具杀虫活性, 而小地老虎对其不敏感的原因之一; 另外, 杠柳新苷P和E对试虫中肠类凝乳胰蛋白酶活性均无明显影响。据此推测, 杠柳活性成分新苷P对东方粘虫中肠弱碱性类胰蛋白酶的激活作用可能是造成试虫中毒的机理之一。     

Overexpression, purification, and refolding of a Porphyromonas gingivalis cysteine protease from Escherichia coli

This paper describes the overexpression of the Rgp-1 (arginine) protease domain from Porphyromonas gingivalis. This protease and the related Kgp (lysine) protease, both of which display trypsin-like specificity, have been implicated as major virulence factors and may play a significant role in the etiology of periodontal disease. Both Rgp-1 and Kgp are initially translated as polyproteins, each containing a protease domain and multiple adhesin domains. The Rgp-1 protease domain was expressed in E. coli, purified, refolded, and assayed for activity. These expression studies demonstrated that prior to the formation of inclusion bodies in the E. coli cytoplasm, the protease was proteolytically active and could hydrolyze a specific synthetic substrate. When the Rgp-1 protease domain was purified from inclusion bodies and refolded, it was found to be autolytically active and displayed specific catalytic activity. This is the first report on the expression and purification of active Rgp-1 from E. coli. Polyclonal antisera raised against recombinant protein recognized the native form of the protease in the P. gingivalis strain W50, indicating that the recombinant protein contained some of the antigenic determinants of the native protease.     

Deletion of the SNP1 trypsin protease from Stagonospora nodorum reveals another major protease expressed during infection

The wheat fungal pathogen Stagonospora nodorum produces an extracellular trypsin-like protease, SNP1, during early stages of hyphal growth on the surface of host leaves and during penetration. Variation of SNP1 mRNA levels and enzyme activity during infection, were correlated with levels of aggressiveness of three wild-type isolates. SNP1 was deleted in two wild-type isolates using a gene replacement strategy. SNP1-deleted mutants completely lacked trypsin activity in vitro and on inoculated wheat leaves, but were not reduced in pathogenicity. SNP1-deleted mutants still have 50% of the total alkaline protease activity of wild-type. This residual activity comes from a previously undetected alkaline protease with subtilisin-like substrate and inhibitor specificities, which is produced in vitro and on host leaves. We hypothesize that this subtilisin protease may act in concert with SNP1 and may compensate for the loss of trypsin protease activity in the SNP1-deletion mutants.     

Cloning of aquaporin-1 of the blue crab, Callinectes sapidus: its expression during the larval development in hyposalinity

ABSTRACT: BACKGROUND: Ontogenetic variation in salinity adaptation has been noted for the blue crab, Callinectes sapidus, which uses the export strategy for larval development: females migrate from the estuaries to the coast to spawn, larvae develop in the ocean, and postlarvae (megalopae) colonize estuarine areas. We hypothesized that C. sapidus larvae may be stenohaline and have limited osmoregulatory capacity which compromises their ability to survive in lower salinity waters. We tested this hypothesis using hatchery-raised larvae that were traceable to specific life stages. In addition, we aimed to understand the possible involvement of AQP-1 in salinity adaptation during larval development and during exposure to hyposalinity. RESULTS: A full-length cDNA sequence of aquaporin (GenBank JQ970426) was isolated from the hypodermis of the blue crab, C. sapidus, using PCR with degenerate primers and 5[PRIME] and 3[PRIME] RACE. The open reading frame of CasAQP-1 consists of 238 amino acids containing six helical structures and two NPA motifs for the water pore. The expression pattern of CasAQP-I was ubiquitous in cDNAs from all tissues examined, although higher in the hepatopancreas, thoracic ganglia, abdominal muscle, and hypodermis and lower in the antennal gland, heart, hemocytes, ovary, eyestalk, brain, hindgut, Y-organs, and gill. Callinectes larvae differed in their capacity to molt in hyposalinity, as those at earlier stages from Zoea (Z) 1 to Z4 had lower molting rates than those from Z5 onwards, as compared to controls kept in 30 ppt water. No difference was found in the survival of larvae held at 15 and 30 ppt. CasAQP-1 expression differed with ontogeny during larval development, with significantly higher expression at Z1-2, compared to other larval stages. The exposure to 15 ppt affected larval-stage dependent CasAQP-1 expression which was significantly higher in Z2- 6 stages than the other larval stages. CONCLUSIONS: We report the ontogenetic variation in CasAQP-1 expression during the larval development of C. sapidus and the induction of its expression at early larval stages in the exposure of hyposalinity. However, it remains to be determined if the increase in CasAQP-1 expression at later larval stages may have a role in adaptation to hyposalinity.     

The Tolkin Gene Is a Tolloid/Bmp-1 Homologue That Is Essential for Drosophila Development

The Drosophila decapentaplegic (dpp) gene, a member of the tranforming growth factor β superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interaction between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp.     

Cloning of a novel protease required for the molting of Locusta migratoria manilensis

Molting is required for progression between larval stages in the life cycle of an insect. The essence of insect molting is the laying down of new cuticle followed by shedding of the old cuticle. Degradation and recycling of old cuticle are brought about by enzymes present in the molting fluid, which fills the space between the old and new cuticle. Here, we describe the cloning of a novel protease gene from Locusta migratoria manilensis, designated as Lm-TSP. The cDNA and its deduced protein sequences were deposited in GenBank (accession numbers EF081255 and ABN13876, respectively). Sequence analysis indicated that Lm-TSP belongs to the trypsin-like serine protease family. We show, by RNA interference (RNAi), that silencing of Lm-TSP leads to dramatic reductions in protease and cuticle-degrading activity of a molting fluid, which leads to molting defects from fourth-instar larvae (L4) to fifth-instar larvae (L5), and between L5 and adult stages. These observations suggest that Lm-TSP plays a critical role in L. migratoria manilensis ecdysis.     

Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes

The secreted Wnt signaling inhibitor Dickkopf1(Dkk1)plays key role in vertebrate head induction.Its receptor Kremen synergizes with Dkkl in Wnt inhibition.Here we have carried out expression and functional studies of the Dkk and Kremen genes in amphioxus(Branchiostoma belcheri).During embryonic and larval development,BbDkk1/2/4 is expressed in the posterior mesoendoderm,anterior somatic mesoderm and the pharyngeal regions.Its expression becomes restricted to the pharyngeal region on the left side at larval stages.In 45 h larvae,BbDkk1/2/4 is expressed specifically in the cerebral vesicle.BbDkk3 was only detected at larval stages in the mid-intestine region.Seven Kremen related genes were identified in the genome of the Florida amphioxus(Branchiostoma floridae),clustered in 4scaffolds,and are designated Kremen1-4 and Kremen-like 1-3,respectively.In B.belcheri,Kremenl is strongly expressed in the mesoendoderm during early development and Kremen3 is expressed asymmetrically in spots in the larval pharyngeal region.In luciferase reporter assays,BbDkk1/2/4 can strongly inhibit Writ signaling,while BbDkk3,BbKremen1 and BbKremen3 can not.No co-operative effect was observed between amphioxus Dkk1/2/4 and Kremens,suggesting that the interaction between Dkk and Kremen likely originated later during evolution.