An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's poreforming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.Using entomopathogens as biopesticides can reduce the use of synthetic pesticides. Entomopathogens have been isolated from soils and the carcasses of insects, although the natural resources of the pathogens are not limited to these. The larvae of Myrmeleontidae insects, referred to as ant lions, suck out the body fluids of their prey. Because ant lions can kill prey larger than themselves, it was postulated that they used toxins. From the regurgitated fluid of Myrmeleon bore larvae, we have purified an insecticidal protein with a molecular mass of 170 kDa (19) and found that it was produced in the larval region from the thorax to the abdomen (33). In addition to the toxin produced by ant lions, insecticidal proteins were found to be produced by bacterial pathogens isolated from M. bore larvae (21, 34). Thus, we have further isolated bacteria from the ant lion's crop, which serves as a reservoir of regurgitated fluid, to evaluate their toxicity to insects. It was found that some bacterial species obtained from the crop exhibit insecticidal actions against Spodoptera litura cutworms when injected (22). Of these, the A3-2 isolate was closely related to Bacillus sphaericus subgroup IIA on the basis of 16S rRNA gene sequencing and DNA-DNA hybridization tests. This Bacillus species is currently used as a biopesticide with mosquitocidal action involving protein toxins, notably, binary toxin, Mtx1, and Mtx2 (2, 6). However, it is not clear whether A3-2 uses such mosquitocidal toxins to kill insects other than mosquitoes.The aim of this study is to determine an insecticidal factor produced by A3-2 and elucidate its mode of action. Here we report that th...
Our data suggest that accumulated neutrophils at the border of the damaged area may contribute to macrophage accumulation at granulation tissue via the production of MCP-1 after liver injury. The plasminogen system is critical for liver repair by facilitating macrophage accumulation and triggering a cascade of subsequent repair events.
Neonicotinoids are a class of insecticides acting selectively on insect nicotinic acetylcholine receptors (nAChRs). These compounds display strong insecticidal activities against various insect pests (including those showing resistance to other classes of insecticides) and good levels of safety in vertebrates, which has led to neonicotinoids showing the fastest growing sales of insecticides worldwide. 1)The targets of neonicotinoids, nAChRs, belong to the cysloop superfamily of ligand-gated ion channels that mediate fast synaptic transmission in both insects and mammals.2) The safety of neonicotinoid insecticides has been shown to stem mainly from their excellent selectivity for insect nAChRs. 3,4)The insecticidal potency is influenced not only by the intrinsic actions on the targets, but also by accessibility to nAChRs and metabolism in insects.5) To date, the nicotinic potencies of many neonicotinoids have been characterized using binding assays to examine the correlation between binding potency and insecticidal potency as well as to clarify the structure-activity relationship. [5][6][7][8][9][10][11] However, relatively few electrophysiological studies have been conducted for such purposes. 3,[12][13][14][15] We have previously investigated the agonist actions of clothianidin and related neonicotinoids on a recombinant fruit fly Da2 (SAD)/chicken b2 hybrid nAChR expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology.16) Neonicotinoids with a cyclic-guanidine (or amidine) group show lower agonist efficacy than those with corresponding acyclic moieties, and the nitromethylene compounds are more potent in terms of the EC 50 in activating the nAChR than the corresponding nitroimine compounds. 15)However, it is not clear whether such structure-activity relationships exist in native insect nAChRs. Therefore, we have investigated the actions of neonicotinoids on nAChRs on the terminal abdominal ganglion (TAG) neurons of American cockroaches using whole-cell patch-clamp electrophysiology. Here we report that the structure-agonist activity relationship observed for the TAG nAChRs resembles that observed for J. Pestic. Sci., 31(1), 35-40 (2006) The actions of neonicotinoid insecticides on nicotinic acetylcholine receptors (nAChRs) in the terminal abdominal ganglion neurons of the American cockroach were investigated using whole-cell patch-clamp electrophysiology. Neonicotinoids possessing a nitromethylene group showed higher agonist affinity than the corresponding nitroimine analogues, whereas compounds with an acyclic guanidine moiety showed greater agonist efficacy than the corresponding cyclic compounds. Imidacloprid showed the lowest agonist efficacy of all neonicotinoids and low concentrations of imidacloprid attenuated acetylcholine-induced currents. However, such blocking actions were minimal with other neonicotinoids. The diverse actions of neonicotinoids on nAChRs, combined with target accessibility based on hydrophobicity, appears to account for their insecticidal potency ...
L‐amino acid oxidases (LAAOs) with broad substrate specificity can be used in the deracemization of D,L‐amino acids (D,L‐AAs) to their D‐enantiomers. Hyper‐thermostable LAAO (HTAncLAAO) was designed through a combination of manual sequence data mining and ancestral sequence reconstruction. Soluble expression of HTAncLAAO (>50 mg/L) can be achieved using an E. coli system. HTAncLAAO, which recognizes seven L‐AAs as substrates, exhibits extremely high thermal stability and long‐term stability; the t1/2 value was 95 °C and <5% activity loss after incubation of the enzyme at 30 °C for 1 week. Deracemization could be achieved at 40 °C with a small amount of enzymes compared to previously reported LAAOs. A total of 0.4 mg (2 U) of HTAncLAAO is enough to deracemize three D,L‐AAs at a preparative scale with high enantiomeric excess (>99 % ee, D‐enantiomer). These results suggest that HTAncLAAO is an excellent biocatalyst to perform this deracemization.
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