A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. A homology search of the deduced amino acid sequence of the protein cDNA revealed 56% homology with Drosophila melanogaster lipase and 21% homology with human lipase. As lipase activity of the protein was confirmed in vitro, this protein was designated Bmlipase-1. Northern blot analysis showed that the Bmlipase-1 gene is expressed in the midgut but not in other tissues, nor is it activated by BmNPV infection. In addition, the Bmlipase-1 gene was shown not to be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that an insect digestive enzyme has potential as a physiological barrier against BmNPV at the initial site of viral infection.Insects exhibit effective immune system measures such as humoral and cellular responses against microbial infection (4). Insect immunity in antibacterial reactions has been the most extensively studied (7,8,13). On the other hand, little is known about insect immunity against viruses (16). Bombyx mori nucleopolyhedrovirus (BmNPV) is a most significant virus in the sericultural industry, often causing severe economic damages. The immune mechanisms of B. mori against this virus remain totally obscure.The infection cycle of BmNPV is mediated by two phenotypically different viral particles: the occlusion-derived virus (ODV) and the budded virus (BV) (9). Occlusion bodies consisting of a crystalline matrix of polyhedron proteins contain ODV particles. When the occlusion bodies are ingested by B. mori larvae, they are dissolved by the alkaline gut juice. The enveloped virions are released and then initiate infection in the midgut columnar epithelial cells. In the case of Autographa californica multicapsid nucleopolyhedrovirus, it was shown that a proportion of the parental virus travels through the midgut epithelial layer, possibly utilizing the plasma membrane reticular system, and enters the hemocoel at the same time, infecting the hemocytes (3). In contrast to ODV, the BV particle consists of a single nucleocapsid surrounded by an envelope acquired as it buds from the plasma membrane of an infected cell and spreads beyond the midgut through the tracheae.Studies on antiviral immunity in insects are still in their infancy, and defense mechanisms at an early stage of viral infection in the alimentary canal remain unknown. The aim of the present study was to determine whether B. mori contains proteins showing anti-BmNPV activity in the gut in order to obtain a clue as to the antiviral mechanisms involved in insect immunity.A protein showing anti-BmNPV was purified through ammonium sulfate fractionation, gel filtration, and reverse-phase high-performance liquid-column chromatography (HPLC) (Fig. 1A). The protein was eluted with 49.5% acetonitrile-0.05% trifluoroacetic acid (TFA) by reverse-phase HPLC. The homogeneity of the antiviral peak fraction was examined both by matrix-as...

A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. The molecular mass of this protein was 24271 Da. Partial N-terminal amino acid sequence of the protein was determined and cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 94% identity with B. mori serine protease so the protein was designated B. mori serine protease-2 (BmSP-2). Analysis of BmSP-2 gene expression showed that this gene is expressed in the midgut but not in other tissues. In addition, BmSP-2 gene was shown to not be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that BmSP-2, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection.

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The photoreaction of a silyl iron complex Cp(CO)2Fe(SiMe3) (1) in acetonitrile in the presence of P(NMeCH2)2(OMe) (L) yielded Cp(CO)LFeMe (2), CpL2FeMe (3), and CpL2Fe(CN) (4), showing that carbon−carbon bond cleavage of acetonitrile was achieved. These C−C bond cleavage products were also obtained in the photoreaction of 1 with 1 equiv of MeCN in THF in the presence of L. The reaction with CD3CN showed that the methyl group on the iron in the products is derived from acetonitrile. The corresponding reaction of Cp(CO)2Fe(ER3) (ER3 = CH3, GeMe3, SnMe3) generated a CO/L exchange complex, Cp(CO)LFe(ER3), showing that a silyl ligand on the iron is indispensable for the C−C bond cleavage of acetonitrile. Theoretical studies on the C−C bond cleavage were performed using the hybrid DFT-B3LYP method. The direct C−C bond oxidative addition of acetonitrile to the 16e species Cp(CO)Fe(SiMe3) expected to readily form from 1 in the photoreaction conditions has a very high activation barrier of 52.7 kcal/mol, suggesting that the oxidative addition is not an appropriate reaction pathway. A more feasible pathway was proposed. The end-on coordination of acetonitrile nitrogen to Cp(CO)Fe(SiMe3), followed by the rearrangement to a CN side-on complex, with the activation energy of 14.8 kcal/mol occurs, and then the insertion of the CN bond into the Fe−Si bond with a small activation energy of 4.0 kcal/mol and the successive C−C bond cleavage of acetonitrile on the Fe coordination sphere with the activation energy of 15.0 kcal/mol take place to give Cp(CO)MeFe(CNSiMe3). The isolation of an iron complex with a methyl group derived from acetonitrile and a silylisocyanide ligand was attained in the photoreaction of Cp(CO)2Fe(SiPh3) in MeCN in the presence of PPh3. The product Cp (PPh3)MeFe(CNSiPh3) was confirmed by the X-ray structure analysis. The reaction mechanism leading to the iron cyanide complex has also been discussed.

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The first catalytic double hydrophosphination of alkynes was achieved by reaction with diarylphosphines in the presence of an iron catalyst. The double hydrophosphination proceeded regioselectively and effectively for various secondary arylphosphines and terminal alkynes to give 1,2-bisphosphinoethane derivatives.

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