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At least 955 records · Page 53Linked to original sources
PubMed2005

Influence of the parasitoid Chelonus inanitus and its polydnavirus on host nutritional physiology and implications for parasitoid development.

Chelonus inanitus is a solitary egg-larval endoparasitoid, which feeds on host haemolymph during its internal phase. Parasitization induces in the host Spodoptera littoralis a precocious onset of metamorphosis and a developmental arrest in the prepupal stage. At this stage the parasitoid larva emerges from the host and consumes it. We show here that parasitization and the co-injected polydnaviruses affect the nutritional physiology of the host mainly in the last larval instar. Polydnaviruses cause a reduced uptake of food and an increase in the concentration of free sugars in the haemolymph and of glycogen in whole body. The parasitoid larva, along with polydnaviruses, causes a reduction of proteins in the host's plasma and an accumulation of lipids in whole body. Dilution of host haemolymph led to a reduced concentration of lipid in parasitoid larvae and a reduced survival rate. Thus, a sufficient concentration of nutrients in the host's haemolymph appears to be crucial for successful parasitoid development. Altogether, the data show that the parasitoid and the polydnavirus differentially influence host nutritional physiology and that the accumulated lipids and glycogen are taken up by the parasitoid in its haematophagous stage as well as through the subsequent external host feeding.

Animal Nutritional Physiological Phenomena↗
PubMed2005

Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells.

BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.

Amino Acid Sequence↗
PubMed2005

Degradation of the S. frugiperda peritrophic matrix by an inducible maize cysteine protease.

A unique 33-kDa cysteine protease (Mir1-CP) rapidly accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by Spodoptera frugiperda and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mir1-CP on the PM in vitro, dissected PMs were treated with purified, recombinant Mir1-CP and the movement of Blue Dextran 2000 across the PM was measured. Mir1-CP completely permeabilized the PM and the time required to reach full permeability was inversely proportional to the concentration of Mir1-CP. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. The lumen side of the PM was more vulnerable to Mir1-CP attack than the epithelial side. Mir1-CP damaged the PM at pH values as high as 8.5 and more actively permeabilized the PM than equivalent concentrations of the cysteine proteases papain, bromelain and ficin. The effect of Mir1-CP on the PMs of Helicoverpa zea, Danaus plexippus, Ostrinia nubilalis, Periplaneta americana and Tenebrio molitor also was tested, but the greatest effect was on the S. frugiperda PM. These results demonstrate that the insect-inducible Mir1-CP directly damages the PM in vitro and is critical to insect defense in maize.

Animals↗
PubMed2006

Effect of Campoletis sonorensis ichnovirus cys-motif proteins on Heliothis virescens larval development.

Polydnaviruses are obligate symbionts of some parasitic hymenopteran wasps responsible for modifying the physiology of their host lepidopteran larvae to benefit the endoparasite. Injection of Campoletis sonorensis ichnovirus (CsIV) into Heliothis virescens larvae alters larval growth, development and immunity but genes responsible for alterations of host physiology are not well described. Recent studies of polydnavirus genomes establish that these genomes encode families of related genes expressed in parasitized larvae. Here we evaluate five members of the CsIV cys-motif gene family for their ability to inhibit growth and development of lepidopteran larvae. To study the function of cys-motif proteins, recombinant proteins were produced from baculovirus expression vectors and injected or fed to H. virescens larvae in diet. rVHv1.1 was identified as the most potent protein tested causing a significant reduction in growth of H. virescens and Spodoptera exigua larvae. H. virescens larvae ingesting this protein also exhibited delayed development, reductions in pupation and increased mortality. Increased mortality was associated with chronic sub-lethal baculovirus infections. Taken together, these data indicate that the cys-motif proteins have pleiotropic effects on lepidopteran physiology affecting both development and immunity.

Amino Acid Motifs↗
PubMed2003

Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells.

Successive viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did not show the accumulation of such concatemers. When introduced into SeUCR1 and SeIZD2109 cells, the non-hr ori concatemers generated in Se301 cells were maintained but did not increase. This suggests that the non-hr ori confers a strong selective advantage in Se301 cells, but not or to a lesser extent in the other cell lines. The cell line-specific accumulation of non-hr ori concatemers might be due to a higher intrinsic recombination frequency in Se301 cells and may reflect tissue related differences involving some host cell factor(s). Since non-hr ori concatemers in Se301 cells were more abundant in intracellular than in extracellular viral DNA preparations, episomal replication and the requirement of a minimal DNA size for packaging into nucleocapsids is hypothesized.

Animals↗
PubMed2005

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression.

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.

Analysis of Variance↗
PubMed2005

Pathogenesis of Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) in susceptible noctuid larvae.

Helicosporidium sp. is a unique, achlorophyllous green alga that has been reported to infect various insect orders, including Lepidoptera, Diptera, and Coleoptera. The infectious cyst stage is ingested by the host, ruptures in the midgut lumen, and releases a filamentous cell. Histopathological examinations using larvae of a susceptible noctuid host, Spodoptera exigua, showed both cysts and filamentous cells affiliated with the microvillar lining of the midgut epithelium. A considerable proportion of the ingested cysts (22-39%) were recovered in feces collected 24 h after ingestion. A small number of filamentous cells passed the midgut epithelium and entered the hemocoel within 4-24 h after cyst ingestion. After 48 h, vegetative cell stages were detected in the hemolymph, followed by a 4- to 5-day period of increasing multiplication. Cyst differentiation in the colonized hemolymph began 6-7 days after the treatment.

Animals↗
PubMed2006

Primary recovery and chromatographic purification of adeno-associated virus type 2 produced by baculovirus/insect cell system.

Adeno-associated virus (AAV) is making its place in gene therapy applications; however, the industry is still facing obstacles in producing a large quantity of highly purified material for clinical studies. Insect cell technology can be used to produce AAV to meet the current demand. During the purification process it was observed that there was a reduced recovery of AAV produced in insect cells, Spodoptera frugiperda (Sf9). It was assumed that the formation of AAV agglomerates and the interaction of AAV with other cellular components were major contributors to this loss. After studying different systems of extraction a sequence of treatment for primary recovery of AAV from cell paste was developed. This sequence was necessary to reduce the AAV losses and to increase the recovery. The purification method avoided the use of ultracentrifugation and adopted chromatographic methods for the purification of AAV. Primary recovery, ion exchange chromatography and hydrophobic interaction chromatography gave an overall yield of 75% from the extracted AAV. The purification process was based on chromatographic methods; therefore, it can be scaled up. Although this method was developed for AAV type 2, it is believed that this method could be modified easily to purify other AAV serotypes.

Animals↗
PubMed2007

Production of sumoylated proteins using a baculovirus expression system.

Spodoptera frugiperda Sf9 cells were found to possess an active endogenous sumoylation system. However, the endogenous sumoylation machinery did not efficiently modify exogenous proteins expressed by infection with recombinant baculoviruses. To overcome this limitation, mammalian sumoylation components were introduced by co-infection with recombinant baculoviruses expressing individual protein components of the sumoylation pathway. Expression of mammalian Ubc9 plus SUMO (either SUMO1 or SUMO3) was necessary and sufficient for active sumoylation of co-infected test proteins. This system provides a simple and convenient means to produce sumoylated mammalian proteins in a eukaryotic environment. Large-scale cultures should provide quantities of sumoylated proteins sufficient for potential purification.

Animals↗
PubMed2005

Expression of SLAM (CDw150) on Sf9 cell surface using recombinant baculovirus mediates measles virus infection in the nonpermissive cells.

Signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been reported as the receptor of measles virus (MV) interacting with MV hemagglutinin (MVH). In this study, we developed a baculovirus-derived vector, the Bacmid-egfp, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the control of the promoter of very late polyhedrin gene from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and employed the recombinant baculovirus to express SLAM in Sf9 (Spodoptera frugiperda) cells and investigate SLAM function. The result showed that the integration of the EGFP expression cassette in the Bac-to-Bac system facilitated research with the system without introducing compromises due to its use. SLAM protein fused to His-tag was expressed in Sf9 cells through the modified Bac-to-Bac system. The expressed SLAM was identified as approximately 46 kDa, and it presented on the cell surface, as revealed by fluorescent immunochemical staining and confocal microscopic analysis. The pull-down assay proved that SLAM protein expressed in this system could interact with MVH protein. After incubating with MV vaccine strain S191, cell fusion was only observed in the Sf9 cells expressing both EGFP and SLAM from recombinant baculovirus rather than those expressing EGFP only from the modified viral vector. Furthermore, MV replicated and induced apoptosis in the Sf9 cells with SLAM expression.

Animals↗
PubMed2005

Expression and purification of active plant diphosphonucleotide phosphatase/phosphodiesterase from baculovirus-infected insect cells.

We have previously purified and characterized a diphosphonucleotide phosphatase/phosphodiesterase (PPD1) from yellow lupin seeds. This report describes an efficient strategy for overexpression in baculovirus-infected Spodoptera frugiperda Sf9 cells and purification of a functional PPD1 enzyme. We tested six variants of recombinant PPD1, differing in secretion peptides, fusion tags, and promoters. The highest expression level of the active PPD1 was achieved when the native signal peptide and the C-terminal V5-6His tag were attached. This recombinant protein was secreted at very high level (18.4 mg/L) to serum-free medium. Single-step purification procedure using metal affinity chromatography resulted in the homogeneous PPD1. The overexpressed protein showed enzymatic activity comparable to the native enzyme isolated previously from plant material. We showed that PPD1, which belongs to purple acid phosphatase family, formed tetrameric structure, which is non-typical for this group of enzymes.

Animals↗
PubMed2005

Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells.

A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.

Animals↗
PubMed2005

High yield of biologically active recombinant human amelogenin using the baculovirus expression system.

The amelogenins are secreted by the ameloblast cells of developing teeth; they constitute about 90% of the enamel matrix proteins and play an important role in enamel biomineralization. Recent evidence suggests that amelogenin may also be involved in the regeneration of the periodontal tissues and that different isoforms may have cell-signalling effects. During enamel development and mineralization, the amelogenins are lost from the tissue due to sequential degradation by specific proteases, making isolation of substantial purified quantities of full-length amelogenin challenging. The aim of the present study was to express and characterize a recombinant human amelogenin protein in the eukaryotic baculovirus system in quantities sufficient for structural and functional studies. Human cDNA coding for a 175 amino acid amelogenin protein was subcloned into the pFastBac HTb vector (Invitrogen), this system adds a hexa-histidine tag and an rTEV protease cleavage site to the amino terminus of the expressed protein, enabling effective one-step purification by Ni2+-NTA affinity chromatography. The recombinant protein was expressed in Spodoptera frugiperda (Sf9) insect cells and the yield of purified his-tagged human amelogenin (rHAM+) was up to 10 mg/L culture. Recombinant human amelogenin (rHAM+) was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, peptide mapping, and MS/MS sequencing. Production of significant amounts of pure, full-length amelogenin opened up the possibility to investigate novel functions of amelogenin. Our recent in vivo regeneration studies reveal that the rHAM+ alone could bring about regeneration of the periodontal tissues; cementum, periodontal ligament, and bone.

Amelogenin↗
PubMed2005

Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells.

Vascular endothelial growth factor (VEGF) is the best characterized multifunctional protein which plays a key role in normal and pathologic angiogenesis. The gene encoding the human VEGF165 was cloned from the ovarian carcinoma cell line (OVCAR3) and expressed in insect cells using the baculovirus expression vector system. The recombinant human VEGF165 (rhVEGF165) protein produced by Sf21 (Spodoptera frugiperda) cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. The rhVEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogeneity by heparin affinity, Resource S and Resource RPC columns. The biological activity of the purified 42-kDa homodimer was shown by the induction of the proliferation of human umbilical vein derived endothelial cells. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 20mg/L) in Sf21 cells.

Animals↗
PubMed2005

Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells.

Sf9, a cell line derived from the lepidopteran insect, Spodoptera frugiperda, is widely used as a host for recombinant glycoprotein expression and purification by baculovirus vectors. Previous studies have shown that this cell line has one or more beta-N-acetylglucosaminidase activities that may be involved in the degradation and/or processing of N-glycoprotein glycans. However, these enzymes and their functions remain poorly characterized. Therefore, the goal of this study was to isolate beta-N-acetylglucosaminidase genes from Sf9 cells, over-express the gene products, and characterize their enzymatic activities. A degenerate PCR approach yielded three Sf9 cDNAs, which appeared to encode two distinct beta-N-acetylglucosaminidases, according to bioinformatic analyses. Baculovirus-mediated expression of these two cDNA products induced membrane-associated beta-N-acetylglucosaminidase activities in Sf9 cells, which cleaved terminal N-acetylglucosamine residues from the alpha-3 and -6 branches of a biantennary N-glycan substrate with acidic pH optima and completely hydrolyzed chitotriose to its constituent N-acetylglucosamine monomers. GFP-tagged forms of both enzymes exhibited punctate cytoplasmic fluorescence, which did not overlap with either lysosomal or Golgi-specific dyes. Together, these results indicated that the two new Sf9 genes identified in this study encode broad-spectrum beta-N-acetylglucosaminidases that appear to have unusual intracellular distributions. Their relative lack of substrate specificity and acidic pH optima are consistent with a functional role for these enzymes in glycoprotein glycan and chitin degradation, but not with a role in N-glycoprotein glycan processing.

Acetylglucosaminidase↗
PubMed2005

Neuropeptides associated with the frontal ganglion of larval Lepidoptera.

The occurrence of neuropeptides in the frontal ganglia of larvae of the tobacco hawkmoth, Manduca sexta, the tomato moth, Lacanobia oleracea and the cotton leafworm, Spodoptera littoralis was investigated using reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and enzyme-linked immunosorbent assay (ELISA). Only three types of peptides could be identified or assigned from frontal ganglion extracts; M. sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT), and F/YXFGL-NH2 allatostatins. The peptide profiles of frontal ganglion of L. oleracea and S. littoralis were similar, with ten identical [M+H]+ ions, seven of which could be assigned to known lepidopteran peptides (Manse-AT, cydiastatin 2, 3, 4 and helicostatin 1, 5, 9). In addition, mass ions corresponding to helicostatin 7 (which was confirmed by MALDI-post source decay analysis) and Manse-AS were present in frontal ganglia of L. oleracea and helicostatin 6 in frontal ganglia of S. littoralis. Only four mass ions from M. sexta frontal ganglia corresponded to known peptides, cydiastatin 3 and 4, helicostatin 1, and Manse-AT. The only difference between the profiles of frontal ganglia from different stages of L. oleracea were mass ions which could not be assigned, and no differences were observed in the allatoregulatory peptides present. In HPLC fractions of M. sexta frontal ganglia, F/YXFGL-NH2 allatostatin-like immunoreactivity was widespread suggesting that more allatostatins were present than were identified.

Animals↗
PubMed2004

Neo-clerodane diterpenes from Teucrium fruticans.

In addition to three out of the four previously reported neo-clerodanes already found in Teucrium fruticans (fruticolone, isofruticolone and 8 beta-hydroxyfruticolone), and 6-acetylteucjaponin B (isolated from T. scordium and T. grisebachii), four new neo-clerodanes, namely 7 beta-hydroxyfruticolone, 11-hydroxyfruticolone, deacetylfruticolone and 6-acetyl-10-hydroxyteucjaponin B were also isolated. The structures were unambiguously elucidated based on extensive NMR spectral studies (one- and two-dimensional experiments). Semi-prep-HPLC proved to be a convenient purification procedure, occasionally being followed by TLC. The new compounds were assayed against Spodoptera littoralis and two of them were shown to have potent antifeedant activity.

Animals↗
PubMed2004

Insect growth inhibition by tocotrienols and hydroquinones from Roldana barba-johannis.

The methanol extract from the aerial parts of Roldana barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and their corresponding acetylated and methylated derivatives showed insecticidal and insect growth regulatory activities against the Fall Armyworm [Spodoptera frugiperda J.E. Smith, (Lepidoptera: Noctuidae)], an important insect pest of corn. The most active compounds were sargachromenol and its acetylated derivative; sargahydroquinoic acid and its acetylated derivative; and a mixture of sargachromenol, sargahydroquinoic acid, and sargaquinoic acid (6:3:1) and the acetylated form of this mixture. All these compounds and mixtures had significant inhibitory effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect. The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. These substances may be useful as natural insecticidal agents.

Animals↗