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At least 109 records · Page 6Linked to original sources
PubMed1996

STAT-like DNA-binding activity in Spodoptera frugiperda cells.

Nuclear extracts from Spodoptera frugiperda (Sf9) cells were shown to contain a factor binding to DNA elements with gamma interferon activated site-like sequences. The DNA-binding activity was shown to be dependent on tyrosine phosphorylation. Hydrodynamic characterization of this entity revealed a Stokes radius of 8.4 nm and a sedimentation coefficient of 5.9 S. The molecular weight was calculated to 209,000. Estimated frictional (f/to) and axial (a/b) ratios indicated an elongated structure of this DNA-binding entity. This DNA-binding factor could represent a dimer of a Sf9 homolog to the mammalian signal transducers and activators of transcription.

Animals↗
PubMed1995

Expression of Trypanosoma congolense antigens in Spodoptera frugiperda insect cells.

Transcripts which encode two metacyclic-form-specific variable surface glycoproteins (mVSGs) of Trypanosoma congolense IL3000 have been cloned into baculovirus expression vectors using a novel transfer vector, pAcL11. One of the recombinant baculoviruses (AcVSG1) expressed a mVSG as a glycoprotein with a signal peptide which was cleaved in this expression system, whereas the other one (AcVSG2) expressed an unprocessed protein. From 1 liter of culture containing 10(9) Spodoptera frugiperda cells infected with the recombinant baculoviruses, 10 and 30 mg of mVSG1 and mVSG2, respectively, were obtained. Monospecific polyclonal antibodies produced by immunization of mice with the recombinant proteins reacted specifically with the respective proteins and showed no cross-reactivities between mVSG1 and mVSG2 in immunoblot assays. The antibodies to each of the proteins stained only the surface of a proportion of intact fixed T. congolense IL3000 metacyclic forms. It was possible to determine from these studies that, on the average, the parasites expressing mVSG1 constitute approximately 45% of the metacyclic population of T. congolense IL3000 maintained in in vitro cultures, whereas those that express mVSG2 constitute approximately 20%.

Amino Acid Sequence↗
PubMed2001

Trypanosoma evansi: cloning and expression in Spodoptera frugiperda [correction of fugiperda] insect cells of the diagnostic antigen RoTat1.2.

A complementary DNA encoding the variant surface glycoprotein (VSG) of Trypanosoma evansi Rode Trypanozoon antigenic type (RoTat)1.2, currently used for experimental serological diagnosis of T. evansi infection in livestock, was cloned as a recombinant plasmid and sequenced. A recombinant baculovirus containing the coding region of RoTat1.2 VSG was constructed to express the protein in Spodoptera frugiperda [corrected] insect cells. From this, sufficient quantities of the recombinant protein are being produced for empirical and wide-scale objective assessment of the diagnostic potential of this antigen. The gene encoding the RoTat1.2 VSG was shown by PCR to be present in the genomes of many different cloned isolates of T. evansi, but not T. brucei, from geographically separate regions of Africa, Asia, and South America. With the recombinant RoTat1.2 at hand, it is now possible to investigate the extent to which epitopes on this VSG are conserved among different T. evansi isolates.

Amino Acid Sequence↗
PubMed1995

Adhesion and cytotoxicity of Bacillus thuringiensis to cultured Spodoptera and Drosophila cells.

Two Bacillus thuringiensis strains were tested for the ability to adhere to cultured Spodoptera and Drosophila insect cells. The wild-type strain is virulent and motile and readily adheres to and kills both types of insect cells. The avirulent mutant strain, which lacks flagella and several other of the proposed virulence factors, does not adhere to the insect cells and does not kill these cells efficiently. Purified flagella bind to insect cells; addition of anti-flagella serum abolishes cell binding and reduces the killing efficiency. Both bacteria and culture supernatant from the wild-type strain, but not from the mutant, are cytotoxic. Of the factors known to be reduced in the avirulent mutant, such as flagellin and phosphatidylcholine (PC) degrading or phosphatidylinositol-specific phospholipase C (PLC), only PC-PLC is shown to be cytotoxic.

Animals↗
PubMed1996

Interactions of Bacillus thuringiensis crystal proteins with the midgut epithelial cells of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Binding of different Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Spodoptera frugiperda larvae was characterized by binding experiments with midgut tissue sections and isolated brush border membrane vesicles. Our results show that ICPs interact with the microvilli of epithelial cells of S. frugiperda in two different ways. The first is typical of highly toxic proteins (like CryIC and CryID); this interaction is saturable and specific. In contrast, some nontoxic proteins (like CryIAb) interact nonspecifically with the microvilli, since the binding of this toxin is not affected by the presence of high concentrations of homologous competitor. The CryIC toxin binds to two brush border proteins of 40 and 44 kDa and the CryIAb toxin binds to a single protein of 150 kDa. Immunological detection of ingested B. thuringiensis ICPs on gut sections of S. frugiperda larvae revealed that CryIC and CryID toxins bound along the epithelial brush border microvilli membrane. Binding of the nontoxic protein CryIAb was also observed in the epithelial brush border membrane of fed larvae, but it was extremely weak, implying that this type of interaction occurs also in vivo although it is not related to toxicity.

Animals↗
PubMed1997

Establishment of a cell line from Spodoptera litura (Lepidoptera: Noctuidae) and replication of S. litura nuclear polyhedrosis virus in vitro.

A new cell line, designated IBL-SLO1A, was established from pupal ovaries of the tobacco cutworm Spodoptera litura. Cells were grown in the TNM-FH insect cell culture medium supplemented with 10% fetal bovine serum. The subculture of cells was initiated with 1-3 x 10(5) cells/ml, a split ratio about 1:10. The growth curve of the cells fit the model of exponential growth (Y = e-0.6750 + 0.0317X R2 = 0. 98), the population doubling time during logarithmic growth at 28 degrees C was 21.9 hr. The number of chromosomes was about 140. Cytopathology characteristics of baculovirus infection were observed when the culture cells were infected with SINPV derived from alkali dissolution of occlusion bodies at 24 hr postinfection (pi). The cells lysed and released occlusion bodies into culture medium at 120 hr pi.

Animals↗
PubMed1999

Spodoptera littoralis type B nucleopolyhedrovirus infection of a grasshopper cell line.

We determined that the type B nucleopolyhedrovirus of the Egyptian cottonworm, Spodoptera littoralis (SpliNPV), can infect a cell line derived from a grasshopper. We compared the infectivity of SpliNPV in two lepidopteran cell lines (Sf9 and Md210) and in a cell line (MSE4) derived from the western migratory grasshopper, Melanoplus sanguinipes (Orthoptera: Acrididae). Both Sf9 and MSE4 cells were permissive for SpliNPV replication and supported production of viable progeny. Md210 cells were nonpermissive for SpliNPV, and although the virus entered into these cells, they supported neither viral replication nor production of viable progeny. Infection of MSE4 cells with SpliNPV resulted in cytopathic effects within 48 h post infection and complete destruction of the cells within 5 days. Both virions and polyhedra were detected within virus-infected MSE4 cells by transmission electron microscopy. Extracellular virions were detected in the culture medium and were infectious to Sf9 cells, indicating that the MSE4 cells supported production of viable virus progeny.

Animals↗
PubMed2000

Biological activity of SeMNPV, AcMNPV, and three AcMNPV deletion mutants against Spodoptera exigua larvae (Lepidoptera: noctuidae).

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigua larvae. In each mutant one gene was deleted by genetic engineering: pp34, coding for the polyhedral membrane; egt, coding for ecdysteroid UDP-glucosyltransferase; or p10, coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD(50)) as well as the highest speed of action (LT(50)) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10 deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect.

Animals↗
PubMed2000

Comparative analysis of the binding of antibodies prepared against the insect Spodoptera exigua and against the mycopathogen Nomuraea rileyi.

Polyclonal antibodies were produced in mice against Spodoptera exigua (beet armyworm) larval hemolymph and hemocytes and against cell wall surfaces of hyphal bodies and hyphae of the entomopathogenic hyphomycete Nomuraea rileyi. In addition to exhibiting strong activity against their original antigenic substrates, all of the antibodies cross-react extensively with other substrates. The hemolymph antibody binds to hemocytes and vice versa, and both antibodies cross-react to the insect fat body basement membrane (extracellular matrix (ECM) and to N. rileyi and Beauveria bassiana (another entomopathogenic fungus) cell wall surfaces (ECM). Likewise, the anti-fungal antibodies cross-react with S. exigua hemolymph and hemocytes, especially the granules that may contain ECM components, and with fat body basement membrane. These cross-reactivities are specific as indicated by negative controls in the microscopy and Western blotting assays. Parallel labeling experiments using Con A suggest that the reactive epitopes contain mannose; however, none of the antibodies bind to mannose residues of nonentomopathogenic Candida albicans or Saccharomyces cerevisiae yeast cells. Thus, these cross-reactivities suggest that the host mimicry expressed by surface components of entomopathogenic fungi represents an important pathogenic determinant.

Animals↗
PubMed2001

Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua.

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.

Animals↗
PubMed2001

Comparison of blastospores of two Paecilomyces fumosoroseus isolates: in vitro traits and virulence when injected into fall armyworm, Spodoptera frugiperda.

Blastospores of two isolates of Paecilomyces fumosoroseus were compared to determine their productivity in vitro and their relative pathogenicity after injection of fall armyworm, Spodoptera frugiperda. Blastospores of less virulent P. fumosoroseus isolate 1576 are smaller than those of isolate 4461, and they germinate and proliferate more slowly in vitro. The pathogenicity of injected blastospores of isolate 1576 against S. frugiperda varies with larval size. In small larvae, percentage mortality was lower among those injected with isolate 1576 than among those injected with isolate 4461. Large larvae were equally susceptible to both isolates. Survival times were higher for isolate 1576 and were higher at lower doses for both isolates. Those larvae injected late in development that ultimately died, regardless of treatment, did not lose weight typical of developing pupae. Injection of large larvae with isolate 4461 resulted in a significantly longer time to pupation among apparently uninfected larvae.

Animals↗
PubMed2001

Expression in Spodoptera frugiperda (Sf21) insect cells of BT-R(1), a cadherin-related receptor from Manduca sexta for Bacillus thuringiensis Cry1Ab toxin.

The cadherin-related receptor of Manduca sexta, BT-R(1), for the Cry1A family of Bacillus thuringiensis insecticidal toxins, was expressed in cultured Spodoptera frugiperda (Sf21) insect cells utilizing the expression vector deltaOp-gp64. Recombinant BT-R(1) was released by the Sf21 cells in soluble form into the culture medium and represents approximately 58% of total BT-R(1) produced by the cells. The soluble protein was purified by affinity chromatography using Cry1Ab toxin coupled to Sepharose 4B. The apparent molecular mass of purified soluble recombinant BT-R(1) is 195 kDa. Radiolabeled toxin bound to purified soluble BT-R(1) with a K(d) value of 1.1 nM, which is similar to that of both membrane-bound BT-R(1) in Sf21 cells and natural BT-R(1) from M. sexta larval midgut tissue. Binding of radiolabeled toxin to soluble BT-R(1) was competitively inhibited by unlabeled Cry1Ab toxin but not by other Cry toxins as was observed also for membrane-bound BT-R(1). The recombinant soluble protein was stable in culture medium for at least 3 days at 27 degrees C and for 7 days at 4 degrees C and exhibited toxin-binding properties similar to the natural protein. Apparently, neither membrane association nor the extent of glycosylation influences the binding affinity and specificity of BT-R(1). Approximately 1 mg of purified BT-R(1) was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) Sf21 cells.

Animals↗
PubMed2002

Optimization of baculovirus-mediated expression and purification of hexahistidine-tagged murine DNA (cytosine-C5)-methyltransferase-1 in Spodoptera frugiperda 9 cells.

Enzymatic DNA methylation of carbon 5 of cytosines is an epigenetic modification that plays a role in regulating gene expression, differentiation, and tumorigenesis. DNA (cytosine-C5)-methyltransferase-1 is the enzyme responsible for maintaining established methylation patterns during replication in mammalian cells. It is composed of a large ( approximately 1100 amino acids (a.a.)) amino-terminal region containing many putative regulatory domains and a smaller ( approximately 500 a.a.) carboxy-terminal region containing conserved, catalytic domains. In this study, murine DNA (cytosine C5)-methyltransferase-1, fused to an amino-terminal hexahistidine tag, was expressed by infecting Spodoptera frugiperda cells for 46 h with a recombinant baculovirus carrying the DNA (cytosine-C5)-methyltransferase-1 cDNA. A total of 3 x 10(8) infected S. frugiperda cells yielded approximately 1 mg of full-length, hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1, which was purified approximately 450-fold from RNase-treated S. frugiperda cell extracts by nickel affinity chromatography. The characterization of hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 through DNA methylation and inhibitor-binding assays indicated that the purified enzyme had at least a 30-fold higher catalytic efficiency with hemimethylated double-stranded oligodeoxyribonucleotide substrates than unmethylated substrates and was most active with small oligodeoxyribonucleotide substrates with a capacity for forming stem-loop structures. The expression and purification procedures reported here differ significantly from the original reports of baculovirus-mediated hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 expression and purification by nickel affinity chromatography and provide a consistent yield of active enzyme.

Animals↗
PubMed1996

Purification of a virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro.

The virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells was separated from the three host nuclear RNA polymerases by DEAE-Sephadex chromatography and then purified through two more steps: heparin-agarose chromatography and glycerol gradient ultracentrifugation. Fractions from each of these purification steps have been assayed in vitro for the ability to perform accurate initiation of transcription on a late (p6.9) and a very late (polyhedrin) template using primer extension analysis. In each case, the ability to accurately initiate transcription of these genes coincided with the virus-induced polymerase activity. Only after the glycerol gradient ultracentrifugation step did significant amounts of nonspecific late initiation occur, but specific late initiation was still readily detectable, suggesting that there is a limited number of late transcription factors, or that the factors are stably bound in a complex. After the glycerol gradient ultracentrifugation step, SDS-PAGE showed fewer than 10 prominent polypeptides remaining in the active fractions, which suggests a high degree of purity of the transcription machinery.

Animals↗
PubMed2001

Identification of a novel occlusion derived virus-specific protein in Spodoptera exigua multicapsid nucleopolyhedrovirus.

Understanding the molecular basis of the distinct biological properties of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), such as its narrow host range and high virulence, requires detailed information on the temporal expression and subcellular localization of SeMNPV gene products. The expression of two unique SeMNPV ORFs, 116 (Se116) and 117 (Se117), which show 45% amino acid similarity, was analyzed. Se116 and Se117 were expressed both in cultured cells and in larvae of S. exigua as polyadenylated transcripts of 0.80 and 0.75 kb, respectively. These transcripts initiated from ATCA(G/T)T promoter motifs, commonly found for baculovirus early genes. Se116 transcripts were detected with increasing abundance from 8 to 48 h p.i., whereas Se117 transcripts were present from 4 h p.i. and most abundantly at 24 h p.i. Western blot analysis of infected Se301 cells revealed 27- and 23-kDa proteins for Se116 and Se117, respectively. C-terminal GFP-fusion proteins of Se116 and Se117 were primarily localized in the nucleus of Se301 cells. When Se301 cells were infected with SeMNPV, both GFP-fusion proteins were localized in the virogenic stroma of the nucleus. While the function of the Se116 protein is still enigmatic, the Se117 protein appeared to be a structural protein associated with nucleocapsids of occlusion-derived SeMNPV virions but not of budded virus.

3' Untranslated Regions↗
PubMed2001

Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome.

The complete Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome contained 139,342 bp with a G+C content of 42.7%, and 141 putative open reading frames (ORFs) or genes of 150 nucleotides or greater that showed minimal overlap. Ninety-six ORFs had homologues in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), 16 had homologues in other baculoviruses, and 29 were unique to SpltMNPV. The homologues of ubiquitin and gp37 are fused in SpltMNPV. The genome lacked a homologue of the major budded virus glycoprotein gene gp64, but it contained a homologue of ORF130 of Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV). There were two homologues of AcMNPV ORF2 (bro gene), and a DnaJ protein gene (SpltORF39) in which the N-terminus showed homologies with the J domain of DnaJ family proteins. Seventeen homologous regions (hrs) were identified, each containing 2-29 palindromic repeats, with an average length of 534 bp and base content (G+C%) of 33.0.

Amino Acid Sequence↗
PubMed1995

Dichlone-induced oxidative stress in a model insect species, Spodoptera eridania.

Southern armyworm, Spodoptera eridania, larvae were provided ad libitum 0.002-0.25% w/w dichlone, 2,3-dichloro-1,4-naphthoquinone (CNQ). Larval mortality occurred in a time-and-dose dependent manner, with an LC17 of 0.01% and an LC50 of 0.26% CNQ at day-5. Extracts of larvae fed control, 0.01, and 0.25% CNQ diets for 5 days were assayed for antioxidant enzymes. While 0.01% CNQ had a mild effect, 0.25% CNQ profoundly increased levels of all antioxidant enzymes that were examined. The increases as compared to control were: 5.3-, 1.9-, 3.2-, 2.6-, 2.8-, and 3.5-fold higher for superoxide dismutase, catalase, glutathione transferase and its peroxidase activity, glutathione reductase and DT-diaphorase, respectively. At 0.01% CNQ, the thiobarbituric acid reactive substances (TBARS) were similar to the control group. However, despite the induction from 0.25% CNQ of all enzymes examined, the lipid peroxidation was not attenuated; the TBARS were 29.7% over the control value. High mortalities and CNQ-induced pathologies reflected in retarded growth, wasting syndrome, and diuresis clearly indicated that the insect sustained severe oxidant-induced injuries before appropriate defenses were fully mobilized. Thus, this quinone causes an oxidative stress in a model insect species analogous to that observed in mammalian species.

Animals↗
PubMed1995

Identification and nucleotide sequence of an ecdysteroid UDP-glucosyltransferase gene of Spodoptera littoralis multicapsid nuclear polyhedrosis virus.

The Spodoptera littoralis multicapsid nuclear polyhedrosis virus (SlMNPV) is a member of the Baculoviridae that shows a distant genetic relationship to the prototype Autographa californica MNPV (AcMNPV). Using an AcMNPV gene-specific probe, we identified and mapped an ecdysteroid UDP-glucosyltransferase (egt) gene in the genome of SlMNPV. Sequence determination of a part from the hybridizing DNA fragment revealed an open reading frame of 1548 nucleotides that exhibits 38% and 44% identity to the egt amino acid sequences of AcMNPV and Lymantria dispar MNPV (LdMNPV), respectively. Sequences flanking the SlMNPV egt gene, including the promoter region, were found to be unique to the virus. The presence of this nonstructural gene in SlMNPV and several other baculoviruses points to the importance of egt for the viral infection process.

Amino Acid Sequence↗