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At least 991 records · Page 55Linked to original sources
PubMed2001

Typical antipsychotics exhibit inverse agonist activity at rat dopamine D1-like receptors expressed in Sf9 cells.

The baculovirus system has been used to express the rat dopamine D1 receptors in Spodoptera frugiperda (Sf9) cells. A panel of typical antipsychotics including, alpha-flupenthixol, fluphenazine and thioridizine were found to inhibit dopamine-dependent stimulation of adenylyl cyclase. However, these compounds were also found to inhibit adenylyl cyclase activity in the absence of agonist in Sf9 cells expressing dopamine D1-like receptors. Therefore, these nonselective dopamine receptor compounds displayed negative intrinsic or inverse agonist activity. None of the compounds tested were neutral antagonists.

Adenylyl Cyclases↗
PubMed2001

Nature of the oligomers formed by muscarinic m2 acetylcholine receptors in Sf9 cells.

Wild-type, FLAG-tagged, and c-myc-tagged muscarinic m2 receptors extracted in digitonin-cholate from singly and co-infected Sf9 (Spodoptera frugiperda) cells were indistinguishable in their binding of [3H]quinuclidinylbenzilate, either before or after purification. The FLAG epitope was found to coimmunoprecipitate with the c-myc epitope when co-infected cells were solubilised in digitonin-cholate, n-dodecyl-beta-D-maltoside or Lubrol-PX. The degree of coprecipitation in digitonin-cholate was unaffected by preincubation of the extract for up to 60 min at 30 degrees C, with or without muscarinic receptor ligands; no coimmunoprecipitation occurred in mixed extracts from singly infected cells. As measured by [3H]quinuclidinylbenzilate, the efficiency of immunoprecipitation from co-infected cells was 87% of that from singly infected cells. The amount of receptor immunoprecipitated from the latter, as determined by densitometry, was 2.3-fold that expected from the loss of binding from the extract. The data suggest that at least some of the receptors were trimeric or larger and that oligomers neither formed nor dissociated under the conditions of the experiments. Also, some receptors appear to be non-functional or latent in digitonin-solubilised extracts.

Animals↗
PubMed1998

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors.

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.

Animals↗
PubMed1999

A threonine residue in the M2 region of the beta1 subunit is needed for expression of functional alpha1beta1 GABA(A) receptors.

Although there is a high degree of homology in the M2 transmembrane segments of alpha1 and beta1 subunits, subunit-specific effects were observed in alpha1beta1 GABA(A) receptors expressed in Spodoptera frugipedra (Sf9) cells when the conserved 13' threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13') was mutated to alanine in the beta1 subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13') was mutated to alanine in the alpha1 subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.

Action Potentials↗
PubMed2003

Further enhancement of baculovirus insecticidal efficacy with scorpion toxins that interact cooperatively.

We have studied whether the cooperative insecticidal effect of certain scorpion toxin pairs, namely either a combination of excitatory and depressant, or alpha and depressant scorpion toxins, would improve the efficacy of Autographa californica nucleopolyhedrovirus (AcMNPV) over a virus expressing only a single toxin, towards Heliothis virescens, Helicoverpa armigera, and Spodoptera littoralis larvae. The best result was achieved by combined expression of the excitatory toxin, LqhIT1, and the depressant toxin, LqhIT2, that provided an ET(50) value of 46.9 h on H. virescens neonates, an improvement of 40% over the efficacy of wild-type AcMNPV, and of 18% and 22% over baculoviruses that express each of the toxins independently. These results demonstrate that significant improvement in efficacy of recombinant baculoviruses is obtainable with toxins that exhibit a cooperative effect, and may contribute to employ baculoviruses to replace hazardous chemicals in insect control.

Animals↗
PubMed1998

Expression of functionally active ATP-sensitive K-channels in insect cells using baculovirus.

We have expressed active ATP-sensitive K-channels (K(ATP) channels) in Spodoptera frugiperda (Sf9) cells using a baculovirus vector. A high yield of active channels was obtained on co-infection with SUR1 and Kir6.2 engineered to contain N- and/or C-terminal tags to permit detection by Western blotting. Channel activity was sensitive to ATP, glibenclamide and diazoxide. Channel activity was also obtained on expression of a C-terminally truncated Kir6.2 (Kir6.2 deltaC26): these channels were blocked by ATP but were insensitive to sulphonylureas. In contrast to Xenopus oocytes and mammalian cells the full length Kir6.2 also gave rise to active channels in Sf9 cells when expressed alone. The highest yield of active K(ATP) channels was obtained on infection with a fusion protein containing SUR1 linked to Kir6.2 deltaC26 via a 6-amino acid linker.

Adenosine Triphosphate↗
PubMed1999

Cell cycle arrest mediated by hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only viral-encoded protein of the hepatitis delta virus (HDV). This protein has been extensively characterized with respect to its biochemical and functional properties. However, the molecular mechanism responsible for persistent HDV infection is not yet clear. Previously, we reported that overexpression of HDAg protects insect cells from baculovirus-induced cytolysis [Hwang, S.B. Park, K.-J. and Kim, Y.S. (1998) Biochem. Biophys. Res. Commun. 244, 652-658]. Here we report that HDAg mediates cell cycle arrest when overexpressed in recombinant baculovirus-infected insect cells. Flow cytometry analysis has shown that HDAg expression in Spodoptera frugiperda cells causes an accumulation of substantial amounts of polyploid DNA in the absence of cell division. This phenomenon may be partly responsible for the persistent infection of chronic HDV patients.

Animals↗
PubMed2002

Parasitoid-mediated transmission of an iridescent virus.

We examined the interaction between an invertebrate iridescent virus (IIV) isolated from Spodoptera frugiperda (J.E. Smith) and the solitary ichneumonid endoparasitoid Eiphosoma vitticolle Cresson. In choice tests, parasitoids examined and stung significantly more virus infected than healthy larvae, apparently due to a lack of defense reaction in virus infected hosts. Parasitoid-mediated virus transmission was observed in 100% of the female parasitoids that stung a virus infected host in the laboratory. Each female parasitoid transmitted the virus to an average (+/-SE) of 3.7+/-0.3 larvae immediately after stinging an infected larva. Caged field experiments supported this result; virus transmission to healthy larvae only occurred in cages containing infected hosts (as inoculum) and parasitoids (as vectors). The virus was highly detrimental to parasitoid development because of premature host death and lethal infection of the developing endoparasitoid. Female parasitoids that emerged from virus infected hosts did not transmit the virus to healthy hosts. We suggest that the polyphagous habits of many noctuid parasitoids combined with the catholic host range of most IIVs may represent a mechanism for the transmission of IIVs between different host species in the field.

Animals↗
PubMed2003

Improving baculovirus resistance to UV inactivation: increased virulence resulting from expression of a DNA repair enzyme.

The use of baculoviruses as biological control agents is hampered by their susceptibility to inactivation by ultraviolet (UV) light. In an attempt to reduce UV inactivation, an algal virus pyrimidine dimer-specific glycosylase, cv-PDG, was expressed in the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV), and the infectivity of recombinant viruses expressing cv-PDG was measured after exposure to UV light. Expression of cv-PDG resulted in a 3-fold decrease in inactivation of budded virus by UV as measured by plaque assay in Spodoptera frugiperda Sf21 cells. However, occluded viruses expressing cv-PDG were not more resistant to UV inactivation than wild type AcMNPV when fed to either S. frugiperda or Trichoplusia ni neonate larvae. Surprisingly, however, viruses expressing cv-PDG showed a significant decrease in both the dose of occluded virus required for oral lethality and the time required for lethality compared to control virus, but these effects were only seen in S. frugiperda and not in T. ni larvae.

Animals↗
PubMed2003

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility.

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.

Animals↗
PubMed2001

Semisynthetic derivatives of ent-kauranes and their antifeedant activity.

Chemical modification of functional groups on linearol yielded several ent-kaurane derivatives. These compounds were tested for their antifeedant activity against larvae of Spodoptera littoralis. Although linearol did not influence the feeding behaviour of larvae, some of its derivatives showed significant antifeedant activity.

Animals↗
PubMed2003

Dihydroisocoumarins and a tetralone from Cytospora eucalypticola.

Two dihydroisocoumarins, 3,5-dimethyl-8-hydroxy-7-methoxy-3,4-dihydroisocoumarin and 3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin were isolated from a culture filtrate of Cytospora eucalypticola, together with three known dihydroisocoumarins and a tetralone derivative. Their structures were determined by spectroscopic methods. These isocoumarins are mildly antifungal, and antibacterial towards gram positive bacteria. A known compound, 5-hydroxymethylmellein, showed mild antifeedant activity towards Spodoptera littoralis.

Animals↗
PubMed2003

Insecticidal pyrido[1,2-a]azepine alkaloids and related derivatives from Stemona species.

Eight new alkaloids, the pyrido[1,2-a]azepines stemokerrin, methoxystemokerrin-N-oxide, oxystemokerrin, oxystemokerrin-N-oxide, and pyridostemin, along with the pyrrolo[1,2-a]azepines dehydroprotostemonine, oxyprotostemonine, and stemocochinin were isolated from four Stemona species together with the known compounds protostemonine, stemofoline, 2'-hydroxystemofoline, and parvistemonine. Their structures were elucidated by 1H and 13C NMR including 2D methods and two key compounds additionally by X-ray diffraction. Besides the formation of a six membered piperidine ring, additional oxygen bridges and N-oxides contributed to structural diversity. The co-occurrence of pyrrolo- and pyridoazepines suggested biosynthetic connections starting from more widespread protostemonine type precursors. Bioassays with lipophilic crude extracts against Spodoptera littoralis displayed very strong insecticidal activity for the roots of S. curtisii and S. cochinchinensis, moderate activity for S. kerrii, but only weak effects for the unidentified species HG 915. The insect toxicity was mainly caused by the accumulation of stemofoline, oxystemokerrin, and dehydroprotostemonine displaying two different modes of action. Based on the various insecticidal activities of 13 derivatives structure-activity relationships became apparent.

Alkaloids↗
PubMed2003

Antioxidant and insect growth regulatory activities of stilbenes and extracts from Yucca periculosa.

The methanol extract from the bark of Yucca periculosa F. Baker afforded 4,4'-dihydroxstilbene, resveratrol and 3,3',5,5'-tetrahydroxy-4-methoxystilbene and had growth regulatory activity against the Fall Army worm (Spodoptera frugiperda J.E. Smith, Lepidoptera:Noctuidae) an insect pest of corn. The most active compound was 3,3',5,5'-tetrahydroxy-4-methoxystilbene which had significant effects at 3 microg/g in diets. In addition to the inhibitory activity on bleaching of crocin induced by alkoxyl radicals, these compounds also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl in TLC autographic and spectrophotometric assays. Our results indicate that these compounds could be involved in interference of sclerotization and moulting. These compounds appear to have selective effects on the pre-emergence metabolism of the insect. The results were fully comparable to known natural insect growth inhibitors such as gedunin and Cedrela extracts and have had a possible role as natural insecticidal agents.

Animals↗
PubMed2003

Antifeedant neo-clerodanes from Teucrium tomentosum Heyne. (Labiatae).

From the acetone extract of Teucrium tomentosum, a new antifeedant neo-clerodane diterpenoid teuctosin (1) was isolated along with teuflin (2), teucrin-H(2) (3), 6beta-hydroxyteuscordin (4), 6beta-acetylteuscordin (5) and montanin-D (6). The structure of the new compound was elucidated comprehensively using 1D and 2D NMR methods and confirmed by X-ray crystallography. All the compounds showed effective antifeedancy against Plutella xylostella and Spodoptera litura at 10 mug/cm(2) of leaf area.

Animals↗
PubMed2000

Inhibition of [(3)H]ponasterone a binding by ecdysone agonists in the intact Sf-9 cell line.

Ecdysone agonists, including dibenzoylhydrazines, significantly inhibited the binding of [(3)H]ponasterone A ([(3)H]PoA) in intact Sf-9 cells (Spodoptera frugiperda). The amount of [(3)H]PoA binding varied in a concentration-dependent manner. According to the IC(50), concentration at which there is 50% inhibition, the order of potency of typical ecdysone agonists is tebufenozide (RH-5992) > methoxyfenozide (RH-2485) > PoA > 20-hydroxyecdysone > cyasterone > RH-5849, makisterone A > or = inokosterone > ecdysone. The ranking is consistent with that obtained from a cultured integument system of the rice stem borer Chilo suppressalis except for methoxyfenozide. Other compounds whose modes of action are different from that of ecdysteroids, for example respiration inhibitors, plant steroid hormones, and chitin synthesis inhibitors, did not inhibit the binding of [(3)H]PoA significantly. The mammalian hormones estradiol and diethylstilbestrol, and a secondary bile acid, lithocholic acid, significantly inhibited the binding of [(3)H]PoA at 25 microM. However, their binding activity in terms of pIC(50) was either very low or not evaluated.

Animals↗
PubMed2003

Providence virus: a new member of the Tetraviridae that infects cultured insect cells.

We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia beta virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology.

Animals↗
PubMed2005

Purification and properties of Rab3 GEP (DENN/MADD).

Rab3A, a member of the Rab3 small GTP-binding protein (G protein) family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and the former is converted to the latter by the action of a GDP/GTP exchange protein (GEP). We have previously purified a GEP from rat brain with lipid-modified Rab3A as a substrate. Purified Rab3 GEP is active on all the Rab3 subfamily members including Rab3A, -3B, -3C, and -3D. Purified Rab3 GEP is active on the lipid-modified form, but not on the lipid-unmodified form. Purified Rab3 GEP is inactive on Rab3A complexed with Rab GDI. The recombinant protein is prepared from the Rab3 GEP-expressed Spodoptera frugiperda cells (Sf9 cells). The properties of recombinant Rab3 GEP, including the requirement for lipid modifications of Rab3A, the substrate specificity, and the sensitivity to Rab GDI, are similar to those of purified Rab3 GEP. Overexpression of Rab3 GEP inhibits Ca(2+)-dependent exocytosis from PC12 cells. On the other hand, Rab3 GEP is identical to a protein named DENN/MADD: differentially expressed in normal versus neoplastic (DENN)/mitogen-activated protein kinase-activating death domain (MADD). Here, we describe the purification method for recombinant Rab3 GEP from Sf9 cells and the functional properties of Rab3 GEP in Ca(2+)-dependent exocytosis by use of the human growth hormone coexpression assay system of PC12 cells.

Animals↗