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At least 775 records · Page 43Linked to original sources
PubMed1989

Cultivation of Blastocrithidia triatomae (Trypanosomatidae) on a cell line of its host Triatoma infestans (Reduviidae).

Blastocrithidia triatomae parasitizes vectors of Chagas' disease and is very difficult to cultivate in conventional media. However, co-cultivation with a cell line of its host Triatoma infestans (TI-32; in Schneider's Drosophila medium supplemented with 20% foetal calf serum and 10% tryptose phosphate broth) led to vigorous growth at 24 or 28 degrees C without an adaptation phase. More than 60 primary cultures were initiated successfully without any failures. Subcultures could be started immediately at weekly intervals. The doubling time was similar in both the primary cultures (41 h) and the 33rd subculture (39 h). The importance of the reduviid cells for B. triatomae became clear after removal of the insect cells, when multiplication of epimastigotes stopped and mainly cysts were formed. Cysts produced in vitro were infective for reduviids. Scanning electron microscopy showed that B. triatomae attached to the host cells, inserted its flagellum into them and destroyed them.

Animals↗
PubMed1990

Autographa californica M nuclear polyhedrosis virus: microtubules and replication.

Progressive reorganization and depolymerization of microtubules corresponded with virus-induced rounding of Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected Spodoptera frugiperda IPLB-Sf-21 cells, suggesting that microtubules were instrumental in maintaining the normal shape of these cells. Depolymerization of all cortical and most of the paranuclear microtubules with colchicine also resulted in cell rounding, confirming this hypothesis. Studies with aphidicolin and cycloheximide indicated the virus-induced effects on the microtubules were mediated by both early and late viral gene products. Microtubules in cells infected with a p10 deletion mutant depolymerized microtubules in a manner similar to those in wild-type virus-infected cells, indicating p10 was not responsible for virus-induced changes in the microtubules. Nevertheless, evidence for the association of p10 and microtubules was obtained by fluorescence microscopy and immunoelectron microscopy. Colchicine depolymerization of microtubules before and throughout infection did not interfere with virus replication, but treatment of cells with taxol, a microtubule-stabilizing agent, both delayed and depressed virus replication. The taxol-induced effect was relieved by the addition of colchicine. These results suggested that AcMNPV-induced depolymerization of microtubules may be a necessary event in, rather than a tangential effect of, virus replication. Attempts to monitor the effects of virus infection on intermediate filaments were unsuccessful due to the lack of cross-reactivity between antibodies to intermediate filament proteins and IPLB-Sf-21 cells, indicating these proteins are not highly conserved in lepidopteran insect cells.

Animals↗
PubMed1994

Photinus pyralis luciferase: vectors that contain a modified luc coding sequence allowing convenient transfer into other systems.

The coding sequence of the Photinus pyralis luciferase (Luc), as contained in the previously described expression vector, pUHC13-1, was modified such that several commonly used restriction sites in the luc gene were removed without changing the primary sequence of the protein. In addition, a Kozak sequence upstream from the ATG start codon was introduced. The resulting plasmid, pUHC131-1, produced increased amounts of Luc activity in HeLa and NIH3T3 cells. The modified luc gene was inserted in both orientations into the multiple cloning site of a commonly used cloning vector. This facilitates transfer of luc into other expression units.

Animals↗
PubMed1996

Molecular cloning and function expression of a diuretic hormone receptor from the house cricket, Acheta domesticus.

Insect diuretic hormones regulate fluid and ion secretion and the receptors with which they interact are attractive targets for new insect control agents. Recently, a diuretic hormone receptor from the moth Manduca sexta was isolated by expression cloning and found to be a member of the calcitonin/secretin/corticotropin releasing factor family of G-protein coupled receptors [Reagan J. D. (1994) J. Biol. Chem. 269, 9-12]. Degenerate oligonucleotides were designed based upon conserved regions in this receptor family and used to isolate a diuretic hormone receptor from the house cricket, Acheta domesticus. The complementary DNA isolated encodes a protein consisting of 441 amino acids with seven putative membrane spanning regions. Interestingly, unlike the M. sexta diuretic hormone receptor, the cricket diuretic hormone receptor contains a putative signal sequence. The receptor shares 53% and 38% sequence identity with the M. sexta diuretic hormone and human corticotropin releasing factor receptors respectively. When expressed in COS-7 cells, the receptor binds A. domesticus diuretic hormone with high affinity and stimulates adenylate cyclase with high potency. Four other insect diuretic hormones are considerably less effective at stimulating adenylate cyclase in COS-7 cells transfected with the receptor. This is in contrast to the M. sexta diuretic hormone receptor which is stimulated by all five insect diuretic hormones with high potency.

Amino Acid Sequence↗
PubMed1999

Immunity proteins from mosquito cell lines include three defensin A isoforms from Aedes aegypti and a defensin D from Aedes albopictus.

An Aedes aegypti mosquito cell line, Aag-2, exhibits a response to immune stimulation that is qualitatively similar to that of C7-10 cultured cells from the related mosquito, Aedes albopictus. Using SDS polyacrylamide gels, we found that a small peptide was preferentially induced by the treatment of growing cells with heat-killed, Gram-positive bacteria. By an analogy with other studies, this small peptide was postulated to be a member of the defensin family of insect immunity peptides. A differential display was used to obtain partial polymerase chain reaction products corresponding to mRNAs that were preferentially expressed in induced cells. One of these products, which contained the partial sequence of a defensin gene, was used to screen cDNA libraries from Ae. aegypti and Ae. albopictus cells. From Ae. aegypti cells, we found two previously described isoforms (A1 and A4) of mosquito defensin A, as well as a new isoform which we defined as A5. From Ae. albopictus cells, we found a new mature mosquito defensin, named D, which contains proline and isoleucine as the final amino acids. In both Ae. aegypti and Ae. albopictus cell lines, the expression of defensin mRNA was visible on Northern blots as early as 3 h after exposure to heat-killed bacteria, and defensin mRNA abundance was maximal at 12-36 h after induction.

Aedes↗
PubMed2000

Overexpression of murine fizzy-related (fzr) increases natural killer cell-mediated cell death and suppresses tumor growth.

Fizzy-related (fzr) is a recently identified 7WD domain family member implicated in cell cycle regulation of Drosophila and yeast. In this study, the murine homologue of fzr was isolated by suppression subtractive hybridization as a gene with decreased expression during malignant progression of a murine B-lymphoma cell line. Retroviral overexpression of fzr in B-lymphoma cells reduced tumor formation. Those tumors that did arise had diminished or extinguished retroviral Fzr. Surprisingly, fzr overexpression dramatically increased B-lymphoma cell susceptibility to natural killer cell (NK) cytotoxicity, a host-resistant mechanism for tumor formation in this model system. These findings implicate fzr as a new category of genes suppressing B-cell tumorigenesis and suggest a novel role for fzr in the target cell interaction with NK cells.

Animals↗
PubMed1994

Inhibition of the Ca(2+)-activated K(+)-channel by sapecin B, an insect antibacterial protein.

Sapecin is an antibacterial protein of the flesh fly and sapecin B is its homologue structurally similar to charybdotoxin of scorpion venom, which is known to be a K+ channel inhibitor. We found that, like charybdotoxin, sapecin B inhibits part of the voltage pulse-induced K+ currents of rat cerebellar Purkinje cells. We suggest that this effect is due to inhibition of the Ca(+)-activated K+ channel. Probably, sapecin B is a naturally occurring K+ channel inhibitor as well as an antibacterial protein.

Animals↗
PubMed2002

Binding of hepatitis C virus-like particles derived from infectious clone H77C to defined human cell lines.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.

Animals↗
PubMed1989

Baculovirus polyhedrin promoter-directed expression of rubella virus envelope glycoproteins, E1 and E2, in Spodoptera frugiperda cells.

To study the capability of Spodoptera frugiperda (fall armyworm; Sf9) cells to synthesize and process mature rubella virus (RV) proteins, a cDNA encoding the structural envelope glycoproteins, E1 (58 kDa) and E2 (42-47 kDa) were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during infection under the transcriptional regulation of the polyhedrin gene promoter. By immunoblot analysis with antibodies directed against purified RV, the individual proteins E1 and E2, and human convalescent serum, a polyprotein precursor migrating with an apparent molecular weight of 90-95 kDa was identified in Sf9 cells infected with the recombinant baculovirus, Ac701-RVE. In addition, two proteins migrating somewhat faster than authentic viral E1 and E2 were resolved. Pulse-chase labeling experiments in the absence and presence of tunicamycin, as well as treatment of the recombinant proteins with endo-beta-N-acetyl-D-glucosaminidase H indicated that the recombinant proteins are glycosylated and that the E1 and E2 apoproteins, respectively, were similar in size as compared to their in vitro synthesized counterparts. The recombinant protein products were further detected by some monoclonal antibodies directed against RV. The results presented here indicate that a polyprotein containing the envelope glycoproteins of RV is expressed and proteolytically cleaved in lepidopteran insect cells to form two proteins which resemble authentic E1 and E2. The baculovirus may therefore be suitable for the abundant expression of RV antigen.

Animals↗
PubMed1980

The development of the female accessory gland in the insect Rhodnius prolixus.

The specialized cell types and two distinct regions of the adult Rhodnius prolixus cement gland develop from a simple pseudostratified epithelial tube during the 20-22 days of the fifth stadium. Feeding initiates the first phase, proliferation. Cells round up and divide tangentially to the lumen. Following the proliferation phase, differentiative mitoses occur and differentiation, resulting in secretory units (consisting of a ductule, gland cell and cuticular lining), ensues in the distal region. Ductule morphogenesis occurs without pseudocilia, thus differing from other insect glands. The complex changes in cell shape and interaction occur during development of the secretory unit. The secretory cell and end-apparatus develop from a double cell unit at the base of elongating ductules. The inner cell produces a complex end-apparatus of epicuticle that mirrors the microvillar pattern and then it degenerates. The ductules are lined by cuticulin and inner epicuticle while the central gland lumen has a layer of endocuticle as well. The epithelium of the proximal region remains simple producing the thick corrugated cuticle characteristic of the adult secretory duct. The mesodermal covering forms a thick longitudinal striated muscle layer that adheres to the epithelium via desmosomes.

Animals↗
PubMed1994

Expression cloning of an insect diuretic hormone receptor. A member of the calcitonin/secretin receptor family.

Insect diuretic hormones and their receptors regulate fluid and ion secretion and thus are attractive targets for the design of novel insect control agents. A complementary DNA clone encoding a corticotropin-releasing factor-related diuretic hormone receptor from the tobacco hornworm Manduca sexta was isolated by expression cloning in COS-7 cells. The receptor consists of 395 amino acids and contains seven putative transmembrane domains. The expressed receptor binds M. sexta diuretic hormone, as well as several related insect diuretic peptides with high affinity. Furthermore, each of these peptides stimulate adenylate cyclase in COS-7 cells transfected with the receptor. The M. sexta diuretic hormone receptor is homologous to the receptors for calcitonin, secretin, vasoactive intestinal peptide, parathyroid hormone, glucagon-like peptide 1, growth hormone-releasing hormone, pituitary adenylate cyclase-activating polypeptide, and glucagon. The M. sexta diuretic hormone receptor is the first nonmammalian member of this family to be identified.

Amino Acid Sequence↗
PubMedDate not supplied

Establishment of two cell lines from embryonic tissue of the tobacco hornworm, Manduca sexta (L).

Two cell lines were derived from primary cultures of embryonic tissue of the tobacco hornworm, Manduca sexta (L.). The cell lines were maintained on hemolymph-free synthetic insect medium. Cytogenetic and immunoligical identification of the lines were carried out. Techniques for obtaining the line and medium, subculturieng and freezing procedures for long-term storage, and the morphological and growth characteristics are described.

Cell Aggregation↗
PubMed2001

Developing transgenic grains with improved oils, proteins and carbohydrates.

DuPont has developed cereals and oilseeds with improved proteins, carbohydrates, and oils for food, feed, and industrial applications. Products which have been or will be introduced include corn and soybeans with increased oil content, improved oil composition, increased amino acid content, altered protein content and functional qualities, altered starch composition, reduced oligosaccharide content, increased sucrose content, and combinations of these traits. These products have been developed using both mutation breeding and molecular biology-based transgenic approaches. We have also worked on improving the underlying technologies in order to accelerate product introductions. Gene discovery has been expedited through a genomics program that now has a database of more than two million sequences from a variety of plants, insects and microbes. Plant cell transformation for elite lines of crop species is being addressed through production laboratories with high throughput processes and through technology improvements. High-throughput, rapid and small-scale assays for biochemical parameters are used to identify plants carrying traits of interest. Small-scale functionality analyses, in which grains are broken down into their component parts and assayed for functional properties, indicate which seeds carry a trait of commercial value. Finally, a number of DNA marker systems are being used to accelerate trait introgression timelines.

Animal Feed↗
PubMed2006

Enzymatic characterization of a trypsin-like serine protease encoded by the genome of cell fusing agent virus.

Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosquito cell line. Based on viral morphology, phenotypic and phylogenetic studies, CFAV had been tentatively assigned to the genus Flavivirus (family Flaviviridae). The determination of the CFAV polyprotein complete sequence showed a putative serine protease domain analogue to the flaviviral NS2B/NS3 complex. This complex had been extensively studied, because it represented one of the main targets for antiflavivirus therapy development. We report herein the biochemical characterization of CFAV DeltaNS2B-NS3pro protease complex. CFAV polyprotein sequence was computationally analysed to identify the amino-acid regions involved in protease activity. We designed, expressed and purified a catalytically active protease whose enzymatic properties were determined using fluorogenic substrates. Our results showed that, despite the low level of conservation of its amino-acid sequence, CFAV protease exhibited physico-chemical properties of other flaviviruses (high pH value requirement for optimal activity, inhibition by salt and preference for substrates featuring a basic residue at P(1) position).

Amino Acid Sequence↗
PubMed1997

Baculovirus transactivator IE1 is functional in mammalian cells.

IE1 of Autographa californica multicapsid nuclear polyhedrosis virus acts as a transactivator of several viral promoters in insect cells. Transient expression assays indicate that IE1 is involved in the activation of the early promoter he65 in both insect TN-368 and mammalian BHK-21 cell lines. IE1 activation of the he65 promoter was compared with IE1 activation of the early 39K promoter. In contrast to the he65 promoter, the 39K promoter was not inducible by IE1 in BHK-21 cells.

Animals↗
PubMed1993

Purification and characterization of recombinant histidine-tagged human platelet 12-lipoxygenase expressed in a baculovirus/insect cell system.

A baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 12-lipoxygenase activity and protein levels were achieved; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in which the (His)6-tagged 12-lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12-hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a Vmax of 2-4 mumol min-1 mg protein-1, Km of 10 microM and kcat of approximately 250 min-1. linoleic acid, on the other hand, was converted to (13S)-13-hydroperoxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same Km. The enzyme was most active between pH 7.5-8 and activity was stimulated significantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75-kDa band in platelets, human erythroleukemia cells and 12-lipoxygenase baculoviral-infected Sf9 cells by immunoblot and immunoprecipitation methods. 12-Lipoxygenase protein represented 0.1% of the total soluble protein in platelet preparations. In immunofluorescence experiments 12-lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12-lipoxygenase should facilitate detailed biochemical structure/function studies.

Animals↗