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At least 649 records · Page 36Linked to original sources
PubMed1976

Comparison of biophysical and morphological properties of occluded and extracellular nonoccluded baculovirus from in vivo and in vitro host systems.

Electron microscopic examination and buoyant density profiles of nonoccluded Rachiplusia ou and Autographa californica nuclear polyhedrosis viruses purified from both infectious insect hemolymph and cell culture medium revealed that the viruses are enveloped, single nucleocapsids. The envelopes exhibited variation in the amount and degree of fit with regard to the nucleocapsids. This was determined by: (i) electron microscopic observations of virus budding from the surface of infected cells; (ii) electron microscopic observations of negatively stained preparations of pelleted, highly purified, nonoccluded enveloped particles; and (iii) the resolution and density distributions of nonoccluded virus in sucrose gradients after centrifugation to equilibrium; all were compared with virus extracted from polyhedra. Peplomers, ovserved on the surface of enveloped nucleocapsids of nonoccluded virus, are not associated with polyhedra-derived virus. Density gradient analysis indicated that virus from insect hemolymph and culture medium exhibited similar densities of approximately 1.17 to 1.18 g/ml. This is significantly different from the buoyant density of an alkali-liberated, enveloped single nucleocapsid (1.20 g/ml). Results of this study show that the nonoccluded forms of two nuclear polyhedrosis viruses from two different sources, hemolymph and cell culture, are similar with regard to several morphological and biophysical characteristics but are quite different from the alkali-liberated, polyhedra-derived form of the virus.

Animals↗
PubMed1989

Receptor activity of rotavirus nonstructural glycoprotein NS28.

Rotavirus morphogenesis involves the budding of subviral particles through the rough endoplasmic reticulum (RER) membrane of infected cells. During this process, particles acquire the outer capsid proteins and a transient envelope. Previous immunocytochemical and biochemical studies have suggested that a rotavirus nonstructural glycoprotein, NS28, encoded by genome segment 10, is a transmembrane RER protein and that about 10,000 Mr of its carboxy terminus is exposed on the cytoplasmic side of the RER. We have used in vitro binding experiments to examine whether NS28 serves as a receptor that binds subviral particles and mediates the budding process. Specific binding was observed between purified simian rotavirus SA11 single-shelled particles and RER membranes from SA11-infected monkey kidney cells and from SA11 gene 10 baculovirus recombinant-infected insect cells. Membranes from insect cells synthesizing VP1, VP4, NS53, VP6, VP7, or NS26 did not possess binding activity. Comparison of the binding of single-shelled particles to microsomes from infected monkey kidney cells and from insect cells indicated that a membrane-associated component(s) from SA11-infected monkey kidney cells interfered with binding. Direct evidence showing the interaction of NS28 and its nonglycosylated 20,000-Mr precursor expressed in rabbit reticulocyte lysates and single-shelled particles was obtained by cosedimentation of preformed receptor-ligand complexes through sucrose gradients. The domain on NS28 responsible for binding also was characterized. Reduced binding of single-shelled particles to membranes was seen with membranes treated with (i) a monoclonal antibody previously shown to interact with the C terminus of NS28, (ii) proteases known to cleave the C terminus of NS28, and (iii) the Enzymobead reagent. VP6 on single-shelled particles was suggested to interact with NS28 because (i) a monoclonal antibody to the subgroup I epitope on VP6 reduced particle binding, (ii) a purified polyclonal antiserum raised against recombinant baculovirus-produced VP6 reduced ligand binding, and (iii) a monoclonal antibody to a conserved epitope on VP6 augmented ligand binding. These experimental data provide support for the hypothesized receptor role of NS28 before the budding stage of rotavirus morphogenesis.

Animals↗
PubMed2002

Characterization of cell lines developed from the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae).

In order to isolate new pathogens (viruses, microsporidia, etc.) or to evaluate the efficiency of some pathogens (serovarieties and mutants of Bacillus thuringiensis, fungi, etc.) in the control of Colorado potato beetle, an economically important pest, we established four cell lines from tissues of this insect. One was initiated from embryonated egg fragments in the M3 medium supplemented with 20% fetal bovine serum (FBS) and then transferred after several passages to the Ex-Cell 400 medium with 20% FBS. Another was initiated from larval hemocytes in Ex-Cell 400 with 5% FBS. Finally, two other cell lines were initiated from adult hemocytes: one in the Ex-Cell 400 with 20% FBS and 1% of lipid mixture and the other in the Ex-Cell 400 with 5% FBS only. These cell lines have been characterized by their morphology with light and electron microscopy, their karyotypes, cell growth, and isozyme analysis. Each cell line differed in morphologic, karyologic, growth, and isozyme patterns. The cell line initiated from embryonated eggs was growing slower than the three initiated from hemocytes. The cytotoxicity of solubilized crystal delta-endotoxins from different B. thuringiensis formulations (M-One, Trident, MYX-1806, Teknar-HPD, and Thuricide) and of destruxins, mycotoxins from Metarhizium anisopliae, was tested on these cell lines. They are sensitive to the solubilized toxins of some strains of B. thuringiensis (serovar. San Diego and serovar. tenebrionis) and to destruxins, and they can be used for the bioassay and detection of toxins and for the study of the mechanism of their action on coleopteran cells.

Animals↗
PubMed2001

Identification and characterization of a novel fibronectin in zebrafish.

Sequence analysis of zebrafish fibronectin (FN) cDNAs indicates that at least two forms of the protein exist in fish. One form (FN1) is very similar to FNs identified in other vertebrates possessing 12 type I, 2 type II, and 17 type III repeats including two alternative splice sites (EIIIA and EIIIB) and a variable region (V). Zebrafish FN1 contains the RGD cell adhesion site in type III(10) and a second cell-binding site (LDV) in the V region. In addition to this conserved form of FN, a novel truncated form of zebrafish FN (FN2) was identified. The predicted structure of FN2 is identical to FN1 at the N-terminal region possessing 9 type I, 2 type II, and the first 3 type III repeats. Following III(3), FN2 contains a unique 20-amino-acid C-terminal tail that is different from the C-terminus of FN1, lacking the two cysteines that are usually involved in the formation of interchain disulfide bonds. Genomic sequence analysis has revealed that FN2 is generated by an alternative RNA splicing pattern that has not been described for FN in other organisms. Reverse transcription-polymerase chain reaction analysis and RNase protection assays reveal that FN2 mRNA is present in the zebrafish embryo throughout development as well as in cultures of an established liver cell line. Experiments conducted with recombinant FN2 synthesized in insect cells demonstrate that the protein promotes the attachment and spreading of fish embryo cells in culture.

Alternative Splicing↗
PubMed1992

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo.

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.

Animals↗
PubMed2005

Cytotoxic clerodane diterpenes from Glossocarya calcicola.

Three clerodane diterpenes were isolated and identified from leaf extract of Glossocarya calcicola. Compound has been characterised as (rel)-10betaH-trans-12xi-(2-methylbut-2(E)-enoyl)-1beta-(isobutanoyl)-6alpha,13xi-dihydroxyclerodan-4(20),8(18)-dien-7,15-dione-15,16-oxide, to which we have assigned the trivial name calcicolin-A. The other two compounds had the same skeletal structure and C-12 substituent but in compound, the C-1 esterifying group becomes 2-methylbut-2(E)-enoic acid and in it becomes 2-methylbutanoic acid. Although anti-insect activity was not observed for G. calcicola, cytotoxicity against insect and human carcinoma cell lines was detected.

Animals↗
PubMed2003

Macrophage responses to silk.

Silk fibers have potential biomedical applications beyond their traditional use as sutures. The physical properties of silk fibers and films make it a promising candidate for tissue engineering scaffold applications, particularly where high mechanical loads or tensile forces are applied or in cases where low rates of degradation are desirable. A critical issue for biomaterial scaffolds is biocompatibility. The direct inflammatory potential of intact silk fibers as well as extracts was studied in an in vitro system. The results indicate that silk fibers are largely immunologically inert in short- and long-term culture with RAW 264.7 murine macrophage cells while insoluble fibroin particles induced significant TNF release. Soluble sericin proteins extracted from native silk fibers did not induce significant macrophage activation. While sericin did not activate macrophages by itself, it demonstrated a synergistic effect with bacterial lipopolysaccharide. The low level of inflammatory potential of silk fibers makes them promising candidates in future biomedical applications.

Animals↗
PubMed2001

Selective inactivation of guanine-nucleotide-binding regulatory protein (G-protein) alpha and betagamma subunits by urea.

G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an alpha subunit and a betagamma dimer. Membrane extraction with 5-7 M urea has been used to uncouple receptors from endogenous G-proteins to permit reconstitution with purified G-proteins. We show that alpha(i) subunits are inactivated with 5 M urea whereas the betagamma dimer requires at least 7 M urea for its inactivation. There is no significant loss of receptors. Surprisingly, Western-blot analysis indicates that the urea-denatured alpha(i) subunit remains mostly membrane-bound and that beta is only partially removed. After 7 M urea treatment, both alpha(i1) and betagamma subunits are required to restore high-affinity agonist binding and receptor-catalysed guanosine 5'-[gamma-thio]triphosphate binding. We demonstrate the generality of this approach for four G(i)-coupled receptors (alpha(2A)-adrenergic, adenosine A1, 5-hydroxytryptamine(1A) and mu-opioid) expressed in insect cells and two mammalian cell lines. Thus a selectivity of urea for G-protein alpha versus betagamma subunits is established in both concentration and mechanism.

Animals↗
PubMed2000

Toward Anopheles transformation: Minos element activity in anopheline cells and embryos.

The ability of the Minos transposable element to function as a transformation vector in anopheline mosquitoes was assessed. Two recently established Anopheles gambiae cell lines were stably transformed by using marked Minos transposons in the presence of a helper plasmid expressing transposase. The markers were either the green fluorescent protein or the hygromycin B phosphotransferase gene driven by the Drosophila Hsp70 promoter. Cloning and sequencing of the integration sites demonstrated that insertions in the cell genome occurred through the action of Minos transposase. Furthermore, an interplasmid transposition assay established that Minos transposase is active in the cytoplasmic environment of Anopheles stephensi embryos: interplasmid transposition events isolated from injected preblastoderm embryos were identified as Minos transposase-mediated integrations, and no events were recorded in the absence of an active transposase. These results demonstrate that Minos vectors are suitable candidates for germ-line transformation of anopheline mosquitoes.

Animals↗
PubMed2006

Reconstitution of invertebrate glutamate receptor function depends on stargazin-like proteins.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are a major subtype of ionotropic glutamate receptors (iGluRs) that mediate rapid excitatory synaptic transmission in the vertebrate brain. Putative AMPARs are also expressed in the nervous system of invertebrates. In Caenorhabditis elegans, the GLR-1 receptor subunit is expressed in neural circuits that mediate avoidance behaviors and is required for glutamate-gated current in the AVA and AVD interneurons. Glutamate-gated currents can be recorded from heterologous cells that express vertebrate AMPARs; however, when C. elegans GLR-1 is expressed in heterologous cells, little or no glutamate-gated current is detected. This finding suggests that other receptor subunits or auxiliary proteins are required for function. Here, we identify Ce STG-1, a C. elegans stargazin-like protein, and show that expression of Ce STG-1 together with GLR-1 and the CUB-domain protein SOL-1 reconstitutes glutamate-gated currents in Xenopus oocytes. Ce STG-1 and homologues cloned from Drosophila (Dro STG1) and Apis mellifera (Apis STG1) have evolutionarily conserved functions and can partially substitute for one another to reconstitute glutamate-gated currents from rat, Drosophila, and C. elegans. Furthermore, we show that Ce STG-1 and Apis STG1 are primarily required for function independent of possible roles in promoting the surface expression of invertebrate AMPARs.

Amino Acid Sequence↗
PubMed1980

Juvenile hormone-binding protein from the cytosol of Drosophila Kc cells.

Insect cells of an established line, Drosophila Kc cells, take up and metabolize juvenile hormone (JH). The cytoplasm of these cells contains a protein that binds JH with specificity, saturability, and high affinity (K(d) = 1.56 x 10(-8) M). The kinetics for the specific binding and dissociation of JH I were independently measured, and the rate constants were found to be k(a) = 1.3 x 10(6) M(-1) min(-1), k(d) = 1.3 x 10(-2) min(-1), respectively. All three juvenile hormones bind to the protein with comparable affinities; the corresponding acid or diol metabolites of JH I are not bound. About 2500 hormone-binding protein molecules are present per cell. The protein has a molecular weight of 80,000 as estimated by gel permeation chromatography and by sucrose gradient sedimentation. The properties of this protein suggest that it functions as a cytoplasmic receptor for juvenile hormone.

Journal Article↗
PubMed1998

The baculovirus anti-apoptotic p35 protein promotes transformation of mouse embryo fibroblasts.

The baculovirus p35 protein is a potent inhibitor of programmed cell death induced by a variety of stimuli in insects, nematodes, and mammalian cell lines. The broad ability of p35 in preventing apoptosis has led us to investigate its effect on mouse embryo fibroblasts in vitro and in vivo. For this purpose, we have used R- cells (3T3-like fibroblasts derived from mouse embryos with a targeted disruption of the insulin-like growth factor I receptor (IGF-IR) genes) and R508 cells (derived from R- and with 15 x 10(3) IGF-IRs per cell). Both cell lines grow normally in monolayer, but they do not form colonies in soft agar, and they are non-tumorigenic in nude mice. We show here that, in addition to its anti-apoptotic effect, p35 causes transformation of R508 cells, as evidenced by the following: 1) decreased growth factor requirements, 2) ability to form foci in monolayer and colonies in soft agar, and 3) ability to form tumors in nude mice. Since R- cells stably transfected with p35 do not transform, our observations suggest that in addition to its effect as an inhibitor of apoptosis, the baculovirus p35 protein has transforming potential that requires the presence of the IGF-IR. The possibility that these two properties could be separated was confirmed by demonstrating that R508 cells expressing another anti-apoptotic protein, Bcl-2, could not form tumors in nude mice.

Animals↗
PubMed1990

Establishment and characterization of a cell line from the mosquito Culex (Culex) theileri (Diptera: Culicidae) and its susceptibility to infection with arboviruses.

The mosquito Culex (Culex) theileri Theobald is probably the major vector of Rift Valley fever (RVF) virus during epizootics on the inland plateau of southern Africa. A cell line was established from the tissues of this mosquito species to facilitate laboratory studies on RVF and other arboviruses. After temperature pretreatment, egg rafts from a laboratory colony were disinfected, mechanically disrupted, and grown in preconditioned Eagles minimum essential medium supplemented with 10% fetal calf serum. The cells were characterized by their protein content, karyology, and their susceptibility to three arboviruses. Although high infectivity titers were obtained, no cytopathic effects were produced by these viruses. The cell line was shown to be free from latent viruses. These results show that this cell line may be employed fruitfully in arbovirus studies.

Animals↗
PubMed1993

Salivary gland material from the sand fly Lutzomyia longipalpis has an inhibitory effect on macrophage function in vitro.

Previous work from our laboratory demonstrated that the infectivity of the protozoan parasite Leishmania major was enhanced in mice if the infecting inoculum contained salivary gland lysates from the sand fly vector Lutzomyia longipalpis. The present study was designed to address the hypothesis that sand fly salivary gland material may function by inhibiting the host immune response. Results indicated that sand fly saliva inhibited the ability of macrophages to present leishmanial antigens to parasite-specific T cells.

Analysis of Variance↗
PubMed1975

Replication of nuclear polyhedrosis virus in a continuous cell culture of Spodoptera frugiperda: microscopy study of the sequence of events of the virus infection.

A microscopy study of the sequence of morphogenic events of Spodoptera frugiperda nuclear polyhedrosis virus infection of S. frugiperda cells is presented which orders the sequence of replication and establishes the time scale within which the events occur. The virus entered the cell by 1 h postinfection and was uncoated. The eclipse period was 9 h and the latent period was 12 h. Polyhedron formation was detected by 18 h postinfection and continued until the deposition of the polyhedron membrane was completed by 48 h postinfection. Aberrant morphogenic characteristics of virus repeatedly passaged in the cell culture were also recorded. Adsorption, envelope morphogenesis, and release mechanisms are discussed in light of other data on in vivo and in vitro baculovirus infections.

Adsorption↗
PubMed2004

trans-Packaged West Nile virus-like particles: infectious properties in vitro and in infected mosquito vectors.

A trans-packaging system for West Nile virus (WNV) subgenomic replicon RNAs (repRNAs), deleted for the structural coding region, was developed. WNV repRNAs were efficiently encapsidated by the WNV C/prM/E structural proteins expressed in trans from replication-competent, noncytopathic Sindbis virus-derived RNAs. Infectious virus-like particles (VLPs) were produced in titers of up to 10(9) infectious units/ml. WNV VLPs established a single round of infection in a variety of different cell lines without production of progeny virions. The infectious properties of WNV and VLPs were indistinguishable when efficiencies of infection of a number of different cell lines and inhibition of infection by neutralizing antibodies were determined. To investigate the usefulness of VLPs to address biological questions in vivo, Culex pipiens quinquefasciatus mosquitoes were orally and parenterally infected with VLPs, and dissected tissues were analyzed for WNV antigen expression. Antigen-positive cells in midguts of orally infected mosquitoes were detected as early as 2 days postinfection and as late as 8 days. Intrathoracic inoculation of VLPs into mosquitoes demonstrated a dose-dependent pattern of infection of secondary tissues and identified fat body, salivary glands, tracheal cells, and midgut muscle as susceptible WNV VLP infection targets. These results demonstrate that VLPs can serve as a valuable tool for the investigation of tissue tropism during the early stages of infection, where virus spread and the need for biosafety level 3 containment complicate the use of wild-type virus.

Animals↗