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At least 937 records · Page 52Linked to original sources
PubMed1999

Expression and characterization of glycolipid-anchored B7-1 (CD80) from baculovirus-infected insect cells: protein transfer onto tumor cells.

Tumor cells can be modified to express immunostimulatory molecules such as B7-1 by protein transfer using purified glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1). In this study recombinant baculovirus encoding GPI-B7-1 (vBacB7-1(GPI)) was established to obtain large quantities of purified GPI-B7-1 to modify tumor cells by protein transfer. vBacB7-1(GPI)-infected insect cells showed high-level cell surface expression of GPI-B7-1 that was susceptible to PIPLC treatment. GPI-B7-1 expressed in insect cells (Bac-GPI-B7-1) mediated T cell proliferation, indicating that the GPI-B7-1 retains costimulatory activity. Moreover, Bac-GPI-B7-1 was completely solubilized in Triton X-100 at 4 degrees C compared to 22% solubilization of GPI-B7-1 expressed in CHOK1 cells, suggesting that GPI-anchored proteins expressed in insect cells may not be clustered into the detergent-insoluble fraction. SDS-PAGE analysis of Bac-GPI-B7-1 showed faster mobility (45 kDa) compared to GPI-B7-1 from CHOK1 (68 kDa) and this difference may be due to a difference in glycosylation. Cell binding assays showed that immunoaffinity-purified Bac-GPI-B7-1 retained its functional ability to bind CD28(+) cells. Moreover, when human tumor cells were incubated with this functionally active purified GPI-B7-1, an efficient transfer of B7-1 onto tumor cells was observed. These results demonstrate that GPI-B7-1 can be expressed in insect cells in a functionally active form and can be used to modify tumor cells for immunotherapeutic applications.

Animals↗
PubMed2001

Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells.

The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.

Animals↗
PubMed1993

Morphogenic capabilities of human immunodeficiency virus type 1 gag and gag-pol proteins in insect cells.

To determine the interaction between the gag precursor and the viral protease and to confirm the role of gag precursor in formation of human immunodeficiency virus type 1 particles, the gag and protease encoding regions of a proviral genome with mutations at the site between p17 and p24 or p24 and p15 were expressed by recombinant baculoviruses under the transcriptional control of the strong polyhedrin promoter. Western blot analyses of the expressed products of p17-p24 mutated viruses revealed that both 41- and 55-kDa proteins were synthesized. However, free p24, p17, and the other smaller cleavage products (p9, p6) could not be detected in infected insect cells. The second recombinant virus (p24-p15) synthesized not only a 55k-Da protein, but also a number of smaller products including a 40k-Da protein, p24, and p17. Examination of the insect cells infected by either of these two recombinant viruses by electron microscopy failed to detect any gag particle formation, although some irregular membrane protrusions and profound distortions of the cell surface were clearly visible in the cells infected with recombinant mutant p17-p24 virus, but not with recombinant p24-p15 mutants. To investigate the morphogenic capability of the gag-pol fusion protein, a mutant gag-pol gene containing an inactive protease as well as a modified gag-pol gene lacking the frameshifting activity were expressed in insect cells. While the inactive protease mutant was capable of forming immature particles that were secreted, the frameshifting mutant synthesized only an aberrant form of gag particles with a large radius of curvature in lieu of spherical particles. However, when this mutant was expressed in insect cells in the presence of a truncated gag protein with M(r) of 46 kDa (lacking only the p6 domain), normal immature particles containing both antigens were formed.

Amino Acid Sequence↗
PubMed1993

Activation and detection of a latent baculovirus resembling Mamestra brassicae nuclear polyhedrosis virus in M. brassicae insects.

A laboratory culture of Mamestra brassicae insects (MbLC) has been found to harbor a latent baculovirus infection. The latent virus was activated by feeding the M. brassicae larvae with either the closely related Panolis flammea nuclear polyhedrosis virus (NPV), or the distantly related Autographa californica NPV. Restriction fragment profiles of the activated virus DNA showed that it is very closely related, if not identical, to M. brassicae NPV. Polymerase chain reaction amplification of polyhedrin gene sequences demonstrated that the latent virus was present throughout the life cycle of the insect; eggs, larvae, pupae, and adults. We failed to detect the presence of a latent virus in a second culture of M. brassicae insects, obtained from the environment and only recently adapted to growth in laboratory conditions, and thus these insects acted as an effective negative control in all the PCR and activation experiments. Using PCR analysis of DNA isolated from dissected tissues of fourth instar MbLC larvae, latent virus sequences were only detected in the fat body. Cell lines established from the isolated MbLC fat body tissue were also shown to harbor the latent virus sequences and should prove useful in further studies to elucidate the mechanisms of latency and virus activation.

Animals↗
PubMed2001

Canine parvovirus capsid assembly and differences in mammalian and insect cells.

We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells.

Animals↗
PubMed2002

The complete genome sequence of Perina nuda picorna-like virus, an insect-infecting RNA virus with a genome organization similar to that of the mammalian picornaviruses.

Perina nuda picorna-like virus (PnPV) is an insect-infecting RNA virus with morphological and physicochemical characters similar to the Picornaviridae. In this article, we determine the complete genome sequence and analyze the gene organization of PnPV. The genome of PnPV consists of 9476 nucleotides (nts) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 8958 nts (2986 codons) flanked by 473 and 45 nt noncoding regions on the 5' and 3' ends, respectively. Northern blotting did not detect the presence of any subgenomic RNA. The PnPV genome codes for four structural proteins (CP1-4), and determination of their N-terminal sequences by Edman degradation, showed that all four are located in the 5' region of the genome. The 3' part of the PnPV genome contains the consensus sequence motifs for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) in that order from the 5' to the 3' end. In all of these characters, the genome organization of PnPV resembles the mammalian picornaviruses and two other insect picorna-like viruses, infectious flacherie virus (IFV) of the silkworm and Sacbrood virus (SBV) of the honeybee. In a phylogenetic tree based on the eight conserved domains in the RdRp sequence, PnPV formed a separate cluster with IFV and SBV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses.

Amino Acid Sequence↗
PubMed1995

Evolution and structural conservation of the control region of insect mitochondrial DNA.

The control regions of mitochondrial DNA of two insects, Schistocerca gregaria and Chorthippus parallelus, have been isolated and sequenced. Their sizes are 752 bp and 1,512 bp, respectively, with the presence of a tandem repeat in C. parallelus. (The sequences of the two repeats are highly conserved, having a homology of 97.5%.) Comparison of their nucleotide sequences revealed the presence of several conserved sequence blocks dispersed through the whole control region, showing a different evolutionary pattern of this region in these insects as compared to that in Drosophila. A highly conserved secondary structure, located in the 3' region near the small rRNA gene, has been identified. Sequences immediately flanking this hairpin structure rather than the sequences of this structure themselves are conserved between S. gregaria/C. parallelus and Drosophila, having a sequence consensus of "TATA" at 5' and "GAA(A)T" at 3'. The motif "G(A)nT" is also present in the 3' flanking sequences of mammalian, amphibian, and fish mitochondrial L-strand replication origins and a potential plant mitochondrial second-strand-replication origin, indicating its universal conservation and functional importance related to replication origins. The stem-and-loop structure in S. gregaria/C. parallelus appears to be closely related to that found in Drosophila despite occupying a different position, and may be potentially associated with a second-strand-replication origin. This in turn suggests that such a secondary structure might be widely conserved across invertebrates while their location in the control region may be variable. We have looked for such a conserved structure in the control regions of two other insects, G. firmus and A. mellifera, whose DNA sequences have been published, and their possible presence is discussed. Mitochondrial control regions characterized to date in five different insect taxa (Drosophila, G. firmus, A. mellifera, S. gregaria, and C. parallelus) may be classed into two distinct groups having different evolutionary patterns. It is observed that tandem repetition of regions containing a probable replication origin occurred in some species from disjunct lineages in both groups, which would be the result of convergent evolution. We also discuss the possibility of a mechanism of "parahomologous recombination by unequal crossing-over" in mitochondria, which can explain the generation of such tandemly repeated sequences (especially the first critical repetition) in the control region of mtDNA, and also their convergent evolution in disjunct biological lineages during evolution.

Animals↗
PubMed1982

Freeze-fracture characteristics of insect gustatory and olfactory sensilla. I. A comparison with vertebrate olfactory receptor cells with special reference to ciliary components.

Freeze-fracture data on antennal olfactory and labellar gustatory sensilla of the blowfly Calliphora vicina were compared with those of vertebrate olfactory organs. Insect antennal and vertebrate olfactory axons have similar diameters and show vesicular expansions; insect labellar axons are on average twice as thick and show no vesicular expansions. Vertebrate olfactory and insect labellar and antennal axons display similar intramembranous particle densities. Antennal axons show particle arrangements, resembling tight-junctions. The few extremely thick axons found in labella and antennae show particle arrangements resembling gap-junctions. In regions, proximal to the pores in the insect sensillar hairs, P-faces of olfactory and gustatory cilia show about 200 particles/microns2. The most proximal and distal portions of the sensory cilia, necklaces and regions in the vicinity of the hair pores respectively, were only encountered in antennal sensilla. P-faces of the ciliary membranes underneath these pores display 1,000-1,200 particles/microns2 in unbranched and branched cilia. These values agree with values found in vertebrate olfactory cilia. It is suggested that these high particle densities are related to entities involved in chemoreceptive activities. Accessory cell micropliae have P-face densities of 2,000-3,000 particles/microns2, values similar to those found in vertebrate supportive cell microvilli. The membranes of the accessory cells display septate-junctions in areas where these cells overlap themselves, each other and in places where they adhere to the exoskeleton or the basement membrane.

Animals↗
PubMed1991

Pharmacology of insect GABA receptors.

A GABA-operated Cl- channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl- channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABAA receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl- channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active at t-butylbicyclophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.

Acetylcholine↗
PubMed1990

A technique for quantitative evaluation of ectoparasitic mites and insects of domestic animals.

A modified KOH dissolution technique using Tween 80 has been developed for the diagnosis and quantitative evaluation of ectoparasitic mites and insects of veterinary importance. Fifty intact skin segments from ten different domestic animals were artificially inoculated in vitro with ten specimens each of the mites Sarcoptes scabiei, Chorioptes bovis, Psoroptes ovis and Demodex caprae and insects Haematopinus eurysternus and Bovicola bovis. Sixty-seven to 88% of the mites and 80-99% of the insects were recovered by this technique. Using this method, parasitic mites and insects were found and identified in all skin scrapings from ten naturally infested animals. Using Cook's dissolution technique in our experimental design, 63.4% of C. bovis and 65% of S. scabiei mites were recovered.

Animals↗
PubMed1996

Immunological response of mice to the bovine respiratory syncytial virus fusion glycoprotein expressed in recombinant baculovirus infected insect cells.

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves. The BRSV genome encodes two major glycoproteins, G and F, which are the major targets for the host antibody response. We have expressed the F glycoprotein in insect cells (Sf9) using a recombinant baculovirus vector. A comparison of the F protein expressed in mammalian and insect cells by SDS-PAGE showed that only part of the baculovirus-produced protein was soluble and processed like the native protein. The antigenicity of the soluble form of the F protein expressed in insect cells was identical to that of the F protein expressed in mammalian cells. Immunization with the F protein expressed in insect cells induced neutralizing antibodies in mice. This antigenic preparation adjuvanted with Quil-A produced an increased neutralizing antibody titer and induced protection.

Animals↗
PubMed1994

Defensive use of an acquired substance (carminic acid) by predaceous insect larvae.

Larvae of two insects, a coccinellid beetle (Hyperaspis trifurcata) and a chamaemyiid fly (Leucopis sp.), feed on cochineal insects and appropriate their prey's defensive chemical, carminic acid, for protective purposes of their own. H. trifurcata discharges the chemical with droplets of blood (hemolymph) that it emits when disturbed; Leucopis sp. ejects the compound with rectal fluid. Ants are thwarted by these defenses, which are compared with the previously-described defense of a pyralid caterpillar (Laetilia coccidivora) that disgorges carminic acid-laden crop fluid. The defensive fluid of all three larvae contains carminic acid at concentrations spanning a range (0.2-6.2%) proven deterrent to ants. Many insects are known to appropriate defensive substances from plants. Insects that acquire defensive chemicals from animal sources may be relatively rare.

Animals↗
PubMedDate not supplied

Degradation of a radiolabeled juvenile hormone analog using two insect species.

A synthetic insect juvenile hormone analog (a juvenoid), ethyl N-[2-[4-[[2,2-(ethylenedioxy)cyclohexyl]methyl]phenox]ethyl]carbam ate, which has displayed high biological activity against different insect species and high stability under field conditions, was selected as a biologically active model compound for a study of a juvenile hormone analog degradation. The biologically active compound itself and its three diversely radiolabeled derivatives were applied to the flesh fly (Sarcophaga bullata) or the tsetse fly (Glossina palpalis), respectively. Monitoring of a fate of the applied juvenile hormone analog was carried out using a detection method of the radioactivity microdistribution within the whole insect body in combination with a radio high performance liquid chromatography (radio-HPLC), both of whole-body extracts made in different, but in advance scheduled, time intervals, and of extracts of insect excreta accumulated over an eight-day experiment.

Animals↗
PubMed1975

On methylating activity of L-(methyl-14C)-methionine in metabolism of phospholipids by insect Ceratitis capitata.

The methylating activity of L-(methyl-14C)-methionine in different stages of developmenent of the insect Ceratitis capitata was studied in a series of in vitro and in vivo experiments. Larval and pharate adult homogenates of the insect were used in the in vitro conditions, and the utilization of the methyl group of methionine for the synthesis of different classes of phospholipids was evaluated. Incorporation of radioactivity in lipids by pharate adult homogenates was significantly higher than that by larval homogenates. In both cases, phosphatidyl ethanolamine showed the highest levels of radioactivity incorporation. Free bases from total lipid hydrolysates were resolved and identified by paper chromatography, and the labeling was investigated by radioactivity scanning of paper chromatograms. Significant differences were observed in the activity of both stages of development of the insect. Larval and pharate adult homogenates incorporated mainly the labeled methyl groups into ethanolamine. Monmethyl ethanolamine was the only methyl derivative that appeared in the hydrolysates of lipids synthesized by larval homogenates, whereas mono-,di-and trimethyl ethanolamine clearly were detected in those synthesized by pharate adult homogeneates. Administration of L-(methyl 14C)-methionine to larvae confirmed the existence of methylation reactions in the metabolic activity of the insect.

Animals↗
PubMed1991

Insect tissue culture systems: models for study of hormonal control of development.

The regulation of growth and development of insects is under endocrine control and involves both juvenile hormones and ecdysteroids. Neuropeptides are master regulators which control the secretion of these hormones. Most experiments in insect endocrinology have been conducted in vivo, but tissue culture methodology is playing an increasing role due to the great interest in simpler model systems for the study of complex processes that occur in vivo. The availability of appropriate media has allowed the culture of a variety of insect organs and cell lines of defined origin which have kept certain properties of the parent tissues. Tissue culture approaches have been useful for studying hormonal control of morphogenetic processes. Cell lines are particularly suited to the study of hormonally regulated mechanisms of macromolecular biosynthesis and gene expression. Thus, the value of in vitro analysis in studies of regulation of hormone production is now recognized. Results obtained from tissue culture allow more precise definition of the hormonal requirements of insect cells and tissues for growth and differentiation and might make possible the discovery of new growth regulators.

Animals↗
PubMed1991

Hormonal effects on insects and other endoparasites in vitro.

Metamorphic and reproductive events in vertebrates and invertebrates are under endocrine control and are often correlated with developmental, behavioral, or reproductive changes in the parasites living in or on these hosts. This paper reviews selected examples of a) host hormone mediated influences on endoparasites in vivo, b) host hormone effects in vitro on protozoan, helminth, and insect endoparasites, and c) identifies possible relationships in hormone effects across parasite taxa. The significance of studies on endoparasites in vitro in relation to the impact of host hormones, antihelminthic, and prophylactic drugs on parasite growth and proliferation will also be addressed. A review of the literature indicates only limited studies have been done in vitro in an attempt to elucidate the bases of reported host hormone influences on endoparasites in vivo. Steroid hormones of hosts seem to stimulate growth, molting or encystment or both of helminth, insect, and protozoan parasites. Vertebrate steroids such as estrogen, testosterone, and progesterone had primarily reproduction- or growth-promoting effects or both on protozoan and nematode parasites. Insect ecdysteroids such as ecdysone, 20-hydroxyecdysone, and makisterone were the most widely studied steroids in vitro and induced growth or molting or both of cestode, nematode, and insect parasite larvae. Although juvenile hormone (JH III) stimulated growth in the protozoan and nematode parasites tested, the analogue methoprene and JH precursors, farnesal, farnesol, and farnesol methyl ether had various effects. Biogenic amines also varied in their effects on the nematode parasites tested, while the peptide hormone, insulin, stimulated growth in the protozoans tested. The evidence for in vitro effects of host hormones on their natural endoparasites is patchy at best. Additional studies are needed to identify the biochemical bases for the numerous host hormone mediated effects on parasites.

Animals↗
PubMedDate not supplied

Molecular correlates of neuronal specificity in the developing insect nervous system.

The development of the nervous system in insects, as in most other higher animals, is characterized by the high degree of precision and specificity with which synaptic connectivity is established. Multiple molecular mechanisms are involved in this process. In insects a number of experimental methods and model systems can be used to analyze these mechanisms, and the modular organization of the insect nervous system facilitates this analysis considerably. Well characterized molecular elements involved in axogenesis are the cell-cell adhesion molecules that underlie selective fasciculation. These are cell-surface molecules that are expressed in a regional and dynamic manner on developing axon fascicles. Secreted molecules also appear to be involved in directing axonal navigation. Nonneuronal cells, such as glia, provide cellular and noncellular substrates that are important pathway cues for neuronal outgrowth. Once outgrowing processes reach their general target regions they make synapses with the appropriate postsynaptic cells. The molecular mechanisms that allow growth cones to recognize their correct target cells are essential for neuronal specificity and are being analyzed in neuromuscular and brain interneuron systems of insects. Candidate synaptic recognition molecules with remarkable and highly restricted expression patterns in the developing nervous system have recently been discovered.

Animals↗
PubMed2006

Biology of the CAPA peptides in insects.

CAPA peptides have been isolated from a broad range of insect species as well as an arachnid, and can be grouped into the periviscerokinin and pyrokinin peptide families. In insects, CAPA peptides are the characteristic and most abundant neuropeptides in the abdominal neurohemal system. In many species, CAPA peptides exert potent myotropic effects on different muscles such as the heart. In others, including blood-sucking insects able to transmit serious diseases, CAPA peptides have strong diuretic or anti-diuretic effects and thus are potentially of medical importance. CAPA peptides undergo cell-type-specific sorting and packaging, and are the first insect neuropeptides shown to be differentially processed. In this review, we discuss the current knowledge on the structure, distribution, receptors and physiological actions of the CAPA peptides.

Amino Acid Sequence↗