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PubMed2000

Extrapancreatic trypsin-2 cleaves proteinase-activated receptor-2.

Proteinase-activated receptors (PARs) are activated by proteolytic removal of a short amino terminal peptide, thus exposing a new amino terminus that functions as a tethered ligand that activates the receptor. With the aim to identify and study potential activators of PAR-2 we have developed a new method to measure proteolytic cleavage of PARs. PAR-2 was tagged with the insulin C-peptide that upon receptor cleavage is released and quantified using an ELISA. The modified receptor, shown to be functional in mouse 3T3 cells, was expressed in an insect cell line and the ability of different proteinases to cleave PAR-2 was studied. Two different mast cell tryptases cleaved PAR-2 in a concentration dependent manner, but were much less potent than pancreatic trypsin and trypsin-2 isolated from a carcinoma cell line. Pancreatic trypsin and trypsin-2 were almost equally effective at cleaving PAR-2 suggesting that extrapancreatic trypsins are potential in vivo activators of PAR-2.

3T3 Cells↗

PubMed1995

Induction of p53-, MDM2-, and WAF1/CIP1-like molecules in insect cells by DNA-damaging agents.

Cellular responses following DNA damage are ubiquitous in the biological world. In response to DNA damage, cell cycle checkpoints are activated, which delay cell cycle progression and most likely serve to allow time for repair. One important checkpoint in mammalian cells, activated in the G1 phase of the cell cycle, is dependent on the p53 tumor suppressor gene product. While p53 is responsible for inducing G1 arrest, the product of the MDM2 gene is believed to alleviate the arrest, allowing continuation of the cell cycle after a transient delay. Inasmuch as MDM2 and WAF1/CIP1 are transactivated by p53, while MDM2 binds to and modulates the activity of p53, a "feedback loop" is thus created. This pathway has been highly conserved in mammalian cells, but its presence outside of vertebrates is unknown. By using human MDM2 and WAF1/CIP1 cDNA probes, and monoclonal antibodies to p53 and Mdm2, we demonstrate in insect cell lines evidence for the existence of p53-, MDM2-, and WAF1/CIP1-like molecules and a p53-regulated pathway following treatment by DNA-damaging agents.

Animals↗

PubMed1998

Structural and ultrastructural changes during the infection of UFL-AG-286 cells with the baculovirus AgMNPV

During infection of the permissive insect cell line UFL-AG-286 by the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) several morphological changes occur. By 12 h postinfection (h p.i.), the infected cells became round and exhibited a decrease in the number of cytoplasmic projections. By 24 h p.i., it was possible to detect a virogenic stroma inside the cell nucleus, and after 48 h p.i., polyhedral inclusion bodies were observed. Some of these morphological modifications are probably due to changes in the cytoskeleton of the cell and this possibility was substantiated by the observation that the distribution of actin and microtubules was dramatically modified upon infection. Several viral-induced proteins were also produced during infection and a sharp decrease in overall protein synthesis was observed. These results are very similar to those obtained with other cell lines infected with different baculoviruses, indicating a similar mechanism of infection. Copyright 1998 Academic Press.

Journal Article↗

PubMed1999

Noxious components of venom from the pupa-specific parasitoid pimpla hypochondriaca

Venom from the endoparasitoid wasp Pimpla hypochondriaca induced a lethal paralysis when injected into the hemocoel of larval, pupal, and adult stages of the tomato moth Lacanobia oleracea and was also toxic to the adult stages of the housefly Musca domestica and the cockroach Blatella germanica. Heating the venom to 58 degreesC did not reduce its potency against M. domestica. Phenoloxidase activity was detected in diluted venom using dihydroxyphenylalanine as the substrate and occurred independently of activators. Enzyme activity was strongly inhibited by 2.0 &mgr;M phenylthiocarbamide (PTC), a known inhibitor of phenoloxidase, and was not detectable in venom that had been heated to 58 degreesC for 10 min. Venom treated to remove phenoloxidase was shown to inhibit phenoloxidase activity in untreated venom, with a potency likely to be sufficient to prevent enzyme activity in the parasitoid's venom sac. The venom was found to be cytotoxic to an insect cell line (SF21) from Spodoptera frugiperda, and this activity was not reduced when the cells were cultured in the presence of 20 &mgr;M PTC. Venom constituents were separated according to size using high-resolution gel filtration. Three venom components: phenoloxidase, a factor which caused paralysis of M. domestica, and a factor which was cytotoxic to SF 21 cells were detected and found to have maximum activity in fractions 19, 40-42, and 50-51, respectively. Phenoloxidase and the factor causing paralysis of M. domestica had estimated apparent molecular masses of 390 and 27 kDa, respectively, while the cytotoxic factor had an apparent molecular mass of less than 13 kDa. Assay for inhibitors of venom phenoloxidase identified two major inhibitory components having highest activity in fractions 48 and 60, respectively. The larger of these two factors had an estimated apparent molecular mass of 12 kDa, but the other factor was considerably smaller than the lowest molecular mass standard (13.7 kDa) used to calibrate the column. Copyright 1999 Academic Press.

Journal Article↗

PubMed2000

Characterization of a Bacillus thuringiensis delta-endotoxin which is toxic to insects in three orders.

We report here the first Bacillus thuringiensis (Bt) toxin which is toxic to insects from three insect orders (Diptera, Coleoptera, and Lepidoptera). An oligonucleotide probe based on the delta-endotoxin N-terminal sequence was used to detect the gene. A 23-kb BamHI fragment containing the intact gene was identified and cloned from Bt strain YBT-226 plasmid DNA into the vector pBluescript II. Through a series of DNA manipulations the size of this fragment was reduced and the gene sequenced. The deduced amino acid sequence gave a predicted molecular mass of 137 kDa and was identical to a cry1Ba protein from Bt subsp. thuringiensis HD-2, which is now designated as Cry1Ba1 under a new classification scheme. This protein also showed 81.6% similarity with the Cry1B protein (Cry1Bb1) from Bt strain EG 5847. When the YBT-226 cry1Ba1 gene was expressed in an acrystalliferous Bt subsp. israelensis strain it produced irregular bipyramidal crystals during sporulation, which reacted specifically with anti-Cry1Ba antiserum. Bioassays using these crystals after purification resulted in significant mortality at low to moderate concentrations to larvae of the house fly (Musca domestica, Diptera), cottonwood leaf beetle (Chrysomela scripta, Coleoptera), and tobacco hornworm (Manduca sexta, Lepidoptera). This broad-spectrum toxicity was not dependent on presolubilization. In assays with insect cell lines not derived from midgut cells, the soluble toxin killed CH1t (Manduca sexta cells) but was inactive against CF1 (Choristoneura fumiferana cells), Aa(s) (Aedes aegypti), and C2 (Culex quinquefasciatus) mosquito cells.

Bacillus thuringiensis↗

PubMed2000

Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication.

Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1). When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged over five times in Ex-Cell 400 to which serum was added; both cell lines produced as much virus as that in SFM. These results suggest that it is not the serum per se but rather some other components which differ between the SFM and the SCM formulations that are responsible for the varied virus production obtained in these studies. The results of these studies suggest that a maintenance and virus production protocol can be developed with Ld cells which could improve overall efficiency of virus production. These studies also suggest that long-term maintenance of cells in SFM was not detrimental to their ability to produce baculoviruses.

Animals↗

PubMed2001

Replication of the gonad-specific virus Hz-2V in Ld652Y cells mimics replication in vivo.

A newly discovered, nonoccluded insect virus, known as gonad-specific virus or Hz-2V, was found to replicate differently in two insect cell lines derived from ovarian tissues (Tn-368 cells from Trichoplusia ni and Ld652Y from Lymantria dispar). Differences between these two cell lines were observed in virus plaque forming ability, rate of viral DNA replication, time course of infectious virus production, and the mechanism of virus release from infected cells. Replication of Hz-2V in Ld652Y cells was more productive and more closely resembled in vivo virus replication.

Animals↗

PubMed1999

The cloning and expression of Pfacs1, a Plasmodium falciparum fatty acyl coenzyme A synthetase-1 targeted to the host erythrocyte cytoplasm.

Plasmodium is unable to carry out de novo fatty acid synthesis and has to obtain these compounds from their host for subsequent activation by thioesterification with coenzyme A. This activity is catalyzed by a fatty acyl-CoA synthetase enzyme (EC 6.2.1.3). Here, we describe a novel gene from P. falciparum whose recombinant purified product from baculovirus-transfected insect cell line had the enzymatic activity of a long-chain fatty acyl-CoA synthetase. It was named pf acs1, since it belongs to a multi-member gene family as revealed by the sequence of several clones and a multi-band pattern in Southern blots. The sequence specifies a product of 820 amino acid residues. It was transcribed and expressed in infected erythrocytes having an apparent molecular mass of 100 kDa. Immuno-labeling of infected erythrocytes with a specific antibody against the carboxy-terminal part of the PfACS1 localized the product early after the erythrocyte invasion in vesicle-like structures budding off the parasitoforous membrane toward the red cell cytoplasm. Its unique carboxy- terminal structure of 70 extra amino acid residues, longer than any other reported acyl-CoA synthetase, is probably related to its localization in the cytoplasm of the host erythrocyte. The phylogenetic relationship among other AMP-forming enzymes, placed PfACS1 closer to Saccharomyces cerevisiae, sharing significant amino acid identities, especially in the conserved signature motif that modulates fatty acid substrate specificity and ATP/AMP-binding domains. Taking into account the importance of this enzymatic activity for the parasite, its extra-cellular location inside the infected erythrocyte, and the divergence with respect to the homologous human enzymes, it may be an important protein as a potential target candidate for chemotherapeutic antimalaria drugs.

Amino Acid Sequence↗

PubMed1994

Overexpression of human DNA topoisomerase I in insect cells using a baculovirus vector.

The 3645-bp human DNA topoisomerase I cDNA isolated by D'Arpa et al. (Proc. Natl. Acad. Sci. USA 85, 1988, 2543-2547) was integrated into the Autographa californica multiple nuclear polyhedrosis virus genome. The recombinant protein was expressed by infecting the SF9 insect cell line with this baculovirus and resulted in a 100-fold overexpression of human DNA topoisomerase I compared to the level found in human cell lines. This 100-kDa recombinant protein has the same electrophoretic mobility as the human DNA topoisomerase I from HeLa cells and is recognized by topoisomerase I-specific monoclonal antibody. The recombinant DNA topoisomerase I was isolated and purified to homogeneity with a two-step fractionation protocol and has a specific activity of 2 x 10(6) U/mg. Enzymatic properties such as stimulation by magnesium and inhibition by camptothecin resemble properties of the enzyme purified from human cell lines.

Animals↗

PubMed1995

Characterization of recombinant heparin cofactor II expressed in insect cells.

Recombinant human heparin cofactor II (rHCII) was expressed as a fully active protein in the High-Five insect cell line. A maximal protein concentration of 6 micrograms/10(6) cells was achieved 2 days postinfection. Approximately 40 micrograms of partially purified rHCII was routinely recovered from 50 ml of media after sequential heparin and Q-Sepharose affinity adsorption. rHCII had a slightly lower apparent molecular weight than blood plasma HCII (pHCII) due to differences in N-glycosylation. Like pHCII, rHCII formed a stable bimolecular complex with thrombin when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The thrombin and chymotrypsin inhibitory properties of rHCII and pHCII were quite similar. In the absence of glycosaminoglycan, the thrombin inhibition rate (k2 x 10(-4) M-1 min-1) was 2.29 +/- 0.36 for rHCII and 3.38 +/- 0.34 for pHCII. Chymotrypsin inhibition rates (k2 x 10(-5) M-1 min-1) were 6.2 +/- 2.0 for rHCII and 8.0 +/- 2.6 for pHCII. In the presence of glycosaminoglycans, the maximal thrombin inhibition rate (k2 x 10(-3) M-1 min-1) for rHCII was 10.4 +/- 2.5 at 100 micrograms/ml heparin and 16.0 +/- 4.3 at 1000 micrograms/ml dermatan sulfate compared to 9.0 +/- 0.7 at 200 micrograms/ml heparin and 18.5 +/- 5.3 at 1000 micrograms/ml dermatan sulfate for pHCII. HCII inhibition of thrombin was blocked by a synthetic sulfated hirudin peptide in both the presence and the absence of glycosaminoglycan. The present report describes for the first time the expression and characterization of HCII in a baculovirus system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rHCII for future structure-function studies.

Animals↗

PubMed1999

Expression of a glycosylphosphatidylinositol-linked Manduca sexta aminopeptidase N in insect cells.

Aminopeptidase N (APN; EC 3.4.11.2) is an exopeptidase that is attached to cell membranes by a hydrophobic amino-terminal stalk in vertebrates or a glycosylphosphatidylinositol (GPI) anchor in insects. In this study, we report the cloning, expression, and characterization of an aminopeptidase N from Manduca sexta midgut. The full-length aminopeptidase N cDNA (APN1a) encodes a 995-amino-acid protein. The predicted amino acid sequence differs by 8 amino acids from M. sexta APN1. These different amino acids do not modify any putative glycosylation or glycosylphosphatidylinositol anchor sites. The full-length cDNA was cloned into an expression plasmid, pHSP-HR5, and transiently expressed in an insect cell line derived from Spodoptera frugiperda (Sf21 cells). Immunoblot analysis with anti-APN antiserum showed that APN1a expressed in Sf21 cells is the same size (120 kDa) as APN found in midgut brush border membranes. After treatment with phosphatidylinositol-specific phospholipase C (PIPLC), anti-cross-reacting determinant antibody specific for PIPLC cleavage products recognized the expressed 120-kDa APN1a, but not endogenous Sf21 proteins, indicating that APN1a has an intact glycosylphosphatidylinositol anchor. These results are evidence that Sf21 cells synthesize few, if any, endogenous GPI-linked proteins. Immunofluorescence staining showed that the expressed APN1a was located on the surface of Sf21 cells.

Amino Acid Sequence↗

PubMed2001

High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus.

A hybrid of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which is infectious to both Spodoptera frugiperda and Bombyx mori, was prepared in our previous study. Two recombinant hybrid baculoviruses, carrying cDNAs of human acidic and basic fibroblast growth factors, respectively, were successfully constructed in this study, for the large-scale production of human aFGF and bFGF using silkworm as host. These recombinant viruses were used to inoculate silkworm larvae. After the infection, the recombinant proteins were not found in the hemolymph. Such nonsecretion from cells has also been observed in the established insect cell lines, Sf21 and Tn-5. Tissue distribution analysis indicated that the expressed products were mainly located in fat body and the production of the recombinant aFGF and bFGF was maximal at around 80 h postinfection. Therefore, silkworm larvae infected with recombinant viruses were dissected and fat bodies were collected for the purification of recombinant aFGF and bFGF. The expression levels in both cases were estimated to be as high as approximately 600-700 microg per larva. Furthermore, the recombinant proteins were characterized and their biological activities were evaluated by in vitro bioassay using cell culture.

Adult↗

PubMed1994

The envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites.

We have previously isolated and characterized two dengue (DEN) 2 viruses mutant in their fusion-from-within (FFWI) phenotype in the insect cell line C6/36. Both viruses lost a potential glycosylation site (Asn-153) in the envelope (E) glycoprotein. To determine whether the change in FFWI phenotype was due to a change in E-glycoprotein glycosylation, we characterized the patterns of glycosylation on the E-glycoprotein of wild-type DEN 1 and DEN 2 viruses. The E-glycoproteins were isolated from purified virus grown in Aedes albopictus C6/36 cells, by use of high-performance size-exclusion chromatography. The tryptic maps of wild-type glycosylated and enzymatically (PNGase F) deglycosylated E-glycoproteins were compared by reverse-phase high-performance liquid chromatography. The DEN 1 virus E-glycoprotein was found to have two peaks in the tryptic map that exhibited shifts after deglycosylation, whereas the DEN 2 virus E-glycoprotein had only one. Besides the potential glycosylation site at Asn-153, both DEN 1 and DEN 2 virus E-glycoproteins have another potential site located at Asn-67. Amino-terminal sequencing of the shifted peaks revealed that DEN 2 virus E-glycoprotein is glycosylated only at Asn-67; however, DEN 1 virus E-glycoprotein is glycosylated at both Asn-67 and Asn-153. These DEN virus serotypes are thus heterogeneous in their use of glycosylation sites. We also determined by a lectin-binding assay that the attached carbohydrates for both viruses were likely to be of the high-mannose type.

Aedes↗

PubMed2004

Invertebrate humoral factors: cytokines as mediators of cell survival.

The presence and the different functional aspects of cytokine-related molecules in invertebrates are described. Cytokine-like factors affect immune functions, such as cell motility, chemotaxis, phagocytosis and cytotoxicity. In particular, cell migration shows a species-specific effect for IL-1alpha and TNF-alpha and a dose-correlated effect for IL-8, PDGF-AB and TGF-beta1. Apart from some exceptions, the phagocytic effect increases significantly at all the concentrations tested and with all the species used. PDGF-AB, TGF-beta1 and IL-8 provoke conformational changes in mollusk immunocytes, involving the signaling transduction pathways of phosphatidylinositol and cAMP. PDGF-AB and TGF-beta1 partially inhibit the induced programmed cell death in an insect cell line, and the survival effect is mediated by the activation of phosphatidylinositol 3-kinase, PKA and PKC. The exogenous administration of these growth factors in an invertebrate wound repair model showed that they are able to control the wound environment and promote the repair process by accelerating the coordinated activities involved. Moreover, IL-1alpha, IL-2 and TNF-alpha are able to induce nitric oxide synthase. PDGF-AB and TGF-beta1 provoke an increase in neutral endopeptidase-24.11 (NEP)-like activity in membrane preparations from mollusk immunocytes, while NEP deactivates the PDGF-AB- and TGF-beta1-induced cell shape changes. Cytokines are also involved in invertebrate stress response in a manner extremely similar to that in vertebrates. Several studies suggest the existence on the mollusk immunocyte membrane of an ancestral receptor capable of binding both IL-2 and CRH. Furthermore, the competition found between CRH and a large number of cytokines supports the idea that invertebrate cytokine receptors show a certain degree of promiscuity. The multiple functions of cytokines detected in invertebrates underline another characteristic of mammalian cytokines, i.e. their great pleiotropicity. Altogether, the studies on the function of the invertebrate humoral factors show a close overlapping with those found in vertebrates, and the hypothesized missing correlation between invertebrate and vertebrate cytokine genes that is emerging from the limited molecular biology data present in literature might represent a very peculiar strategy followed by Nature in the evolution of cytokines.

Animals↗

PubMed1993

Multiple conductance states of newly formed single gap junction channels between insect cells.

Two cells of an insect cell line (Aedes albopictus, clone C6/36) were pushed together to form a cell pair while the intercellular current flow was monitored. This approach enabled us to study the formation of gap junction channels and explore their electrical properties. We found that the single channels exhibit multiple conductance states. The conductance of a fully open channel was 365 pS; the subconductance steps were 1/7 to 1/5 of the maximal conductance. The voltage gradient across the junction did not influence the conductance of fully open channels, but affected the dwell time at particular conductance states. The latter provides an explanation for the voltage-dependent conductance of gap junction membranes seen in these cells. The very first channel opening always was slow (15-50 ms), suggesting the involvement of a mechanism different from conventional channel gating.

Aedes↗

PubMed1990

Chitin metabolism: a target for drugs against parasites.

Chitin is an important component of the exoskeleton of arthropods and of the egg shell in nematodes, but it does not occur in vertebrates. Therefore, it represents a useful target for drugs against ectoparasitic crustaceans, insects and endoparasitic nematodes. In this review we describe the basic characteristics of chitin, chitin synthesis and degradation and the hormonal regulation of chitin metabolism. Substances interfering with chitin metabolism like benzoylphenyl-urea derivatives but also some recently detected compounds are described. The necessity for a more detailed understanding of chitin metabolism and the establishment of better model systems, like e.g. chitin producing insect cell lines, is stressed and some examples are given in this review.

Animals↗

PubMed1986

Further characterization of an alkaline protease activity associated with iridescent virus type 6. Brief report.

Iridescent virus type 6 infecting Galleria mellonella larvae contained an associated alkaline protease activity which appeared to be tightly bound to the virions and essentially localized on the outside of the viral particle. Under alkaline conditions the virus was degraded by proteolytic cleavage of viral envelope proteins. Proteolytic activity was not present in virus propagated in a permissive insect cell line or when purified from isolated larval fat-bodies instead from whole larvae. The results suggest that the protease associated with Iridescent virus type 6 is of larval origin.

Cell Compartmentation↗

PubMed1987

Factors to consider in performing survival studies with insect cells.

Insect cell lines are not well-suited to colony formation in liquid medium following low-density cell plating. The present studies demonstrate that the time of addition of fetal bovine serum to the culture medium and the number of gamma-irradiated feeder cells added to each plate are important factors in developing a useful colony formation assay. TN-368 lepidopteran and WR69-DM-1 dipteran cell lines were used for these experiments. Both cell types display increased plating efficiencies if serum is added to the medium one or more days prior to plating as compared to adding serum immediately before plating. Growth curves obtained by seeding cells at higher densities also indicate that cell growth is slightly better if serum is added one or more days before seeding. These findings are especially important for survival and toxicity studies because the results demonstrate that even seemingly minor factors involved in cell survival assays may benefit treated cells to a greater degree than untreated control cells, thus providing an erroneous assessment of cell survival.

Animals↗