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PubMed1991

Replacement of vertebrate serum with lipids and other factors in the culture of invertebrate cells, tissues, parasites, and pathogens.

Culture medium supplementation with vertebrate serum results in the selection of fibroblastoid insect cell lines and a general decline during continuous subculturing of both morphologic and functional differentiation of the surviving cells. Essential lipid mixtures can substitute for vertebrate serum in the culture of insect and some vertebrate cells, tissues, parasites, and pathogens. The provision of sterols and essential (with nonessential) polyunsaturated fatty acids as phospholipids in oxidation-protected peptoliposomes or proteoliposomes allows cells in culture to duplicate in vivo specific membranes more accurately. Such lipid-corrected membranes allow cultured cells to communicate with neighboring cells through the extracellular matrix, effectively transmit hormonal signals directly and via receptor control, and respond with various tissue-specific functions and differentiation states as directed.

Animals↗

PubMed1987

Activation of an insect baculovirus promoter in mammalian cells by adenovirus functions.

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in insect cell lines in culture. In mammalian cells, however, the virus cannot be propagated. AcNPV DNA does not replicate or persist and is not transcribed in mammalian cells (Tjia et al., 1983). In insect cells productively infected with AcNPV, at least two major late viral gene products have been recognized, the polyhedrin, which makes up the bulk of the polyhedral inclusion bodies in infected cell nuclei, and a 10,000 Da protein (p10) of unknown function. The p10 promoter has been fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as a reporter gene (Knebel et al., 1985). Activity of this construct can be elicited in AcNPV-infected insect cells but not in uninfected insect cells or in mammalian cells. Presumably, the late p10 promoter requires other AcNPV gene products for activity. When the pAcp10-CAT construct is transfected into BHK21 hamster cells at about 18 h after infection with human adenovirus type 5 (Ad5), the insect AcNPV promoter is transactivated in cells of the heterologous mammalian species. The results of S1 protection analyses on the RNA from Ad5-infected and pAcp10-CAT transfected cells reveal that the p10 promoter is used for initiation of transcription. Similarly, the p10 insect virus promoter is activated in BHK21 hamster cells cotransfected with the HindIII-G fragment of adenovirus type 2 (Ad2) DNA which contains the E1A and parts of the E1B region in the left terminal 7.8% of the Ad2 genome. Moreover, in human 293 cells or in BHK297-C131 hamster cells, which both carry and constitutively express the E1 region of Ad5 DNA, the pAcp10-CAT construct is also expressed, and similarly in HE7 hamster cells which carry appreciable portions of the Ad2 genome (Klimkait and Doerfler, 1985). It is concluded that adenovirus functions are capable of transactivating a heterologous insect virus promoter in mammalian cells.

Adenoviridae↗

PubMed2001

3-Hydroxy-3-methylglutaryl coenzyme A synthase-1 of Blattella germanica has structural and functional features of an active retrogene.

Blattella germanica has two cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase genes, HMG-CoA synthase-1 and -2. HMG-CoA synthase-1 gene shows several features of processed genes (retroposons): it contains no introns but has a short direct-repeat sequence (ATTATTATT) at both ends. An atypical feature is the presence at both ends of the gene of short inverse repeats flanked by direct repeats. There is neither a TATA box nor a CAAT box in the 5' region. Comparative analysis with other species suggests that the HMG-CoA synthase-1 gene derives from HMG-CoA synthase-2. Cultured embryonic B. germanica UM-BGE-1 cells express HMG-CoA synthase-1 but not HMG-CoA synthase-2, suggesting that the intron-less gene is functional. In addition, it can complement MEV-1 cell line, which is auxotrophic for mevalonate. We show that compactin and mevalonate do not significantly affect the mRNA levels of HMG-CoA synthase-1 in UM-BGE-1 cells. Compactin induces a 6.7-fold increase in HMG-CoA reductase activity, which is restored to normal levels by mevalonate. HMG-CoA synthase activity is not modified by either of these effectors, suggesting that the mevalonate pathway in this insect cell line is regulated by post-transcriptional mechanisms affecting HMG-CoA reductase but not HMG-CoA synthase.

Animals↗

PubMed2001

Biosynthesis and subcellular localization of a lepidopteran insect alpha 1,2-mannosidase.

Like lower and higher eucaryotes, insects have alpha 1,2-mannosidases which function in the processing of N-glycans. We previously cloned and characterized an insect alpha 1,2-mannosidase cDNA and demonstrated that it encodes a member of a family of N-glycan processing alpha 1,2-mannosidases (Kawar, Z., Herscovics, A., Jarvis, D.L., 1997. Isolation and characterisation of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology 7, 433-443). These enzymes have similar protein sequences, require calcium for their activities, and are sensitive to 1-deoxymannojirimycin, but can have different substrate specificities and intracellular distributions. We recently determined the substrate specificity of the insect alpha 1,2-mannosidase, SfManI (Kawar, Z., Romero, P., Herscovics, A., Jarvis, D.L., 2000. N-glycan processing by a lepidopteran insect and 1,2-mannosidase. Glycobiology 10, 347-355). Now, we have examined the biosynthesis and subcellular localization of SfManI. We found that SfManI is partially N-glycosylated and that N-glycosylation is dramatically enhanced if the wild type sequon is changed to one that is highly utilized in a mammalian system. We also found that an SfManI-GFP fusion protein had a punctate cytoplasmic distribution in insect cells. Colocalization studies indicated that this fusion protein is localized in the Golgi apparatus, not in the endoplasmic reticulum or lysosomes. Finally, N-glycosylation had no influence over the substrate specificity or subcellular localization of SfManI.

Animals↗

PubMed1999

Transformed Lepidopteran insect cells: new sources of recombinant human tissue plasminogen activator.

Stable transformation was used to generate a cloned insect cell line (Bm5 silkmoth cells) over-expressing human tissue plasminogen activator (tPA). This cell line expressed 135 microg/mL single chain tPA in serum-free medium in static culture with a maximum specific activity of 120 IU/microg. In serum-containing medium, this line expressed 160 microg/mL of combined single-chain tPA, two-chain tPA, and a higher molecular weight SDS-stable tPA complex in suspension cultures with a maximum specific activity of 255 IU/microg. Approximately 100 copies of the tPA cDNA were randomly integrated into each Bm5 cell. For secretion of recombinant tPA from Bm5 cells, the native human tPA signal peptide is as effectively recognized as an insect specific signal peptide derived from a silkmoth chorion gene. Finally, stably transformed polyclonal populations of Bm5, High Five, and Sf21 cells expressing tPA were generated and compared for relative tPA expression.

Animals↗

PubMedDate not supplied

Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection.

Pluronic F-68 is a widely used protective agent in sparged animal cell bioreactors. In this study, the attachment-independent Spodoptera frugiperda Sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. First, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. Supplementing the medium with 0.2% (w/v) Pluronic F-68, however, fully protected the cells. Next, cell growth was compared in two airlift bioreactors with similar geometry but different sparger design; one of these bioreactors consisted of a thin membrane distributor, while the other consisted of a porous stainless steel distributor. The flow rates and bubble sizes were comparable in the two bioreactors. Supplementing the medium with 0.2% (w/v) Pluronic F-68 provided full protection to cells growing in the bioreactor with the membrane distributor but provided essentially no protection in the bioreactor with the stainless steel distributor. These results strongly suggest that cell damage can occur in the vicinity of the gas distributor. In addition, these results demonstrate that bubble size and gas flow rate are not the only important considerations of cell damage in sparged bioreactors. A model of cell death in sparged bioreactors is presented.

Animals↗

PubMed1988

Regulation of host RNA levels during baculovirus infection.

During infection of the permissive host insect cell line Spodoptera frugiperda IPLB-SF-21 by the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV), the levels of host actin, histone, and heat shock 70 (hsp70) RNAs are reduced substantially. Reduction of the host RNA levels occurs primarily during a narrow window of the replication process, from approximately 12 to 18 hr postinfection (p.i.), corresponding to the phase in which the extracellular form of the virus buds into the media. A late viral protein appears to be required for this reduction since cycloheximide, an inhibitor of cytosolic protein synthesis, and aphidicolin, an inhibitor of host and viral DNA polymerases, inhibit the reduction of actin and histone RNA levels. A cDNA corresponding to the carboxyl half of the S. frugiperda mitochondrial cytochrome c oxidase subunit III (COIII) gene was isolated, sequenced, and characterized. Two differentially regulated mitochondrial transcripts of this gene are observed. The level of the larger of these transcripts, which is dependent on active cytosolic protein synthesis, is reduced during virus infection in a fashion similar to that of the nuclear host genes. The smaller COIII transcript is stable until at least 24 hr p.i. but the level of this RNA eventually declines by 48 hr p.i.

Actins↗

PubMed2001

Functional Analysis of Helicase Gene Promoter and Homologous Region 3 Enhancer in Bombyx mori Nuclear Polyhedrosis Virus.

The promoter of the helicase gene, including 510 bp upstream of ATG,was cloned and sequenced, and was found that it had both early and late RNA initiation sites. The initiation codon ATG was deleted by using point mutation. Luciferase gene, as a reporter gene, was fused with the promoter region to construct the plsmid pBm hel 510 luc. When pBm hel 510 luc was transfected into Bm-5 and Sf-21 cell lines, the helicase gene promoter was recognized by cellular RNA polymerase and transactivated by viral factors. Baculovirus homologous regions (hrs) act as viral DNA replication start sites, which also have been shown to alter the rate of transcription for cis-linked promoters. BmNPV hr3 was cloned into a downstream site of luc gene, to study the effect of this enhancer on hel 510 promoter activity. The transient expression in transfected insect cell lines and silkworm larvae indicated that hr 3 could enhance the transcriptional level of hel 510 promoter by about 7 000 and 1 000 fold, respectively.

Journal Article↗

PubMed2000

A cell-line-specific defect in the intracellular transport and release of assembled retroviral capsids.

Retrovirus assembly involves a complex series of events in which a large number of proteins must be targeted to a point on the plasma membrane where immature viruses bud from the cell. Gag polyproteins of most retroviruses assemble an immature capsid on the cytoplasmic side of the plasma membrane during the budding process (C-type assembly), but a few assemble immature capsids deep in the cytoplasm and are then transported to the plasma membrane (B- or D-type assembly), where they are enveloped. With both assembly phenotypes, Gag polyproteins must be transported to the site of viral budding in either a relatively unassembled form (C type) or a completely assembled form (B and D types). The molecular nature of this transport process and the host cell factors that are involved have remained obscure. During the development of a recombinant baculovirus/insect cell system for the expression of both C-type and D-type Gag polyproteins, we discovered an insect cell line (High Five) with two distinct defects that resulted in the reduced release of virus-like particles. The first of these was a pronounced defect in the transport of D-type but not C-type Gag polyproteins to the plasma membrane. High Five cells expressing wild-type Mason-Pfizer monkey virus (M-PMV) Gag precursors accumulate assembled immature capsids in large cytoplasmic aggregates similar to a transport-defective mutant (MA-A18V). In contrast, a larger fraction of the Gag molecules encoded by the M-PMV C-type morphogenesis mutant (MA-R55W) and those of human immunodeficiency virus were transported to the plasma membrane for assembly and budding of virions. When pulse-labeled Gag precursors from High Five cells were fractionated on velocity gradients, they sedimented more rapidly, indicating that they are sequestered in a higher-molecular-mass complex. Compared to Sf9 insect cells, the High Five cells also demonstrate a defect in the release of C-type virus particles. These findings support the hypothesis that host cell factors are important in the process of Gag transport and in the release of enveloped viral particles.

Animals↗

PubMed2003

A cell-based high-throughput screening system for detecting ecdysteroid agonists and antagonists in plant extracts and libraries of synthetic compounds.

Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.

Animals↗

PubMed1993

Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells.

Deciphering the roles of the individual subunits of the heteromeric Na,K-ATPase in the structure, function, and assembly of this enzyme has been complicated because most expression systems have endogenous levels of Na,K-ATPase activity. This problem has become even more significant in light of the recent identification of multiple isoforms for both the alpha and beta subunits. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, affords two distinct advantages for the expression of the Na,K-ATPase; some insect cells have little or no levels of Na,K-ATPase, and baculovirus-infected cells produce extremely high levels of foreign protein. We have made two separate recombinant baculoviruses containing the rodent alpha 1 or beta 1 cDNAs and used them to infect the insect cell line Sf-9. The infected Sf-9 cells produce Na,K-ATPase subunit protein on the order of 5-10 micrograms of protein/ml of cultured cells. The rodent alpha 1 polypeptide produced in the Sf-9 cells is indistinguishable electrophoretically and antigenically from the native subunit. The expressed beta 1 subunit is also antigenically identical but has a higher electrophoretic mobility due to differential glycosylation by the infected Sf-9 cell. In contrast to other systems, when expressed alone, each individual Na,K-ATPase subunit is targeted to the infected Sf-9 plasma membrane. In contrast, when infected with a virus that induces the heavy chain of murine IgG, the infected Sf-9 cell retains the polypeptide in the endoplasmic reticulum. However, when both IgG light and heavy chains are expressed, the polypeptides are properly processed and secreted. When the Na,K-ATPase alpha 1 and beta 1 polypeptides are simultaneously expressed, they form detergent-resistant complexes that are functional. Ouabain-sensitive ATPase activity on the order of 5 mumol Pi/mg/h in infected Sf-9 membranes was dependent on the expression of both the alpha 1 and beta 1 subunits. Sodium-dependent phosphorylated intermediates were detected that were potassium- and ouabain-sensitive. No increase in ouabain-sensitive activity or phosphorylated intermediates was detected when either subunit was expressed alone. The alpha 1 beta 1-coinfected cells were also able to transport ions, as detected in 86Rb uptake experiments. Thus, the recombinant Na,K-ATPase expressed in insect cells is biologically active and is suitable for structural and functional analysis.

Animals↗

PubMed1999

Visualization of a recombinant gene protein in the baculovirus expression vector system using confocal scanning laser microscopy.

Expression of the recombinant protein beta-galactosidase in the Spodoptera frugiperda Sf-9 insect cell line infected by the Autographa californica nuclear polyhedrosis virus expressing beta-galactosidase (AcNPV-betagal) was visualized using confocal scanning laser microscopy with fluorescent staining of both the recombinant protein and the cell nucleus. The average size of the insect cells and the intracellular DNA concentration both increased markedly, respectively reading 3.8- and 2.3-fold the values before infection. The average beta-galactosidase activity began to increase at 20-24 h post infection and finally reached 1.9 x 10(4) units/ml. As the post infection time increased, the stained nucleus images expanded and spread broadly. Beta-galactosidase was first identified by fluorescent staining at 12 h post-infection, filled the cell at 27 h, began to be released at 36 h, and finally spread out of the cell. The locations of the nucleus and expressed beta-galactosidase were identified from computerized tomograms and 3-dimensional images.

Journal Article↗

PubMedDate not supplied

Two lepidopteran cell lines stably transformed by the abc transporter gene pdr5 show tolerance to diacetoxyscirpenol.

The pleiotropic drug resistance 5 gene (pdr5) encodes a multidrug membrane transporter and plays a very important role in the efflux of a broad range of chemicals in yeast cells. To study the possible function of pdr5 in insect cells, two stably pdr5-transformed lepidopteran insect cell lines, Sf21 and CF-203, were developed. Transcripts of pdr5 were detected in these two lines using Northern blotting and RT-PCR analysis. When cells were treated with the protein synthesis inhibitor diacetoxyscirpenol, the transformed Sf21 and CF-203 cell lines showed increased tolerance to this chemical. However, unlike in yeast cells, ecdysone agonist RH5992 could not be excluded by PDR5, probably because of low expression levels or imperfect incorporation of the recombinant protein in these transformed cell lines.

ATP-Binding Cassette Transporters↗

PubMed1994

Production and characterization of recombinant mouse brain-derived neurotrophic factor and rat neurotrophin-3 expressed in insect cells.

Bioactive brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were produced using the baculovirus expression system and purified to homogeneity using ion-exchange and reversed-phase chromatography. Yields of purified neurotrophin-3 (300-500 micrograms/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20-50 micrograms/L). Improved production of BDNF (150-200 micrograms/L) was achieved by expressing BDNF from a chimeric prepro-NGF/mature BDNF construct using the Trichoplusia ni insect cell line. Tn-5B1-4. Examination of the distribution of BDNF protein from both the non-chimeric prepro-BDNF and the chimeric prepro-NGF/mature BDNF viruses in Sf-21- and Tn-5B1-4-infected cells suggests a specific deficiency in the Tn-5B1-4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N-terminal amino acid sequencing of purified recombinant BDNF and neurotrophin-3 demonstrated that the insect cells processed their precursors to the correct N-terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS.

Animals↗

PubMed1998

Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells.

An established lepidopteran insect cell line (Sf9) was cotransfected with expression plasmids encoding neomycin phosphotransferase and bovine beta 1,4-galactosyltransferase. Neomycin-resistant transformants were selected, assayed for beta 1,4-galactosyltransferase activity, and the transformant with the highest level of enzymatic activity was characterized. Southern blots indicated that this transformed Sf9 cell derivative contained multiple copies of the galactosyltransferase-encoding expression plasmid integrated at a single site in its genome. One-step growth curves showed that these cells supported normal levels of baculovirus replication. Baculovirus infection of the transformed cells stimulated beta 1,4-galactosyltransferase activity almost 5-fold by 12 h postinfection. This was followed by a gradual decline in activity, but the infected cells still had about as much activity as uninfected controls as late as 48 h after infection and they were able to produce a beta 1,4-galactosylated virion glycoprotein during infection. Infection of the transformed cells with a conventional recombinant baculovirus expression vector encoding human tissue plasminogen activator also resulted in the production of a galactosylated end-product. These results demonstrate that stable transformation can be used to add a functional mammalian glycosyltransferase to lepidopteran insect cells and extend their N-glycosylation pathway. Furthermore, stably-transformed insect cells can be used as modified hosts for conventional baculovirus expression vectors to produce foreign glycoproteins with "mammalianized" glycans which more closely resemble those produced by higher eucaryotes.

Animals↗

PubMed1986

Structurally related Bacillus thuringiensis delta-endotoxins display major differences in insecticidal activity in vivo and in vitro.

Many strains within the 22 serotypes of Bacillus thuringiensis produce crystal delta-endotoxins with slight differences in their insecticidal toxicity spectrum in vivo. Since the basis of this specificity is unknown, we chose to compare the activity of delta-endotoxins from three strains: B. thuringiensis var. kurstaki HD-1, var. aizawai HD-249 and var. thuringiensis HD-350, both in vivo and on insect cell lines in vitro. Immunoblotting with antisera to activated var. kurstaki P1 lepidopteran toxin revealed antigenic cross-reaction with the 130 X 10(3) Mr toxin of var. aizawai, and with polypeptides of 130 and 138 (X 10(3)) Mr from var. thuringiensis. In addition, crystals from var. kurstaki and var. aizawai contained an antigenically related 63 X 10(3) Mr protein that did not cross-react with antisera to the 130 X 10(3) Mr component. Bioassays on Pieris brassicae larvae (Lepidoptera) and Aedes aegypti larvae (Diptera) indicated that the 130 X 10(3) Mr protein of var. kurstaki, and the 138 plus 130 X 10(3) Mr components of var. thuringiensis killed only P. brassicae, while the 130 X 10(3) Mr protein of var. aizawai and the 63 X 10(3) Mr proteins of var. aizawai and var. kurstaki were toxic to both P. brassicae and A. aegypti. Activation of the 130 and 138 (X 10(3)) Mr proteins of the three varieties of B. thuringiensis with insect gut proteases yielded active products of 50-60 (X 10(3)) Mr. Assay of these products on a range of lepidopteran and dipteran cell lines revealed very different toxicity spectra: var. kurstaki killed only one lepidopteran line, var. thuringiensis killed two lepidopteran lines, while var. aizawai was cytolytic to all of the lepidopteran and most of the dipteran cell lines tested, reflecting its broader spectrum in vivo. Thus we have shown that antigenic cross-reaction of B. thuringiensis delta-endotoxins does not necessarily imply a similar toxicity spectrum in vivo or in vitro.

Bacillus thuringiensis↗

PubMed2000

Biochemical characterization of CD39L4.

Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components in these physiological systems. CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.

Adenosine Triphosphatases↗

PubMed1986

Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases.

The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect-specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines.

Animals↗