Anopheles gambiae odorant binding protein crystal complex with the synthetic repellent DEET: implications for structure-based design of novel mosquito repellents.

Insect odorant binding proteins (OBPs) are the first components of the olfactory system to encounter and bind attractant and repellent odors emanating from various sources for presentation to olfactory receptors, which trigger relevant signal transduction cascades culminating in specific physiological and behavioral responses. For disease vectors, particularly hematophagous mosquitoes, repellents represent important defenses against parasitic diseases because they effect a reduction in the rate of contact between the vectors and humans. OBPs are targets for structure-based rational approaches for the discovery of new repellent or other olfaction inhibitory compounds with desirable features. Thus, a study was conducted to characterize the high resolution crystal structure of an OBP of Anopheles gambiae, the African malaria mosquito vector, in complex with N,N-diethyl-m-toluamide (DEET), one of the most effective repellents that has been in worldwide use for six decades. We found that DEET binds at the edge of a long hydrophobic tunnel by exploiting numerous non-polar interactions and one hydrogen bond, which is perceived to be critical for DEET's recognition. Based on the experimentally determined affinity of AgamOBP1 for DEET (K (d) of 31.3 µΜ) and our structural data, we modeled the interactions for this protein with 29 promising leads reported in the literature to have significant repellent activities, and carried out fluorescence binding studies with four highly ranked ligands. Our experimental results confirmed the modeling predictions indicating that structure-based modeling could facilitate the design of novel repellents with enhanced binding affinity and selectivity.

Percutaneous bronchial artery embolization in the management of massive hemoptysis in chronic lung diseases. Immediate and long-term outcomes.

AIM: Bronchial artery embolization (BAE) is a well-established, non-surgical procedure in the emergency treatment of massive hemoptysis. This study aims to evaluate the immediate and long-term prognosis of BAE for the management of massive hemoptysis in our center. METHODS: Twenty consecutive patients (mean age: 59+/-14 years) with massive hemoptysis, underwent BAE with microspheres (Embospheres BioSphere Medical SA, Paris, France), polyvinyl alcohol particles (PVA, Ivalon, Cathmed Science; Paris, France) or/and steel coils (Cook, Denmark) after thoracic aortography and diagnostic selective and superselective catheterization of bronchial arteries and systemic collateral vessels in the bleeding lung area. Hemoptysis was due to bronchiectasis (55%), non-operable aspergillomas (15%), active tuberculosis (15%), malignancy (10%) and cystic fibrosis (5%). Mean duration of follow-up was 29+/-18 months. The recurrent-free time was calculated with Kaplan-Meier analysis. RESULTS: Immediate control of bleeding was achieved in all patients. Recurrent cases of hemoptysis were observed in 6/20 patients (30%) within 3 years and 4 of them (66.6%) occurred early in the first 3 months. Recurrent-free time was 9 months (standard error: 4) (95% confidence interval: 0-17). Repeated interventions were required in all early recurrences, due to either recanalization of the occluded arteries or non-bronchial systemic artery supply. Combined use of PVA and coils was proved effective in these cases. No serious complications were observed. CONCLUSION: BAE is an effective and safe intervention in cases of massive hemoptysis. However, recurrences are common and long-term follow-up is considered important with a view to perform repeated interventions with combination of embolic materials.

Specific interactions among odorant-binding proteins of the African malaria vector Anopheles gambiae.

In this report we present results from a comprehensive study undertaken toward the identification of proteins interacting with odourant-binding proteins (OBPs) of the African malaria vector Anopheles gambiae with a focus on the interactions among different OBPs. From an initial screen for proteins that interact with a member of the Plus-C group of OBPs, OBP48, which is primarily expressed in female antennae and downregulated after a blood meal, a number of interacting proteins were identified, which included five classic OBPs and OBP48 itself. The interacting OBPs as well as a number of other classic and Plus-C group OBPs that were not identified in the initial screen, were expressed in lepidopteran cells and subsequently examined for in vitro interactions in the absence of exogenously added ligands. Co-immunoprecipitation and chemical cross-linking studies suggest that OBP48 is capable of homodimerizing, heterodimerizing and forming higher order complexes with those examined examples of classical OBPs identified in the initial screen but not with other classical or Plus-C group OBPs that failed to appear in the screen. The latter OBPs are, however, also capable of forming homodimers in vitro and, at least in the case of two examined classic OBPs, heterodimers as well. These results suggest a previously unsuspected potential of nonrandom combinatorial complexity that may be crucial for odour discrimination by the mosquito.

A virus essential for insect host-parasite interactions encodes cystatins.

Cotesia congregata is a parasitoid wasp that injects its eggs in the host caterpillar Manduca sexta. In this host-parasite interaction, successful parasitism is ensured by a third partner: a bracovirus. The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The C. congregata bracovirus (CcBV) is injected at the same time as the wasp eggs in the host hemolymph. Expression of viral genes alters the caterpillar's immune defense responses and developmental program, resulting in the creation of a favorable environment for the survival and emergence of adult parasitoid wasps. Here, we describe the characterization of a CcBV multigene family which is highly expressed during parasitism and which encodes three proteins with homology to members of the cystatin superfamily. Cystatins are tightly binding, reversible inhibitors of cysteine proteases. Other cysteine protease inhibitors have been described for lepidopteran viruses; however, this is the first description of the presence of cystatins in a viral genome. The expression and purification of a recombinant form of one of the CcBV cystatins, cystatin 1, revealed that this viral cystatin is functional having potent inhibitory activity towards the cysteine proteases papain, human cathepsins L and B and Sarcophaga cathepsin B in assays in vitro. CcBV cystatins are, therefore, likely to play a role in host caterpillar physiological deregulation by inhibiting host target proteases in the course of the host-parasite interaction.

Functional expression of mammalian opioid receptors in insect cells and high-throughput screening platforms for receptor ligand mimetics.

Lepidopteran cell lines have been engineered to constitutively express high levels of mouse delta opioid receptors either alone or in combination with human Galpha16 protein. Biochemical and pharmacological studies demonstrate that these lines contain all the mediator G proteins and downstream effectors required for opioid receptor function, including phospholipase C, and that expression of exogenous Galpha16 does not contribute significantly to increased receptor responses upon activation. The activation of the phospholipase C pathway in the transformed cells upon stimulation with known receptor ligands results in easily and quantitatively measurable increases in free intracellular calcium, which can be monitored by automated fluorescent methods, while the addition of specific antagonists blocks the agonist-induced responses. Therefore, the transformed lepidopteran cell lines can be used as sensitive high-throughput screening platforms for fast detection of delta opioid receptor ligand mimetics (agonists and antagonists) in collections of natural products and synthetic compounds.

The orphan nuclear receptors BmE75A and BmE75C of the silkmoth Bombyx mori: hornmonal control and ovarian expression.

The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the stimulation of ovarian follicle development in the silkmoth, Bombyx mori. To understand better the mechanism by which 20E regulates silkmoth oogenesis, Bombyx homologs of the ecdysone-inducible orphan nuclear receptor E75 (BmE75) were cloned and their expression was analyzed in developing ovaries and staged follicles during metamorphosis. Of the two BmE75 isoforms isolated, only the A-isoform (BmE75A) has been identified previously in lepidopteran insects. BmE75C, on the other hand, shows significant sequence homology in its N-terminus to the Drosophila E75C isoform. Northern blot analysis shows unique expression patterns for each isoform mRNA during ovarian development. While the A-isoform seems to be mainly implicated in the earlier stages of the ecdysone response during previtellogenesis and vitellogenesis, expression of the C-isoform becomes strongly induced in an ecdysteroid-independent fashion at the transition from vitellogenesis to choriogenesis. Our data indicate a complex regulation of the expression of the BmE75 gene during oogenesis and postulate a new role for the BmE75C receptor at the end of vitellogenesis and the beginning of choriogenesis.

The orphan nuclear receptor BmHR3A of Bombyx mori: hormonal control, ovarian expression and functional properties.

Ovarian development in the domesticated silkmoth, Bombyx mori, is induced by the molting hormone 20-hydroxy-ecdysone (20E) shortly after larval-pupal ecdysis. Studies of the ecdysone response in Drosophila and other insects have shown that 20E exerts its effects initially by the induction of a small number of early genes, including the orphan nuclear receptors HR3, that transduce and amplify the hormone signal. Here we show that the silkmoth orphan receptor BmHR3A acts in the 20E-induced regulatory cascade in the ovary during pupal and pharate adult development in a manner different than that observed in the classical ecdysone regulatory hierarchy in Drosophila salivary glands at the end of the third instar. While other isoforms of BmHR3 are induced as early gene products in the ecdysone response, BmHR3A is induced 2 days after 20E administration in the silkmoth ovary and, thus, behaves as late product. The period of accumulation of BmHR3A in ovarian follicular cells occurs during vitellogenesis and coincides with the period of transcriptional expression of the ESP (egg-specific protein) gene, whose product constitutes a major component of the egg yolk, while it is reciprocal to the period of expression of BmGATAbeta, a gene encoding a regulator of late chorion gene expression. Bandshift experiments demonstrate that BmHR3A binds specifically to RORE (Retinoic acid-related Orphan receptor Response Element)-like sequences in the promoters of both genes, thus suggesting a direct role for BmHR3A in regulating the expression of BmGATAbeta and ESP genes during vitellogenesis. Finally, we show that BmHR3A functions as a constitutive transcriptional activator in a B. mori derived cell line. We propose that BmHR3A may function as a regulator of vitellogenesis in the silkmoth ovary.

Secretion of cytoplasmic and nuclear proteins from animal cells using novel secretion modules.

Production of recombinant proteins that are not secreted outside the producing cells usually requires purification steps that can result in significant yield reductions and loss of biological activity. Using insect cells as a model system to devise the means for secreting recombinant proteins that are not normally destined for secretion outside the producing cells, we initially examined the ability of an insect-specific signal peptide sequence to direct secretion of two intracellular proteins (the cytoplasmic enzyme chloramphenicol acetyl transferase [CAT] and the nuclear protein Bombyx mori chorion factor 1 [BmCF1]) expressed in transfected silkmoth cells. Although this signal sequence functioned efficiently as a chimera with normally secreted proteins, it failed to secrete CAT and BmCF1, suggesting that additional signals are required for passage of these polypeptides through the secretion pathway. For this reason, we also generated a secretion module consisting of the secreted protein juvenile hormone esterase (JHE), a spacer region containing a histidine tag and an endopeptidase cleavage site, to which coding sequences of choice can be cloned as C-terminal extensions. In C-terminal fusions with the CAT and BmCF1 open reading frames, the N-terminal JHE moiety was able to provide all the signals necessary for secretion of CAT and BmCF1 into the extracellular environment. The histidine tag present in the spacer region allowed purification of fusion proteins by metal affinity chromatography under nondenaturing conditions, and the enteropeptidase cleavage site was recognized and cleaved by the cognate protease causing the release of the intracellular proteins from the secretion module. We also show that another secreted protein, human granulocyte-macrophage colony stimulating factor (GM-CSF) can substitute for JHE in the secretion module and that these secretion modules can function in mammalian cells.

Mariner (Mos1) transposase and genomic integration of foreign gene sequences in Bombyx mori cells.

Widespread occurrence in insects and the capacity to transpose in the absence of host-derived factors means that mariner-like elements are considered to be attractive candidates for the development of a universal insect genetic transformation system. Here we show that the Mos1 mariner element of Drosophila mauritiana is capable of mediating excision and transposition events in a silkmoth (Bombyx mori) derived tissue culture cell line (Bm5 cells). Plasmid rescue assays, in combination with Southern hybridization and polymerase chain reaction (PCR) analyses, confirm that the Mos1 transposase can mediate excision of DNA sequences, inserted between terminally repeated sequences recognized by the transposase, and integration into the chromosomal DNA of the Bm5 cells. In addition to chromosomal integration events, inter- and intraplasmid transposition and target element excision events were also detected. Approximately 50% of the plasmids recovered from plasmid rescue assays were found to contain the 'signature' of Mos1-specific excision and/or integration events, indicating that the mariner transposase functions efficiently in the Bombyx cells. Because mariner-induced excision and integration events are strictly dependent on the presence of a co-transfected Mos1 transposase expression vector, it is clear that the multiple copies of endogenous mariner-like elements (Bmmar1) that exist in the Bombyx genome are neither functional nor do they interfere with the efficiency of the transposition process. Thus, the Mos1 element and, probably, mariner elements, in general, hold great promise for the development of genetic transformation systems for lepidopteran insects.

Determinants of the functional interaction between the soluble GM-CSF receptor and the GM-CSF receptor beta-subunit.

The GM-CSF receptor consists of a GM-CSF specific low affinity alpha-subunit (GMRalpha) and a beta-subunit (betac) that associates with GMRalpha in the presence of GM-CSF to form a high-affinity complex. A splice variant soluble isoform of GMRalpha (solalpha) consists of the extracellular domain of GMRalpha and a unique 16-amino acid C-terminal domain. Exogenously administered solalpha is unable to associate with betac on the cell surface either in the presence or absence of GM-CSF. However, paradoxically, co-expression of solalpha with betac results in the ligand-independent association of solalpha with betac on the cell surface via the C-terminal domain of solalpha. To study the interaction and functional characteristics of the solalpha-betac complex we engineered a soluble betac-subunit (ECDbeta) and expressed it alone and with solalpha. Co-expressed but not independent sources of solalpha and ECDbeta could be co-precipitated in the absence of ligand demonstrating the extracellular domain of betac was sufficient for association with solalpha upon co-expression. However, independent sources of solalpha could associate with ECDbeta in the presence of GM-CSF as could a C-terminal deficient solalpha mutant (ECDalpha) and the addition of ECDbeta to ECDalpha and GM-CSF was associated with a conversion from a low- to high-affinity ligand-receptor complex.

Minimal domain requirement for cation transport by the potassium-dependent Na/Ca-K exchanger. Comparison with an NCKX paralog from Caenorhabditis elegans.

The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient. Three mammalian rod NCKX cDNAs have been cloned to date, but quantitative analysis of NCKX function in heterologous systems has proven difficult. Here, we describe a simple system for quantitative analysis of NCKX function; stable transformation of cultured insect cells with the novel pEA1/153A vector containing NCKX cDNAs was combined with measurements of potassium-dependent (45)Ca uptake in sodium-loaded cells. We carried out structure-function studies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassium-dependent calcium transport and without affecting key properties of the potassium binding site; 2) the affinity of NCKX for potassium was about 10-fold greater in choline medium when compared with lithium medium; this shift was observed in rod outer segments or in cells expressing full-length rod NCKX, the above deletion mutant, or a distantly related NCKX paralog cloned from Caenorhabditis elegans. We conclude that the potassium binding site is highly conserved among members of the NCKX family and is formed by residues located within the two sets of transmembrane spanning segments in the NCKX sequence.

Use of flow cytometry to rapidly optimize the transfection of animal cells.

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.

Screening of transformed insect cell lines for recombinant protein production.

Nine insect cell lines were evaluated for their potential as host systems for recombinant protein production using a new expression vector permitting the continuous high-level expression of secreted glycoproteins by transformed insect cells (Farrell et al., 1998). As a means of preliminary screening, all nine insect cell lines were transfected with the green fluorescence protein. Growth in static and suspension culture was then examined as a further method of screening. On the basis of their transfection efficiencies and cell growth characteristics, five insect cell lines, Bm5, High Five, IPLB-LdFB, IZD-MB-0503, and Sf-21, were selected for stable transformation to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). These five cell lines were stably transformed using an antibiotic resistance scheme and evaluated as a polyclonal population. Increasing the antibiotic concentration was found to cause not only a decrease in the specific growth rate but also an increase in the specific protein production rate and final GM-CSF concentration. The transformed High Five cells exhibited by far the greatest specific protein production rate of 5.1 x 10(-)(6) microgram/(cell.h), resulting in the highest final GM-CSF concentration of 22.8 mg/L when grown in static culture. One cloned High Five cell line produced a GM-CSF concentration of 46 mg/L in static culture and 27 mg/L in suspension culture.

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.

The p95 gene of Bombyx mori nuclear polyhedrosis virus: temporal expression and functional properties.

As part of our effort to identify baculovirus proteins acting as transcriptional regulators, we have characterized a gene, p95, of Bombyx mori nuclear polyhedrosis virus (BmNPV) that encompasses an open reading frame for a putative 95-kDa polypeptide (P95). The N-terminal half of the conceptually translated P95 contains two zinc finger-type DNA-binding motifs, and its C terminus contains a proline-rich region reminiscent of transcriptional activation regions. Northern blot analysis indicates that two mRNA species, 3.5 and 1.7 kb in size, are transcribed from the p95 gene at different times postinfection. These two mRNA species are produced by differential polyadenylation site usage. While the longer transcript can encode the P95 protein, the shorter one may encode a prematurely terminated version of the P95 polypeptide produced by ribosome frameshifting occurring at heptanucleotide "slippage" sites located near the relevant polyadenylation site. Transcription of the p95 gene is initiated at a proximal site located 70 nucleotides upstream of the translation start codon of P95, a middle site located 170 nucleotides from the start codon, and a set of three closely spaced distal sites located 385, 390, and 409 nucleotides from the translation start codon. The middle and distant initiation sites are utilized before and after BmNPV DNA replication, while transcripts initiated at the proximal site occur largely during the late and very late stages of viral infection. Transient-expression assays indicate that P95 can stimulate gene expression driven by the promoter of its own gene and the promoter of the cytoplasmic actin gene of B. mori. The P95-mediated trans activation can be further augmented by BmIE1, an immediate-early gene product of BmNPV. In contrast to the case with the actin promoter, however, the promoter of the p95 gene can be trans activated by the product of its own gene only in the presence of BmIE1. Our data suggest that proteins P95 and BmIE1 of BmNPV and, by analogy, those of other baculoviruses may interact with each other and synergize to potentiate transcription.

The orphan receptor BmHNF-4 of the silkmoth Bombyx mori: ovarian and zygotic expression of two mRNA isoforms encoding polypeptides with different activating domains.

Two silkmoth nuclear receptor isoforms, BmHNF-4a and BmHNF-4b, that are related to the mammalian orphan receptor HNF-4, were characterized. Their characterization revealed that they differ from each other only in their 5' UTR and N-terminus of the predicted polypeptides. In ovarian tissue, the two receptors are expressed as a delayed response to 20-hydroxy-ecdysone and their expression increases during vitellogenesis. BmHNF-4 mRNA is localized in the cytoplasm of follicular cells and a binding activity that recognizes a mammalian HNF-4 response element is present in follicular cell nuclear extracts. BmHNF-4 mRNA is also present in the oocyte, the unfertilized egg and the early embryo, thus displaying a behavior reminiscent of maternal mRNA. Both mRNA isoforms are found in the embryo following fertilization and their abundance is modulated during ensuing embryogenesis. In contrast to the rather limited distribution of HNF-4 in mammalian tissues, BmHNF-4 is expressed in most larval and pharate adult tissues of the silkmoth.

High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector.

An expression cassette for continuous high-level expression of secreted glycoproteins by transformed lepidopteran insect cells has been developed as an alternative to baculovirus and mammalian cell expression systems. The expression cassette utilizes the promoter of the silkmoth cytoplasmic actin gene to drive expression from foreign gene sequences, and also contains the ie-1 transactivator gene and the HR3 enhancer region of BmNPV to stimulate gene expression. Using an antibiotic-resistance selection scheme, we have cloned a Bm5 (silkmoth) cell line overexpressing the secreted glycoprotein juvenile hormone esterase (JHE-KK) at levels of 190 mg/L in batch suspension cultures. A baculovirus (AcNPV) expressing the same gene under the control of the p10 promoter of AcNPV produced only 4 mg/L active JHE in static cultures of infected Sf21 cells. A cloned Bm5 cell line overexpressing a soluble isoform of the alpha-subunit of the granulocyte-macrophage colony stimulating factor receptor (solGMRalpha) was also generated and produced five times more solGMRalpha in static cultures than a cloned BHK cell line obtained by transformation with a recombinant expression cassette utilizing the human cytomegalovirus (CMV) enhancer-promoter system. Finally, we show that recombinant protein expression levels in transformed Bm5 cells remain high in serum-free media, that expression is stable even in the absence of antibiotic selection, and that lepidopteran cells other than Bm5 may be used equally efficiently with this new expression cassette for producing recombinant proteins.

Characterization of a domain of the genome of BmNPV containing a functional gene for a small capsid protein and harboring deletions eliminating three open reading frames that are present in AcNPV.

The nucleotide sequence of a 1.5-kb fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been determined. An open reading frame (ORF) has been identified, which encodes a protein of a predicted molecular mass of 15 kDa (p15). Analysis of the p15 sequence revealed that it shares significant similarities with a number of previously characterized viral capsid proteins. Two mRNAs, 0.7- and 1.3-kb, are transcribed from the p15 gene. The 0.7-kb transcript appears before BmNPV DNA replication, while the 1.3-kb transcript is transcribed only after viral DNA replication. The transcription start sites for the 0.7- or 1.3-kb transcripts have been localized into two areas mapping 114 and 557/559 bp upstream of the p15 translation initiation codon, respectively, and the two transcripts appear to share a common poly(A) addition site located 114 bp downstream of the translation termination codon. Comparisons of the nucleotide sequence of the 1.5-kb BmNPV fragment with that of another isolate of BmNPV and with Autographa californica NPV (AcNPV) genomic DNA reveal that the corresponding region of AcNPV encompasses 4.1 kb of DNA and three additional ORFs that are absent from the BmNPV genome. These findings raise questions about the origin and functional relevance of the putative polypeptides encoded by the three ORFs of AcNPV.

A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.