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PubMed1999

Real-time fluorimetric analysis of gramicidin D- and alamethicin-induced K+ efflux from Sf9 and Cf1 insect cells.

Gramicidin D and alamethicin are pore-forming peptides which exhibit lethal properties against a large spectrum of cells. Despite a wealth of experimental data from artificial membranes, the time course and quantitative analysis of the activity of these ionophores are not well described in living cells. In the present study, the newly described fluorescent dye CD-222 was used to monitor extracellular potassium ion concentration and report the effects of these antibiotics on the K+ permeability of the plasma membrane of Spodoptera frugiperda (Sf9) and Choristoneura fumiferana (Cf1) insect cells. Both peptides induced a rapid efflux of intracellular K+ as a consequence of ion channel formation in the cell membrane. K+ efflux began without any measurable delay. While the final extracellular K+ concentration was unaffected by ionophore concentration, the rate of K+ efflux was dose dependent. Using a model describing the partition of the peptides in lipid membranes, the K+ efflux kinetic parameters were determined for both cell types and both pore formers. The proposed stoichiometry for the channel formed by gramicidin in living cells is in good agreement with the two-monomers model based on data from artificial membrane systems. The K+-permeable channel formed by alamethicin in insect cells appears to involve three monomers.

Alamethicin↗
PubMed2000

Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells.

Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides. Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc. On the basis of these results and on the structure of an eight-cysteine domain from fibrillin-1, we present a model of glycosylated CR3 and discuss the role of glycosylation in eight-cysteine domain protein-protein interactions.

Amino Acid Sequence↗
PubMedDate not supplied

Osmolarity effects on observed insect cell size after baculovirus infection are avoided using growth medium for sample dilution.

Rates of cell size increase are an important measure of success during the baculovirus infection process. Batch and fed batch cultures sustain large fluctuations in osmolarity that can affect the measured cell volume if this parameter is not considered during the sizing protocol. Where osmolarity differences between the sizing diluent and the culture broth exist, biased measurements of size are obtained as a result of the cell osmometer response. Spodoptera frugiperda (Sf9) cells are highly sensitive to volume change when subjected to a change in osmolarity. Use of the modified protocol with culture supernatants for sample dilution prior to sizing removed the observed error during measurement.

Animals↗
PubMedDate not supplied

Cell cycle progression in serum-free cultures of Sf9 insect cells: modulation by conditioned medium factors and implications for proliferation and productivity.

Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (micro(N, max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining micro(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase micro(N,max), decrease the time to reach micro(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.

Animals↗
PubMedDate not supplied

Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture.

Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 10(5) cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of beta-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production.

Alkaline Phosphatase↗
PubMedDate not supplied

Purification and characterization of an anti-apoptotic protein isolated from Lonomia obliqua hemolymph.

Previously it was reported that supplementation of insect cell culture with Lonomia obliqua hemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58-63). In this work the anti-apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf-9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti-apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf-9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliqua hemolymph is due to the presence of potent anti-apoptotic factors.

Animals↗
PubMedDate not supplied

Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity.

The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Low passage (LP) cells at passages 20-45 were compared to high passage (HP) cells at passages >100. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. LP cells passed a switch in growth kinetics, characterized by a shorter lag phase and a higher growth rate, after 30-40 passages. After this point, CM lost its stimulating effect on proliferation. HP cells displayed a still shorter lag phase and reached the maximum cell density 24-48 earlier than LP cells. HP cells also exhibited higher specific productivity of recombinant protein compared to LP cells, when infected with baculovirus during the initial 48 h of culture. The specific productivity of LP cells was decreased by 30-50% by addition of 20% CM or 10 kDa filtrated CM, whereas addition of CM to cells having passed the switch in growth kinetics had no negative effect on productivity. Cell cycle analysis showed that a large proportion of HP cells, >60%, was transiently arrested in G2/M after inoculation. In LP cultures this proportion was lower, 40-45%, and addition of CM decreased the arrested population further. This correlated to the cell size, the HP cells being the largest: HP cells > LP > LP + 20% CM > LP + 20% 10 kDa filtrated CM. Since the degree of synchronization in G2/M correlated to the productivity, yeastolate limitation was used to achieve 85% G2/M synchronized cells. In this culture the specific productivity was maintained during a prolonged production phase and a 69% higher volumetric yield was obtained. The results suggest that a decreasing degree of synchronization during the course of culture partly explains the cell-density-dependent drop in productivity in Sf9 cells.

Animals↗
PubMedDate not supplied

Production of functional hepatocyte growth factor (HGF) in insect cells infected with an HGF-recombinant baculovirus in a serum-free medium.

Three insect cell lines, SL-7B cells derived from Spodoptera litura, Sf9, and High Five (Hi-5) cells, were used for the production of pro-hepatocyte growth factor (pro-HGF). Cells were cultured and then infected with a recombinant HGF-containing baculovirus in a serum-free medium. In SL-7B cells, pro-HGF is synthesized and excreted from the cells and late in infection is converted to a heterodimeric form of HGF even when the cells are grown in serum free medium. Conversion of a single-chain form of HGF (pro-HGF) into an HGF heterodimer was unexpected, as pro-HGF is normally cleaved by a serum protease called HGF activator. The proliferation activity of heparin-affinity-purified HGF from serum-free culture supernatant of SL-7B cells is comparable to that obtained from HGF converted by serum proteases, suggesting that SL-7B cells produce a functionally analogous protease to correctly process pro-HGF. This work reports, for the first time, on the feasibility of properly processing pro-HGF to form functional HGF by proteases from invertebrate cells in serum-free media. Avoiding the supplementation of sera provides the advantages of a low production cost, zero contamination of infectious agents from sera, and simple downstream product purification. Experimental results further demonstrate that the conversion of pro-HGF by insect cells is cell-line-dependent, because proteases in Hi-5 or Sf9 cells could not process pro-HGF as efficiently and properly as those in SL-7B cells.

Animals↗
PubMed2004

Selenium biotransformations in an insect ecosystem: effects of insects on phytoremediation.

Phytoremediation of selenium-contaminated soils may be influenced by higher trophic levels including insects. We examined how selenium affects the behavior, survival, and development of the wasp parasitoid Cotesia marginiventris, parasitizing its natural host, the beet armyworm Spodoptera exigua, feeding on alfalfa, Medicago sativa, irrigated with water containing selenate. X-ray absorption spectroscopy was used to quantify the selenium chemical forms in each trophic level. Alfalfa partially transformed selenate to organoselenium. S. exigua contained only organoselenium, both directly absorbed from M. sativa and transformed from selenate. C. marginiventris cocoons collected shortly after larval emergence contained only organoselenium derived from the host. The surprising finding of trimethylselenonium-like species in adult parasitoids and the cocoons from which they emerged suggests that adults and pharates can detoxify excess selenium through methylation and volatilization. Adult parasitoids do not discriminate against selenium-containing alfalfa, even though alfalfa generates selenium volatiles. Parasitoids raised on selenium-fed larvae emerged later and pupae weighed less than their selenium-free counterparts. We conclude therefore that C. marginiventris can be used to control S. exigua damage to M. sativa being used to remove selenium from soils. Moreover, the presence of such insects may improve phytoremediation by increasing biotransformation of inorganic selenium and release of volatile selenium species.

Animals↗
PubMed2002

Insect antifeedants from tropical plants II: structure of zumsin.

A novel A-seco limonoid was isolated from methanolic extract of Croton jatrophoides and designated as zumsin. This compound showed potent antifeedant activity against two lepidopteran larvae, pink bollworm, Pectinophora gossypiella (PC(50) = 1 microg/cm(2), PC(95) = 8 microg/cm(2)), and fall armyworm, Spodoptera frugiperda (PC(50) = 2 microg/cm(2), PC(95) = 16 microg/cm(2)). The structure of zumsin was determined as 1 using a variety of spectroscopic methods including nuclear magnetic resonance, mass spectrometry, and circular dichroism. The structure consists of an A'-B trans-fused ring while dumsin (2), a constituent of the same source, maintains an A'-B cis-fused ring, and suggests two unique biosynthetic processes after A ring oxidative expansion.

Animals↗
PubMed2002

Feeding deterrence and contact toxicity of Stemona alkaloids-a source of potent natural insecticides.

On the basis of chronic feeding bioassays with neonate larvae of Spodoptera littoralis reared on an artificial diet, the methanolic leaf and root extracts from Stemona collinsae displayed very high insect toxicity compared to those of two Aglaia species, a commercial Pyrethrum extract, and azadirachtin, whereas S. tuberosa extracts demonstrated low activity in roots and no activity in leaves. Beyond that, in leaf disk choice tests against fifth instar larvae, S. collinsae showed strong antifeedant activity, whereas S. tuberosa was characterized by remarkable repellency. The anti-insect properties of both species were based on pyrrolo[1,2-a]azepine alkaloids, from which didehydrostemofoline (asparagamine A) was the major compound of the roots of S. collinsae, exhibiting the highest toxicity in feeding assays. Saturation and hydroxylation of the side chain in the co-occurring stemofoline and 2'-hydroxystemofoline, respectively, led to an increasing loss of activity. Contact toxicity tests with stemofoline and didehydrostemofoline exhibited even higher activities than those of Pyrethrum extract. Tuberostemonine was the dominating alkaloid in the roots of S. tuberosa, showing outstanding repellency but no toxic effects.

Aglaia↗
PubMed2003

Antifeedant activity of some pentacyclic triterpene acids and their fatty acid ester analogues.

The 3-O-fatty acid ester derivatives (C(12)-C(18)) of two pentacyclic triterpenic acids, ursolic acid and oleanolic acid, were synthesized under mild esterification conditions in excellent yields (80-85%) and screened for their antifeedant activity, together with the parent acids, against the agricultural pest tobacco caterpillar larvae (Spodoptera litura F) in a no-choice laboratory study. The Urs-12-ene-28-carboxy-3beta-octadecanoate and olean-12-ene-28-carboxy-3beta-hexadecanoate were found to exhibit exceptionally potent antifeedant activities at 50 microg/cm(2) concentration, even after 48 h.

Animals↗
PubMed2003

6beta-hydroxygedunin from Azadirachta indica. Its potentiation effects with some non-azadirachtin limonoids in neem against lepidopteran larvae.

The biological activity of 6beta-hydroxygedunin isolated from Azadirachta indica A. Juss. was assessed using the gram pod borer, Helicoverpa armigera (Hubner), and Asian armyworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), alone and in combination with other limonoids, gedunin, salannin, nimbinene, and azadirachtin. The compound exhibited growth inhibitory activity in artificial diet bioassays, with 24.2 and 21.5 ppm, respectively, inhibiting growth by 50%. This efficacy was higher in comparison to gedunin (EC(50) = 50.8 and 40.4 ppm), salannin (EC(50) = 74.5 and 72.0 ppm), and nimbinene (EC(50) = 391.4 and 404.5 ppm). Azadirachtin, however, remained the most active neem allelochemical against both insect species. Nutritional assays clearly demonstrated that, though relative consumption and growth rates of fourth instar larvae were reduced, gedunin-type compounds induced physiological toxicity, evident by reduced efficiency of conversion of ingested food (ECI) in feeding experiments. Salannin and nimbinene, on the contrary, induced concentration-dependent feeding deterrence only. In feeding experiments, combinations of the compounds revealed that when azadirachtin was present in a mixture, EC(50) values did not deviate from the individual efficacy of azadirachtin (0.26 and 0.21 ppm, respectively) against H. armigera and S. litura larvae. However, a combination without azadirachtin did show a potentiation effect with potent EC(50) values among structurally different molecules, i.e., when salannin or nimbinene was combined with 6beta-hydroxygedunin or gedunin rather than structurally similar salannin + nimbinene or 6beta-hydroxygedunin + gedunin. Obviously, azadirachtin being the most active compound in neem is not synergized or influenced by any other limonoid, but other non-azadirachtin limonoids were more potent in specific combinations vis-à-vis the structural chemistry of the compound. It is obvious from the present study that potentiation among non-azadirachtin limonoids having explicitly two different modes of action, such as feeding deterrence and physiological toxicity, may be playing a significant role in the potentiation effect.

Animals↗
PubMed2002

Insect antifeedant activity of tetranortriterpenoids from the Rutales. A perusal of structural relations.

Structure-related insect antifeedant relationship of 56 limonoids (both natural and modified) from the plants belonging to the order Rutales was attempted considering substitution patterns, oxidation states, and hydrophobicity, as well as distant geometry derived through conformational analysis on molecular modeling. Orientation of the furan and hydroxylation at specific carbon sites have been shown to influence the antifeedancy against the fall armyworm, Spodoptera litura.

Animals↗
PubMed2004

Differentially enhanced insect resistance, at a cost, in Arabidopsis thaliana constitutively expressing a transcription factor of defensive metabolites.

A transgenic line of Arabidopsis thaliana constitutively expressing a conserved MYB transcription factor of phenylpropanoid biosynthesis resulting in solid-purple leaves had significantly increased resistance to leaf feeding by first instar fall armyworms (Spodoptera frugiperda), but no enhanced resistance to cabbage looper (Trichoplusia ni) larvae, when compared to wild type plants. However, inflorescence and silique (seed pod) production were significantly reduced by 22 and 52%, respectively, in the transgenic line compared to wild type plants. Reduction in feeding by S. frugiperda was significantly positively correlated with reduction in weights of survivors, but both were negatively correlated with the concentration of anthocyanins. These results indicate that a single gene regulator can activate a defensive pathway sufficient to produce increased resistance to insects but that this activation confers a cost in plant productivity.

Animals↗
PubMed2005

Insect repellent/antifeedant activity of 2,4-methanoproline and derivatives against a leaf- and seed-feeding pest insect.

2,4-methanoproline is a natural product isolated from the seeds of Ateleia herbert smithii Pittier that was formerly suggested to have insect repellent/antifeedant activity; however, this was not tested quantitatively. In this study the insect repellent/antifeedant potency of methanoproline was measured against larvae of the cotton leafworm, Spodoptera littoralis (Boisd.), and adults of the cowpea weevil, Callosobruchus maculatus (F.). In addition, several N-alkyl, amino, and nitrile derivatives of methanoproline with varying stereodemanding substituents were synthesized and also tested. It was shown that in S. littoralis methanoproline itself did not show any significant activity but that derivatives 5, 7, 8, and 10 did show a reasonable repulsive/antifeedant activity that was comparable to the commercial repellent DEET. A significant repellent activity was scored for methanoproline in adults of C. maculatus that was similar to DEET.

Animals↗
PubMed2004

Extremely potent antifeedant neo-clerodane derivatives of scutecyprol A.

Two known neo-clerodane diterpenoids, scutecyprol A (1) and scutalbin C (2), have been isolated from the acetone extract of the aerial parts of Scutellaria sieberi. The antifeedant activity of scutecyprol A (1), of its 15-oxo derivative (3), and of several halohydrins (4-9), synthesized starting from compounds 1 and 3, against Spodoptera littoralis have been determined and structure-antifeedant relationships are discussed.

Animals↗
PubMed2005

Antifungal and insect antifeedant 2-phenylethanol esters from the liverwort Balantiopsis cancellata from Chile.

A chemical study of a dichloromethane extract of Balantiopsis cancellata led to the isolation of four known 2-phenylethanol esters (1-4) and a phenylethanediol benzoate (5). Antifeedant activity toward Spodoptera littoralis (disk-choice bioassay) and growth inhibition of the phytopathogen Cladosporium herbarum in TLC-bioautography assays were determined. The results show that the antifeedant and antifungal activity of the extract is attributable mainly to the trans-beta-methylthioacrylate 4.

Animals↗