Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists such as fenofibrate are used to treat dyslipidemia. Although fenofibrate is considered safe in humans, it is known to cause hepatocarcinogenesis in rodents. To evaluate untargeted metabolic profiling as a tool for gaining insight into the underlying pharmacology and hepatotoxicology, Fischer 344 male rats were dosed with 300 mg/kg/day of fenofibrate for 14 days and the urine and plasma were analyzed on days 2 and 14. A combination of liquid and gas chromatography mass spectrometry returned the profiles of 486 plasma and 932 urinary metabolites. Aside from known pharmacological effects, such as accelerated fatty acid beta-oxidation and reduced plasma cholesterol, new observations on the drug's impact on cellular metabolism were generated. Reductions in TCA cycle intermediates and biochemical evidence of lactic acidosis demonstrated that energy metabolism homeostasis was altered. Perturbation of the glutathione biosynthesis and elevation of oxidative stress markers were observed. Furthermore, tryptophan metabolism was up-regulated, resulting in accumulation of tryptophan metabolites associated with reactive oxygen species generation, suggesting the possibility of oxidative stress as a mechanism of nongenotoxic carcinogenesis. Finally, several metabolites related to liver function, kidney function, cell damage, and cell proliferation were altered by fenofibrate-induced toxicity at this dose.
Skeletal muscles undergo progressive atrophy and pathologic remodeling with age. To better understand muscle senescence, we used metabolomic profiling to characterize ‘adult’ (15‐month‐old; n=8) and ‘old’ (32‐month‐old; n=8) FBN male rats. Gastrocnemius (gastroc) and soleus muscles were analyzed, as well as plasma. Compared to adult gastroc, old gastroc showed evidence of altered glucose metabolism, including accumulation of glycolytic, glycogenolytic, and pentose phosphate pathway intermediates. Pyruvate was elevated with age, yet tricarboxylic acid (TCA) cycle intermediates were reduced, and nicotinamide adenine dinucleotide was reduced 82% (p<0.0001). Indicative of muscle atrophy, 3‐methylhistidine and free amino acids were elevated in old gastroc. The monounsaturated fatty acids oleate, cis‐vaccenate, and palmitoleate, also increased in old gastroc. Old soleus showed reductions in glycolytic and TCA cycle intermediates, and 39% (p<0.0001) reduction in flavin adenine dinucleotide. Age‐related plasma biomarkers showing the largest % increases included glycocholate (218%), heme (122%), 1,5‐anhydroglucitol (119%), 1‐palmitoleoyl‐glycerophosphocholine (95%), palmitoleate (64%), and creatine (53%). These changes are consistent with reduced insulin sensitivity in aging. In sum, energetic dysfunction appears central to pathological metabolic processes in aging skeletal muscle.
Summary Spike mosses ( Selaginellaceae ) represent an ancient lineage of vascular plants in which some species have evolved desiccation tolerance (DT). A sister‐group contrast to reveal the metabolic basis of DT was conducted between a desiccation‐tolerant species, Selaginella lepidophylla , and a desiccation‐sensitive species, Selaginella moellendorffii , at 100% relative water content (RWC) and 50% RWC using non‐biased, global metabolomics profiling technology, based on GC/MS and UHLC/MS/MS 2 platforms. A total of 301 metabolites, including 170 named (56.5%) and 131 (43.5%) unnamed compounds, were characterized across both species . S. lepidophylla retained significantly higher abundances of sucrose, mono‐ and polysaccharides, and sugar alcohols than did S. moellendorffii . Aromatic amino acids, the well‐known osmoprotectant betaine and flavonoids were also more abundant in S. lepidophylla . Notably, levels of γ‐glutamyl amino acid, linked with glutathione metabolism in the detoxification of reactive oxygen species, and with possible nitrogen remobilization following rehydration, were markedly higher in S. lepidophylla . Markers for lipoxygenase activity were also greater in S. lepidophylla , especially at 50% RWC. S. moellendorffii contained more than twice the number of unnamed compounds, with only a slightly greater abundance than in S. lepidophylla . In contrast, S. lepidophylla contained 14 unnamed compounds of fivefold or greater abundance than in S. moellendorffii , suggesting that these compounds might play critical roles in DT. Overall, S. lepidophylla appears poised to tolerate desiccation in a constitutive manner using a wide range of metabolites with some inducible components, whereas S. moellendorffii mounts only limited metabolic responses to dehydration stress.
<p>Supplementary Figure 3. HEK293 cells were transiently transfected with either CV or L2HGDH cDNA and TET1 cDNA. Protein lysates were harvested and immunoblotted for the indicated proteins.</p>
Both the bacterium Photorhabdus luminescens alone and its symbioticPhotorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity ofPhotorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD50) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity. Both the bacterium Photorhabdus luminescens alone and its symbioticPhotorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity ofPhotorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD50) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity. Southern corn rootworm polyacrylamide gel electrophoresis matrix-assisted laser desorption ionization time of flight Entomopathogenic nematodes of the genera Steinernemaand Heterorhabditis have been used for the biological control of soil dwelling pests that include weevils and lepidopteran species (1Klein M.G. Gaugler R. Kaya H.K. Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, FL1990: 195-214Google Scholar, 2Qin X.X. Kao R.T. Yang H.W. Zhang G.Y. For. Res. 1988; 1: 179-185Google Scholar, 3Wang J.X. Li L.Y. Rev. Nematol. 1987; 10: 483-489Google Scholar). Strong et al. (4Strong D.R. Maron J.L. Connors P.G. Whipple A. Harrison S. Jefferies R.L. Oecologia. 1995; 104: 85-92Crossref PubMed Scopus (107) Google Scholar, 5Strong D.R. Kaya H.K. Whipple A.L. Child S. Kraig S. Bondonno M. Dyer K. Maron J.L. Oecologia. 1996; 108: 167-173Crossref PubMed Scopus (89) Google Scholar) have provided the best illustration of entomopathogenic nematode predation in the environment by their documentation of the ecological relationship betweenHeterorhabditis hepialus and the ghost moth caterpillar,Hepialus californicus. The mechanism by which the entomopathogenic nematodes are able to predate and reproduce in the host involves a mutualistic relationship between the nematode and its symbiotic bacteria, Photorhabdus sp. andXenorhabdus sp. (6Akhurst R.J. Dunphy G.B. Beckage N. Thompson S. Federici B. Entomopathogenic Nematodes in Biological Control. 2. Academic Press, Inc., New York1993: 75-87Google Scholar, 7Forst S. Nealson K. Microbiol. Rev. 1996; 60: 21-43Crossref PubMed Google Scholar). Bovien (8Bovien P. Vidensk. Medd. Dan. Naturhist. Foren. Khobenhavn. 1937; 101: 1-114Google Scholar) had postulated an association between a steinernematid species and a bacterium during the 1930's. 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Recently, increased support for the separation of these two genera was obtained employing 16 S ribosomal DNA analysis (13Szallas E. Koch C. Fodor A. Burghardt J. Buss O. Szentirmai A. Nealson K. Stackedbrandt E. Int. J. Syst. Bacteriol. 1997; 47: 402-407Crossref PubMed Scopus (52) Google Scholar). Photorhabdus is represented by a single species,Photorhabdus luminescens, so named because of its bioluminescent nature. This phenotype is unique among the Enterobacteriaceae and other bacteria of terrestrial origin. Once an infective juvenile nematode has penetrated the host hemocoel, the bacterial symbiont is released from the nematode gut, septicemia becomes established, and insect death occurs within 48 h. Although the nematodes may play a role in insect death, in most cases the bacteria alone are sufficient to cause insect mortality following injection into the hemocoel (14Gotz P. Boman A. Boman H.G. Proc. R. Soc. Lond. 1981; 212: 333-350Google Scholar, 15Balcerzak M. Acta Parasitol. Pol. 1991; 36: 175-181Google Scholar). The importance of these bacteria in the life cycle of the nematode has been well documented using axenically reared nematodes (14Gotz P. Boman A. Boman H.G. Proc. R. Soc. Lond. 1981; 212: 333-350Google Scholar, 16Gerritsen L.J.M. Smits P.H. Fundam. Appl. Nematol. 1993; 16: 367-373Google Scholar). Together with the lack of evidence for the free living existence of this bacterium, it has been postulated that the symbiotic association is essential for the survival of both nematode and its symbiotic bacteria. However, one possible exception is the report by Farmer et al. (17Farmer III, J.J. Jorgensen J.H. Grimont P.A. Akhurst R.J. Poinar Jr., G.O. Ageron E. Pierce G.V. Smith J.A. Carter G.P. Wilson K.L. Hickman-Brenner F.W. J. Clin. Microbiol. 1989; 27: 1594-1600Crossref PubMed Google Scholar) who isolatedP. luminescens strains from clinical samples with no apparent nematode association (18Boemare N. Laumond C. Mauleon H. Biocon. Sci. Technol. 1996; 6: 333-345Crossref Scopus (97) Google Scholar). The precise set of mechanisms by which the symbiotic bacterium is able to circumvent the defense host systems in the hemocoel is still under investigation. It has been suggested that the virulence events that lead to bacterial proliferation could involve multiple factors, such as secretion of lipases and proteases, the release of lipopolysaccharide molecules, and the anti-hemocytic properties of the bacterial cell surface (19Akhurst R.J. Boemare N.E. Gaugler R. Kaya H. Entomopathogenic Nematodes in Biological Control. CRC Press Inc., Boca Raton, FL1990: 75-87Google Scholar, 20Nealson K.H. Schmidt T.M. Bleakley B. Gaugler R. Kaya H.K. Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, FL1990: 271-284Google Scholar, 21Dunphy G.B. Webster J.M. J. Gen. Microbiol. 1991; 134: 1017-1028Google Scholar, 22Jarosz J. Balcerzak M. Skrzypek H. Entomophaga. 1991; 36: 361-368Crossref Scopus (10) Google Scholar, 23Dunphy G.B. Webster J.M. J. Invertebr. Pathol. 1991; 58: 40-51Crossref Scopus (83) Google Scholar, 24Schmidt T.M. Bleakley B. Nealson K.H. Appl. Environ. Microbiol. 1988; 54: 2793-2797Crossref PubMed Google Scholar, 25Akhurst R.J. Dunphy G. Beckage N. Thompson S. Federici B. Parasites and Pathogens of Insects. 2. Academic Press, New York1993: 1-23Google Scholar, 26Akhurst R.J. Bedding R. Akhurst R. Kaya H. Nematodes and the Biological Control of Insect Pests. CSIRO Publications, Melbourne, Australia1993: 127-136Google Scholar, 27Milstead J.E. J. Invertebr. Pathol. 1979; 33: 324-327Crossref Scopus (30) Google Scholar, 28Poinar Jr., G.O. Thomas G.M. J. Invertebr. Pathol. 1967; 9: 510-514Crossref Scopus (46) Google Scholar). Recently, Clarke and Dowds (29Clarke D.J. Dowds B.C.A. J. Invertebr. Pathol. 1995; 66: 149-155Crossref Scopus (53) Google Scholar) speculated that a secreted lipase is responsible for the insecticidal activity observed against Galleria mellonella. In addition to secreted factors, these bacteria are known to produce intracellular inclusion bodies similar to those produced by Bacillus thuringiensis (30Couche G. Gregson R. J. Bacteriol. 1987; 169: 5279-5288Crossref PubMed Google Scholar, 31Bowen D. Ensign J. Proc. Mtg. Am. Soc. Microbiol. 1987; 183: I-66Google Scholar). But unlike Bt endotoxin crystals they are not insecticidal and are thought to provide amino acid nutrients for the emerging nematodes (32Bintrim S.B. Ensign J.C. J. Bacteriol. 1998; 180: 1261-1269Crossref PubMed Google Scholar). In other studies, Bowen (33.Bowen, D., Characterization of a High Molecular Weight Insecticidal Protein Complex Produced by the Entomopathogenic Bacterium Photorhabdus luminescens.Ph.D. thesis, 1995, University of Wisconsin, Madison.Google Scholar) reported that a soluble protein fraction derived from P. luminescensculture medium possessed sufficient insecticidal activity to killManduca sexta upon injection. As part of our efforts to find suitable insecticidal proteins that could be employed to produce insect-resistant plants, we initiated a study to further characterize the nature of the oral insecticidal activity. It was found that ofP. luminescens W-14 fermentation broth showed excellent potency against Southern corn rootworm (SCR)1 neonates, a surrogate maize pest. We describe here the isolation and characterization of two distinct but structurally similar protein toxins that are highly potent against SCR larvae. We also show that toxin could be further processed and activated by protease cleavage. Stock inoculum of P. luminescens strain W-14 (ATCC accession number 55397) was produced by inoculating 175 ml of 2% Proteose Peptone number 3 (PP3) (Difco) liquid media with a primary variant subclone in a 500-ml flask and incubated for 16 h at 28 °C on a rotary shaker at 150 rpm. The production broth was achieved by inoculation of 1.75 ml of the stock inoculum into fresh PP3 medium in 500-ml flasks (175 ml of culture/flask). After inoculation, the culture was incubated at 28 °C for 24 h as above. Following incubation, the broth was centrifuged at 2,600 × g for 1 h at 10 °C and vacuum filtered through Whatman GF/D (2.7 μm) and GF/B (1 μm) glass filters to remove debris. The broth was then used for the studies described here. Protein fractions were diluted into 10 mm sodium phosphate buffer, pH 7.0, and applied directly in 40-μl aliquots to diet plate wells (surface area 1.5 cm2) containing artificial diet (34Rose R.I. McCabe J.M. J. Econ. Entomol. 1973; 66: 398-400Crossref Google Scholar). The diet plate was then allowed to air dry in a sterile flow-hood. The wells were then infested with single, neonate Diabrotica undecimpunctate howardi (Southern corn rootworm) hatched from surface sterilized eggs. The plates were sealed, placed in a humidified growth chamber, and maintained at 27 °C for 3–5 days. Mortality was then scored. For quantitation of toxin potency, 16 insects per toxin dose were used, and assays were repeated 2–4 separate times. LC50 was determined using the toxin concentration needed to cause 50% insect mortality. Unless noted, the isolation protocol entailed starting with 5 liters of broth that was concentrated using an Amicon (Beverly, MA) spiral ultrafiltration cartridge Type S1Y100 (molecular mass cut off 100 kDa) attached to an Amicon M-12 filtration device. The retentate was diafiltered with 10 mm sodium phosphate, pH 7.0 (Buffer A), until the absorbance of filtrate at 280 nm was below 0.1. The retentate was then applied at 5 ml/min to a Poros 50 HQ (PerSeptive Biosystem, Framingham, MA) strong anion exchange column (1.6 × 15 cm). The column was washed with 5 bed volumes of Buffer A and then eluted with 0.4m NaCl in Buffer A. The biologically active fraction, determined by SCR bioassays, was loaded in 20-ml aliquots onto a gel filtration column Sepharose CL-4B (2.6 × 100 cm) which was equilibrated with Buffer A. The protein was eluted in Buffer A at a flow rate of 0.75 ml/min. The biologically active fractions were pooled and applied to a Mono-Q 10/10 column equilibrated with 20 mm Tris-HCl, pH 7.0 (Buffer B), at a flow rate of 1 ml/min. The column was washed with Buffer B until the optical density at 280 nm returned to baseline level. The proteins bound to the column were eluted with a linear gradient of 0 to 1m NaCl in Buffer B at 2 ml/min for 60 min. Two-ml fractions were collected and activity was determined by a dilution series of each fraction in the bioassay. Two activity peaks against SCR were observed and were named A (fraction 14 to 19) and B (fraction 20 to 25). Activity peaks A and B were pooled separately and both peaks were further purified using the procedure described below. Solid (NH4)2SO4 was added to the above protein fractions containing either toxin A or B to a final concentration of 1.7 m. Proteins were then applied to a phenyl-Superose 10/10 column equilibrated with 1.7 m(NH4)2SO4 in 50 mmpotassium phosphate buffer, pH 7 (Buffer C), at 1 ml/min. After washing the column with 10 ml of Buffer C, proteins bound to the column were eluted with a linear gradient of 1.7 m(NH4)2SO4, 50 mmpotassium phosphate, pH 7.0, to 25% ethylene glycol, 25 mmpotassium phosphate, pH 7.0, at 1 ml/min for 120 min. Fractions were dialyzed overnight against Buffer A. The most active factions were pooled and applied to a Mono Q 5/5 column which was equilibrated with Buffer B at 1 ml/min. The proteins bound to the column were eluted at 1 ml/min by a linear gradient of 0 to 1m NaCl in Buffer B. For the final step of purification, the most biologically active fractions were pooled, brought to a final concentration of 1.7m with solid (NH4)2SO4, and applied to a phenyl-Superose 5/5 column equilibrated with Buffer C at 1 ml/min. Proteins bound to the column were then eluted with a linear gradient of Buffer C to 10 mm potassium phosphate, pH 7.0, at 0.5 ml/min for 60 min. Fractions were dialyzed overnight against Buffer A. The final purified protein from activity peak A and activity peak B from the Mono Q 10/10 column was named toxin A and toxin B, respectively. The purified toxins were found stable at 4 °C for over 6 months and were used for all the characterizations reported in this paper. The native molecular mass was estimated by gel filtration on either a Superdex 200 HR 10/30 column or a Sepharose CL-4B column (1.6 × 50 cm) as specified in each experiment. The columns were calibrated with a mixture of known molecular mass standards. Protein concentrations were determined according to the method of Bradford (35Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with bovine serum albumin as standard. Native and SDS-PAGE analyses were performed on either 10% or 4–20% gradient gels (36Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Western blotting was performed using ECL Western blotting detection reagent according to the manufacturer's instructions (Amersham). The purified toxin A and toxin B, as well as partially purified active gel filtration fractions, were separated on a 10% SDS-PAGE gel and blotted to a Bio-Rad polyvinylidene difluoride membrane according to the manufacturer's procedure. The protein bands were localized by staining with Amido Black for 1 min (0.1% Amido Black 10 B (Sigma) in 10% acetic acid) followed by destaining for 1 min with 5% acetic acid. Blots were sent for NH2-terminal sequencing at Cambridge ProChem (Lexington, MA). NH2-terminal sequences are described under "Results." Protein molecular mass using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy was determined on a Voyager Biospectrometry workstation with delayed extraction technology (PerSeptive Biosystems, Framingham, MA). Typically, the protein of interest (100–500 pmol in 5 μl) was mixed with 1 μl of acetonitrile and dialyzed for 0.5 to 1 h on a Millipore VS filter having a pore size of 0.025 μm (Millipore Corp., Bedford, MA). Dialysis was performed by floating the filter on water followed by adding the protein/acetonitrile mixture as a droplet to the filter surface. After dialysis, the dialyzed protein was removed using a pipette and then mixed with a matrix consisting of sinapinic acid and trifluoroacetic acid according to the manufacturers' instructions. The protein and matrix (4 μl total volume) were allowed to co-crystallize on a ∼3 cm (2Qin X.X. Kao R.T. Yang H.W. Zhang G.Y. For. Res. 1988; 1: 179-185Google Scholar) gold-plated sample plate (PerSeptive Biosystems). Excitation of the crystals and subsequent mass analysis was performed using the following conditions: laser setting of 3050, pressure of 4.55e-07, low mass gate of 1500.0, negative ions off; accelerating voltage of 25,000, grid voltage of 90.0%, guide wire voltage of 0.010%, linear mode, and a pulse delay time of 350 ns. The genes for toxin A and toxin B described in this paper were subsequently cloned (gene cloning to be presented elsewhere). Unique gene sequences were selected for peptides A1, A2, and B2 (peptides to be described under "Results"). The following peptides were synthesized according to the deduced amino acids sequences: NPNNSSNKLMFYPVYQYSGNT (for peptide A1), VSQGSGSAGSGNNNLAFGAG (for peptide A2), and FDSYSQLYEENINAGEQRA (peptide B2). The corresponding antibodies to the above three peptides were generated in Genemed Biotechnology Inc. (San Francisco, CA). The crude sera were purified using a SulfoLinkTM Coupling Gel column (Pierce). Each column was prepared by immobilizing a specific peptide to the gel following the protocol described by the manufacturer. The respective antisera was applied to the column at the rate of 0.5 ml/min. The column was subsequently washed with phosphate-buffered saline, pH 7.6. The purified antibodies were eluted from the column using 0.05 m sodium acetate, pH 3.0, and they were immediately neutralized to pH 7.0 with 1 m Tris, pH 8.0. The three purified peptide-specific antibodies were named synA1Ab, synA2Ab, and synB2Ab, and specifically recognized peptide A1, A2, and B2, respectively. The standard reaction consisted of 40 μg of purified P. luminescens W-14 toxin B protease as specified under "Results," and 0.1 m Tris buffer, pH 8.0, in a total volume of 100 μl. For control reactions, protease was omitted. The reaction mixtures were incubated at 37 °C overnight. At the end of the reaction, 10 μl was removed and then boiled with an equal volume of 2 × SDS-PAGE sample buffer for SDS-PAGE analysis. The remaining 90 μl of reaction mixture was serially diluted with 10 mm sodium phosphate buffer, pH 7.0, and analyzed by SCR bioassay. The fermentation broth of P. luminescensW-14 strain presented a broad spectrum of oral activity against a variety of insects (data not shown), including SCR. Because of our interest in controlling corn rootworm in maize, SCR bioassays were used to follow the Photorhabdus insecticidal activity in this study. Consistent with prior studies by Ensign (33.Bowen, D., Characterization of a High Molecular Weight Insecticidal Protein Complex Produced by the Entomopathogenic Bacterium Photorhabdus luminescens.Ph.D. thesis, 1995, University of Wisconsin, Madison.Google Scholar), it was found that the majority of the activity was retained upon extensive dialysis or upon concentration with devices containing 100-kDa molecular mass filters, indicating that the majority of activity was associated with large molecular mass material. Because of the large size, an Amicon M-12 filtration unit equipped with a 100-kDa membrane was used to enrich activity from 5 to 10 liters of broth. Following a single step elution from an ion-exchange column, the bulk of the activity was further resolved by gel filtration using a preparative Sepharose CL-4B column (Fig. 1 A). The native molecular mass of the activity appeared to be in the range of 700 to 900 kDa, as judged by comparison to the migration of molecular weight standards. SDS-PAGE analysis of this active protein fraction indicated the presence of more than 10 major peptides (Fig. 1 B). It was found that essentially all of the insecticidal activity could be eliminated at temperatures above 60 °C or upon treatment with protease K. Similar results were obtained with broth samples (data not shown). These results are consistent with the toxic activity being proteinaceous. In order to further determine the biochemical nature of the protein toxin, samples were treated with a variety of reversible and irreversible protease inhibitors which included E-64 (cysteine inhibitor), 3,4-dichloroisocoumarin (serine inhibitor), leupeptin (serine inhibitor), and pepstatin (aspartic inhibitor). In all cases, no effects were observed on the biological activity of the toxin. In addition, aliquots of toxin complex treated with inhibitors of metalloenzymes, including 1,10-phenanthroline and EDTA, did not affect the insecticidal potency. As shown in Table I, NH2-terminal amino acid analysis of selected peptides from the SDS-PAGE (Fig. 1 B) demonstrated that each was distinct, but several peptides appeared to have substantial sequence similarity (50–67%). The amino acid sequence for a 64-kDa peptide corresponded to groEL, an Escherichia coli chaperonin (37Hemmingsen S.M. Woolford C. van der Vies S.M. Tilly K. Dennis D.T. Georgopoulos C.P. Hendrix R.W. Ellis R.J. Nature. 1988; 333: 330-334Crossref PubMed Scopus (934) Google Scholar), whose concentration varied for each preparation. The occurrence of related peptides suggested that several toxins may be present in the 860-kDa fraction.Table INH2-terminal amino acid sequences of peptides in 860-kDa toxin fractionPeptide molecular massAmino acid sequenceaThe symbols used are: *, N/D/P/R/E amino acid; -, no determination; |, identical amino acids; ∶, similar amino acids.201 kDaFIQGYSDLFGN-A∶ | | |∶ |190 kDaLIGYNNQFSG*A175 kDaMQNSQTFSVGEL||∶| |∶∶ |165 kDaMQDSPEVSITTL175 kDaMLSTMEKQLNESQRDA108 kDaMNLASPLISRTE80 kDaMINLDINEQNKIMVV68 kDaSESLFTQTLKEA-RDALVA64 kDaAAKDVKFGSDARVKMLRGVN|||||||| |||||||||||groELAAKDVKFGNDARVKMLRGVN61 kDaAGDTANIGD–FLPa The symbols used are: *, N/D/P/R/E amino acid; -, no determination; |, identical amino acids; ∶, similar amino acids. Open table in a new tab The 860-kDa fraction was applied to a Mono Q column and resolved by a linear salt gradient. It was found that the insect activity was broadly eluted throughout the gradient. Each fraction was bioassayed by serial dilution in order to identify those with the highest SCR activity. Two peaks were identified with high SCR toxicity: one that eluted at approximately 0.2 mNaCl (peak A) and the other at approximately 0.3 m NaCl (peak B) (Fig. 2). Each activity peak was pooled separately and further purified by a series of hydrophobic and ion-exchange chromatography columns. The toxin purified from peak A was denoted toxin A while the toxin from peak B was named toxin B. Both purified toxin A and toxin B contained two predominant bands on a 4–20% SDS-PAGE (Fig. 3 A). The peptides were named A1 and A2 for the large peptides, and A2 and B2 for the small peptides in toxin A and toxin B, respectively.Figure 3Characterization of isolated toxins. A, 4–20% SDS-PAGE of purified toxin A (lane 1) and toxin B (lane 2). 5 and 2 μg of protein were applied to lanes 1 and 2, respectively. The protein molecular weight marker is shown to the left of the sample lanes. B, 2–15% native PAGE of purified toxin A (lane 1) and toxin B (lane 2). 5 and 2 μg of protein were applied to lanes 1 and 2, respectively. C, estimation of the native molecular mass of toxin A and toxin B by gel filtration on Superdex 200 10/30 column. The protein markers used were: thyroglobulin, 670 kDa; bovine γ-globulin, 158 kDa; chicken ovalbumin, 44 kDa; equine myoglobin, 17 kDa; vitamin B-12, 1.3 kDa.View Large Image Figure ViewerDownload (PPT) The subunit molecular weights of both toxins were assessed by MALDI-TOF mass spectroscopy and SDS-PAGE as presented in TableII. When the SDS-PAGE gel was quantified by densitometry, the relative band intensity of the large and small peptides in both toxin preparations was found to be approximately 3 to 1 ratio, respectively. Given their molecular masses, these results suggested that these subunits were represented in the purified toxins in equal molar amounts. Toxin A and toxin B showed single stained bands in native PAGE, with toxin A migrating further than toxin B (Fig.3 B). Using gel filtration on Superdex S-200, the native molecular mass of toxin A or toxin B was measured to be approximately 860 kDa (Fig. 3 C). This was re-confirmed by gel filtration on a Sepharose CL-4B column (data not shown). Treatment of the 860-kDa fraction with high salt or non-ionic detergents failed to affect the mobility of the toxin activity on gel filtration. The insecticidal activity (LD50) of toxin A and toxin B against SCR was determined to be 5 and 87 ng/cm2 diet, respectively (Fig.4). Toxin A was also highly potent against tobacco hornworm (M. sexta), a lepidopteran species, however, for toxin B only growth inhibition was detectable against tobacco hornworm.Table IIAnalysis of toxin A and toxin B peptides by MALDI-TOF MS and SDS-PAGEPeptideSDS-PAGEMALDI-TOF MSA1188,000 Da208,186 DaA256,000 Da63,544 DaB1201,000 Da206,473 DaB258,000 Da63,520 Da Open table in a new tab Amino-terminal amino acid sequencing was performed on toxin A and toxin B subunits. The NH2-terminal sequence of B1 was identical to the sequence for the 201-kDa peptide presented in Table I, however, no amino-terminal sequence was obtained for the large subunit of toxin A. High amino acid identity (∼82%) was found between the NH2 terminus of peptides A2 and B2, the smaller peptides of the two toxins (TableIII).Table IIIComparison of NH2-terminal amino acid sequences of small subunits of toxin A and toxin BPeptide A2LRSANTLTDLFLPQ||∶|| |||||Peptide B2ANSLTALFLPQNSRKThe symbols used are: |, identical amino acids; ∶, similar amino acids. Open table in a new tab The symbols used are: |, identical amino acids; ∶, similar amino acids. Since toxin B was an order of magnitude less active than toxin A against SCR neonates, the possibility that the activity observed in toxin B was due to a small amount of contamination of toxin A in the final purified toxin B material was examined by Western analysis using antibodies specific to synthetic peptides corresponding to either toxin A or toxin B. Three peptide-specific antibodies used for analysis were synA1Ab and synA2Ab, which specifically recognized peptides A1 and A2 in toxin A, respectively, and synB2Ab, which specifically recognized peptide B2 in toxin B. Western analysis of purified toxin A and toxin B (5 μg of protein/lane) was performed using the above three antibodies. As shown in Fig. 5, immunological analysis showed that only toxin A but not toxin B reacted when either synA1Ab or synA2Ab was used (Fig. 5, A and B). Conversely, only toxin B but not toxin A reacted when synB2Ab was used (Fig. 5 C). This indicated that no detectable cross-contamination existed in the purified toxin A and toxin B fractions. Therefore, the insecticidal activities observed for each toxin were independent of each other. To eliminate the possibility that protease activity was responsible for the insecticidal activity, samples of isolated toxins were examined for enzymatic activity using fluorescein isothiocyanate-derivatized substrates. Compared with controls and endogenous proteases, no significant protease activity was found for either toxin fraction (data not shown). A second biological activity that has been associated with certain classes of bacterial toxins is the ability to lyse different cell types. In order to examine the general lytic properties of these toxins, in vitro assays were performed using rabbit erythrocytes incubated with up to 0.7 and 2 μg of purified toxin A or toxin B, respectively. Using protein sample buffer as controls, no cell lysis was found with either toxin, while erythrocytes treated with the appropriate units of α-hemolysin from Staphylococcus aureus, a positive control, were comple
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