The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.
Research conducted during past decades to reduce the level of the tobacco specific nitrosamine N-nitrosonornicotine (NNN) and its precursor nornicotine in tobacco yielded identification of three tobacco genes encoding for cytochrome P450 nicotine demethylases converting nicotine to nornicotine. We carried out trials to investigate the effect of using tobaccos containing three non-functional nicotine demethylase genes on the selective reduction of NNN in cigarette tobacco filler and mainstream smoke. Our results indicate that the presence of non-functional alleles of the three genes reduces the level of nornicotine and NNN in Burley tobacco by 70% compared to the level observed in currently available low converter (LC) Burley tobacco varieties. The new technology, named ZYVERT™, does not require a regular screening process, while a yearly selection process is needed to produce LC Burley tobacco seeds for NNN reduction. The reduction of NNN observed in smoke of blended prototype cigarettes is proportional to the inclusion level of tobacco having ZYVERT™ technology. Inclusion of Burley tobacco possessing the new trait into a typical American blend resulted in a selective reduction of NNN in cigarette smoke, while the levels of other Harmful and Potentially Harmful Constituents (HPHC) currently in the abbreviated list provided by the US Food and Drug Administration are statistically equivalent in comparison with the levels obtained in reference prototype cigarettes containing LC Burley.
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. However, it was not until 1966 that Poinar (9Poinar Jr., G.O. Nematologica. 1966; 12: 105-108Crossref Scopus (72) Google Scholar) reported that a single species of bacterium in the family Enterobacteriaceae was present in the anterior region of the infective nematode of this species. Since then investigators have shown thatSteinernema species carry bacteria of the genusXenorhabdus while Heterorhabditis nematodes harbor species of the genus Photorhabdus (10Thomas G.M. Poiner Jr., G.O. Int. J. Syst. Bacteriol. 1979; 29: 352-360Crossref Scopus (249) Google Scholar, 11Akhurst R.J. Boemare N.E. J. Gen. Micro. 1988; 134: 1835-1845PubMed Google Scholar). In 1993, Photorhabdus bacteria were proposed by Boemare and colleagues (12Boemare N. Akhurst R.J. Mourant R.G. Int. J. Syst. Bacteriol. 1993; 43: 249-255Crossref Scopus (330) Google Scholar) for re-classification as a distinct genus fromXenorhabdus, based on a variety of phenotyptic, ecological, and molecular studies. 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
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMetalloporphyrin DNA interactions: insights from NMR studies of oligodeoxyribonucleotidesJames A. Strickland, Luigi G. Marzilli, W. David Wilson, and Gerald ZonCite this: Inorg. Chem. 1989, 28, 23, 4191–4198Publication Date (Print):November 1, 1989Publication History Published online1 May 2002Published inissue 1 November 1989https://pubs.acs.org/doi/10.1021/ic00322a005https://doi.org/10.1021/ic00322a005research-articleACS PublicationsRequest reuse permissionsArticle Views66Altmetric-Citations21LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Abstract : Human Area Networks represent an emerging field of communications technology with the potential to offer significant advantages to military operations. This thesis explores and defines Human Area Networks (HAN) and addresses how HANs relate to existing network topologies as well as the various terminologies associated with HANs. The focus of research addresses the notion of as an event and attempts to relate the various interpretations of touch networking to HANs while describing a preliminary architecture through the use of multiple scenarios and use cases, quality attributes, and functional requirements. Additionally, this thesis explores the opportunities associated with one particular implementation of HAN: Intrabody Communications (IBC), and proposes an implementation plan for conceptual IBC devices. Ultimately, this thesis demonstrates the potential value of IBC and HANs in a Joint Tactical scenario with recommendations for iteratively evaluating the techniques, tactics and procedures (TTP) in an incremental manner that seamlessly evolves with technology advancements.
Abstract Factors influencing the binding of tetracationic porphyrin derivatives to DNA have been comprehensively evaluated by equilibrium dialysis, stopped‐flow kinetics, etc., for meso ‐tetrakis (4‐ N ‐methylpyridiniumyl)porphyrin TMpyP(4). Technical difficulties have previously precluded a comprehensive study of metalloporphyrins. Since electrostatic interactions with the DNA and metal derivatization of the porphyrins have important consequences, we have investigated in greater detail two isomers of TMpyP(4) { meso ‐tetra‐kis(3‐ N ‐methylpyridiniumyl)porphyrin, [TMpyP (3)] and meso ‐tetrakis (2‐ N ‐methylpyridiniumyl)porphyrin [TMpyP(2)]} in which the position of the charged centers has been varied. A comprehensive study of the Cu(II) derivatives, e.g., CuTMpyP(4), was possible since the difficulties encountered previously with Ni (II) compounds were not a problem with Cu(II) porphyrins [J. A. Strickland, L. G. Marzilli, M. K. Gay, and W. D. Wilson (1988) Biochemistry 27 , 8870–8878]. At 25°C, the apparent equilibrium constants [ K obs ] decreased with increasing [Na + ] for all porphyrins. The K obs values were comparable for TMpyP(4) and TMpyP(3) binding to either polyd(G‐C)·polyd(G‐C) [poly[d(G‐C) 2 ]] or poly[d(A‐T)·poly[d(A‐T)]][poly[d(A‐T) 2 ]]. For the copper (II) porphyrins, the K obs values were about fivefold greater. The K obs value for CuTMpyP(2) binding to poly[d(G‐C) 2 ] was too small to measure under typical salt conditions; however, K obs for CuTMpyP(4) or CuTMpyP(3). Application of the condensation theory for polyelectrolytes suggests about three charge interactions when CuTMpyP(4), CuTMpyP(3), and TMpyP(4) bind to poly[d(G‐C) 2 ] or poly[d(A‐T) 2 ], a result comparable to that reported for TMpyP(4). At 20°C and 0.115 M [Na + ], incorporation of copper decreased the rates of dissociation from poly[d(A‐T) 2 ] by a 100‐fold compared to those reported for TMpyP(4) but had little effect on the rates of dissociation from poly[d(G‐C) 2 ]. Also, movement of the H 3 CN + group from the fourth to the third position of the pyridinium ring enhanced the rates of dissociation from poly[d(A‐T) 2 ] but decreased the rates of dissociation from poly[d(G‐C) 2 ]. From polyelectrolyte theory, the [Na + ] dependence of the dissociation rates from poly[d(G‐C) 2 ] is consistent with intercalative binding, while that for poly[d(A‐T) 2 ] is consistent with an outside binding model. For calf thymus [CT]DNA at 20°C, a greater decrease in the AT than in the GC imino 1 H‐nmr signal was observed upon addition of CuTMpyP(2), suggesting selective outside binding to the AT regions. Flow dichroism experiments with calf thymus DNA revealed a small reduced dichroism [ red D] value with CuTMpyP(2), indicative of disordered, outside binding. However, a large red D with CT DNA was found for CuTMpyP(4) and CuTMpyP(3), suggesting ordered intercalative binding. Titrations of closed circular superhelical DNA (CCS DNA) with CuTMpyP(4) and CuTMpyP(3) produced large increases in the solution reduced viscosity (SRV), indicative of unwinding. Twice the concentration of TMpyP(4) was needed for a similar effect, a result suggesting either that CuTMpyP (4) and CuTMpyP(3) intercalate into more sites, or that they produce more unwinding per site. Alternatively, CuTMpyP(4) and CuTMpyP(3) could unwind CCS DNA through a nonintercalative binding mode. Addition of CuTMpyP(4) or CuTMpyP(3) increased the SRV of poly[d(A‐T) 2 ], poly[d(G‐C) 2 ], and a variety of native linear DNAs varying in percentage GC. However, CuTMpyP(4) decreased the SRV of poly[d (A)]·poly[d(T)]. Whereas the viscosity increases with poly[d(G‐C) 2 ] probably result from intercalation, the unusual increase in the SRV of poly[d(A‐T) 2 ] could arise from conversion of a small amount of hairpin to the B form or from some other type of cooperative conformational change. Together these results suggest generally more favorable interactions with DNA of the copper porphyrins than the analogous metal‐free porphyrins. However, our evidence clearly rules out appreciably greater GC vs AT selectivity for the copper porphyrins. Electrostatic interactions with DNA are similar for the TMpyP(4) and TMpyP(3) species, but are evidently much less favorable for the TMpyP(2) porphyrins. Finally, we find no clear evidence for additional binding modes for copper porphyrins.
Traditional breeding and molecular approaches have been used to develop tobacco varieties with reduced nicotine and secondary alkaloid levels. However, available low-alkaloid tobacco varieties have impaired leaf quality likely due to the metabolic consequences of nicotine biosynthesis downregulation. Recently, we found evidence that the unbalanced crosstalk between nicotine and polyamine pathways is involved in impaired leaf ripening of a low-alkaloid (LA) Burley 21 line having a mutation at the Nic1 and Nic2 loci, key biosynthetic regulators of nicotine biosynthesis. Since the Nic1 and Nic2 loci are comprised of several genes, all phenotypic changes seen in LA Burley 21 could be due to a mixture of genetics-based responses. Here, we investigated the commercial burley variety TN90 LC and its transgenic versions with only one downregulated gene, either putrescine methyl transferase (PMT-RNAi) or PR50-protein (PR50-RNAi). Nicotine levels of cured lamina of TN90 LC, TN90 PMT-RNAi and TN90 PR50-RNAi, were 70.5 ± 3.8, 2.4 ± 0.5, and 6.0 ± 1.1 mg/g dry weight, respectively. Low-alkaloid transgenic lines showed delayed leaf maturation and impaired leaf quality. We analyzed polyamine contents and ripening markers in wild-type TN90 control plants (WT) and the two transgenic lines. The ripening markers revealed that the PMT-RNAi line showed the most pronounced impaired leaf maturation phenotype at harvest, characterized by higher chlorophyll (19%) and glucose (173%) contents and more leaf mesophyll cells per area (25%), while the ripening markers revealed that maturation of PR50-RNAi plants was intermediate between PMT-RNAi and WT lines. Comparative polyamine analyses showed an increase in free and conjugated polyamines in roots of both transgenic lines, this being most pronounced in the PMT-RNAi plants. For PMT-RNAi plants, there were further perturbations of polyamine content in the leaves, which mirrored the general phenotype, as PR50-RNAi transgenic plants looked more similar to the WT than PMT-RNAi transgenic plants. Activity of ornithine decarboxylase, the enzyme that catalyzes the committing step of polyamine biosynthesis, was significantly higher in roots and mature leaves of PMT-RNAi plants in comparison to WT, while there was no increase observed for arginine decarboxylase. Treatment of both transgenic lines with polyamine biosynthesis inhibitors decreased the polyamine content and ameliorated the phenotype, confirming the intricate interplay of polyamine and nicotine biosynthesis in tobacco and the influence of this interplay on leaf ripening.
Journal Article The 85-kD Crystalline cysteine protease inhibitor from potato contains eight cystatin domains Get access Terence A. Walsh, Terence A. Walsh DowElanco Biotechnology Lab, P.O. Box 68955, Indianapolis, IN 46268-1053 Search for other works by this author on: Oxford Academic PubMed Google Scholar James A. Strickland James A. Strickland DowElanco Biotechnology Lab, P.O. Box 68955, Indianapolis, IN 46268-1053 Search for other works by this author on: Oxford Academic PubMed Google Scholar Protein Engineering, Design and Selection, Volume 6, Issue Supplement, 1993, Page 53, https://doi.org/10.1093/protein/6.Supplement.53-b Published: 01 January 1993
