Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were observed in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these two species. In addition, we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics.

Escherichia coli and Sf9 Contaminant Databases to Increase Efficiency of Tandem Mass Spectrometry Peptide Identification in Structural Mass Spectrometry Experiments.

Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no Spodoptera frugiperda (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for Escherichia coli. For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and E. coli NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.

Osmolality as a Novel Mechanism Explaining Diet Effects on the Outcome of Infection with a Blood Parasite.

Recent research has suggested that the outcome of host-parasite interactions is dependent on the diet of the host, but most previous studies have focused on "top-down" mechanisms, i.e., how the host's diet improves the host immune response to drive down the parasite population and improve host fitness. In contrast, the direct impacts of host nutrition on parasite fitness and the mechanisms underpinning these effects are relatively unexplored. Here, using a model host-pathogen system (Spodoptera littoralis caterpillars and Xenorhabdus nematophila, an extracellular bacterial blood parasite), we explore the effects of host dietary macronutrient balance on pathogen growth rates both in vivo and in vitro, allowing us to compare pathogen growth rates both in the presence and absence of the host immune response. In vivo, high dietary protein resulted in lower rates of bacterial establishment, slower bacterial growth, higher host survival, and slower speed of host death; in contrast, the energy content and amount of carbohydrate in the diet explained little variation in any measure of pathogen or host fitness. In vitro, we show that these effects are largely driven by the impact of host dietary protein on host hemolymph (blood) osmolality (i.e., its concentration of solutes), with bacterial growth being slower in protein-rich, high-osmolality hemolymphs, highlighting a novel "bottom-up" mechanism by which host diet can impact both pathogen and host fitness.

Binding specificity of ostreolysin A6 towards Sf9 insect cell lipids.

Oyster mushrooms (Pleurotus spp.) have recently been shown to produce insecticidal bi-component protein complexes based on the aegerolysin proteins. A role for these proteins is thus indicated for defence and protection of the mushroom, and we propose their use as new environmentally friendly bioinsecticides. These aegerolysin-based protein complexes permeabilise artificial lipid vesicles through aegerolysin binding to an insect-specific sphingolipid, ceramide phosphoethanolamine (CPE), and they are cytotoxic for the Spodoptera frugiferda (Sf9) insect cell line. Tandem mass spectrometry analysis of the Sf9 lipidome uncovered lipids not previously reported in the literature, including in particular C14 sphingosine-based CPE molecular species, which comprised ~4 mol% of the whole lipidome. Further analysis of the lipid binding specificity of an aegerolysin from P. ostreatus, ostreolysin A6 (OlyA6), to lipid vesicles composed of commercial lipids, to lipid vesicles composed of the total lipid extract from Sf9 cells, and to HPLC-separated Sf9 cell lipid fractions containing ceramides, confirmed CPE as the main OlyA6 receptor, but also highlighted the importance of membrane cholesterol for formation of strong and stable interactions of OlyA6 with artificial and natural lipid membranes. Binding assays performed with glycan arrays and surface plasmon resonance, which included invertebrate-specific glycans, excluded these saccharides as potential additional OlyA6 receptors.

Characterization of the active fragments of Spodoptera litura Lebocin-1.

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.

Detoxification of Plant Aromatic Abietanoids via Cleavage of the Benzene Ring into 11,12-Seco-diterpene Polyenes by a Specialist Insect of Leucosceptrum canum.

Leaves of Leucosceptrum canum harbor abundant toxic aromatic abietanoids, and they are rarely attacked by insect herbivores, except for the larvae of Nacna malachitis. The excrements of the insect that fed on L. canum leaves were investigated, leading to the isolation and identification of two unprecedented 11,12-seco-abietane diterpene polyenes: nacnabietanins A (1) and B (2). This discovery heralds a unique detoxification mechanism of plant aromatic abietanoids by insects through enzymatic cleavage of stable benzene rings into more easily degraded polyenes.

Biocontrol potential of methyl chavicol for managing Spodoptera frugiperda (Lepidoptera: Noctuidae), an important corn pest.

Synthetic insecticides applied to control Spodoptera frugiperda (Lepidoptera: Noctuidae) can have negative impacts on environment and human health. Botanical essential oils can be sources of organic molecules with biocontrol potential and advantages, such as minor impacts on the selection of resistant pest insects and low toxicity to humans. The aim of this study was to investigate the biocontrol action of essential oils from Brazilian species and methyl chavicol compounds on the development and metabolism of S. frugiperda. Essential oils of Eremanthus erythropappus (Asteraceae), Ocimum selloi, Hyptis suaveolens, and Hyptis marrubioides (Lamiaceae) were distilled by the steam distillation method and analyzed by gas chromatograph techniques. The essential oils were incorporated into an artificial diet (at 1, 2, and 4 mg mL-1) and offered to S. frugiperda caterpillars. Larvae of S. frugiperda at 48 h of age were fed an artificial diet containing the major constituent of O. selloi (methyl chavicol). The major compounds of the essential oils were methyl chavicol for O. selloi, α-bisabolol for E. erythropappus, bicyclogermacrene for H. suaveolens, and ß-thujone for H. marrubioides. O. selloi caused 100% mortality in S. frugiperda larvae at a concentration of 1 mg mL-1 after 48 h. H. marrubioides essential oil caused 100% mortality in larvae at a concentration of 4 mg mL-1 after 48 h. O. selloi and H. marrubioides inhibited acetylcholinesterase (AchE) activity in 72.87% and 81.69% of larvae, respectively. O. selloi presented the highest toxicity to S. frugiperda and the lowest inhibition of AchE. Methyl chavicol was lethal to all larvae within 24 h at a concentration of 0.92 mg mL-1 of diet. Methyl chavicol showed the best insecticidal activity and potential to be used as a natural insecticide to control S. frugiperda.

Incorporating G-C Pair-Recognizing Guanidinium into PNAs for Sequence and Structure Specific Recognition of dsRNAs over dsDNAs and ssRNAs.

Recognition of RNAs under physiological conditions is important for the development of chemical probes and therapeutic ligands. Nucleobase-modified dsRNA-binding PNAs (dbPNAs) are promising for the recognition of dsRNAs in a sequence and structure specific manner under near-physiological conditions. Guanidinium is often present in proteins and small molecules for the recognition of G bases in nucleic acids, in cell-penetrating carriers, and in bioactive drug molecules, which might be due to the fact that guanidinium is amphiphilic and has unique hydrogen bonding and stacking properties. We hypothesized that a simple guanidinium moiety can be directly incorporated into PNAs to facilitate enhanced molecular recognition of G-C pairs in dsRNAs and improved bioactivity. We grafted a guanidinium moiety directly into a PNA monomer (designated as R) using a two-carbon linker as guided by computational modeling studies. The synthetic scheme of the PNA R monomer is relatively simple compared to that of the previously reported L monomer. We incorporated the R residue into various dbPNAs for binding studies. dbPNAs incorporated with R residues are excellent in sequence specifically recognizing G-C pairs in dsRNAs over dsDNA and ssRNAs. We demonstrated that the R residue is compatible with unmodified T and C and previously developed modified L and Q residues in dbPNAs for targeting model dsRNAs, the influenza A viral panhandle duplex structure, and the HIV-1 frameshift site RNA hairpin. Furthermore, R residues enhance the cellular uptake of PNAs.

Structure and Composition of Native Membrane Derived Polymer-Supported Lipid Bilayers.

Over the last two decades, supported lipid bilayers (SLBs) have been extensively used as model systems to study cell membrane structure and function. While SLBs have been traditionally produced from simple lipid mixtures, there has been a recent surge in compositional complexity to better mimic cellular membranes and thereby bridge the gap between classic biophysical approaches and cell experiments. To this end, native cellular membrane derived SLBs (nSLBs) have emerged as a new category of SLBs. As a new type of biomimetic material, an analytical workflow must be designed to characterize its molecular composition and structure. Herein, we demonstrate how a combination of fluorescence microscopy, neutron reflectometry, and secondary ion mass spectrometry offers new insights on structure, composition, and quality of nSLB systems formed using so-called hybrid vesicles, which are a mixture of native membrane material and synthetic lipids. With this approach, we demonstrate that the nSLB formed a continuous structure with complete mixing of the synthetic and native membrane components and a molecular stoichiometry that essentially mirrors that of the hybrid vesicles. Furthermore, structural investigation of the nSLB revealed that PEGylated lipids do not significantly thicken the hydration layer between the bilayer and substrate when on silicon substrates; however, nSLBs do have more topology than their simpler, purely synthetic counterparts. Beyond new insights regarding the structure and composition of nSLB systems, this work also serves to guide future researchers in producing and characterizing nSLBs from their cellular membrane of choice.

Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds.

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.

Mutational disruption of the ABCC2 gene in fall armyworm, Spodoptera frugiperda, confers resistance to the Cry1Fa and Cry1A.105 insecticidal proteins.

The use of Bt proteins in crops has revolutionized insect pest management by offering effective season-long control. However, field-evolved resistance to Bt proteins threatens their utility and durability. A recent example is field-evolved resistance to Cry1Fa and Cry1A.105 in fall armyworm (Spodoptera frugiperda). This resistance has been detected in Puerto Rico, mainland USA, and Brazil. A S. frugiperda population with suspected resistance to Cry1Fa was sampled from a maize field in Puerto Rico and used to develop a resistant lab colony. The colony demonstrated resistance to Cry1Fa and partial cross-resistance to Cry1A.105 in diet bioassays. Using genetic crosses and proteomics, we show that this resistance is due to loss-of-function mutations in the ABCC2 gene. We characterize two novel mutant alleles from Puerto Rico. We also find that these alleles are absent in a broad screen of partially resistant Brazilian populations. These findings confirm that ABCC2 is a receptor for Cry1Fa and Cry1A.105 in S. frugiperda, and lay the groundwork for genetically enabled resistance management in this species, with the caution that there may be several distinct ABCC2 resistances alleles in nature.

A mechanically strengthened polyacrylamide gel matrix fully compatible with electrophoresis of proteins and nucleic acids.

Polyacrylamide gel electrophoresis is a universal tool in a biochemist's toolkit for protein and nucleic acid separation and subsequent visualisation and analysis. The standard formulation of polyacrylamide gels consists of acrylamide (ACM) monomer crosslinked with bisacrylamide (MBA) which creates a gel with excellent sieving properties but which is mechanically fragile and prone to tearing during post-electrophoresis manipulations involved in visualisation and analysis. By adding a poly(ethylene oxide) macro-crosslinker to the standard gel formulation, we have created a tough gel matrix that can be used to fractionate proteins and nucleic acids by polyacrylamide gel electrophoresis. The protein and nucleic acid resolving capabilities and performance during staining and electroblotting of the tough gel matrix rivals that of conventional acrylamide/bisacrylamide gels. The tough gel matrix is resistant to tear and remarkably elastic, capable of stretching to over four times its original length before breaking, and represents a significant improvement over standard polyacrylamide gel formulations.

Novel Peptidase Kunitz Inhibitor from Platypodium elegans Seeds Is Active against Spodoptera frugiperda Larvae.

A novel Kunitz-type inhibitor from Platypodium elegans seeds (PeTI) was purified and characterized. The mass spectrometry analyses of PeTI indicated an intact mass of 19 701 Da and a partial sequence homologous to Kunitz inhibitors. PeTI was purified by ion exchange and affinity chromatographies. A complex with a 1:1 ratio was obtained only for bovine trypsin, showing a Ki = 0.16 nM. Stability studies showed that PeTI was stable over a wide range of temperature (37-80 °C) and pH (2-10). The inhibitory activity of PeTI was affected by dithiothreitol (DTT). Bioassays of PeTI on Spodoptera frugiperda showed negative effects on larval development and weight gain, besides extending the insect life cycle. The activities of digestive enzymes, trypsin and chymotrypsin, were reduced by feeding larvae with 0.2% PeTI in an artificial diet. In summary, we describe a novel Kunitz inhibitor with promising biotechnological potential for pest control.

Purification and properties of the chymotrypsin inhibitor from wild emmer wheat (Triticum dicoccoides) of Israel and its toxic effect on beet armyworm, Spodoptera exigua.

A novel chymotrypsin inhibitor, which detected in the seed of wild emmer wheat (Triticum dicoccoides), was purified by ion-exchange chromatography, affinity chromatography and Ultracentrifugation. On the basis of its specificity, this inhibitor was named WeCI (wild emmer chymotrypsin inhibitor). SDS-PAGE analysis displayed that the purified WeCI is a single chain polypeptide with a molecular weight of approximately 13kDa. The inhibition constants (Ki) for amylase and bovine pancreatic chymotrypsin were 1.12×10-9M and 2.41×10-9M, respectively. Automated sequencing and mass spectrometry analyses revealed that WeCI is a neutral monomeric protein consisting of 119 residues. In vitro, WeCI strongly suppressed bovine pancreatic chymotrypsin as well as chymotrypsin-like activities separated from the midgut of the beet armyworm Spodoptera exigua. No inhibitory activities were found against bovine pancreatic trypsin, bacterial subtilisin, or porcine pancreatic elastase. The primary structure of WeCI was markedly similar (46-95%) to those of several proteins belonging to the wheat crop chymotrypsin/α-amylase inhibitor superfamily and displayed the typical sequence motif of the α-amylase inhibitor-seed storage protein group. WeCI significantly inhibited the growth and development of Spodoptera exigua, dependent on inhibitor concentration. WeCI significantly increased the mortality rate of Spodoptera exigua and caused a significant decrease in its fertility.

Bitrophic and Tritrophic Effects of Transgenic cry1Ab/cry2Aj Maize on the Beneficial, Nontarget Harmonia axyridis (Coleoptera: Coccinellidae).

Harmonia axyridis (Pallas) is a common and abundant predator in China and may be exposed to Cry toxins that are produced in Bt crops either by feeding on plant parts or by feeding on target or nontarget herbivorous insects. A new Bt maize line, expressing the Cry1Ab/Cry2Aj fused protein, has been developed and should be rigorously assessed for the ecological risks on the natural enemy. Laboratory experiments were carried out to study the effects of this Bt maize on nontarget predator H. axyridis via bitrophic interaction of adult H. axyridis feeding on Bt maize pollen and tritrophic interaction of H. axyridis consuming the lepidopteran prey. Spodoptera exigua (Hübner) neonate larvae were used to transfer Bt protein because they could survive after ingesting transgenic cry1Ab/cry2Aj maize kernels in the previous study. ELISA bioassays confirmed that the Bt protein could be transferred, but diluted through Bt maize-prey-predator. Life history parameters such as survival, development, weight, fecundity, and egg hatching rate were not significantly different when H. axyridis consumed prey that had been reared on Bt maize compared with prey reared on a nontransformed parental control. Furthermore, feeding directly on Bt maize pollen also had no detrimental effects on fitness, survival, and weight of female and male adults. In conclusion, our results indicate that transgenic cry1Ab/cry2Aj maize poses no ecological risks on the nontarget predator H. axyridis.

Better cold tolerance of Bt-resistant Spodoptera exigua strain and the corresponding cold-tolerant mechanism.

Spodoptera exigua is a secondary target pest of Bt cotton commercialized in China. With the continuous adoption of Bt cotton, populations of S. exigua have gradually increased. However, the cold tolerance ability of Bt-resistant S. exigua and the effect of continuous Bt diet on anti-cold materials are unknown. In our study, it was found that Bt-resistant S. exigua (Bt10) developed better with shorter larval and pupal duration and higher pupation rate compared to CK at the suboptimal low temperature. The supercooling points and freezing points of the Bt-resistant S. exigua strain were determined, and body water content and anti-cold materials such as total sugar, trehalose and glycogen, glycerol and fat were examined to explore the effect of Bt toxin on overwintering and on population increase. The results showed that the supercooling point and the freezing point of the Bt-resistant S. exigua pupae were both significantly lower than that of the Bt-susceptible strain. No difference was found in the body water content of pupae and adults between the two strains. Total sugar content of the Bt-resistant strain at both the pupal and adult stages was higher than that of the susceptible strain at the corresponding stages, and glycogen content of the Bt-resistant strain at the larval stage was higher than that of the susceptible larval S. exigua. Fat content of the Bt-resistant larvae, pupae and adults was for each higher than that of the susceptible strain, but the difference was not significant except for that of the 3rd instar larvae. Glycerol content of the Bt-resistant strain at larval, pupal and adult stages was for each higher than that of the corresponding life stages of the susceptible strain. It can be seen that more glycerol was accumulated in Bt-resistant S. exigua. The results indicate that Bt-resistant S. exigua has better cold tolerance. The contents of the anti-freeze substances of progeny, especially glycerol, were increased after previous generations were continuously fed on Bt protein, which means that the Bt-resistant secondary target pests could more easily overcome the overwinter season and become a source of crop damage the following year.

Quantitative proteomic analysis of the fall armyworm saliva.

Lepidopteran larvae secrete saliva on plant tissues during feeding. Components in the saliva may aid in food digestion, whereas other components are recognized by plants as cues to elicit defense responses. Despite the ecological and economical importance of these plant-feeding insects, knowledge of their saliva composition is limited to a few species. In this study, we identified the salivary proteins of larvae of the fall armyworm (FAW), Spodoptera frugiperda; determined qualitative and quantitative differences in the salivary proteome of the two host races-corn and rice strains-of this insect; and identified changes in total protein concentration and relative protein abundance in the saliva of FAW larvae associated with different host plants. Quantitative proteomic analyses were performed using labeling with isobaric tags for relative and absolute quantification followed by liquid chromatography-tandem mass spectrometry. In total, 98 proteins were identified (>99% confidence) in the FAW saliva. These proteins were further categorized into five functional groups: proteins potentially involved in (1) plant defense regulation, (2) herbivore offense, (3) insect immunity, (4) detoxification, (5) digestion, and (6) other functions. Moreover, there were differences in the salivary proteome between the FAW strains that were identified by label-free proteomic analyses. Thirteen differentially identified proteins were present in each strain. There were also differences in the relative abundance of eleven salivary proteins between the two FAW host strains as well as differences within each strain associated with different diets. The total salivary protein concentration was also different for the two strains reared on different host plants. Based on these results, we conclude that the FAW saliva contains a complex mixture of proteins involved in different functions that are specific for each strain and its composition can change plastically in response to diet type.

Molecular dynamics and protein interaction studies of lipopeptide (Iturin A) on α- amylase of Spodoptera litura.

The small mottled willow moth (Spodoptera litura) is one of the best-known agricultural pest insects. To understand the insecticidal activity, we have selected iturin A compound produced by Bacillus amyloliquefaciens RHNK22 which showed the strongest and most common inhibitory effect on the Spodoptera litura protein. In this work we have identified the action of iturin A on α- amylase is a major digestive enzyme of Spodoptera litura using docking studies. A 3D model of α- amylase from Spodoptera litura was generated using 2HPH as a template with the help of Modeller7v7. With the aid of the molecular mechanics and molecular dynamics methods, the final model is obtained and is further checked by Procheck and Verify 3D graph programs, which showed that the final refined model is reliable. With this model, a adjustable docking study was performed with iturin A using GOLD software. The results indicated that ARG 18, THR15, LEU42 in α- amylase are important determinant residues in binding as they have strong hydrogen bonding interactions with iturin A. These hydrogen binding interactions play an important role for the stability of the complex.

Inducible De Novo Biosynthesis of Isoflavonoids in Soybean Leaves by Spodoptera litura Derived Elicitors: Tracer Techniques Aided by High Resolution LCMS.

Isoflavonoids are a characteristic family of natural products in legumes known to mediate a range of plant-biotic interactions. For example, in soybean (Glycine max: Fabaceae) multiple isoflavones are induced and accumulate in leaves following attack by Spodoptera litura (Lepidoptera: Noctuidae) larvae. To quantitatively examine patterns of activated de novo biosynthesis, soybean (Var. Enrei) leaves were treated with a combination of plant defense elicitors present in S. litura gut content extracts and L-α-[13C9, 15N]phenylalanine as a traceable isoflavonoid precursor. Combined treatments promoted significant increases in 13C-labeled isoflavone aglycones (daidzein, formononetin, and genistein), 13C-labeled isoflavone 7-O-glucosides (daidzin, ononin, and genistin), and 13C-labeled isoflavone 7-O-(6″-O-malonyl-ß-glucosides) (malonyldaidzin, malonylononin, and malonylgenistin). In contrast levels of 13C-labeled flavones and flavonol (4',7-dihydroxyflavone, kaempferol, and apigenin) were not significantly altered. Curiously, application of fatty acid-amino acid conjugate (FAC) elicitors present in S. litura gut contents, namely N-linolenoyl-L-glutamine and N-linoleoyl-L-glutamine, both promoted the induced accumulation of isoflavone 7-O-glucosides and isoflavone 7-O-(6″-O-malonyl-ß-glucosides), but not isoflavone aglycones in the leaves. These results demonstrate that at least two separate reactions are involved in elicitor-induced soybean leaf responses to the S. litura gut contents: one is the de novo biosynthesis of isoflavone conjugates induced by FACs, and the other is the hydrolysis of the isoflavone conjugates to yield isoflavone aglycones. Gut content extracts alone displayed no hydrolytic activity. The quantitative analysis of isoflavone de novo biosynthesis, with respect to both aglycones and conjugates, affords a useful bioassay system for the discovery of additional plant defense elicitor(s) in S. litura gut contents that specifically promote hydrolysis of isoflavone conjugates.

Comparison of gating dynamics of different IP3R channels with immune algorithm searching for channel parameter distributions.

The gating properties of the inositol 1, 4, 5-trisphosphate (IP3) receptor (IP3R) are determined by the binding and unbinding capability of Ca2+ ions and IP3 messengers. With the patch clamp experiments, the stationary properties have been discussed for Xenopus oocyte type-1 IP3R (Oo-IP3R1), type-3 IP3R (Oo-IP3R3) and Spodoptera frugiperda IP3R (Sf-IP3R). In this paper, in order to provide insights about the relation between the observed gating characteristics and the gating parameters in different IP3Rs, we apply the immune algorithm to fit the parameters of a modified DeYoung-Keizer model. By comparing the fitting parameter distributions of three IP3Rs, we suggest that the three types of IP3Rs have the similar open sensitivity in responding to IP3. The Oo-IP3R3 channel is easy to open in responding to low Ca2+ concentration, while Sf-IP3R channel is easily inhibited in responding to high Ca2+ concentration. We also show that the IP3 binding rate is not a sensitive parameter for stationary gating dynamics for three IP3Rs, but the inhibitory Ca2+ binding/unbinding rates are sensitive parameters for gating dynamics for both Oo-IP3R1 and Oo-IP3R3 channels. Such differences may be important in generating the spatially and temporally complex Ca2+ oscillations in cells. Our study also demonstrates that the immune algorithm can be applied for model parameter searching in biological systems.