Native PAGE Assay Research Articles

Resistance in Spodoptera litura (F.) to Insecticides and Detoxification Enzymes

Standard leaf dip bioassay experiments were conducted to study the insecticide resistance in Spodoptera litura (F.) in soybean. Chlorantraniliprole was the most toxic while, cypermethrin and organophosphates (profenophos and triazophos) recorded least toxicity against S. litura populations. Field population of S. litura showed the highest resistance to cypermethrin with resistance ratios (RRs) ranging from 244 to 376. The field populations collected from three locations of Southern Rajasthan exhibited low resistance to newer insecticides, chlorantraniliprole and spinetoram with Lab-SS strain. All the enzyme activities were significantly higher in the whole larval extracts and midgut extracts of S. litura larvae when compared to the Lab-SS strain. Analysis of the carboxyl esterase enzyme in the native PAGE assay revealed that the electrophoretic profiles showed presence of different band regions (EST 1 to EST 7) with esterase activity in the field populations along with the laboratory susceptible strain. The susceptibility of three field populations to different group of insecticides along with the profile of detoxification enzymes indicate the need to formulate a region-specific insecticide resistance management (IRM).

A Native PAGE Assay for the Biochemical Characterization of G Protein Coupling to GPCRs.

G protein-coupled receptors (GPCRs) are a large family of membrane-embedded receptors that have diverse roles in physiology and are major drug targets. GPCRs transduce an agonist binding signal across the membrane to activate intracellular heterotrimeric G proteins. The dynamic nature of the receptors and the complexity of their interactions with agonists and G proteins present significant challenges for biochemical studies. Most biochemical/biophysical methods that have been employed to study GPCR-G protein coupling require purified receptors and are technically difficult. Here, we provide a protocol for a relatively simple and time- and cost-effective membrane protein native PAGE assay, to visualize and biochemically characterize agonist-dependent coupling of detergent-solubilized GPCRs to purified G protein surrogate "mini-G" proteins, which stabilize the receptor in an active state. The assay was developed for our studies of the calcitonin receptor-like receptor, a class B GPCR that mediates the actions of calcitonin gene-related peptide and adrenomedullin peptide agonists. It does not require a purified receptor and it can be used in a screening format with transiently-transfected adherent mammalian cell cultures, to quickly identify detergent-stable complexes amenable to study, or in a quantitative format with membrane preparations, to determine apparent affinities of agonists for the mini-G-coupled receptor and apparent affinities of mini-G proteins for the agonist-occupied receptor. The latter provides a partial measure of agonist efficacy. The method should be applicable to other GPCRs, and has the potential to be adapted to the study of other challenging membrane proteins and their complexes with binding partners. Graphic abstract: Visualizing agonist-dependent mini-G protein coupling and determining apparent binding affinities using the native PAGE assay quantitative formats.

Functional characterization of STATa/b genes encoding transcription factors from Branchiostoma belcheri

The signal transducer and activator of transcription (STAT), as an important transcription factor of the Janus kinase (JAK)-STAT signaling pathway, is pivotal for development and immunity and well documented in vertebrates. However, the STAT gene has not been reported in chordate amphioxus (Branchiostoma belcheri). In this study, we firstly identify and characterize two STAT genes from Branchiostoma belcheri (designed as AmphiSTATa and AmphiSTATb). Secondly, our results reveal that AmphiSTATa is clustered with vertebrate STAT1, STAT2, STAT3 and STAT4, whereas AmphiSTATb is grouped with STAT5 and STAT6 based on phylogenetic analysis. Thirdly, AmphiSTATa and AmphiSTATb are found to widely express in five representative tissues of amphioxus (gill, hepatic cecum, intestine, muscle and notochord) by RT-qPCR analysis. Importantly, both AmphiSTATa and AmphiSTATb can be involved in innate immune responses to LPS stimulation. Fourthly, we demonstrate that AmphiSTATa and AmphiSTATb can form homodimers or heterodimers by Co-IP and Native-PAGE assay, and that AmphiSTATa and AmphiSTATb proteins can also distribute in cytoplasm and nucleus by the subcellular localization. Taken together, our findings not only reveal the roles of AmphiSTATa and AmphiSTATb in amphioxus innate immune responses to LPS stimulation, but provide a new insight into further elucidating the evolution and function of STATs in animals.

Biochemical characterization of G protein coupling to calcitonin gene–related peptide and adrenomedullin receptors using a native PAGE assay

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein-coupled receptor (CLR), which heterodimerizes with three receptor activity-modifying proteins (RAMP1-3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein-tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

Orchestration of Cu-Zn SOD and class III peroxidase with upstream interplay between NADPH oxidase and PM H+-ATPase mediates root growth in Vigna radiata (L.) Wilczek

Post-germination plant growth depends on the regulation of reactive oxygen species (ROS) metabolism, spatiotemporal pH changes and Ca+2 homeostasis, whose potential integration has been studied during Vigna radiata (L.) Wilczek root growth. The dissipation of proton (H+) gradients across plasma membrane (PM) by CCCP (protonophore) and the inhibition of PM H+-ATPase by sodium orthovanadate repressed SOD (superoxide dismutase; EC 1.15.1.1) activity as revealed by spectrophotometric and native PAGE assay results. Similar results derived from treatment with DPI (NADPH oxidase inhibitor) and Tiron (O2ˉ scavenger) denote a functional synchronization of SOD, PM H+-ATPase and NOX, as the latter two enzymes are substrate sources for SOD (H+ and O2ˉ, respectively) and are involved in a feed-forward loop. After SOD inactivation, a decline in apoplastic H2O2 content was observed in each treatment group, emerging as a possible cause of the diminution of class III peroxidase (Prx; EC 1.11.1.7), which utilizes H2O2 as a substrate. In agreement with the pivotal role of Ca+2 in PM H+-ATPase and NOX activation, Ca+2 homeostasis antagonists, i.e., LaCl3 (Ca+2 channel inhibitor), EGTA (Ca+2 chelator) and LiCl (endosomal Ca+2 release blocker), inhibited both SOD and Prx. Finally, a drastic reduction in apoplastic OH (hydroxyl radical) concentrations (induced by each treatment, leading to Prx inhibition) was observed via fluorometric analysis. A consequential inhibition of root growth observed under each treatment denotes the importance of the orchestrated functioning of PM H+-ATPase, NOX, Cu-Zn SOD and Prx during root growth. A working model demonstrating postulated enzymatic synchronization with an intervening role of Ca+2 is proposed.

Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity

Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1H NMR and 31P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F0 sector, and thereby increase ATP synthesis.

Esterases are responsible for malathion resistance in Anopheles stephensi: A proof using biochemical and insecticide inhibition studies.

Increase in prevalence and intensity of insecticide-resistance in vectors of vector-borne diseases is a major threat to sustainable disease control; and, for their effective management, studies on resistance mechanisms are important to come out with suitable strategies. Esterases are major class of detoxification enzymes in mosquitoes, which confers protection against insecticides in causing resistance. This study was aimed at biochemical characterization of esterases responsible for malathion resistance in Anopheles stephensi mosquitoes, along with its validation through biochemical techniques and native-PAGE assays. Laboratory maintained susceptible and resistant An. stephensi mosquitoes were used for assessing the activity and effect of α - and β -esterases on malathion. Bioassay, synergist bioassay, biochemical assay and native- PAGE were employed to characterize the role of esterases in conferring malathion-resistance. Notably significant (p < 0.0001) enhancement in α - and β -esterases activity was observed with 2-fold increase in resistant An. StephensiGOA compared to susceptible An. StephensiBB. native-PAGE depicted two major bands 'a' (Rf = 0.80) and 'b' (Rf = 0.72) in susceptible An. stephensiBB , while one intense band 'b' (Rf = 0.72) was visible in resistant An. stephensiGOA. Inhibition assay revealed complete inhibition of α - and β -esterases activity in presence of 1 mM malathion in susceptible strain compared to observed partial inhibition in resistant strain on native-PAGE. This study provides a better understanding on the role of esterase enzyme (carboxylesterase) in conferring malathion-resistance in An. stephensi mosquitoes, as evident from the native-PAGE assay results. The study results could be used in characterizing the resistance mechanisms in vectors and for suggesting alternative chemical insecticide based resistance management strategies for effective vector-borne disease control.

The auxin conjugate indole-3-acetyl-aspartate affects responses to cadmium and salt stress in Pisum sativum L.

The synthesis of IAA-amino acid conjugates is one of the crucial regulatory mechanisms for the control of auxin activity during physiological and pathophysiological responses. Indole-3-acetyl-aspartate (IAA-Asp) is a low molecular weight amide conjugate that predominates in pea (Pisum sativum L.) tissues. IAA-Asp acts as an intermediate during the auxin degradation pathway. However, some recent investigations suggest a direct signaling function of this conjugate in various processes. In this study, we examine the effect of 100μM IAA-Asp alone and in combination with salt stress (160mM NaCl) or heavy metal stress (250μM CdCl2) on H2O2 concentration, protein carbonylation as well as catalase and ascorbate (APX) and guaiacol peroxidase (GPX) activities in 7-day-old pea seedlings. As revealed by spectrophotometric analyses, IAA-Asp increased the carbonylated protein level and reduced the H2O2 concentration. Moreover, IAA-aspartate potentiated the effect of both Cd2+ ions and NaCl on the H2O2 level. The enzymatic activities (catalase and peroxidases) were examined using spectrophotometric and native-PAGE assays. IAA-Asp alone did not affect catalase activity, whereas the two peroxidases were regulated differently. IAA-Asp reduced the APX activity during 48h cultivation. APX activity was potentiated by IAA-Asp+NaCl after 48h. Guaiacol peroxidase activity was diminished by all tested compounds. Based on these results, we suggest that IAA-Asp can directly and specifically affect the pea responses to abiotic stress.

The c-Fos and c-Jun from Litopenaeus vannamei play opposite roles in Vibrio parahaemolyticus and white spot syndrome virus infection

Growing evidence indicates that activator protein-1 (AP-1) plays a major role in stimulating the transcription of immune effector molecules in cellular response to an incredible array of stimuli, including growth factors, cytokines, cellular stresses and bacterial and viral infection. Here, we reported the isolation and characterization of a cDNA from Litopenaeus vannamei encoding the full-length c-Fos protein (named as Lvc-Fos). The predicted amino acid sequences of Lvc-Fos contained a basic-leucine zipper (bZIP) domain, which was characteristic of members of the AP-1 family. Immunoprecipitation and native-PAGE assays determined that Lvc-Fos could interact with the Lvc-Jun, a homolog of c-Jun family in L. vannamei, in a heterodimer manner. Further investigation demonstrated that Lvc-Fos and Lvc-Jun were expressed in all tested tissues and located in the nucleus. Real-time RT-PCR analysis showed both Lvc-Fos and Lvc-Jun in gills were up-regulated during Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges. In addition, reporter gene assays indicated Lvc-Fos and Lvc-Jun could activate the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp, as well as WSSV immediate early (IE) genes wsv069 and wsv249, in a different manner. Knockdown of Lvc-Fos or Lvc-Jun by RNA interference (RNAi) resulted in higher mortalities of L. vannamei after infection with V. parahaemolyticus, suggesting that Lvc-Fos and Lvc-Jun might play protective roles in bacterial infection. However, silencing of Lvc-Fos or Lvc-Jun in shrimp caused lower mortalities and virus loads under WSSV infection, suggesting that Lvc-Fos and Lvc-Jun could be engaged for WSSV replication and pathogenesis. In conclusion, our results provided experimental evidence and novel insight into the roles of L. vannamei AP-1 in bacterial and viral infection.

Characterization of amino acid residues essential for tetramer formation and DNA-binding activity of ssDNA-binding protein of Mycobacterium tuberculosis

Mycobacterium tuberculosis Rv0054 encodes a single-stranded DNA-binding protein (MtbSSB) that is essential for survival of the human pathogen and causative agent of tuberculosis. The function of MtbSSB has been proposed to be different from its E. coli homolog. However, the critical amino acid residues of MtbSSB and their regulatory effects on DNA-binding ability remain to be clearly characterized. In this study, using a frequency-controlled random mutagenesis method (FRM), mutant libraries of MtbSSB were successfully constructed. On the whole, 146 single, double, and triple MtbSSB mutants, which covered 89% of the amino acid residues along the whole MtbSSB gene, were isolated. Using bacterial two-hybrid assays in combination with native PAGE assays, four new mutants, E62G, D104N, E94G/T137N, and S130P/G153N were found to totally or partially lose their ability to form tetramer. Three novel mutants, E62G, D104N, and E94G/T134N, were characterized to have a much lower ssDNA-binding activity, while one mutant, F21L, was found to have a significantly higher activity through both electrophoretic mobility shift and surface plasmon resonance assays. Interestingly, three amino acid residues, E62, D104, and E94, were found to regulate both oligomerization and ssDNA-binding activity of MtbSSB. Our work provides an important resource and should help improve the understanding of the biochemical mechanisms and structure-function relationship of the DNA-binding protein in this important human pathogen.

Danthron Functions as a Retinoic X Receptor Antagonist by Stabilizing Tetramers of the Receptor

Retinoic X receptor (RXR) is a promising target for drug discovery against cancer and metabolic syndromes. Here, we identified a specific RXRα antagonist, danthron, from the traditional Chinese medicine rhubarb. Danthron repressed all tested RXRα-involved response element transcription, including the RXRE, PPRE, FXRE, and LXRE. Results from native PAGE and isothermal titration calorimetry (ITC)-based assays indicated that danthron bound to the tetrameric RXRα-LBD in a specific stoichimetric ratio, and such a binding could influence the corepressor SMRT affinity to the receptor. Additionally, a unique tetrameric structure of the apo-RXRα ligand-binding domain (LBD) was determined, which exhibited a larger tetramer interface and different ligand-binding pocket size compared with the one previously reported. Together with the biochemical and biophysical results, the determined crystal structure of danthron-soaked RXRα-LBD suggested a new mechanism for danthron antagonism to tetrameric RXRα. Moreover, the in vivo efficient improvement of insulin sensitivity by danthron was observed in diet-induced obese (DIO) mice. Thus, our findings were expected to supply new insights into the structural basis of RXRα antagonist for its further potential therapeutic application.

Escherichia Coli Redox Enzyme Maturation Proteins, TorD and DmsD Interact with GTP as Shown by Native Page Assays

The twin-arginine translocation (Tat) system can transport fully folded proteins across the cytoplasmic membrane. Transport is dependent on a twin-arginine motif within a cleavable signal peptide. Substrates for the Tat system include redox proteins, necessary for growth during anaerobic respiration. The transport of the catalytic subunits responsible for N- and S-oxide respiration, DmsAB and TorA are dependent on DmsD and TorD respectively. Both are system specific chaperones termed redox enzyme maturation proteins (REMP) and are predicted to play multiple roles in the maturation and export of redox enzymes, including the known role for binding to the twin-arginine motif. Upon successful translocation and signal peptide cleavage, the REMPs remain in the cytoplasm. The question arises as to what governs binding and release of the signal peptide in order for translocation to occur. Here, we propose that REMPs may function like general chaperones like DnaK/DnaJ which exhibit nucleotide binding and hydrolysis for substrate release. It is speculated that TorD binds GTP or the molybdopterin dinucleotide with low affinity. Furthermore, GTP binding sites have been predicted for Shewanella typhimurium TorD (1N1C.pdb) and Salmonella typhimurium DmsD (1S9U.pdb) structures. We have developed a native PAGE assay, showing both DmsD and TorD exhibiting different banding patterns in the presence of GTP, while being unaffected by ATP. Preliminary results indicate that the presence of the NTP counter ion Mg2+ in addition to GTP does not change the banding patterns. DmsD and TorD share the same highly alpha-helical structure. Therefore we have done structural alignments to the E. coli DmsD (3EFP.pdb) structure and mapped the residues predicted to bind GTP to identify targets for mutagenesis. Our work aims to definitively answer the question if these chaperones indeed bind GTP.

Purification and Characterization of the Lipase from Marine Vibrio fischeri

Lipolytic enzymes from marine microbes have been the focus of intense and growing research. The bioluminescencebacterium Vibrio fischeri was produced lipase enzyme when the medium contained specific substrate. The lipase waspurified from the concentrated culture supernatant. The most active fractions were obtained using the technique ofprecipitation with ammonium sulphate. The precipitated fraction was purified by desalting and ion exchangechromatography. The purified active fraction exhibiting final specific activity of 121U/mg and characterized; theoptimum pH was likely between 7 to 8, the optimum temperature was 30°C and about 80 % of activity at 5°C. Theenzyme was very stable at the pH 8, at the temperature 30°C. The enzyme was monomeric protein having molecularmass of 57 KDa estimated by native PAGE assay.

Purification and Partial Characterization of Esterase from Marine Vibrio fischeri

Lipolytic enzymes from marine microbes have been the focus of intense and growing research. The bioluminescence bacterium Vibrio fischeri was produced esterase enzyme when the medium contained specific substrate. The esterase was purified from the concentrated culture supernatant. The most active fractions were obtained using the technique of precipitation with 1N HCl. The precipitated fraction was purified by ion exchange chromatography (DEAE-Cellulose) and gel filteration chromatography (Sephadex G200). The purified active fraction exhibiting final specific activity of 300U/mg and characterized; the optimum pH was 7.5, the optimum temperature was 30°C. The enzyme was very stable at the temperature 30°C and at wide range of pH. The enzyme was monomeric protein having molecular mass of 37 KDa estimated by native PAGE assay.

Interactions between homopolymeric amino acids (HPAAs)

Many human proteins contain consecutive amino acid repeats, known as homopolymeric amino acid (HPAA) tracts. Some inherited diseases are caused by proteins in which HPAAs are expanded to an excessive length. To this day, nine polyglutamine-related diseases and nine polyalanine-related diseases have been reported, including Huntington's disease and oculopharyngeal muscular dystrophy. In this study, potential HPAA-HPAA interactions were examined by yeast two-hybrid assays using HPAAs of approximately 30 residues in length. The results indicate that hydrophobic HPAAs interact with themselves and with other hydrophobic HPAAs. Previously, we reported that hydrophobic HPAAs formed large aggregates in COS-7 cells. Here, those HPAAs were shown to have significant interactions with each other, suggesting that hydrophobicity plays an important role in aggregation. Among the observed HPAA-HPAA interactions, the Ala28-Ala29 interaction was notable because polyalanine tracts of these lengths have been established to be pathogenic in several polyalanine-related diseases. By testing several constructs of different lengths, we clarified that polyalanine self-interacts at longer lengths (>23 residues) but not at shorter lengths (six to approximately 23 residues) in a yeast two-hybrid assay and a GST pulldown assay. This self-interaction was found to be SDS sensitive in SDS-PAGE and native-PAGE assays. Moreover, the intracellular localization of these long polyalanine tracts was also observed to be disturbed. Our results suggest that long tracts of polyalanine acquire SDS-sensitive self-association properties, which may be a prerequisite event for their abnormal folding. The misfolding of these tracts is thought to be a common molecular aspect underlying the pathogenesis of polyalanine-related diseases.

RNA Recognition Mechanism of the Minimal Active Domain of the Human Immunodeficiency Virus Type-2 Nucleocapsid Protein

NCp8 of HIV-2 contains two CCHC-type zinc fingers connected by a linker, and is involved in many critical steps of the virus life cycle. It was previously shown that the first zinc finger flanked by the linker is the minimal active domain for specific binding to viral RNA. In our previous study, we determined the three-dimensional structure of NCp8-f1, including the minimal active domain, and found that a hydrogen bond between Asn(11) N(delta)H and Arg(27) O stabilized the conformation of the linker in the vicinity of the zinc finger [Kodera et al. (1998) Biochemistry 37, 17704-17713]. In this study, RNA binding activities of NCp8-f1 and three types of its mutant peptides were analysed by native PAGE assay. The activity and three-dimensional structure of NCp8-f1/N11A, in which alanine is substituted for Asn(11) thereby affecting the conformation of the linker, was analyzed and compared with those of NCp8-f1. We demonstrated that the existence of Arg(4) and/or Lys(5) and Arg(26) and/or Arg(27) were necessary for binding RNA. Furthermore, the linker's flexible orientation, which is controlled by the hydrogen bond between Asn(11) N(delta)H and Arg(27) O, appears to be a structural basis for NCp8 existing as a multi-functional protein.

Tobacco Nectaries Express a Novel NADPH Oxidase Implicated in the Defense of Floral Reproductive Tissues against Microorganisms

Hydrogen peroxide produced from the nectar redox cycle was shown to be a major factor contributing to inhibition of most microbial growth in floral nectar; however, this obstacle can be overcome by the floral pathogen Erwinia amylovora. To identify the source of superoxide that leads to hydrogen peroxide accumulation in nectary tissues, nectaries were stained with nitroblue tetrazolium. Superoxide production was localized near nectary pores and inhibited by diphenylene iodonium but not by cyanide or azide, suggesting that NAD(P)H oxidase is the source of superoxide. Native PAGE assays demonstrated that NADPH (not NADH) was capable of driving the production of superoxide, diphenyleneiodonium chloride was an efficient inhibitor of this activity, but cyanide and azide did not inhibit. These results confirm that the production of superoxide was due to an NADPH oxidase. The nectary enzyme complex was distinct by migration on gels from the leaf enzyme complex. Temporal expression patterns demonstrated that the superoxide production (NADPH oxidase activity) was coordinated with nectar secretion, the expression of Nectarin I (a superoxide dismutase in nectar), and the expression of NOX1, a putative gene for a nectary NADPH oxidase that was cloned from nectaries and identified as an rbohD-like NADPH oxidase. Further, in situ hybridization studies indicated that the NADPH oxidase was expressed in the early stages of flower development although superoxide was generated at later stages (after Stage 10), implicating posttranslational regulation of the NADPH oxidase in the nectary.

Role of Cyclophilin B in Activation of Interferon Regulatory Factor-3

IRF-3 is a member of the interferon regulatory factors (IRFs) and plays a principal role in the induction of interferon-beta (IFN-beta) by virus infection. Virus infection results in the phosphorylation of IRF-3 by IkappaB kinase epsilon and TANK-binding kinase 1, leading to its dimerization and association with the coactivators CREB-binding protein/p300. The IRF-3 holocomplex translocates to the nucleus, where it induces IFN-beta. In the present study, we examined the molecular mechanism of IRF-3 activation. Using bacterial two-hybrid screening, we isolated molecules that interact with IRF-3. One of these was cyclophilin B, a member of the immunophilins with a cis-trans peptidyl-prolyl isomerase activity. A GST pull-down assay suggested that one of the autoinhibition domains of IRF-3 and the peptidyl-prolyl isomerase domain of cyclophilin B are required for the binding. A knockdown of cyclophilin B expression by RNA interference resulted in the suppression of virus-induced IRF-3 phosphorylation, leading to the inhibition of the subsequent dimerization, association with CREB-binding protein, binding to the target DNA element, and induction of IFN-beta. These findings indicate that cyclophilin B plays a critical role in IRF-3 activation.

Immunological assessment of an insecticide resistance-associated esterase in the Western corn rootworm

In previous investigations, we have determined that organophosphate resistance in the western corn rootworm, Diabrotica virgifera virgifera, is at least partially attributed to a group of non-specific carboxylesterases referred to as group II. Antiserum raised against a purified 66-kDa group II esterase is specific for the denatured enzyme. This antiserum reacts similarly with both beetle homogenates from resistant and susceptible populations, although there is much higher signal intensity in immunoblots of resistant relative to susceptible beetles. These results suggest that overproduction of group II esterases is the underlying basis of esterase-mediated resistance in D. v. virgifera by demonstrating that (1) group II esterases are immunologically indistinguishable between the resistant and susceptible populations, and (2) the intensity differences are due to increased group II esterase proteins in the resistant population. The diagnostic potential of immunological-based assays was tested with a traditional diagnostic concentration bioassay and a biochemical-based native PAGE assay. Significant correlations were observed among all three diagnostic assays (regression coefficients ranging from 0.95 to 0.96). These results demonstrate the importance of the 66-kDa protein as a resistance-associated biochemical marker, thus emphasizing the potential for 66-kDa protein-targeted immunoassays in resistance monitoring programs.

Shared Protein Components of SINE RNPs

The heterogeneous, short RNAs produced from the high, copy, short mobile elements (SINEs) interact with proteins to form RNA–protein (RNP) complexes. In particular, the BC1 RNA, which is transcribed to high levels specifically in brain and testis from one locus of the ID SINE family, exists as a discrete RNP complex. We expressed a series of altered BC1, and other SINE-related RNAs, in several cell lines and tested for the mobility of the resulting RNP complexes in a native PAGE assay to determine which portions of these SINE RNAs contribute to protein binding. When different SINE RNAs were substituted for the BC1 ID sequence, the resulting RNPs exhibited the same mobility as BC1. This indicates that the protein(s) binding to the ID portion of BC1 is not sequence specific and may be more dependent upon the secondary structure of the RNA. It also suggests that all SINE RNAs may bind a similar set of cellular proteins. Deletion of the A-rich region of BC1 RNA has a marked effect on the mobility of the RNP. Rodent cell lines exhibit a slightly different mobility for this shifted complex when compared to human cell lines, reflecting evolutionary differences in one or more of the protein components. On the basis of mobility change observed in RNP complexes when the A-rich region is removed, we decided to examine poly(A) binding protein (PABP) as a candidate member of the RNP. An antibody against the C terminus of PABP is able to immunoprecipitate BC1 RNA, confirming PABP's presence in the BC1 RNP. Given the ubiquitous role of poly(A) regions in the retrotransposition process, these data suggest that PABP may contribute to the SINE retrotransposition process.