[Show abstract][Hide abstract] ABSTRACT: Expression of chitinase is developmentally regulated in insects in consonance with their molting process. During the larval-larval metamorphosis in Helicoverpa armigera, chitinase gene expression varies from high to negligible. In the five-day metamorphic course of fifth-instar larvae, chitinase transcript is least abundant on third day and maximal on fifth day. MicroRNA library prepared from these highest and lowest chitinase-expressing larval stages resulted in isolation of several miRNAs. In silico analysis of sequenced miRNAs revealed three miRNAs having sequence similarity to 3'UTR of chitinase. Gene-targeted specific action of these miRNAs, was investigated by luciferase reporter having 3'UTR of chitinase. Only one of three miRNAs, miR-24, inhibited luciferase expression. Further, a day-wise in vivo quantification of miR-24 in fifth-instar larvae revealed a negative correlation with corresponding chitinase transcript abundance. The force-feeding of synthetic miR-24 induced significant morphological aberrations accompanied with arrest of molting. These miR-24 force-fed larvae revealed significantly reduced chitinase transcript abundance.
[Show abstract][Hide abstract] ABSTRACT: Anopheles culicifacies is the main vector for transmission of Plasmodium vivax malaria in the Indian subcontinent. A strain of An. culicifacies isolated from its natural niche displayed complete refractoriness to P. vivax by melanotic encapsulation of ookinetes. Prophenoloxidases are key components of the phenoloxidase cascade that leads to recognition and melanization of invading organisms. We isolated and cloned prophenoloxidase-encoding acppo6 gene of An. culicifacies and analyzed its expression profile under various regimens of immune challenge. The acppo6 was differentially expressed during various stages of larval development. The acppo6 transcription was also up-regulated in response to bacteria and Plasmodium vinckei petteri challenge. The transcript levels of the acppo6 gene were higher in naive adult refractory female mosquitoes as compared with female susceptible mosquitoes. Furthermore, the induction of acppo6 in the susceptible strain upon Plasmodium infection was negligible as compared with that of the refractory strain. The observation is suggestive of the role of acppo6 in effectuating a melanotic response in Plasmodium-incompetent naturally occurring refractory An. culicifacies strain.
No preview · Article · Nov 2010 · Journal of Medical Entomology
[Show abstract][Hide abstract] ABSTRACT: Serine proteases are a class of proteolytic enzymes that are synthesized as enzymically inactive zymogens and when required
in the cell, they are activated by the removal of proregion. The role of proregions as potent and specific inhibitors of their
associated protease has been established. Here, we investigated the inhibition of a recombinantly expressed and refolded Anopheles
ulicifacies serine protease (ACSP) that was isolated from the body tissue of an Indian malaria vector, A. culicifacies by its own N-terminally located 19 amino acid residue propeptide. The synthetic peptide identical to the propeptide, its
three deletion mutants and leupeptin (a general serine protease inhibitor) were tested invitro for their inhibitory activity towards recombinant ACSP. Amongst the five peptides tested, leupeptin displayed maximum inhibition
closely followed by native propeptide. The reduction or loss of inhibitory potential of deletion mutants of propeptide revealed
the importance of charged residues present in the propeptide for inhibition of the cognate enzyme.
No preview · Article · Mar 2008 · International Journal of Peptide Research and Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Insecticidal crystal proteins produced by strains of Bacillus thuringiensis cause larval death upon interaction with specific receptors located at the midgut epithelium of susceptible insects. Large quantities of easily purified aminopeptidase and cadherin-like Cry toxin receptors can facilitate the further study of Cry toxin binding and pore formation. Here, we report the solubilisation and purification of aminopeptidase N from Spodoptera litura (SlAPN). Recombinantly expressed and membrane anchored aminopeptidase N showed differential solubilisation with various ionic and nonionic detergents. The N-lauryl sarcosine (NLS)-solubilised SlAPN was purified to near homogeneity by anion exchange and gel filtration chromatography and refolded to its catalytically active form. The optimized purification regimen lead to >90% purification of the catalytically active SlAPN with 11% recovery and 9-folds purification. The interaction of purified SlAPN with biologically active Cry1C protein has been qualitatively and quantitatively characterized. By ligand blotting experiment, we demonstrated the linearity of interaction of the two purified proteins and lack of interaction of SlAPN with structurally divergent nontoxic Cry1Ac protein. The equilibrium dissociation constant (K(D)) of purified SlAPN for Cry1C was calculated by ELISA (90nM). Interaction of enzymatically inactive SlAPN with Cry1C and catalytic activity of APN-Cry1C complex suggested that the catalytic site and toxin-binding sites of SlAPN do not overlap.
No preview · Article · Aug 2007 · Protein Expression and Purification
[Show abstract][Hide abstract] ABSTRACT: Background
The main vector for transmission of malaria in India is the Anopheles culicifacies mosquito species, a naturally selected subgroup of which is completely refractory (R) to transmission of the malaria parasite, Plasmodium vivax;
Here, we report the molecular characterization of a serine protease (acsp30)-encoding gene from A. culicifacies, which was expressed in high abundance in the refractory strain compared to the susceptible (S) strain. The transcriptional upregulation of acsp30 upon Plasmodium challenge in the refractory strain coincided with ookinete invasion of mosquito midgut. Gene organization and primary sequence of acsp30 were identical in the R and S strains suggesting a divergent regulatory status of acsp30 in these strains. To examine this further, the upstream regulatory sequences of acsp30 were isolated, cloned and evaluated for the presence of promoter activity. The 702 bp upstream region of acsp30 from the two strains revealed sequence divergence. The promoter activity measured by luciferase-based reporter assay was shown to be 1.5-fold higher in the R strain than in the S. Gel shift experiments demonstrated a differential recruitment of nuclear proteins to upstream sequences of acsp30 as well as a difference in the composition of nuclear proteins in the two strains, both of which might contribute to the relative abundance of acsp30 in the R strain;
The specific upregulation of acsp30 in the R strain only in response to Plasmodium infection is suggestive of its role in contributing the refractory phenotype to the A. culicifacies mosquito population.
Full-text · Article · May 2007 · BMC Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The application of Bacillus thuringiensis is revised, with description of its biology, ecology and potentials for insects management. Co-evolution of the crystal proteins
structure with insect hosts is reviewed, with data on the classification and nomenclature. The function of various Cry proteins
and their role in insect parasitism are described together with the mechanism of action of the toxins. Applications for control
of mosquitoes and blackflies and formulations are reviewed. Factors related to the development of resistance and potentials
in the integrated pest management are discussed.
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) has emerged as a powerful tool to rapidly analyze gene functions in a wide variety of eukaryotic organisms as well as in cultured cell lines. We demonstrate here that RNAi can be applied to study the function of a transgene expressed in an insect cell line (Spodoptera frugiperda, Sf21). The aminopeptidase N gene (apn) targeted for silencing in the present study was isolated from the midgut of Spodoptera litura larvae and expressed in Sf21 cells using baculovirus expression system. The recombinant APN protein expressed at the surface of Sf21 cells was shown to interact with insecticidal crystal protein, Cry1C, by in vitro experiments. The exogenous addition/transfection of APN dsRNA or siRNA in the cultured cells resulted in partial/complete inhibition of expression of apn leading to the loss of toxin binding to the transgene expressing cells. These experiments highlighted the usefulness of RNAi as a tool to study the function of an expressed transgene in insect cell line and to study the specificity of receptor-ligand interaction.
No preview · Article · Aug 2004 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.
Full-text · Article · Jan 2004 · Microbiology and Molecular Biology Reviews
[Show abstract][Hide abstract] ABSTRACT: Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth. Unlike delta-endotoxins which are produced as parasporal inclusion bodies within the cell during sporulation, VIP is secreted into the culture medium. Here we report the relocation of the expression of VIP into the mother cell compartment in a manner similar to well-characterized Cry proteins. Relocation of VIP is directed to mother cell by placing its synthesis under sporulation-dependent promoters, BtI and BtII. The insertion of cry preferred transcription termination sequence at the 3(') region and a STAB-SD sequence at the 5(') region of the gene provided stability to the vip transcript and enhanced its yield. The demonstrated expression of VIP within the cells in the form of inclusion bodies would facilitate development of a suitable formulation for the application of this class of insecticidal proteins in the field.
Full-text · Article · Nov 2003 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect.
[Show abstract][Hide abstract] ABSTRACT: Insecticidal crystal proteins of Bacillus thuringiensis bind to receptors in the midgut of susceptible insects leading to pore formation and death of the insect. The identity of the receptor is not clearly established. Recently a direct interaction between a cloned and heterologously expressed aminopeptidase (slapn) from Spodoptera litura and the Cry1C protein was demonstrated by immunofluorescence and in vitro ligand blot interaction. Here we show that administration of slapn double-stranded RNA to S. litura larvae reduces its expression. As a consequence of the reduced expression, a corresponding decrease in the sensitivity of these larvae to Cry1C toxin was observed. The gene silencing was retained during the insect's moulting and development and transmitted to the subsequent generation albeit with a reduced effect. These results directly implicate larval midgut aminopeptidase N as receptor for Bacillus thuringiensis insecticidal proteins.
No preview · Article · Jan 2003 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 971 Recombinantly expressed isoenzymic aminopeptidases from Helicoverpa armigera (American cotton bollworm) midgut display differential interaction with closely related Bacillus thuringiensis insecticidal proteins Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of amino-peptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoerpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these
Full-text · Article · Jan 2003 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: Malaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double-stranded RNA of cysteine protease genes (falcipain-1 and -2) in the malaria parasite, Plasmodium falciparum. Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain double-stranded (ds)RNAs are reminiscent of the effects observed upon administering E-64, a cysteine protease inhibitor. The parasites treated with falcipain's dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are broken into short interference RNAs approximately 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence-specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.
Full-text · Article · Oct 2002 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series
of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report
the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of
a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining
of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing
S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol
moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies
and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to
the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.
Full-text · Article · Oct 2002 · Applied and Environmental Microbiology