Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal beta 1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 x 10(6) cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) crossreacted with re-PNA. The subunit molecular weight (30 kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.

Interrogating an insect society

Insect societies such as those of ants, bees, and wasps consist of 1 or a small number of fertile queens and a large number of sterile or nearly sterile workers. While the queens engage in laying eggs, workers perform all other tasks such as nest building, acquisition and processing of food, and brood care. How do such societies function in a coordinated and efficient manner? What are the rules that individuals follow? How are these rules made and enforced? These questions are of obvious interest to us as fellow social animals but how do we interrogate an insect society and seek answers to these questions? In this article I will describe my research that was designed to see answers from an insect society to a series of questions of obvious interest to us. I have chosen the Indian paper wasp Ropalidia marginata for this purpose, a species that is abundantly distributed in peninsular India and serves as an excellent model system. An important feature of this species is that queens and workers are morphologically identical and physiologically nearly so. How then does an individual become a queen? How does the queen suppress worker reproduction? How does the queen regulate the nonreproductive activities of the workers? What is the function of aggression shown by different individuals? How and when is the queen's heir decided? I will show how such questions can indeed be investigated and will emphasize the need for a whole range of different techniques of observation and experimentation.

Characterization of the genome of Oryctes baculovirus, a viral biocide of the insect pest Oryctes rhinoceros

Oryctes baculovirus is a viral biocide exploited for the control of the insect pest Oryctes rhinoceros. We have recently established a physical map of the genome of the Indian isolate of Oryctes baculovirus (OBV-KI). Here we examine the genomic relatedness between OBV-KI and OBV-PV505, the type isolate (originally from the Philippines), by DNA reassociation kinetics and by the use of restriction endonucleases. On the basis of differences in restriction-enzyme profiles between the two genomes, and previously reported differences in protein profiles and antigenic makeup, we propose the taxonomic status of a variant of Oryctes baculovirus for the Indian isolate.

Detection of a CpA methylase in an insect system: characterization and substrate specificity

A cytosine-specific DNA methyltransferase (EC 2.1.1.37) has been purified to near homogeneity from a mealybug (Planococcus lilacinus). The enzyme can methylate cytosine residues in CpG sequences as well as CpA sequences. The apparent molecular weight of the enzyme was estimated as 135,000 daltons by FPLC. The enzyme exhibits a processive mode of action and a salt dependance similar to mammalian methylases. Mealybug methylase exhibits a preference for denatured DNA substrates.

Piezoelectrically actuated insect scale flapping wing

An energy method is used in order to derive the non-linear equations of motion of a smart flapping wing. Flapping wing is actuated from the root by a PZT unimorph in the piezofan configuration. Dynamic characteristics of the wing, having the same size as dragonfly Aeshna Multicolor, are analyzed using numerical simulations. It is shown that flapping angle variations of the smart flapping wing are similar to the actual dragonfly wing for a specific feasible voltage. An unsteady aerodynamic model based on modified strip theory is used to obtain the aerodynamic forces. It is found that the smart wing generates sufficient lift to support its own weight and carry a small payload. It is therefore a potential candidate for flapping wing of micro air vehicles.

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian Cobra) venom

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue. (C) Munksgaard 1995.

Characterization of Major Zinc Containing Myonecrotic and Procoagulant Metalloprotease `Malabarin' from Non Lethal Trimeresurus malabaricus Snake Venom with Thrombin Like Activity: Its Neutralization by Chelating Agents

A major myonecrotic zinc containing metalloprotease `malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu-Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A alpha followed by B beta subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Changing resonator geometry to boost sound power decouples size and song frequency in a small insect

Despite their small size, some insects, such as crickets, can produce high amplitude mating songs by rubbing their wings together. By exploiting structural resonance for sound radiation, crickets broadcast species-specific songs at a sharply tuned frequency. Such songs enhance the range of signal transmission, contain information about the signaler's quality, and allow mate choice. The production of pure tones requires elaborate structural mechanisms that control and sustain resonance at the species-specific frequency. Tree crickets differ sharply from this scheme. Although they use a resonant system to produce sound, tree crickets can produce high amplitude songs at different frequencies, varying by as much as an octave. Based on an investigation of the driving mechanism and the resonant system, using laser Doppler vibrometry and finite element modeling, we show that it is the distinctive geometry of the crickets' forewings (the resonant system) that is responsible for their capacity to vary frequency. The long, enlarged wings enable the production of high amplitude songs; however, as a mechanical consequence of the high aspect ratio, the resonant structures have multiple resonant modes that are similar in frequency. The drive produced by the singing apparatus cannot, therefore, be locked to a single frequency, and different resonant modes can easily be engaged, allowing individual males to vary the carrier frequency of their songs. Such flexibility in sound production, decoupling body size and song frequency, has important implications for conventional views of mate choice, and offers inspiration for the design of miniature, multifrequency, resonant acoustic radiators.

Biomechanics of Substrate Boring by Fig Wasps: Role of Zinc in Insect Cuticle

There are many biomechanical challenges that a female insect must meet to successfully oviposit and ensure her evolutionary success. These begin with selection of a suitable substrate through which the ovipositor must penetrate without itself buckling or fracturing. The second phase corresponds to steering and manipulating the ovipositor to deliver eggs at desired locations. Finally, the insect must retract her ovipositor fast to avoid possible predation and repeat this process multiple times during her lifetime. From a materials perspective, insect oviposition is a fascinating problem and poses many questions. Specifically, are there diverse mechanisms that insects use to drill through hard substrates without itself buckling or fracturing? What are the structure-property relationships in the ovipositor material? These are some of the questions we address with a model system consisting of a parasitoid fig wasp - fig substrate system. To characterize the structure of ovipositors, we use scanning electron microscopy with a detector to quantify the presence of transition elements. Our results show that parasitoid ovipositors have teeth like structures on their tips and contain high amounts of zinc as compared to remote regions. Sensillae are present along the ovipositor to aid detection of chemical species and mechanical deformations. To quantify the material properties of parasitoid ovipositors, we use an atomic force microscope and show that tip regions have higher modulus as compared to remote regions. Finally, we use videography to show that ovipositors buckle during oviposition and estimate the forces needed to cause substrate boring based on Euler buckling analysis. Such methods may be useful for the design of functionally graded surgical tools.

Conformational Diversity in Contryphans from Conus Venom: cis-trans Isomerisation and Aromatic/Proline Interactions in the 23-Membered Ring of a 7-Residue Peptide Disulfide Loop

Conformational diversity or shapeshifting in cyclic peptide natural products can, in principle, confer a single molecular entity with the property of binding to multiple receptors. Conformational equilibria have been probed in the contryphans, which are peptides derived from Conus venom possessing a 23-membered cyclic disulfide moiety. The natural sequences derived from Conus inscriptus, GCV(D)LYPWC* (In936) and Conus loroisii, GCP(D)WDPWC* (Lo959) differ in the number of proline residues within the macrocyclic ring. Structural characterisation of distinct conformational states arising from cis-trans equilibria about Xxx-Pro bonds is reported. Isomerisation about the C2-P3 bond is observed in the case of Lo959 and about the Y5-P6 bond in In936. Evidence is presented for as many as four distinct species in the case of the synthetic analogue V3P In936. The Tyr-Pro-Trp segment in In936 is characterised by distinct sidechain orientations as a consequence of aromatic/proline interactions as evidenced by specific sidechain-sidechain nuclear Overhauser effects and ring current shifted proton chemical shifts. Molecular dynamics simulations suggest that Tyr5 and Trp7 sidechain conformations are correlated and depend on the geometry of the Xxx-Pro bond. Thermodynamic parameters are derived for the cis trans equilibrium for In936. Studies on synthetic analogues provide insights into the role of sequence effects in modulating isomerisation about Xxx-Pro bonds.

Isolation and characterization of biofumigant from leaves of Lantana camara for control of stored grain insect pests

Due to environmental concerns, health hazards to man and the evolution of resistance in insect pests, there have been constant efforts to discover newer insecticides both from natural sources and by chemical synthesis. Natural sources for novel molecules hold promise in view of their eco-friendly nature, selectivity and mammalian safety. We have isolated one natural bioactive molecule from the leaves of Lantana camara named Coumaran, based on various physical-chemical and spectroscopic techniques (IR, H-1 NMR, C-13 NMR and MS). Coumaran is highly toxic and very low concentration is needed for control of stored product insects. This molecule has potent grain protectant potential and caused significant reduction in F1 progeny of all the three species in the treated grain and the progeny was completely suppressed at 30 mu g/l. The differences in germination between the control and treated grains were not significant. The lack of any adverse effect of Coumaran on the seed germination is highly desirable for a grain protectant, becoming a potential source of biofumigant for economical and environmentally friendly pest control strategies against stored grain pests during storage of grains or pulses. (C) 2013 Elsevier B.V. All rights reserved.