Purification and characterization of an insect haemolymph protein promoting in vitro replication of the Bombyx mori nucleopolyhedrovirus.
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We have identified a novel protein that promotes Bombyx mori nucleopolyhedrovirus (BmNPV) replication in vitro. This protein was purified from heat-treated haemolymph of B. mori larvae by gel filtration and ion exchange chromatography, and designated as promoting protein (PP). The molecular mass of native PP estimated by column chromatography and that of denatured PP estimated by SDS-PAGE were 9600 Da and 15200 Da, respectively, suggesting that native PP is composed of a single polypeptide and may behave in the column as if it is a smaller protein because of its conformation and/or adsorptive nature. Addition of the PP to the culture medium of SES-BoMo-15A cells derived from B. mori embryos resulted in the strong promotion of BmNPV replication. The promoting activity positively correlated with the amount of PP in the culture medium up to 1 microg/ml, above which maximum virus replication occurred and resulted in the highest budded virus production and polyhedrin promoter-mediated luciferase gene expression of 10000-fold and 6000-fold higher than those without PP, respectively. A cDNA encoding the PP precursor (prePP) was successfully cloned and sequenced. Comparison between the amino acid sequence deduced from the nucleotide sequence of prePP cDNA and the N-terminal 18 amino acids determined for the purified PP indicated that the prePP (154 amino acids) consisted of a mature PP polypeptide (136 amino acids) with a signal sequence at the N terminus. Recombinant PP expressed from the cDNA using a baculovirus vector was similar in molecular mass, immunoreactivity and promoting activity to the native PP. (+info)
Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella.
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Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors. (+info)
Haemolymph Mg(2+) regulation in decapod crustaceans: physiological correlates and ecological consequences in polar areas.
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Reptant decapod crustaceans are almost absent from the Southern Ocean south of the Antarctic Convergence. We tested the hypothesis that this may be due to the reduced ability of this group to regulate Mg(2+) levels in the haemolymph ([Mg(2+)](HL)). Mg(2+) acts as an anaesthetic in marine invertebrates and its level is higher in Reptantia (crabs such as Cancer spp., Chionoecetes spp., Maja spp., 30-50 mmol l(-)(1)) than in Natantia (prawns such as Pandalus spp., Palaemon spp., Crangon spp., 5-12 mmol l(-)(1)). We varied [Mg(2+)](HL) in three species of reptant decapod crustaceans, Carcinus maenas, Hyas araneus and Eurypodius latreillei, and investigated heart rate, the rate of oxygen consumption and levels of spontaneous and forced activity at different temperatures. The rate of oxygen consumption and heart rate increased significantly with reduction in [Mg(2+)](HL) over the entire temperature range investigated in E. latreillei. In H. araneus, an increase in metabolic and heart rates compared with control values was found only at temperatures below 2 degrees C. Forced and spontaneous activity levels increased significantly in the group of [Mg(2+)](HL)-reduced animals below 0 degrees C, at which control animals were mostly inactive. At a reduced [Mg(2+)](HL) of 5-12 mmol l(-)(1), which is the [Mg(2+)](HL) of caridean shrimps in the Southern Ocean, Q(10) and activation energy were reduced for all these variables and extended the temperature range over which physiological functions were maintained. We suggest that the high [Mg(2+)](HL) in Reptantia causes relaxation of the animals and reduces their scope for activity, especially at temperatures below 0 degrees C. The hypothesis that the synergistic effects of high [Mg(2+)](HL) and low temperature probably prevented the Reptantia from recolonizing the permanently cold water of polar areas is discussed. (+info)
A tubular network associated with the brush-border surface of the Aedes aegypti midgut: implications for pathogen transmission by mosquitoes.
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The mosquito Aedes aegypti is capable of transmitting a variety of pathogens to man and to other vertebrates. The midgut of this insect has been well-studied both as the tissue where the first contact occurs between ingested pathogens and the insect host, and as a model system for blood meal digestion in blood-sucking insects. To understand better the nature of the midgut surface encountered by parasites or viruses, we used scanning electron microscopy to identify the most prominent structures and cell morphologies on the luminal midgut surface. The luminal side of the midgut is a complex and layered set of structures. The microvilli that are found on most, but not all, cells are covered by a network of fine strands that we have termed the microvilli-associated network (MN). The MN strands are membranous, as shown by a membrane bilayer visible in cross sections of MN strands at high magnification in transmission electron micrographs. The MN is found in blood-fed as well as unfed mosquitoes and is not affected by chitinase treatment, suggesting that it is not related to the chitinous peritrophic membrane that is formed only after blood feeding. The cells in the midgut epithelium have two distinct morphologies: the predominant cell type is densely covered with microvilli, while cells with fewer microvilli are found interspersed throughout the midgut. We used lectins to probe for the presence of carbohydrates on the midgut surface. A large number of lectins bind to the luminal midgut surface, suggesting that a variety of sugar linkages are present on the structures visualized by electron microscopy. Some of these lectins partially block attachment of malaria ookinetes to the midgut surface in vitro. Thus, the mosquito midgut epithelium, like the lining of mammalian intestines, is complex, composed of a variety of cell types and extensively covered with surface carbohydrate that may play a role in pathogen attachment. (+info)
Molecular characterization of an additional shrimp hyperglycemic hormone: cDNA cloning, gene organization, expression and biological assay of recombinant proteins.
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The crustacean eyestalk CHH/MIH/GIH neurohormone gene family represents a unique group of neuropeptides identified mainly in crustaceans. In this study, we report the cloning and characterization of the cDNA and the gene encoding the hyperglycemic hormone (MeCHH-B) of the shrimp Metapenaeus ensis. The amino acid sequence of MeCHH-B shows 85% identity to that of MeCHH-A (formerly MeCHH-like neuropeptide). Two separate but identical MeCHH-B genes were identified in the genome of shrimp by library screening and they are located on different CHH gene clusters. The organization of the MeCHH-B gene is identical to other members of the CHH/MIH/GIH neurohormone family. MeCHH-B is expressed at a constant level in the eyestalks of juveniles and mature females. Unlike the MeCHH-A gene, a low level of MeCHH-B transcripts can also be detected in the central nervous system. Interestingly, the expression pattern of MeCHH-B in the eyestalk of vitellogenic females is reversed to that of the MeCHH-A gene. At the middle stage of gonad maturation, a minimum level of MeCHH-B transcript was recorded and a maximum level of MeCHH-A transcript was detected. Recombinant proteins for MeCHH-A and MeCHH-B were produced by a bacterial expression system. The hemolymph glucose level of bilaterally eyestalk-ablated shrimp increased two-fold 1 h after the rCHH injection and then returned to normal after 2 h. The hyperglycemic effect of these fusion proteins is comparable to that of de-stalked shrimp injected with crude extract from a single sinus gland. (+info)
Bacterial-injection-induced syntheses of N-beta-alanyldopamine and Dopa decarboxylase in the hemolymph of coleopteran insect, Tenebrio molitor larvae.
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Injection of Escherichia coli into larvae of the coleopteran Tenebrio molitor resulted in the appearance of a dopamine-like substance on the electrochemical detector. To characterize this dopamine-like substance, we purified it to homogeneity from the immunized hemolymph and determined its molecular structure to be N-beta-alanyldopamine using the liquid chromatographic/tandem mass spectrometric method. Chemically synthesized N-beta-alanyldopamine showed the same retention time on HPLC as the purified N-beta-alanyldopamine from immunized larvae. To elucidate the molecular mechanism of N-beta-alanyldopamine synthesis in vivo, we examined the enzyme activity of Dopa decarboxylase against E. coli-injected hemolymph of T. molitor larvae. The enzyme activity of Dopa decarboxylase increased dramatically approximately 8 h after injection; Dopa decarboxylase activity of injected larvae being 10-times higher than naive larvae after 24 h. To evaluate the extent of quantitative changes of Dopa decarboxylase in response to bacterial challenge, Tenebrio Dopa decarboxylase was purified to homogeneity from the whole larvae and a cDNA clone for Tenebrio Dopa decarboxylase was isolated. RNA blot hybridization revealed that expression of the Dopa decarboxylase gene was activated transiently 3-8 h after E. coli challenge. Immunoprecipitation experiments showed that Tenebrio Dopa decarboxylase was detected from 8 to 24 h in E. coli-injected larval extract. Thus, bacterial injection into T. molitor larvae might induce transcriptional activation of a Dopa decarboxylase gene, and then synthesis of N-beta-alanyldopamine. The synthesized N-beta-alanyldopamine might be used as a substrate by phenoloxidase during melanin synthesis in the humoral defense response or the melanotic encapsulation reaction of the cellular defense response. (+info)
Octopamine mimics the effects of parasitism on the foregut of the tobacco hornworm Manduca sexta.
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The parasitic braconid wasp Cotesia congregata lays its eggs inside the body of the larval stage of its host, the moth Manduca sexta. The Cotesia congregata larvae develop within the hemocoel of their host until their third instar, when they emerge and spin cocoons and pupate on the outer surface of the caterpillar. From this time until their death approximately 2 weeks later, the Manduca sexta larvae show striking behavioral changes that include dramatic declines in spontaneous activity and in the time spent feeding. Coincident with these behavioral changes, it is known that octopamine titers in the hemolymph of the host become elevated by approximately 6.5-fold. Octopamine is an important modulator of neural function and behavior in insects, so we examined hosts for neural correlates to the behavioral changes that occur at parasite emergence. We found that, in addition to the changes reported earlier, after parasite emergence (post-emergence), Manduca sexta larvae also showed marked deficits in their ability to ingest food because of a disruption in the function of the frontal ganglion that results in a significant slowing or the absence of peristaltic activity in the foregut. This effect could be produced in unparasitized fifth-instar larvae by application of blood from post-emergence parasitized larvae or of 10(-6)mol l(-1)d,l-octopamine (approximately the level in the hemolymph of post-emergence larvae). In contrast, blood from parasitized larvae before their parasites emerge or from unparasitized fifth-instar larvae typically had no effect on foregut activity. The effects of either post-emergence parasitized blood or 10(-6)mol l(-1) octopamine could be blocked by the octopamine antagonists phentolamine (at 10(-5)mol l(-1)) or mianserin (at 10(-7)mol l(-1)). (+info)
Immunological detection of serpin in the fall webworm, Hyphantria cunea and its inhibitory activity on the prophenoloxidase system.
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We previously identified a serine type protease inhibitor (serpin) cDNA, using PCR-based differential display, in the fall webworm which was up-regulated following a bacterial challenge (Shin et al., 1998). The serpin cDNA was inserted into an expression vector and the serpin protein was expressed in Escherichia coli. In order to investigate the action of serpin in vivo, we examined the concentration of serpin protein in the larvae of Hyphantria cunea by Western blot analysis using a polyclonal antibody raised in a rabbit injected with recombinant serpin. H. cunea serpin was found mainly in the plasma with a molecular mass of 56.6 kDa on SDS-PAGE followed by Western blot analysis. The concentration of serpin in the plasma was slightly increased following bacterial challenge. A new 50.5 kDa (approx.) band was detected post E. coli and distilled water injection. Both E. coli and distilled water injection induced increased phenoloxidase (PO) activity in the plasma, although E. coli injection produced a larger increase in activity. Hyphantria serpin probably participates in negative regulation of the prophenoloxidase (proPO) cascade. Recombinant serpin inhibits PO activity in the hemocyte lysate fraction activated by LPS. There is a similarity between the P2-P2' region (NKFG) of the serpin reactive site loop and the S2-S2' region (NRFG) of the insect proPO maturation site. This indicates a form of competitive inhibition of serpin against a protease involved in the activation of proPO. A tyrosine residue in the P11 region of serpin, which is conserved in the S11 regions of all known proPOs maturation sites, provides further support for this hypothesis. (+info)