A high-affinity juvenile hormone binding protein was purified from the hemolymph of the tobacco hornworm, Manduca sexta, employing ammonium sulfate precipitation and affinity and sizeseparation chromatography. The naturally occurring enantiomer of juvenile hormone III (107?) was converted to juvenile hormone III acid and then covalently attached to aminohexyl-Sepharose 4B. Hemolymph from early fifth stadium (60 h postecdysis) larvae was used as the source of hJHBP. The yield of hJHBP was approximately 25% of the starting material, with 3.5 mg of highly purified, biologically active hJHBP recovered from 100 mL of hemolymph. Binding parameters were examined using equilibrium dialysis and highly purified, enantiomerically correct juvenile hormone I and II and racemic JH III. The equilibrium dissociation constants for juvenile hormone I and II were approximately 6 X 10-10 M at 4 °C, while racemic juvenile hormone III displayed an equilibrium dissociation constant of 1.9 X 10~9M. At25 °C the equilibrium dissociation constant for juvenile hormone I was 1.6 X 10-9 M. Half-times of dissociation were also determined for the three homologs.
Polyclonal antibodies made against 86 kDa (86 k), 80 kDa (80 k) and 54 kDa (54 k) vitellins of Oxya japonica japonica are used for Western blotting. Anti‐80k vitellin antibody is cross‐reacted with a 95 kDa (95 k) vitellin. While 95 k vitellin is present both in the female hemolymph and in the oocyte, 80 k vitellin is detected only in the oocyte and the laid egg. In the growing oocytes, as 95 k vitellin is faded out gradually, 80 k vitellin is accumulated increasingly, indicating postendocytic processing of 95 k vitellin brings 80 k vitellin. Further conforming the hypothesis, partial digestion of 95 k vitellin with pepsin and α‐chymotrypsin makes several protein bands of molecular weight around 80 kDa. Thus, the 95k vitellin may have a cleavage site (s) to produce 80 k vitellin which forms fairly stable tertiary structure. In the reduced condition (20 mM glutathion), both 95 k and 80 k vitellins were digested throughly by endogenous proteinase at pH 4. Both 86 k and 54 k vitellins, respectively, show no apparent molecular weight changes in the growing oocyte and in the hemolymph.
A new putative transposon was identified in the tobacco budworm, Heliothis virescens. This transposon was characterized as a full length CORE-SINE (65 bp of "CORE" core specific nucleotide short interspersed elements) that resembled sequences from three other lepidopterans and humans. In particular, the A-box and B-box regions of this sequence most closely conformed to the signature of CORE-SINEs from widely divergent species. This CORE-SINE was present as a polymorphism in a hypervariable region of the gene hscp, which is the target of pyrethroid insecticides and other xenobiotics in the nerve axon. We described this new putative transposon as Noct-1 due to its presence in a noctuid moth. This is the first description of a full-length CORE-SINE with the A-box, B-box, target site duplication, and candidate core domain from an insect.
We cloned a full-length tyrosine hydroxylase cDNA from the integument of the diamondback moth, Plutella xylostella. In the phylogenetic tree, tyrosine hydroxylase (PxTH) clustered with the other lepidopteran THs. Serine residues in the PxTH sequence, namely Ser(24), Ser(31), Ser(35), Ser(53), and Ser(65), were predicted to be the target sites for phosphorylation based on PROSITE analysis. In particular, Ser(35) of PxTH is highly conserved across a broad phylogenetic range of animal taxa including rat and human. Western blot analysis using both PxTH-Ab1 and PxTH-Ab2 polyclonal antibodies verified the expression of PxTH in all life cycle stages of P. xylostella, namely the larval, pupal, and adult stages. To examine the possible immune function of PxTH in P. xylostella, PxTH gene expression was investigated by RT-PCR and western blotting analysis after challenging P. xylostella with bacteria. PxTH expression was elevated 1 h post-infection and was continued till 12 h of post-infection relative to control larvae injected with sterile water.
In this study, the endocellulase gene from Monochamus saltuarius (MsGHF5) was transformed into Escherichia coli (RosettaBlue(DE3)pLysS strain), and induced by IPTG. The molecular weight of recombinant MsGHF5 (rMsGHF5) was 78 kDa and was expressed as a fusion protein with maltose binding protein in pMAL-c2 expression vector. Native-PAGE was conducted with 0.1% carboxymethyl cellulose as a substrate, and the zymogenic bands were observed. The Michaelis constant and maximum velocity of rMsGHF5 were 0.199 mg/mL and 0.034 μmol/ min/mL, respectively. The optimal condition for rMsGHF5 occurred at pH 5 and 30°C. Fe 2+ and Mn 2+ stimulated the activity of rMsGHF5 by 167 and 114% respectively, whereas Cu 2+ , Hg 2+ and Zn 2+ inhibited its activity.
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