Eiichi Kuwano

Control mechanism of diapause of the pharate first-instar larvae of the silkmoth Antheraea yamamai.

Abstract The application of an imidazole derivative (KK-42), body ligation and extirpation of specific ganglia suggest a new endocrine mechanism responsible for the regulation of diapause in pharate first-instar larvae of insects. The present results indicate that a humoral repressive factor originates in the region of the mesothorax and that a humoral maturation factor is released from the region of the 2nd to 5th abdominal segments. Our data suggest that the former inhibits the action of the latter during diapause.

Cholesterol esterase accelerates intestinal cholesterol absorption

Mechanisms of acceleration of cholesterol absorption by cholesterol esterase were investigated in various experimental conditions. Lymphatic recovery of cholesterol intubated as a micellar solution containing phosphatidylcholine (PC) into the duodenum was enhanced by the co-administration of cholesterol esterase in rats drained of bile and pancreatic juice. However, no accelerated incorporation was observed when cholesterol was solubilized in PC-depleted micelles. Cholesterol esterase dose-dependently accelerated the incorporation of cholesterol into differentiated Caco-2 cells, only when cholesterol was solubilized in PC-containing micelles. The accelerated incorporation of cholesterol into Caco-2 cells by cholesterol esterase disappeared when the enzyme was preincubated with a suicide inhibitor of cholesterol esterase. Cholesterol esterase has an activity as phospholipase A(2). When 10% of PC in bile salt micelles was replaced by lysophosphatidylcholine (lysoPC), the incorporation of cholesterol into Caco-2 cells was significantly accelerated. Cholesterol esterase enhanced the incorporation of micellar cholesterol into brush border membranes prepared from the rat jejunum. The addition of cholesterol esterase to bile salt micelles accelerated the release of micellar cholesterol in a dose-dependent manner, only when the micelles contained PC. These observations strongly suggest that cholesterol esterase hydrolyzes PC in bile salt micelles and thereby, accelerating the release of cholesterol from bile salt micelles. This may be a major cause of the acceleration of cholesterol absorption by cholesterol esterase.

Inhibitory Action of an Imidazole Compound on Ecdysone Synthesis in Prothoracic Glands of the Silkworm, Bombyx mori

The precocious pupation was induced either by allatectomy at the time of third ecdysis or by topical application of an imidazole compound (KK‐42; 1‐benzyl‐5‐[(E)‐2, 6‐dimethyl‐1, 5‐heptadienyl] imidazole) to the fourth (penultimate) instar larvae of the silkworm, Bombyx mori. However, the critical period for KK‐42 treatment in induction of precocious pupation was longer than that for allatectomy. The effects of KK‐42 depended on the doses applied and a half‐maximum dose was estimated to be approx. 10 μg/larva. KK‐42 suppressed the increase in hemolymph ecdysteroid titres leading to larval ecdysis in controls. Ecdysteroid levels remained at low levels for about 6 days after the treatment, followed by an increase toward precocious pupation. When the prothoracic glands from the mature fifth instar larvac were incubated in vitro in Graces medium containing various concentrations of KK‐42, secretion of ecdysone into the medium was suppressed depending upon the doses of KK‐42 added and a half‐inhibition concentration was estimated to be approx. 1 nM. Thus, KK‐42 was shown to be an inhibitory agent to ecdysteroid secretion in silkworm larvae.

Termination of diapause in pharate first-instar larvae of the gypsy moth Lymantria dispar japonica by an imidazole derivative KK-42

1-Benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole (KK-42) terminated diapause in pharate first-instar larvae of the gypsy moth, Lymantria dispar japonica. This effect of KK-42 was not prevented with juvenile hormone III and its analogs (methoprene and S-31183). In addition, free ecdysteroid titers were lower in diapausing eggs and the eggs treated with KK-42. These results indicate that KK-42 has the same function as in the breakdown of diapause of pharate first-instar larvae in the wild silkmoth, Antheraea yamamai.

Structure/activity studies on 1,5-disubstituted imidazoles as inhibitors of juvenile hormone biosynthesis in isolated corpora allata of the cockroach diploptera punctata

Abstract The activity of eight substituted imidazoles as inhibitors of juvenile hormone III synthesis in the cockroach, Diploptera punctata , was measured in a short-term in vitro radiochemical assay. The potency of these compounds (IC 50 of juvenile hormone III synthesis) ranged from 80 n M for KK-98 to 48 μ M for KK-71. For some compounds (KK-71, KK-51), inhibition of juvenile hormone III synthesis was not accompanied by an accumulation of the immediate precursor methyl farnesoate within the glands. For other compounds (KK-83, KK-96, KK-98) significant accumulation (i.e., more than 100 pmol/pair) of methyl farnesoate was observed. The results suggest that some substituted imidazole compounds are selective inhibitors of the corpus allatum methyl farnesoate epoxidase at low concentrations, but all inhibit methyl farnesoate synthesis at higher concentrations. Experiments with farnesol and farnesoic acid, two precursors of juvenile hormone III synthesis, suggested that the imidazole compounds may inhibit farnesoic acid O -methyl transferase in addition to their predicted inhibition of the epoxidase.

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai : Cloning and expression pattern in pharate first instar larvae in relation to diapause

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.

Titres of biogenic amines and ecdysteroids: effect of octopamine on the production of ecdysteroids in the silkworm Bombyx mori

At day two, a sharp peak of octopamine (OA) was observed in last instar female Bombyx mori larvae. This peak also appeared in male larvae a day later than in females at day three. An OA peak was also observed before the 3rd ecdysis. However, no OA peaks were observed in 4th instar larvae. At day eight and nine of the 5th instar, another OA peak was observed for male and female, respectively. A peak of tyramine (TA) was found at day one followed by a peak of OA at day two in 3rd instar larvae. At day two, a day before OA peak, a peak of TA was observed for male insects and before the 2nd peak of OA, TA titre was also high in 5th instar larvae. Immediately after 3rd ecdysis, a high titre of DL-beta-(3,4-dihydroxyphenyl)alanine (DOPA) was observed, followed by a peak of dopamine (DA) at day five. A peak of DOPA was found at day one followed by a peak of DA at day two in 3rd instar larvae. Similarly, a small peak of DOPA was observed at day two, followed by an increase of DA at days eight and nine after the 4th ecdysis. Ecdysteroid peaks were observed just before the 3rd and 4th ecdysis and an ecdysteroid titre increased after the start of spinning. The effects of OA and JH on production of ecdysteroids by prothoracic glands (PGs) were examined in order to identify neuromediators responsible for triggering pupation in B. mori larvae. Exogeneous OA (10-100 mM) reduced and 10 &mgr;M OA stimulated the production of ecdysteroids in the presence and absence of brain extracts by PGs in the final instar (day five) of B. mori in vitro. Meanwhile, exogeneous JHI (10 &mgr;g/ml) stimulated and at 5 &mgr;g/ml it reduced production of ecdysteroids in the presence of brain extracts. Gramine, an OA antagonist, delayed pupation when applied in the diet. Thus, OA may produce some biological effects on the programming of larval-pupal development.

Effects of 20-hydroxyecdysone and KK-42 on chitinase and β-N-acetylglucosaminidase during the larval-pupal transformation of Bombyx mori

Abstract The appearance of chitinolytic enzymes, chitinase and β-N-acetylglucosaminidase, in integuments of fifth-larval instars of the silkworm, Bombyx mori, was investigated by injection of 20-hydroxyecdysone into the hemolymph of the ligated larvae, and by topical application of an imidazole compound (KK-42, 1-benzyl-5[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole) along the dorsal vessel of the larvae at the beginning of spinning behavior. 20-Hydroxyecdysone induced both enzyme activities. However, the induction patterns were different between two types of chitinolytic enzymes. Chitinase was rapidly induced only by high hormone levels (30 μg/insect, 7.5 μg/g live wt) and soon decreased, while β-N-acetylglucosaminidase was gradually induced even by low hormone levels (6 μg/insect, 1.5 μg/g live wt). KK-42 suppressed both the larval-pupal transformation and appearance of chitinolytic enzymes. Application of KK-42 (50 μg/insect) caused 1-day delay in β-N-acetylglucosaminidase and 2-day delay in chitinase. It was shown by immunoblotting and activity staining that the appearance of the enzyme activities was associated with that of the respective enzyme molecules. The molecular species of β-N-acetylglucosaminidase appeared was mainly the 67.5 kDa subunit. In the case of chitinase, several molecular species including active forms (88 and 65 kDa) and zymogenic form (about 215 kDa) were observed. These results suggest that β-N-acetylglucosaminidase is induced in an active form by relatively low ecdysteroid levels, whereas chitinase is induced through activation of the zymogen by higher levels of hormone.

Three-dimensional common-feature hypotheses for octopamine agonist 2-(arylimino)imidazolidines

Three-dimensional pharmacophore hypotheses were built from a set of 10 octopamine (OA) agonist 2-(Arylimino)imidazolidines (AIIs), 2-(Arylimino)thiazolidines (AITs) and 2-(Arylimino)oxazolidines (AIOs). Among the 10 common-featured models generated by program Catalyst/HipHop, a hypothesis including a ring aromatic (RA), a positive ionizable (PI) and three hydrophobic aliphatic (HpAl) features was considered to be important in evaluating the OA-agonist activity. Active OA agonist 2,6-Et2 AII mapped well onto all the RA, PI and HpAl features of the hypothesis. On the other hand, less active compounds were shown to be difficult to achieve the energetically favorable conformation which is found in the active molecules in order to fit the 3-D common-feature pharmacophore models. Taken together, 2,6-Et2-Ph and foramidine structures are important as OA agonists. The present studies on OA agonists demonstrate that a RA, a PI and three HpAl sites located on the molecule seem to be essential for OA-agonist activity.

Determination by LC-MS of Juvenile Hormone Titers in Hemolymph of the Silkworm, Bombyx mori

Juvenile hormone (JH) I, II and III in the hemolymph of the silkworm, Bombyx mori were quantified by liquid chromatography-mass spectrometry (LC-MS). JHs were treated with methanol and trifluoroacetic acid to convert into JH methoxyhydrines (JH-MHs). The key to the analytical condition for JH-MHs was the addition of 5 µM sodium acetate to the eluting solution. Each JH-MH was observed as the sodium adduct ion with good sensitivity. This improved method enabled the titration of JH I, II and III in hemolymph of the silkworm to be monitored from the 3rd instar through to the early pupal stage. A peak of JH I was observed immediately after ecdysis in the 3rd and 4th instar stages. The JH I titer sharply decreased on day 1 and reached the lowest level before ecdysis, but there was no peak at the beginning of the 5th stadium, and no apparent increase was observed until pupation.