Biomedical subjects

Yoshiaki Nakagawa

Publications and source records attributed to Yoshiaki Nakagawa.

At least 19 recordsLinked to original sources

PubMed2006

Estimation of the hydrophobicity of 2,4-diphenyl-1,3-oxazoline analogs and QSAR analysis of their ovicidal activity against Tetranychus [corrected] urticae.

Partition coefficients of six 2-phenyl-1,3-oxazoline congeners containing 2-I, 2-NO2, 2-CF3, 2,6-(CH3)2, 2,6-F2, and 2-F-6-Cl substitutions on the phenyl moiety were measured in a 1-octanol/water system using the flask-shaking method. The effect on the hydrophobicity (LogP) of substituents on the phenyl moiety of 2-phenyl-1,3-oxazolines linearly correlated with that of benzamide congeners. logP values of other 2-(substituted phenyl)-1,3-oxazoline analogs were empirically estimated from the corresponding substituted benzamides. The ovicidal activity of 2-(substituted phenyl)-4-phenyl-1,3-oxazoline analogs against the two-spotted spider mite Tetranychus [corrected] urticae was quantitatively analyzed using the classical QSAR (Hansch-Fujita) method. Results showed that ovicidal activity increases with hydrophobicity. The introduction of inductive electron-withdrawing groups at ortho-positions increased ovicidal activity, but addition of steric bulk was unfavorable. Substitution at either the meta- or para-position was detrimental to the acaricidal activity.

Animals↗

PubMed2005

Synthesis of 26,27-bisnorcastasterone analogs and analysis of conformation-activity relationship for brassinolide-like activity.

Three castasterone (CS) derivatives with varied side-chain moieties, 26,27-bisnorcastasterone (20S-bisnorCS), 20-epi-26,27-bisnorcastasterone (20R-bisnorCS), and 21,26,27-trisnorcastasterone (trisnorCS), were synthesized stereoselectively from either stigmasterol or dehydroisoandrosterone. The 50% effective doses (ED50, nmol/plant) in the concentration-response curve for brassinolide-like activity in the rice lamina inclination assay were determined to be 0.020 nmol (pED50 = 10.7) for 20S-bisnorCS, 3.2 nmol (pED50 = 8.5) for 20R-bisnorCS, and 2.0 nmol (pED50 = 8.7) for trisnorCS. An analog containing an ester linkage between the steroid and the side-chain moiety of 20S-bisnorCS was also synthesized and its activity was evaluated to be 3.2 nmol (pED50 = 8.5), being equipotent to 20R-bisnorCS and trisnorCS. The activity of 20S-bisnorCS was 1/40 that of CS. The conformation analysis was conducted using a systematic search, showing that the activity decreases with an increase in the degree of freedom of the side chain of the steroidal skeleton.

Brassinosteroids↗

PubMed2005

High-throughput screening of ecdysone agonists using a reporter gene assay followed by 3-D QSAR analysis of the molting hormonal activity.

In this study, 172 diacylhydrazine analogs were examined for their ability to activate an ecdysone (molting hormone)-dependent reporter gene in a silkworm (Bombyx mori) cell-based high-throughput screening assay. The measured EC(50) values (concentration required to cause an effect in 50% of the cells) were used to construct a 3-D QSAR model that describes the ecdysone agonist activities of the diacylhydrazine analogs. Of these compounds, 14 exhibited no activity and were excluded from the 3-D QSAR analysis. The resulting equation described approximately 74% of the activity for 158 compounds. The final equation consisted of 42% electrostatic and 58% steric effects (r(2) = 0.74 and q(2) = 0.45). Comparative molecular field analysis (CoMFA) was used to visualize the steric and electrostatic potential fields that were favorable and unfavorable for biological activity. Of particular interest was the observation that the hydrophobic parameter (logP) was not necessary for describing the observed activities, although previous studies have cited the importance of hydrophobic parameters in both classical and 3-D QSAR analyses of these compounds. Modeling studies of the B. mori ecdysone receptor supported the observed physicochemical parameters required for activity reported by the CoMFA models. Comparison of the present analysis with those performed using other lepidopteran assay systems evidenced a high degree of correlation (r(2) = 0.81 for a Sf-9 cell-based assay and r(2) = 0.89 for a Chilo suppressalis integument-based assay), indicating that it is valid to compare the results generated with the B. mori cell-based system to those generated with previous lepidopteran assays. This novel assay system is amendable to a high-throughput screening format and should greatly increase our ability to discover novel agonists of molting hormone (ecdysone) activity.

Amino Acid Sequence↗

PubMed2005

Classical and three-dimensional QSAR for the inhibition of [3H]ponasterone A binding by diacylhydrazine-type ecdysone agonists to insect Sf-9 cells.

The activity of 52 diacylhydrazine congeners was evaluated by measuring the inhibition of the incorporation of [3H]ponasterone A into intact Sf-9 cells. Eleven compounds were newly synthesized in this study. Results showed that the substitution of the 2-CH3 or 3-OCH3 moiety of methoxyfenozide with other groups or the removal of either group was unfavorable to the activity. The activity was quantitatively analyzed using both classical QSAR (Hansch-Fujita) and three-dimensional QSAR methods (comparative molecular field analysis, CoMFA). Sterically favorable fields were observed at the 3- and 4-positions of the benzene ring opposite from the t-butyl group (B-ring), and a sterically unfavorable field was evidenced at the 2-position. Another sterically unfavorable field developed surrounding the favorable field observed at the 4-position of the B-ring. Electrostatically negative fields were observed near the CO moiety, above the benzene ring, and at the 4-position of the B-ring. The optimum hydrophobicity of compounds in terms of their logP values was calculated to be approximately 4.1. Results of the three dimensional structure-activity relationship analyses were consistent with those obtained from the previously reported classical QSAR for 2-chlorobenzoyl analogs containing various para-substituents. The high activity of potent insecticides such as tebufenozide and chromafenozide were rationalized by CoMFA. Thus, this CoMFA result will be useful in the design of new compounds and in understanding the molecular mechanism of the ligand-receptor interactions.

Animals↗

PubMed2005

Nonsteroidal ecdysone agonists.

Nonsteroidal ecdysone agonists are novel compounds that have become attractive candidates not only as pest control agents in agriculture but also as tools for research. Their narrow spectrum of activity makes them relatively safe as pesticides, and their mode of action as ligands for gene expression has found application in gene therapy and inducing transgenic gene expression in plants. These diacylhydrazines (DAHs) are potent nonsteroidal ecdysone agonists, and four of them, tebufenozide, methoxyfenozide, chromafenozide, and halofenozide, have been developed as insecticides. Although these compounds are very toxic to insects, they are safe for mammals and are environmentally benign. Their action on insects is also selective, the first three are effective against Lepidoptera but weakly active or inactive on Diptera and Coleoptera. On the other hand, halofenozide is effective on Coleoptera but mildly active on Lepidoptera. Previous reviews on ecdysone agonists have concentrated on the biological response of some DAHs and their effects on pests. In this review, the chemistry, biological effects and their modes of action at the molecular level will be covered. In addition, a few studies on other nonsteroidal ecdysone agonists, such as 3,5-di-tert-butyl-4-hydroxy-N-iso-butylbenzamide, acylaminoketones, and benzoyl-1,2,3,4-tetrahydroquinolines, will be briefly reviewed.

Animals↗

PubMed2005

Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata.

cDNA cloning of ecdysone receptor (EcR) and ultraspiracle (USP) of the coleopteran Colorado potato beetle Leptinotarsa decemlineata (LdEcR and LdUSP) was conducted. Amino-acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects, especially the coleopteran yellow mealworm Tenebrio molitor. Northern hybridization analysis showed a 12.4-kb message for the LdEcR A-isoform, a 10.5-kb message for the LdEcR B1-isoform and a 5.7-kb message for the LdUSP, in fat body, gut, integument, testis and ovaries. In developmental profile studies, expression of both the LdEcR and LdUSP transcript in integument changed dramatically. In gel mobility shift assays, in vitro translated LdEcR alone bound weakly to the pal1 ecdysone response element, although LdUSP alone did not, and this binding was dramatically enhanced by the addition of LdUSP. LdEcR/LdUSP complex also showed significant binding to an ecdysone agonist, ponasterone A (K(D) = 2.8 nm), while LdEcR alone showed only weak binding (K(D) = 73.4 nm), and LdUSP alone did not show any binding. The receptor-binding affinity of various ecdysone agonists to LdEcR/LdUSP was not correlated to their larvicidal activity to L. decemlineata. From these results, it was suggested that multiple factors including the receptor binding affinity are related to the determination of the larvicidal activity of nonsteroidal ecdysone agonists in L. decemlineata.

Amino Acid Sequence↗

PubMed2004

Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity.

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2alpha,3alpha,20,22-tetrahydroxy-5alpha-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 microM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 microM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 microM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.

Animals↗

PubMed2004

Synthesis of brassinosteroids of varying acyl side chains and evaluation of their brassinolide-like activity.

Brassinosteroids containing various side chain moieties were synthesized and their activity was determined as the reciprocal logarithm of the ED(50) (50% effective dose per plant in moles) in the rice lamina inclination assay using synergist indole-3-acetic acid (IAA). The introduction of a hydroxyl group in the alpha-position to the carbonyl group of the ester structure significantly enhanced the activity. 2alpha,3alpha-Dihydroxy-17beta-[(2R,3S)-2-hydroxy-3-methylpentanoyl]oxy-B-homo-7-oxa-5alpha-androstan-6-one showed the highest activity, for which the pED(50) was determined to be 10.5 under synergistic conditions with IAA. Under identical conditions, the pED(50) values of brassinolide and castasterone were determined to be 13.6 and 12.3 respectively. With respect to the alpha-carbon of the acyl moiety, the R-form was 10 times more potent than the corresponding S-form. Substituting the terminal structure (Et) of the side chain to that of the most potent compound, brassinolide (i-Pr), did not increase the activity.

Biological Assay↗

PubMed2003

A cell-based high-throughput screening system for detecting ecdysteroid agonists and antagonists in plant extracts and libraries of synthetic compounds.

Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.

Animals↗

PubMed2003

Use of classical and 3-D QSAR to examine the hydration state of juvenile hormone esterase inhibitors.

Carboxylesterases are important enzymes in the metabolism of numerous pharmaceuticals and agrochemicals. They are of importance in many detoxification pathways, but their endogenous role remains unclear. The most potent esterase inhibitors found to date are trifluoromethylketone (TFK) containing compounds, which have been shown to inhibit both mammalian and insect esterases at the low nM level. The detailed mechanism by which these compounds inhibit the enzyme is still unclear. They are highly hydrated in aqueous solutions, but their mechanism of inhibition suggests that inhibition occurs through the ketone, not the hydrated gem-diol. Some studies have stated that the ketone is the inhibitor, while others have reported the gem-diol as the active form. Using juvenile hormone esterase (JHE) as a model system, we examined this question using both classical QSAR and 3-D QSAR with comparative molecular field analysis (CoMFA). Classical QSAR analyses demonstrated the high dependence of inhibitor potency upon log P as well as the limitations of sterically unfavorable substituents. The ketone form of the inhibitor consistently provided improved correlations over the gem-diol, with the final equations describing 72 and 69% of inhibitor activity, respectively, for 97 compounds. Initial CoMFA analyses for the ketone provided a significant equation for 108 compounds (q(2)=0.412, m=6); however all cross-validated values for the gem-diol form of the inhibitors were not statistically significant (q(2)<0.3). Inclusion of hydrophobicity descriptors in both CoMFA equations increased their significance; however the final ketone equation (q(2)=0.500, m=7) was still statistically improved over the gem-diol (q(2)=0.506, m=8). These results support those obtained for the classical QSAR analysis and further illustrate the importance of log P in the inhibition mechanism of these inhibitors. The CoMFA models also identified novel target areas for the synthesis of new JHE inhibitors. These results suggest that the ketone is the active form of TFK-containing inhibitors.

Carboxylic Ester Hydrolases↗

PubMed2003

Correlations of the electrophysiological activity of neonicotinoids with their binding and insecticidal activities.

The electrophysiological actions of various neonicotinoids, including substituted benzyl derivatives, against recombinant Drosophila SAD/chicken beta2 hybrid nicotinic acetylcholine receptor (nAChR) were measured to analyze the relationships between the in vivo (insecticidal) and in vitro (binding and agonist) activities. Most of the neonicotinoids tested were capable of inducing inward currents by activating the hybrid nAChRs expressed in Xenopus laevis oocytes, whereas some compounds had no agonist activity and only blocked the acetylcholine-induced currents. Variations in the agonist activity were well correlated with those in the binding potency evaluated using [3H]imidacloprid as well as insecticidal activities.

Algorithms↗

PubMed2003

Insecticidal activity and nicotinic acetylcholine receptor binding of dinotefuran and its analogues in the housefly, Musca domestica.

The insecticidal activity of dinotefuran and 23 related compounds against the housefly, Musca domestica (L) was measured by injection with metabolic inhibitors. Dinotefuran was less active than imidacloprid and clothianidin by a factor of 10 in molar concentrations. Their binding activities to the fly-head membrane preparation were measured by using [125I]alpha-bungarotoxin ([125I]alpha-BGTX) and [3H]imidacloprid ([3H]IMI) as radioligands. The activity of some selected compounds measured with [3H]IMI was 10(4)-fold higher than that measured with [125I]alpha-BGTX. With [3H]IMI as a radioligand, dinotefuran was 13-fold less active than imidacloprid. The inhibitory effect of dinotefuran on the binding of [3H]IMI to the membrane preparation was in a competitive manner. Quantitative analysis of the insecticidal activity of the test compounds with the binding activity measured with [3H]IMI showed that the higher the binding activity, the higher was the insecticidal activity.

Animals↗

PubMed2003

Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis.

cDNA for ultraspiracle (USP) from the lepidopteran rice stem borer Chilo suppressalis was cloned using PCR techniques. The deduced amino acid sequence of C. suppressalis USP (CsUSP) was very similar to those of other lepidopteran USPs, especially to the Manduca sexta USP-2 isoform. Northern hybridization analysis detected a 6.5-kb message in the epidermis, fat body, and midgut of wandering larvae. CsUSP mRNA expression in the epidermis varied little during the last larval instar. Gel mobility shift assays showed that in vitro translated C. suppressalis ecdysone receptor (CsEcR) and CsUSP proteins bound to the Pal1 or Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. In a ligand-receptor binding assay, [(3)H]ponasterone A ([(3)H]PoA) did not bind to individual CsEcR or CsUSP protein, but bound strongly to the CsEcR/CsUSP complex. [(3)H]PoA binding to CsEcR/CsUSP complex was competed by 20-hydroxyecdysone and a non-steroidal ecdysteroid agonist, RH-5992, but not by cholesterol, indicating that compounds with molting hormone activity against C. suppressalis can bind specifically to the CsEcR/CsUSP complex.

Amino Acid Sequence↗

PubMed2003

Binding affinity of nonsteroidal ecdysone agonists against the ecdysone receptor complex determines the strength of their molting hormonal activity.

N-tert-Butyl-N,N'-dibenzoylhydrazine and its analogs are nonsteroidal ecdysone agonists that exhibit insect molting hormonal and larvicidal activities. The interaction mode of those ecdysone agonists with the heterodimer of the ecdysone receptor and ultraspiracle has not been fully elucidated. We expressed the ecdysone receptor B1 and the ultraspiracle of the lepidopteran, Chilo suppressalis, using an in vitro transcription/translation system and confirmed, using gel-shift assays, that the proteins function as ecdysone receptors. We also analyzed their ligand-binding affinity. A potent ecdysteroid, ponasterone A, specifically bound to the ecdysone receptor with low affinity (KD = 55 nm), and the specific binding was dramatically increased (KD = 1.2 nm) in the presence of the ultraspiracle. For seven nonsteroidal ecdysone agonists and five ecdysteroids, the binding activity to the in vitro-translated ecdysone receptor-ultraspiracle complex was linearly correlated with the binding activity to the inherent receptor protein in the cell-free preparation of C. suppressalis integument. The binding to the ecdysone receptor-ultraspiracle complex for a series of compounds was highly correlated with their molting hormonal activity, indicating that the binding affinity of nonsteroidal ecdysone agonists to the ecdysone receptor-ultraspiracle complex primarily determines the strength of their molting hormonal activity.

Animals↗

PubMed2003

Herpes simplex esophagitis from 1307 autopsy cases.

BACKGROUND AND AIMS: The aim of the present study was to clarify relationship between macroscopic and microscopic features of herpes simplex esophagitis (HSE), and localization of herpes simplex virus type 1 (HSV-1)-infected cells in esophageal lesions from autopsy cases of histopathologically proven HSE. METHODS: The study comprised morphological, immunohistochemical, cultural and electron microscopic investigations for 24 HSE patients collected from 1307 serial autopsy cases in the past 10 years. RESULTS: Macroscopic characteristics of HSE were divided into three types. Types I and II showed small punched-out lesions with and without raised margins, respectively. Type III revealed that multiple ulcers became confluent like a map. Microscopic findings showed that the HSV infection process seemed to begin in squamous epithelium and to induce vertical cellular change into the intact epithelial layer. Intranuclear inclusion bodies were observed at the centers of lesions, and ballooning changes of squamous cells at the margins. Both localization of HSV-1-infected cells and presence of HSV-1 in the esophageal lesions were confirmed with immunohistochemical staining, viral culture and electron microscopy. CONCLUSION: The present results suggest that the macroscopic and microscopic postmortem features of HSE may provide useful information for clinical diagnosis of HSE.

Adult↗

PubMed2002

Use of ab initio calculations to predict the biological potency of carboxylesterase inhibitors.

Carboxylesterases are important enzymes responsible for the hydrolysis and metabolism of numerous pharmaceuticals and xenobiotics. These enzymes are potently inhibited by trifluoromethyl ketone containing (TFK) inhibitors. We demonstrated that the ketone hydration state was affected by the surrounding chemical moieties and was related to inhibitor potency, with inhibitors that favored the gem-diol conformation exhibiting greater potency. Ab initio calculations were performed to determine the energy of hydration of the ketone, and the values were correlated with esterase inhibition data for a series of carboxylesterase inhibitors. This system was examined in three different mammalian models (human liver microsomes, murine liver microsomes, and commercial porcine liver esterase) and in an insect enzyme preparation (juvenile hormone esterase). In all cases, the extent of ketone hydration was strongly correlated with biological potency. Our results showed a very strong correlation with the extent of hydration, accounting for 94% of activity for human liver microsome esterase inhibition (p < 0.01). The atomic charge on the carbon atom of the carbonyl group in the TFK also strongly correlated with inhibitor potency, accounting for 94% of inhibition activity in human liver microsomes (p < 0.01). In addition, we provide crystallographic evidence of intramolecular hydrogen bonding in sulfur-containing inhibitors and relate these data to gem-diol formation. This study provides insight into the mechanism of carboxylesterase inhibition and raises the possibility that inhibitors that too strongly favor the gem-diol configuration have decreased potency due to low rate of ketone formation.

Animals↗

PubMed2002

Quantitative structure-activity studies of insect growth regulators: XIX. Effects of substituents on the aromatic moiety of dibenzoylhydrazines on larvicidal activity against the beet armyworm Spodoptera exigua.

Larvicidal activity against the beet armyworm, Spodoptera exigua (Hübner), was measured after topical treatment for a series of N-tert-butyl-dibenzoylhydrazines having various substituents in the benzoyl (A-ring) moiety closer to the tert-butyl group, the other benzoyl (B-ring) moiety being unsubstituted. The effects of substituents on the larvicidal activity were analyzed using the classical quantitative structure-activity relationship (QSAR) procedure. Introduction of hydrophobic substituents with a small volume into any position was favourable to activity. The existence of electron-withdrawing substituents at ortho positions was also favourable to activity. For multi-substituted compounds, physico-chemically unknown unfavourable factors were suggested to remain after separating common substituent effects derived from QSAR for mono-substituted analogues. With the exception of the unsubstituted compound RH-5849, the effect of substituents in the A-ring moiety on the larvicidal activity was similar to those found with the lepidopteran rice stem borer, Chilo suppressalis (Walker). The larvicidal activity of RH-5849 against S exigua was significantly lower than the value predicted from the correlation between activities against S exigua and C suppressalis. Topical treatment with piperonyl butoxide, a synergist inhibiting oxidative metabolism, slightly enhanced the larvicidal activity of RH-5849 against S exigua.

Algorithms↗

PubMed2002

Nicotinic acetylcholine receptor binding of imidacloprid-related diaza compounds with various ring sizes and their insecticidal activity against Musca domestica.

Fifteen 5-substituted 1-(6-chloro-3-pyridylmethyl)-2-nitromethylene-1,3- diazacyclohexanes and three other related compounds having a five- or seven-membered ring were synthesized and their biological activities were measured in vivo and in vitro. The insecticidal (in vivo) activity was evaluated against houseflies Musca domestica L under synergistic conditions with propargyl propyl phenyl phosphonate and piperonyl butoxide. The binding activity of each compound to nicotinic acetylcholine receptor in vitro was measured using [125I] alpha-bungarotoxin. The insecticidal activities of the unsubstituted diazacyclohexane analogues were slightly higher than those of the imidazolidine analogues, but the enlargement of ring size to diazacycloheptane lowered the activity. Substitution of 1,3-diazacyclohexane or imidazolidine rings was not generally favourable for the activity, but the unsubstituted 1,3-diazacyclohexane analogue showed the highest binding activity. Ring substitutions and ring enlargement decreased the activity 100-30,000-fold.

Animals↗