Root System Depth in Arabidopsis Is Shaped by EXOCYST70A3 via the Dynamic Modulation of Auxin Transport.

Root system architecture (RSA), the distribution of roots in soil, plays a major role in plant survival. RSA is shaped by multiple developmental processes that are largely governed by the phytohormone auxin, suggesting that auxin regulates responses of roots that are important for local adaptation. However, auxin has a central role in numerous processes, and it is unclear which molecular mechanisms contribute to the variation in RSA for environmental adaptation. Using natural variation in Arabidopsis, we identify EXOCYST70A3 as a modulator of the auxin system that causes variation in RSA by acting on PIN4 protein distribution. Allelic variation and genetic perturbation of EXOCYST70A3 lead to alteration of root gravitropic responses, resulting in a different RSA depth profile and drought resistance. Overall our findings suggest that the local modulation of the pleiotropic auxin pathway can gives rise to distinct RSAs that can be adaptive in specific environments.

Genotypes, Networks, Phenotypes: Moving Toward Plant Systems Genetics.

One of the central goals in biology is to understand how and how much of the phenotype of an organism is encoded in its genome. Although many genes that are crucial for organismal processes have been identified, much less is known about the genetic bases underlying quantitative phenotypic differences in natural populations. We discuss the fundamental gap between the large body of knowledge generated over the past decades by experimental genetics in the laboratory and what is needed to understand the genotype-to-phenotype problem on a broader scale. We argue that systems genetics, a combination of systems biology and the study of natural variation using quantitative genetics, will help to address this problem. We present major advances in these two mostly disconnected areas that have increased our understanding of the developmental processes of flowering time control and root growth. We conclude by illustrating and discussing the efforts that have been made toward systems genetics specifically in plants.

Aminooxy-naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l-tryptophan aminotransferase: structure-activity relationships.

We previously reported l-α-aminooxy-phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure-activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole-3-acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2-(aminooxy)-3-(naphthalen-2-yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin-deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia-lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them 'pyruvamine'.

Genetic control of root growth: from genes to networks.

BACKGROUND: Roots are essential organs for higher plants. They provide the plant with nutrients and water, anchor the plant in the soil, and can serve as energy storage organs. One remarkable feature of roots is that they are able to adjust their growth to changing environments. This adjustment is possible through mechanisms that modulate a diverse set of root traits such as growth rate, diameter, growth direction and lateral root formation. The basis of these traits and their modulation are at the cellular level, where a multitude of genes and gene networks precisely regulate development in time and space and tune it to environmental conditions. SCOPE: This review first describes the root system and then presents fundamental work that has shed light on the basic regulatory principles of root growth and development. It then considers emerging complexities and how they have been addressed using systems-biology approaches, and then describes and argues for a systems-genetics approach. For reasons of simplicity and conciseness, this review is mostly limited to work from the model plant Arabidopsis thaliana, in which much of the research in root growth regulation at the molecular level has been conducted. CONCLUSIONS: While forward genetic approaches have identified key regulators and genetic pathways, systems-biology approaches have been successful in shedding light on complex biological processes, for instance molecular mechanisms involving the quantitative interaction of several molecular components, or the interaction of large numbers of genes. However, there are significant limitations in many of these methods for capturing dynamic processes, as well as relating these processes to genotypic and phenotypic variation. The emerging field of systems genetics promises to overcome some of these limitations by linking genotypes to complex phenotypic and molecular data using approaches from different fields, such as genetics, genomics, systems biology and phenomics.

Regeneration of the Cardiac Conduction System by Adipose Tissue-Derived Stem Cells.

BACKGROUND: Adipose tissue is one of the sources of mesenchymal stem cells, which have the potential to differentiate into various types of cells, including myocytes. Whether brown adipose tissue (BAT)-derived cells might differentiate into the cardiac pacemaking-conducting cells, and have the potential to regenerate the cardiac conduction system (CCS), is investigated in this study. METHODS AND RESULTS: BAT was isolated from the interscapular area of mice and enzymatically digested before culture. Round or fusiform cells showed spontaneous beating at 4-7 days after culturing of BAT-derived cells. Reverse transcriptase-polymerase chain reaction analysis and immunocytochemical analysis revealed that BAT-derived cells expressed several cardiomyocytes, the CCS and pacemaker (PM) cell marker genes and proteins. Patch-clamp techniques revealed that spontaneous electrical activity and the shape of the action potential showed properties of cardiac PM cells. Next, a complete atrioventricular (AV) block was created in mice and green fluorescent protein-positive (GFP (+)) BAT-derived cells were injected intramyocardially around the AV node. At 1 week after transplantation, 50% of BAT-derived cells injected mice showed a sinus rhythm or a 2:1 AV block. Immunohistochemical analysis revealed that injected GFP (+) cells were engrafted and some GFP (+) cells co-expressed several cardiac PM cell marker proteins. CONCLUSIONS: BAT-derived cells differentiate into the CCS and PM-like cells in vitro and in vivo, and may become a useful cell source for arrhythmia therapy.

A Multiplexed, Time-Resolved Assay of Root Gravitropic Bending on Agar Plates.

The ability of roots to orient their growth relative to the vector of gravity, root gravitropism (positive gravitropism), is observed in root systems of higher plants and is an essential part of plant growth and development. While there are various methods for quantifying root gravitropism, many methods that can efficiently measure gravitropism at a reasonable throughput do not yield temporal resolution of the process, while methods that allow for high-temporal resolution are often not suitable for an efficient measurement of multiple roots. Here, we describe a method to analyze the root gravitropism activity at an increased throughput with a fine time-resolution using Arabidopsis thaliana plants.

Virtual screening for ligands of the insect molting hormone receptor.

Insect growth is regulated by the orchestrated event of ecdysteroids and their receptor proteins. Agonists/antagonists of ecdysteroid receptor are predicted to disrupt normal growth, providing good candidates of new insecticides. A database of over 2 million compounds was subjected to a shape-based virtual screening cascade to identify novel nonsteroidal hits similar to the known EcR ligand ponasterone A. Testing revealed micromolar hits against two strains of insect cells. Docking experiments against EcR were used to support the predicted binding mode of these ligands based on their overlay to ponasterone A.

Auxin biosynthesis inhibitors, identified by a genomics-based approach, provide insights into auxin biosynthesis.

Despite its importance in plant growth and development, the auxin biosynthetic pathway has remained elusive. In this study, we analyzed hormone series transcriptome data from AtGenExpress in Arabidopsis and found that aminoethoxyvinylglycine (AVG) had the strongest anti-auxin activity. We also identified other effective compounds such as L-amino-oxyphenylpropionic acid (AOPP) through additional screening. These inhibitors shared characteristics in that they inhibited pyridoxal enzymes and/or aminotransferases. They reduced endogenous IAA levels in both monocots and dicots. L-AOPP inhibited root development of Arabidopsis in main root elongation, gravitropism, root skewing and root hair formation. This inhibition was generally recovered after exogenous IAA treatment, and the recovery was almost completely to the level of non-inhibited seedlings. The compounds inhibited conversion from tryptophan to indole-3-pyruvic acid in enzyme extracts from Arabidopsis and wheat. Our data collectively suggest that the inhibitors directly blocked auxin biosynthesis, and that the major target site was tryptophan aminotransferase. This enzyme probably makes up one of the major biosynthesis pathways conserved among higher plants. Each inhibitor, however, demonstrated a different action spectrum in shoot and root of rice and tomato, indicating diversity in biosynthesis pathways between organs and species. Our results provide novel insights into auxin biosynthesis and action, and uncover structural characteristics of auxin biosynthesis inhibitors.

Effects of nicorandil on the cAMP-dependent Cl- current in guinea-pig ventricular cells.

In guinea-pig cardiomyocytes, a cAMP-dependent Cl(-) current (I(Cl,cAMP)) flows through a cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which belongs to a family of the ATP-binding cassette (ABC) proteins. Although several K(+)-channel openers and sulfonylurea ATP-sensitive K(+) (K(ATP))-channel blockers reportedly inhibit I(Cl,cAMP), effects of nicorandil on the Cl(-) current have not been evaluated. This study was conducted to examine the effects of nicorandil on I(Cl,cAMP) in isolated guinea-pig ventricular cells using patch clamp techniques. Nicorandil in concentrations higher than 300 microM enhanced the I(Cl,cAMP) preactivated by 0.1 microM isoproterenol. The isoproterenol-induced I(Cl,cAMP) was inhibited by 100 microM glibenclamide, but not by 100 microM pinacidil. SNAP (S-nitroso-N-acetyl-D,L-penicillamine, 10 microM), a nitric oxide (NO) donor, similarly enhanced the isoproterenol-induced I(Cl,cAMP). However, SG-86, a denitrated metabolite possessing K(+ )channel-opening action, failed to enhance the Cl(-) current. When the I(Cl,cAMP) was activated by 3-isobutyl-1-methylxanthine (IBMX, 30 microM), either nicorandil or SNAP failed to enhance the isoproterenol-induced I(Cl,cAMP). Thus, nicorandil enhances I(Cl,cAMP) in guinea-pig cardiomyocytes through an increase in intracellular cGMP, although direct modulation of I(Cl,cAMP) by NO cannot be completely excluded.

Characterization of juvenile hormone epoxide hydrolase and related genes in the larval development of the silkworm Bombyx mori.

Juvenile hormone epoxide hydrolases (JHEHs) are a family of enzymes that hydrolyze juvenile hormones (JHs). They are important in terms of organ-specific regulation and irreversible degradation. In contrast to three JHEH genes (jheh) in Drosophila melanogaster and five jheh in Tribolium castaneum, only one jheh gene has been reported to date in lepidopteran insects. By searching a genome database of the silkworm, KAIKOBLAST, five JHEH-related genes (jheh-r), in addition to Bmjheh, were found. Developmental changes in mRNA expression were brought about revealing several unique patterns for each of jheh-r as to developmental stages and organ-specificity. Recombinant proteins of JHEH-r were expressed using a baculovirus system to evaluate their enzymatic activities. Three of the five JHEH-r recombinant proteins had JH hydrolytic activities. This is the first report on lepidopteran jheh-related genes and also provides the comprehensive analysis of multiple jheh-related genes in an insect species with respect to their functions in enzyme activities.

Purification and cDNA cloning of LaIT2, a novel insecticidal toxin from venom of the scorpion Liocheles australasiae.

The novel insecticidal toxin, LaIT2, was isolated from venom of the scorpion Liocheles australasiae. The amino acid sequence of LaIT2 was determined by an Edman degradation analysis and subsequent cDNA cloning. LaIT2 is composed of 59 amino acids with three disulfide bridges, and shares sequence similarity to the scorpion beta-KTx peptides.

Molecular cloning of the ecdysone receptor and the retinoid X receptor from the scorpion Liocheles australasiae.

cDNAs of the ecdysone receptor and the retinoid X receptor were cloned from the Japanese scorpion Liocheles australasiae, and the amino acid sequences were deduced. The full-length cDNA sequences of the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor were 2881 and 1977 bp in length, respectively, and the open reading frames encoded proteins of 560 and 414 amino acids. The amino acid sequence of the L. australasiae ecdysone receptor was similar to that of the ecdysone receptor-A of the soft tick, Ornithodoros moubata (68%) and to that of the ecdysone receptor-A1 of the lone star tick, Amblyomma americanum (66%), but showed lower similarity to the ecdysone receptors of Orthoptera and Coleoptera (53-57%). The primary sequence of the ligand-binding region of the L. australasiae ecdysone receptor was highly homologous to that of ticks (85-86%). The amino acid sequence of the L. australasiae retinoid X receptor was also homologous to the amino acid sequence of ultraspiracles of ticks (63%) and insects belonging to the orders Orthoptera and Coleoptera (60-64%). The identity of both the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor to their lepidopteran and dipteran orthologs was less than 50%. The cDNAs of both the L. australasiae ecdysone receptor (L. australasiae ecdysone receptor-A) and the L. australasiae retinoid X receptor were successfully translated in vitro using a rabbit reticulocyte lysate system. An ecdysone analog, ponasterone A, bound to L. australasiae ecdysone receptor-A (K(D) = 4.2 nM), but not to L. australasiae retinoid X receptor. The L. australasiae retinoid X receptor did not enhance the binding of ponasterone A to L. australasiae ecdysone receptor-A, although L. australasiae retinoid X receptor was necessary for the binding of L. australasiae ecdysone receptor-A to ecdysone response elements.

Long-term endothelin a receptor blockade inhibits electrical remodeling in cardiomyopathic hamsters.

BACKGROUND: The endothelin (ET) system is activated in failing hearts. Congestive heart failure frequently is associated with ventricular arrhythmias, which may result from electrical remodeling such as changes of ionic current density and heterogeneous action potential prolongation. We examined the effects of long-term ET(A) receptor blockade on the electrophysiological properties of ventricular cells, the surface ECG, and the survival in BIO 14.6 cardiomyopathic hamsters. METHODS AND RESULTS: Membrane currents and action potentials were recorded from left ventricular cells isolated from normal F1beta hamsters and cardiomyopathic BIO 14.6 hamsters untreated and chronically treated with TA-0201, an ET(A) receptor antagonist. In ventricular cells of untreated BIO 14.6 hamsters, the action potential duration was prolonged and the densities of the L-type Ca2+ current (I(Ca,L)), the transient outward current (I(to)), the delayed rectifier K+ current (I(K)), and the inward rectifier K+ current (I(K1)) were decreased compared with those of F1beta hamsters. Long-term treatment with the ET(A) receptor antagonist significantly attenuated action potential duration prolongation and reduction of I(to), I(K), and I(Ca,L) in BIO 14.6 ventricular cells. Long-term ET(A) receptor blockade prevented the QT prolongation and ventricular arrhythmias and improved the survival rate in the cardiomyopathic hamsters. CONCLUSIONS: Long-term treatment with an ET(A) antagonist inhibits electrical remodeling such as downregulation of K+ and Ca2+ currents, action potential prolongation, and the increased QT interval and thereby suppresses ventricular arrhythmias in cardiomyopathic hearts. ET(A) receptor blockade may provide a new strategy for the prevention of ventricular arrhythmias associated with heart failure.

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.

ClC-3B, a novel ClC-3 splicing variant that interacts with EBP50 and facilitates expression of CFTR-regulated ORCC.

We have cloned ClC-3B, a novel alternative splicing variant of ClC-3 (ClC-3A) that is expressed predominantly in epithelial cells. ClC-3B has a different, slightly longer C-terminal end than ClC-3A and contains a consensus motif for binding to the second PDZ (PSD95/Dlg/ZO-1) domain of the epithelium-specific scaffolding protein EBP50. Both in vitro and in vivo binding assays demonstrate interaction between ClC-3B and EBP50. C127 mouse mammary epithelial cells transfected with ClC-3B alone showed diffuse immunoreactivity for ClC-3B in the cytoplasmic region. In contrast, when EBP50 was cotransfected with ClC-3B, strong immunoreactivity for ClC-3B appeared at the leading edges of membrane ruffles. Patch-clamp experiments revealed that cotransfection of ClC-3B and EBP50 resulted in a remarkable increase in outwardly rectifying Cl- channel (ORCC) activities at the leading edges of membrane ruffles in C127 cells. The electrophysiological properties of the ClC-3B-induced ORCCs are similar to those of ORCCs described in native epithelial cells. When cystic fibrosis transmembrane conductance regulator (CFTR) was cotransfected with ClC-3B and EBP50, ClC-3B-dependent ORCCs were activated via the protein kinase A-dependent pathway. These findings indicate that ClC-3B is itself a CFTR-regulated ORCC molecule or its activator.

Beating is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes.

Cell transplantation could be a potential therapy for heart damage. Skeletal myoblasts have been expected to be a good cell source for autologous transplantation; however, the safety and efficacy of their transplantation are still controversial. Recent studies have revealed that skeletal muscle possesses the stem cell population that is distinct from myoblasts. To elucidate whether skeletal muscle stem cells can transdifferentiate into cardiomyocytes, we cocultured skeletal muscle cells isolated from transgenic mice expressing green fluorescent protein with cardiomyocytes of neonatal rats. Skeletal muscle-derived cells expressed cardiac-specific proteins such as cardiac troponin T and atrial natriuretic peptide as well as cardiac-enriched transcription factors such as Nkx2E (formerly called Csx/Nkx2.5) and GATA4 by coculture with cardiomyocytes. Skeletal muscle-derived cells also expressed cadherin and connexin 43 at the junctions with neighboring cardiomyocytes. Cardiomyocyte-like action potentials were recorded from beating skeletal muscle-derived cells. Treatment of nifedipine or culture in Ca2+-free media suppressed contraction of cardiomyocytes and inhibited skeletal muscle cells to express cardiac-specific proteins. Cyclic stretch completely restored this inhibitory effect. These results suggest that some part of skeletal muscle cells can transdifferentiate into cardiomyocytes and that direct cell-to-cell contact and contraction of neighboring cardiomyocytes are important for the transdifferentiation.

From phenotypes to causal sequences: using genome wide association studies to dissect the sequence basis for variation of plant development.

Tremendous natural variation of growth and development exists within species. Uncovering the molecular mechanisms that tune growth and development promises to shed light on a broad set of biological issues including genotype to phenotype relations, regulatory mechanisms of biological processes and evolutionary questions. Recent progress in sequencing and data processing capabilities has enabled Genome Wide Association Studies (GWASs) to identify DNA sequence polymorphisms that underlie the variation of biological traits. In the last years, GWASs have proven powerful in revealing the complex genetic bases of many phenotypes in various plant species. Here we highlight successful recent GWASs that uncovered mechanistic and sequence bases of trait variation related to plant growth and development and discuss important considerations for conducting successful GWASs.

Inhibitory effect of the class III antiarrhythmic drug nifekalant on HERG channels: mode of action.

Nifekalant is a class III antiarrhythmic drug that has been shown to be effective against ventricular tachyarrhythmias in experimental animals and humans. We examined the detailed electrophysiological effects of nifekalant on human-ether-a-go-go-related gene (HERG) channels expressed in Xenopus oocytes. Nifekalant inhibited the HERG current in a concentration-dependent manner with an IC(50) value of 7.9 microM although the drug did not inhibit the minK current in Xenopus oocytes, suggesting selective inhibition of the rapid component of the delayed rectifier K(+) current (I(Kr)) in cardiomyocytes. Nifekalant showed a higher binding affinity for the open state than for the inactive state of HERG channels. Nifekalant inhibited HERG channels in a frequency-dependent manner. The onset of the blockade was rapid but the recovery from the block was slow. Nifekalant modified the voltage dependence and kinetics of HERG channel gating. Thus, nifekalant inhibits HERG channels in a voltage-dependent and frequency-dependent manner, and the inhibitory effect may underlie the clinical efficacy of the drug against ventricular tachyarrhythmias.

Inhibitory effects of AMP 579, a novel cardioprotective adenosine A1/A2A receptor agonist, on native IKr and cloned HERG current.

We investigated the effects of 1S-[1a,2b,3b,4a(S*)]-4-[7-[[1-[(3-chloro-2-thienyl)methylpropyl]propyl-amino]-3H-imidazo[4,5-b] pyridyl-3-yl]-N-ethyl-2,3-dihydroxycyclopentane carboxamide (AMP 579), a novel cardioprotective adenosine A(1)/A(2A) receptor agonist, on the rapid and slow components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in guinea-pig ventricular myocytes and on the human ether-a-go-go-related gene (HERG) channel expressed in human embryonic kidney (HEK 293) cells. Whole-cell current and membrane potential were recorded using patch-clamp techniques. In guinea-pig ventricular myocytes, AMP 579 inhibited I(Kr) in a concentration-dependent manner with IC(50) value of 15.2 microM, when I(Kr) was blocked by chromanol 293B. On the contrary, AMP 579 (10 microM) did not affect I(Ks) in the presence of the I(Kr) blocker E-4031. The former effect of AMP 579 was unaffected by either the selective adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine or the non-selective adenosine A(1)/A(2) receptor antagonist 8-sulphophenyltheophylline. Moreover, AMP 579-induced inhibition of I(Kr) was not voltage- and frequency-dependent. In HEK 293 cells expressing HERG channels, AMP 579 (10 microM) significantly blocked the HERG current at +10 mV by 34.9+/-7.0% (n=4, p<0.05), and the degree of inhibition was comparable with that observed in guinea-pig ventricular myocytes (36.8+/-6.0%, n=4). AMP 579 (10 microM) significantly inhibited the L-type Ca(2+) current (I(Ca)) by 41.0+/-6.8% (n=5, p<0.05), which was unaffected by 8-sulphophenyl-theophylline. Consequently, despite its inhibitory actions on I(Kr) or HERG current, the drug significantly shortened the action potential duration measured at 90% repolarization from 275.6+/-19.4 to 208.3+/-18.6 ms (n=4, p<0.05). Thus, AMP 579 inhibits both native I(Kr) and cloned HERG channels with additional inhibitory effect of I(Ca), and such inhibitory effects may at least partially underlie the observed antifibrillatory action of the drug during myocardial ischemia/reperfusion.

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.