Effect of heat-moisture treated brown rice crackers on postprandial flow-mediated dilation in adults with mild endothelial dysfunction.

Background: Endothelial dysfunction is an early pathophysiological feature and independent predictor of a poor prognosis in most forms of cardiovascular disease. We evaluated the effect of brown rice crackers (BR-C) on endothelial function. Methods: Effect of heat-moisture treated (HMT) -BR-C on postprandial flow-mediated dilation (FMD) in adults with mild endothelial dysfunction was compared with that of BR-C and white rice crackers (WR-C) in 12 adults with mild endothelial dysfunction (less than 7.0% of FMD) by a randomized, single-blind, three-treatment three-period crossover trial (UMIN 000034898). Since we considered that the FMD increase was associated with the treatment of HMT-BR-C, we examined the effect of three possible factors: postprandial glucose levels, polyphenol content, and polyphenol release from the food matrix. Results: Mean pre-intake baseline FMD values of HMT-BR-C, BR-C, and WR-C were 4.9%, 5.1%, and 4.9%, respectively, and those values 1 h post-intake were 6.3%, 5.1%, and 4.8%, respectively. There was no difference in intergroup comparisons of FMD using Dunnett's multiple comparison test. There was a significant increase in FMD only in HMT-BR-C in intragroup comparisons (P = 0.042 by paired-t test). In comparison with BR-C, no significant difference was noted in the postprandial glucose level nor in the content of total polyphenols and ferulic acid derivatives in HMT-BR-C. However, the 70% ethanol extracted from HMT-BR-C contained a significantly larger amount of free and bound ferulic acids than from BR-C. Conclusion: HMT-BR-C intake increased the postprandial FMD response.

Evaluation of Fructo-, Inulin-, and Galacto-Oligosaccharides on the Maillard Reaction Products in Model Systems with Whey Protein.

The present study aimed to investigate the effects of fructo-, inulin-, and galacto-oligosaccharides (FOS, IOS, and GOS) on forming the Maillard reaction products such as browning, α-dicarbonyl compounds, and advanced glycation end products (AGEs). The model solutions at pH 6.8 containing each carbohydrate (mono-, di-, and oligosaccharides) and whey protein were incubated at 50 °C for 8 weeks. In the IOS model, sugars of DP3 or larger were significantly decreased at 4 weeks, whereas at 6 weeks in the FOS model. The residual amount of GOS after 8 weeks was higher than FOS and IOS; however, a large amount of 3-deoxyglucosone was formed compared to the other models. Nε-Carboxymethyllysine (CML) concentrations in oligosaccharide models were about half of those in monosaccharide and lactose models. The highest concentrations of glyoxal- and methylglyoxal-derived hydroimidazolones 3 (G-H3 and MG-H3) were observed in the IOS model, indicating the involvement of fructose units linked by ß-2 → 1 bonds. G-H3 and MG-H3 quantification could be a useful indicator to reflect the modification of an arginine residue by fructose if used acid-hydrolysis for AGE analysis. CML, G-H3, and MG-H3 were considerably formed even in the FOS model, which has no reducing terminal site, suggesting that degradation products of oligosaccharides probably participated in the formation of AGEs.

Evaluation of Blood-Brain Barrier Permeability of Polyphenols, Anthocyanins, and Their Metabolites.

Anthocyanins (ACs) are able to protect neurons against ß-amyloid-induced neurotoxicity. In this study, we evaluated blood-brain barrier (BBB) permeability of these compounds using a model kit to clarify the mechanism of AC on the brain. Black currant or strawberry AC extract was orally administrated to male Wistar rats. The urine and extirpated brain were collected before and after administration and analyzed quantitatively by liquid chromatography-tandem mass spectrometry. After administration of AC, several phenolic acids were detected in the urine samples. Further, AC and some AC metabolites were found in the brain tissue. BBB permeabilities of these compounds were much lower than the positive control. Epigallocatechin, daidzein, genistein, equol, and nobiletin presented high BBB permeability, whereas apigenin, luteolin, quercetin, and kaempferol showed medium permeability, and epicatechin, rutin, fisetin, resveratrol, and curcumin BBB permeation was neglected. These results suggested that ACs were difficult to cross BBB into the brain and ACs were not directly associated with the prevention of ß-amyloid-induced neurotoxicity.

Identification of dipyrrolone pigments and their precursors formed in the Maillard reaction of carnosine and pentose under weakly acidic conditions.

Colored compounds formed by the Maillard reaction of carnosine with xylose or glucose were investigated in this study. Yellow pigments showing an absorption maximum at 450 nm were found in a heated solution of carnosine with xylose at pH 5.0. These pigments were then isolated and identified as dicarnosyl-dipyrrolones A and B. The generation of dipyrrolones in the absence of lysine suggests that dipyrrolone pigments can be formed by pentose as well as every amino compound such as amino acids, peptides and proteins possessing a free amino group. Analysis of α-dicarbonyls using LC-MS/MS showed that pentosone, 1-deoxypentosone, 3-deoxypentosone (3-DP), and methylglyoxal were predominantly generated via degradation of Amadori compounds. Also, a potential formation pathway of dypyrrolones was established, indicating that an Amadori compound that could form 3-DP is likely to play a role as a main precursor for dipyrrolones.

Stress-derived reactive oxygen species enable hemocytes to release activator of growth blocking peptide (GBP) processing enzyme.

Insect cytokine growth blocking peptide (GBP) is synthesized as an inactive precursor, termed proGBP, that is normally present in a significant concentration in the hemolymph of non-stressed animals (Hayakawa, 1990, 1991). Under stress conditions, proGBP is instantly processed to active GBP by a serine protease and this is thought to be an important initial step for insects to cope with stress-induced adverse effects via GBP-induced physiological changes. However, the detailed mechanism underlying proteolytic processing of hemolymph proGBP in insects under stress conditions remains unknown. Here we demonstrated that proGBP processing requires ROS-induced release of a proteinaceous factor from hemocytes that activates the inactive proGBP processing enzyme. The release of the activator protein from hemocytes is initiated by an elevation of the cytoplasmic Ca2+ concentration induced by ROS. Therefore, we concluded that stress-induced activation of proGBP requires ROS-dependent stimulation of an intracellular calcium signaling pathway in hemocytes, followed by release of the hemocyte proteinaceous factor that specifically activates the proGBP processing enzyme.

N-acetyl-l-tyrosine is an intrinsic triggering factor of mitohormesis in stressed animals.

Under stress conditions, mitochondria release low levels of reactive oxygen species (ROS), which triggers a cytoprotective response, called "mitohormesis". It still remains unclear how mitochondria respond to stress-derived stimuli and release a low level of ROS. Here, we show that N-acetyl-l-tyrosine (NAT) functions as a plausible intrinsic factor responsible for these tasks in stressed animals. NAT is present in the blood or hemolymph of healthy animals, and its concentrations increase in response to heat stress. Pretreatment with NAT significantly increases the stress tolerance of tested insects and mice. Analyses using Drosophila larvae and cultured cells demonstrate that the hormetic effects are triggered by transient NAT-induced perturbation of mitochondria, which causes a small increase in ROS production and leads to sequential retrograde responses: NAT-dependent FoxO activation increases in the gene expression of antioxidant enzymes and Keap1. Moreover, we find that NAT represses tumor growth, possibly via the activation of Keap1. In sum, we propose that NAT is a vital endogenous molecule that could serve as a triggering factor for mitohormesis.

Therapeutic Effects of Anthocyanins for Vision and Eye Health.

Anthocyanin (AC) is widely used as supplement of eye health in Europe and in East Asia. In this review, I describe AC effects to clarify the mechanism is important in order to understand the effects of AC on vision health. The bioavailability of AC is quite low but, reported as intact form and many kinds of metabolite. And AC passes through the blood-aqueous fluid barrier and blood-retinal barrier. In vitro study, AC had a relaxing effect on ciliary muscle which is important to treat both myopia and glaucoma. And AC stimulate the regeneration of rhodopsin in frog rod outer segment. Furthermore, AC could inhibit the axial length and ocular length elongation in a negative lens-induced chick myopia model. In addition, we summarized clinical studies of AC intake improved dark adaptation and transient myopic shift and the improvement on retinal blood circulation in normal tension glaucoma patients.

Repeated phenotypic selection for cuticular blackness of armyworm larvae decreased stress resistance.

Armyworm Mythimna separata larvae show changes in cuticle darkening depending on population densities and are roughly categorized into two phenotypes, a pale brown solitary type and black-colored gregarious type. Although the color difference in both larval types is apparent, it remains ambiguous whether any change in physiological traits accompanies the cuticle darkening. To answer this query, we repeated genetic selection of the blackness phenotype over one hundred generations in our laboratory colony and produced a black-colored (BL) strain. Comparison between non-selected control (CTL) and BL strains revealed an increased fecundity and adult life span in the BL strain compared with the CTL strain. In contrast, BL strain larvae were found to be significantly more sensitive to heat stress than those in the CTL strain. Hemolymph reactive oxygen species (ROS) levels were higher in the BL strain than in the CTL strain under both non-stress and heat stress conditions. Antioxidant activities of the hemolymph were not significantly different between the two strains under non-stress condition, but the activities increased to higher levels in the CTL strain than those in the BL strain after heat stress. Activities and gene expression levels of antioxidant enzymes such as catalase and superoxide dismutase (SOD) in the fat body were significantly higher in CTL strain larvae than in BL strain larvae after heat treatment. Thermal stress tolerance of the offspring of crossings between the two strains showed a tolerance level almost equivalent to the maternal one: the cross between CTL females and BL males produced offspring with the higher tolerance compared with the oppositely crossed offspring. Expression levels of the antioxidant enzyme genes of the former offspring were found to be similar to those of CTL strain. These results indicate a trade-off between reproductive activity and stress resistance: the BL strain had acquired high reproductivity but had lost stress tolerance through repeated genetic selection. Furthermore, the present genetic analyses demonstrated that the phenotype of stress tolerance is derived from the maternal parent.

Impaired Chylomicron Assembly Modifies Hepatic Metabolism Through Bile Acid-Dependent and Transmissible Microbial Adaptations.

The mechanisms by which alterations in intestinal bile acid (BA) metabolism improve systemic glucose tolerance and hepatic metabolic homeostasis are incompletely understood. We examined metabolic adaptations in mice with conditional intestinal deletion of the abetalipoproteinemia (ABL) gene microsomal triglyceride transfer protein (Mttp-IKO), which blocks chylomicron assembly and impairs intestinal lipid transport. Mttp-IKO mice exhibit improved hepatic glucose metabolism and augmented insulin signaling, without weight loss. These adaptations included decreased BA excretion, increased pool size, altered BA composition, and increased fibroblast growth factor 15 production. Mttp-IKO mice absorb fructose normally but are protected against dietary fructose-induced hepatic steatosis, without weight loss or changes in energy expenditure. In addition, Mttp-IKO mice exhibit altered cecal microbial communities, both at baseline and following fructose feeding, including increased abundance of Bacteroides and Lactobacillus genera. Transplantation of cecal microbiota from chow-fed Mttp-IKO mice into antibiotic-treated wild-type recipients conferred transmissible protection against fructose-induced hepatic steatosis in association with a bloom in Akkermansia and increased Clostridium XIVa genera, whose abundance was positively correlated with fecal coprostanol and total neutral sterol excretion in recipient mice. However, antibiotic-treated Mttp-IKO mice were still protected against fructose-induced hepatic steatosis, suggesting that changes in microbiota are not required for this phenotype. Nevertheless, we found increased abundance of fecal Akkermansia from two adult ABL subjects with MTTP mutations compared to their heterozygous parents and within the range noted in six healthy control subjects. Furthermore, Akkermansia abundance across all subjects was positively correlated with fecal coprostanol excretion. Conclusion: The findings collectively suggest multiple adaptive pathways of metabolic regulation following blocked chylomicron assembly, including shifts in BA signaling and altered microbial composition that confer a transmissible phenotype.

Submucosal fat accumulation in human colorectal tissue and its association with abdominal obesity and insulin resistance.

BACKGROUND: In addition to visceral fat, peripheral ectopic fat accumulation is suggested to play a role in the pathophysiology of metabolic syndrome, which is known to be associated with not only cardiovascular diseases and type 2 diabetes mellitus but also colorectal cancer. OBJECTIVE: This study aims to clarify whether there is ectopic fat accumulation in human colorectal tissue in association with metabolic syndrome or its components such as abdominal obesity and insulin resistance. METHODS: Lipid contents of colorectal tissue were measured in 27 patients with colorectal polyp excised endoscopically. In addition, lipid droplets were immunohistochemically estimated using anti-perilipin antibody in 32 patients with colorectal cancer resected surgically. RESULTS: Increasing tissue triglyceride/phospholipid ratio was associated with increasing body mass index, fasting plasma insulin level and homeostasis model assessment as an index of insulin resistance (HOMA-IR), and also decreasing serum adiponectin level. Lipid droplets were observed in the submucosal region of colorectal tissue. The amount of lipid droplets was associated with increasing body mass index, waist circumference and visceral fat area. CONCLUSION: This study showed the presence of submucosal fat accumulation in human colorectal tissue and its association with abdominal obesity and insulin resistance.

Identification of a cytokine combination that protects insects from stress.

Growth-blocking peptide (GBP) and stress-responsive peptide (SRP) are insect cytokines whose expression levels are elevated by various stressful conditions such as parasitization and high or low temperatures. Both GBP and SRP are synthesized as precursors and released into the hemolymph, where they are enzymatically processed to active peptides. Injection of active GBP or SRP into early last instar larvae elicits a reduction in feeding and consequent growth retardation in the armyworm Mythimna separata. Although such functions are thought to benefit insects under stressful conditions by affecting their physiologies and behaviors, the relationship between GBP and SRP remains elusive. Here we show that heat stress-induced reactive oxygen species (ROS) elevated hemolymph GBP, which activated SRP transcription and increased the SRP concentration in the hemolymph. Injection of both GBP and SRP elevated hemolymph antioxidant levels. We found that simultaneous increases in both active cytokines occurred in the larval hemolymph from 2 to 3 h after heat stress or H2O2 injection, suggesting a synergic action of the two factors. This speculation was confirmed by demonstrating that co-injection of GBP and SRP caused a more severe reduction in appetite and growth retardation than injection of an individual peptide alone. However, injection of GBP together with SRP did not elevate SRP expression at all, indicating the effect of negative feedback regulation. Furthermore, SRP RNAi larvae showed higher body weights compared to controls, and GBP-induced growth retardation was partially abrogated in SRP RNAi larvae. These results led us to conclude that GBP is an upstream cytokine in the regulation of SRP expression and that these cytokines synergistically retard larval growth by repressing feeding activities when insects are exposed to stress conditions.

A gene-driven recovery mechanism: Drosophila larvae increase feeding activity for post-stress weight recovery.

Recovery from weight loss after stress is important for all organisms, although the recovery mechanisms are not fully understood. We are working to clarify these mechanisms. Here, we recorded enhanced feeding activity of Drosophila melanogaster larvae from 2 to 4 h after heat stress at 35°C for 1 h. During the post-stress period, expression levels of sweet taste gustatory receptor genes (Grs), Gr5a, Gr43a, Gr64a, and Gr64f, were elevated, whereas bitter taste Grs, Gr66a, and Gr33a, were decreased in expression and expression of a non-typical taste receptor Gr, Gr68a, was unchanged. Similar upregulation of Gr5a and downregulation of Gr66a was recorded after cold stress at 4°C. Expression levels of tropomyosin and ATP synthase ß subunit were significantly increased in larval mouth parts around 3 to 5 h after the heat stress. We infer that up-regulation of post-stress larval feeding activity, and weight recovery, is mediated by increasing capacity for mouth part muscular movements and changes in taste sensing physiology. We propose that Drosophila larvae, and likely insects generally, express an efficient mechanism to recover from weight loss during post-stress periods.

Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress.

A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.

Heat stress hardening of oriental armyworms is induced by a transient elevation of reactive oxygen species during sublethal stress.

Pre-exposure to mild heat stress enhances the thermotolerance of insects. Stress hardening is a beneficial physiological plasticity, but the mechanism underlying it remains elusive. Here we report that reactive oxygen species (ROS) concentrations were quickly and transiently elevated in the armyworms, Mythimna separata, by exposing them to 40°C, but not other tested temperatures. Larvae exposed to 40°C had subsequently elevated antioxidant activity and the highest survival of all tested heating conditions. The elevation of ROS after lethal heating at 44°C for 1 h was approximately twofold compared to heating at 40°C. Injection of an optimal amount of hydrogen peroxide (H2 O2 ) similarly caused sequential elevation of ROS and antioxidant activity in the test larval hemolymph, which led to significantly enhanced survival after lethal heat stress. The H2 O2 -induced thermotolerance was abolished by coinjection of potent antioxidants such as ascorbic acid or N-acetylcysteine. Both preheating at 40°C and H2 O2 injection enhanced expression of genes encoding superoxide dismutase 1, catalase, and heat shock protein 70 in the fat body of test larvae, indicating the adequate heat stress induced a transient elevation of ROS, followed by upregulation of antioxidant activity. We infer that thermal stress hardening is induced by a small timely ROS elevation that triggers a reduction-oxidation signaling mechanism.

Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source.

Rice straw was evaluated as a carbon source for the fungi, Trichoderma reesei and Humicola insolens, to produce enzymes for rice straw hydrolysis. The enzyme activity of T. reesei and H. insolens cultivated in medium containing non-treated rice straw were almost equivalent to the enzyme of T. reesei cultivated in Avicel medium, a form of refined cellulose. The enzyme activity of T. reesei cultivated in medium containing NH4OH-treated rice straw was 4-fold higher than enzyme from cultures grown in Avicel medium. In contrast, H. insolens enzyme from cultures grown in NH4OH-treated rice straw had significantly lower activity compared with non-treated rice straw or Avicel. The combined use of T. reesei and H. insolens enzymes resulted in a significant synergistic enhancement in enzymatic activity. Our data suggest that rice straw is a promising low-cost carbon source for fungal enzyme production for rice straw hydrolysis.

Characterization of Venom and Oviduct Components of Parasitoid Wasp Asobara japonica.

During natural parasitization, Asobara japonica wasps introduce lateral oviduct (LO) components into their Drosophila hosts soon after the venom injection to neutralize its strong toxicity; otherwise, the host will die. Although the orchestrated relationship between the venom and LO components necessary for successful parasitism has attracted the attention of many researchers in this field, the molecular natures of both factors remain ambiguous. We here showed that precipitation of the venom components by ultracentrifugation yielded a toxic fraction that was inactivated by ultraviolet light irradiation, boiling, and sonication, suggesting that it is a virus-like entity. Morphological observation of the precipitate after ultracentrifugation showed small spherical heterogeneous virus-like particles 20-40 nm in diameter. The venom's detrimental effect on D. melanogaster larvae was not directly neutralized by the LO components but blocked by a hemolymphal neutralizing factor activated by the LO factor. Furthermore, we found that A. japonica venom and LO components acted similarly on the larvae of the common cutworm Spodoptera litura: the venom injection caused mortality but coinjection of the LO factor protected S. litura larvae from the venom's toxicity. In contrast, D. ficusphila and D. bipectinata, which are closely related to D. melanogaster but non-habitual host species of A. japonica, were not negatively affected by A. japonica venom due to an intrinsic neutralizing activity in their hemolymph, indicating that these species must have acquired a neutralizer of A. japonica venom during evolution. These results give new insights into the characteristics of both the venom and LO components: A. japonica females have utilized the virus-like toxic venom factor to exploit a wider range of host species after the evolutionary process enabled them to use the LO factor for activation of the host hemolymph neutralizer precursor, although the non-habitual host Drosophila species possess an active intrinsic neutralizer in their hemolymph.

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

Function of desiccate in gustatory sensilla of drosophila melanogaster.

Desiccate (Desi), initially discovered as a gene expressing in the epidermis of Drosophila larvae for protection from desiccation stress, was recently found to be robustly expressed in the adult labellum; however, the function, as well as precise expression sites, was unknown. Here, we found that Desi is expressed in two different types of non-neuronal cells of the labellum, the epidermis and thecogen accessory cells. Labellar Desi expression was significantly elevated under arid conditions, accompanied by an increase in water ingestion by adults. Desi overexpression also promoted water ingestion. In contrast, a knockdown of Desi expression reduced feeding as well as water ingestion due to a drastic decrease in the gustatory sensillar sensitivity for all tested tastants. These results indicate that Desi helps protect insects from desiccation damage by not only preventing dehydration through the integument but also accelerating water ingestion via elevated taste sensitivities of the sensilla.

Bacteria Endosymbiont, Wolbachia, Promotes Parasitism of Parasitoid Wasp Asobara japonica.

Wolbachia is the most widespread endosymbiotic bacterium that manipulates reproduction of its arthropod hosts to enhance its own spread throughout host populations. Infection with Wolbachia causes complete parthenogenetic reproduction in many Hymenoptera, producing only female offspring. The mechanism of such reproductive manipulation by Wolbachia has been extensively studied. However, the effects of Wolbachia symbiosis on behavioral traits of the hosts are scarcely investigated. The parasitoid wasp Asobara japonica is an ideal insect to investigate this because symbiotic and aposymbiotic strains are available: Wolbachia-infected Tokyo (TK) and noninfected Iriomote (IR) strains originally collected on the main island and southwest islands of Japan, respectively. We compared the oviposition behaviors of the two strains and found that TK strain females parasitized Drosophila melanogaster larvae more actively than the IR strain, especially during the first two days after eclosion. Removing Wolbachia from the TK strain wasps by treatment with tetracycline or rifampicin decreased their parasitism activity to the level of the IR strain. Morphological and behavioral analyses of both strain wasps showed that Wolbachia endosymbionts do not affect development of the host female reproductive tract and eggs, but do enhance host-searching ability of female wasps. These results suggest the possibility that Wolbachia endosymbionts may promote their diffusion and persistence in the host A. japonica population not only at least partly by parthenogenesis but also by enhancement of oviposition frequency of the host females.

Identification of two juvenile hormone inducible transcription factors from the silkworm, Bombyx mori.

Juvenile hormone (JH) regulates many physiological processes in insects. However, the signal cascades in which JH is active have not yet been fully elucidated, particularly in comparison to another major hormone ecdysteroid. Here we identified two JH inducible transcription factors as candidate components of JH signaling pathways in the silkworm, Bombyx mori. DNA microarray analysis showed that expression of two transcription factor genes, E75 and Enhancer of split mß (E(spl)mß), was induced by juvenile hormone I (JH I) in NIAS-Bm-aff3 cells. Real time RT-PCR analysis confirmed that expression of four E75 isoforms (E75A, E75B, E75C and E75D) and E(spl)mß was 3-8 times greater after JH I addition. Addition of the protein synthesis inhibitor cycloheximide did not suppress JH-induced expression of the genes, indicating that they were directly induced by JH. JH-induced expression of E75 and E(spl)mß was also observed in four other B. mori cell lines and in larval hemocytes of final instar larvae. Notably, E75A expression was induced very strongly in larval hemocytes by topical application of the JH analog fenoxycarb; the level of induced expression was comparable to that produced by feeding larvae with 20-hydroxyecdysone. These results suggest that E75 and E(spl)mß are general and direct target genes of JH and that the transcription factors encoded by these genes play important roles in JH signaling.