Ethnic-specific genetic susceptibility loci for endometriosis in Taiwanese-Han population: a genome-wide association study.

Endometriosis is a common gynecological disorder affecting around 10% of reproductive-age women. Although many hypotheses were proposed, genetic alteration has been considered as one of the key factors promoting pathogenesis. Due to racial/ethnic disparities in the process of hormone regulation and nutrition metabolism, a genome-wide association study (GWAS) with 2794 cases and 27,940 controls was conducted in a Taiwanese-Han population. Our study identified five significant susceptibility loci for endometriosis, and three of them, WNT4 (on the 1p36.12), RMND1 (6q25.1), and CCDC170 (6q25.1), have been previously associated with endometriosis across different populations, including European and Japanese descent cohorts. Other two including C5orf66/C5orf66-AS2 (5q31.1) and STN1 (10q24.33) are newly identified ones. Functional network analysis of potent risk genes revealed the involvement of cancer susceptibility and neurodevelopmental disorders in endometriosis development. In addition, long non-coding RNAs (lncRNAs) C5orf66 and C5orf66-AS2 can interact with many RNA-binding proteins (RBPs) which can influence RNA metabolic process, mRNA stabilization, and mRNA splicing, leading to dysregulation in tumor-promoting gene expression. Those findings support clinical observations of differences in the presentation of endometriosis in Taiwanese-Han population with higher risks of developing deeply infiltrating/invasive lesions and the associated malignancies.

In Silico and In Vitro Screening of Serine Racemase Agonist and In Vivo Efficacy on Alzheimer's Disease Drosophila melanogaster.

The NMDA receptor hypofunction has been implicated in schizophrenia, memory impairment, and Alzheimer's disease. Modulating the abundance of D-serine, a co-agonist of the NMDA receptor, is a strategy to treat symptoms of the NMDA receptor hypofunction. In contrast to D-amino acid oxidase (DAAO) inhibitors, which aim at decreasing the loss of D-serine, this study tried to identify serine racemase (SRR) agonists, which boost the conversion of L-serine to D-serine. We used holo and apo structures of human SRR for the molecular docking against the National Cancer Institute (NCI) and ZINC compound databases and validated their efficacy by in vitro SRR activity assay. We identified NSC294149 (2-amino-3-(3-nitroimidazo[1,2-a]pyridin-2-yl)sulfanylpropanoic acid) as a potential SRR agonist and confirmed its amelioration of the hazard ratio of survival of the AD model of fruit fly (Drosophila melanogaster). These results suggest that the SRR agonist could be a drug design target against the NMDA receptor hypofunction symptoms.

Ergosta-7,9(11),22-trien-3ß-ol Rescues AD Deficits by Modulating Microglia Activation but Not Oxidative Stress.

Ergosta-7,9(11),22-trien-3ß-ol (EK100) was isolated from the Taiwan-specific medicinal fungus Antrodia camphorata, which is known for its health-promotion and anti-aging effects in folk medicine. Alzheimer's disease (AD) is a major aging-associated disease. We investigated the efficacy and potential mechanism of ergosta-7,9(11),22-trien-3ß-ol for AD symptoms. Drosophila with the pan-neuronal overexpression of human amyloid-ß (Aß) was used as the AD model. We compared the life span, motor function, learning, memory, oxidative stress, and biomarkers of microglia activation and inflammation of the ergosta-7,9(11),22-trien-3ß-ol-treated group to those of the untreated control. Ergosta-7,9(11),22-trien-3ß-ol treatment effectively improved the life span, motor function, learning, and memory of the AD model compared to the untreated control. Biomarkers of microglia activation and inflammation were reduced, while the ubiquitous lipid peroxidation, catalase activity, and superoxide dismutase activity remained unchanged. In conclusion, ergosta-7,9(11),22-trien-3ß-ol rescues AD deficits by modulating microglia activation but not oxidative stress.

Ergosta-7, 9 (11), 22-trien-3ß-ol Interferes with LPS Docking to LBP, CD14, and TLR4/MD-2 Co-Receptors to Attenuate the NF-κB Inflammatory Pathway In Vitro and Drosophila.

Ergosta-7, 9 (11), 22-trien-3ß-ol (EK100) was isolated from Cordyceps militaris, which has been used as a traditional anti-inflammatory medicine. EK100 has been reported to attenuate inflammatory diseases, but its anti-inflammatory mechanism is still unclear. We were the first to investigate the effect of EK100 on the Toll-like receptor 4 (TLR4)/nuclear factor of the κ light chain enhancer of B cells (NF-κB) signaling in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells and the green fluorescent protein (GFP)-labeled NF-κB reporter gene of Drosophila. EK100 suppressed the release of the cytokine and attenuated the mRNA and protein expression of pro-inflammatory mediators. EK100 inhibited the inhibitor kappa B (IκB)/NF-κB signaling pathway. EK100 also inhibited phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (Akt) signal transduction. Moreover, EK100 interfered with LPS docking to the LPS-binding protein (LBP), transferred to the cluster of differentiation 14 (CD14), and bonded to TLR4/myeloid differentiation-2 (MD-2) co-receptors. Compared with the TLR4 antagonist, resatorvid (CLI-095), and dexamethasone (Dexa), EK100 suppressed the TLR4/AKT signaling pathway. In addition, we also confirmed that EK100 attenuated the GFP-labeled NF-κB reporter gene expression in Drosophila. In summary, EK100 might alter LPS docking to LBP, CD14, and TLR4/MD-2 co-receptors, and then it suppresses the TLR4/NF-κB inflammatory pathway in LPS-stimulated RAW264.7 cells and Drosophila.

Far-infrared Radiation Improves Motor Dysfunction and Neuropathology in Spinocerebellar Ataxia Type 3 Mice.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine neurodegenerative disease resulting from the misfolding and accumulation of a pathogenic protein, causing cerebellar dysfunction, and this disease currently has no effective treatments. Far-infrared radiation (FIR) has been found to protect the viability of SCA3 cells by preventing mutant ataxin-3 protein aggregation and promoting autophagy. However, this possible treatment still lacks in vivo evidence. This study assessed the effect of FIR therapy on SCA3 in vivo by using a mouse model over 28 weeks. Control mice carried a healthy wild-type ATXN3 allele that had a polyglutamine tract with 15 CAG repeats (15Q), whereas SCA3 transgenic mice possessed an allele with a pathological polyglutamine tract with expanded 84 CAG (84Q) repeats. The results showed that the 84Q SCA3 mice displayed impaired motor coordination, balance abilities, and gait performance, along with the associated loss of Purkinje cells in the cerebellum, compared with the normal 15Q controls; nevertheless, FIR treatment was sufficient to prevent those defects. FIR significantly improved performance in terms of maximal contact area, stride length, and base support in the forepaws, hindpaws, or both. Moreover, FIR treatment supported the survival of Purkinje cells in the cerebellum and promoted the autophagy, as reflected by the induction of autophagic markers, LC3II and Beclin-1, concomitant with the reduction of p62 and ataxin-3 accumulation in cerebellar Purkinje cells, which might partially contribute to the rescue mechanism. In summary, our results reveal that FIR confers therapeutic effects in an SCA3 transgenic animal model and therefore has considerable potential for future clinical use.

Treatment with Caffeic Acid and Resveratrol Alleviates Oxidative Stress Induced Neurotoxicity in Cell and Drosophila Models of Spinocerebellar Ataxia Type3.

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a polyglutamine (polyQ) repeat in the protein ataxin-3 which is involved in susceptibility to mild oxidative stress induced neuronal death. Here we show that caffeic acid (CA) and resveratrol (Res) decreased reactive oxygen species (ROS), mutant ataxin-3 and apoptosis and increased autophagy in the pro-oxidant tert-butyl hydroperoxide (tBH)-treated SK-N-SH-MJD78 cells containing mutant ataxin-3. Furthermore, CA and Res improved survival and locomotor activity and decreased mutant ataxin-3 and ROS levels in tBH-treated SCA3 Drosophila. CA and Res also altered p53 and nuclear factor-κB (NF-κB) activation and expression in tBH-treated cell and fly models of SCA3, respectively. Blockade of NF-κB activation annulled the protective effects of CA and Res on apoptosis, ROS, and p53 activation in tBH-treated SK-N-SH-MJD78 cells, which suggests the importance of restoring NF-κB activity by CA and Res. Our findings suggest that CA and Res may be useful in the management of oxidative stress induced neuronal apoptosis in SCA3.

Autophagy and its link to type II diabetes mellitus.

Autophagy, a double-edged sword for cell survival, is the research object on 2016 Nobel Prize in Physiology or Medicine. Autophagy is a molecular mechanism for maintaining cellular physiology and promoting survival. Defects in autophagy lead to the etiology of many diseases, including diabetes mellitus (DM), cancer, neurodegeneration, infection disease and aging. DM is a metabolic and chronic disorder and has a higher prevalence in the world as well as in Taiwan. The character of diabetes mellitus is hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and failure of producing insulin on pancreatic beta cells. In T2DM, autophagy is not only providing nutrients to maintain cellular energy during fasting, but also removes damaged organelles, lipids and miss-folded proteins. In addition, autophagy plays an important role in pancreatic beta cell dysfunction and insulin resistance. In this review, we summarize the roles of autophagy in T2DM.

Far-infrared radiation protects viability in a cell model of Spinocerebellar Ataxia by preventing polyQ protein accumulation and improving mitochondrial function.

Far infrared radiation (FIR) is currently investigated as a potential therapeutic strategy in various diseases though the mechanism is unknown. Presently, we tested if FIR mediates beneficial effects in a cell model of the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3). SCA3 is caused by a mutation leading to an abnormal polyglutamine expansion (PolyQ) in ataxin-3 protein. The consequent aggregation of mutant ataxin-3 results in disruption of vital cell functions. In this study, neuroblastoma cells (SK-N-SH) was transduced to express either non-pathogenic ataxin-3-26Q or pathogenic ataxin-3-78Q proteins. The cells expressing ataxin-3-78Q demonstrated decreased viability, and increased sensitivity to metabolic stress in the presence rotenone, an inhibitor of mitochondrial respiration. FIR exposure was found to protect against these effects. Moreover, FIR improved mitochondrial respiratory function, which was significantly compromised in ataxin-3-78Q and ataxin-3-26Q expressing cells. This was accompanied by decreased levels of mitochondrial fragmentation in FIR treated cells, as observed by fluorescence microscopy and protein expression analysis. Finally, the expression profile LC3-II, Beclin-1 and p62 suggested that FIR prevent the autophagy inhibiting effects observed in ataxin-3-78Q expressing cells. In summary, our results suggest that FIR have rescuing effects in cells expressing mutated pathogenic ataxin-3, through recovery of mitochondrial function and autophagy.

Cross-species identification of in silico microsatellite biomarkers for genetic disease.

Microsatellites appear widely in genomes of diverse species. Variants of repeat number of microsatellites often correlate with risks of genetic disorder or severity of diseases. Using cross-species comparison, the proposed system comprehensively verifies microsatellites of specific genes related to 16 genetic disorders. Genomic information retrieved from 14 frequently used model organisms in biomedical study was thoroughly analyzed, emphasizing conserved and diverse traits. Features of microsatellite sequences among different organisms, including appearing frequency, position, pattern and distribution, could be determined automatically for stating genetically functional conservation and evolutionary correlation. This research found that among mammals and fishes, the microsatellite sequences are conserved in the genes of epidermal growth factor receptor, ataxia telangiectasia mutated and androgen receptor corresponding to cancers, ataxia telangiectasia and hepatocellular carcinoma, respectively. Still, except fruit fly conserved CAG repeats in Huntington and Spinocerebellar ataxia type 2 genes, no microsatellites were conserved in those genes linked to neurological/neurodegenerative disorders among mammal and fish species. In comparison of mammalian species, microsatellite biomarkers identified from 17 genetic disorder-related genes revealed high repeat conservation, especially in human, gorilla and macaque. Obviously, this comparative analysis illustrates microsatellite repeats affecting genetic disorders, highly correlated to evolutionary distance of species. Chief contribution of this in silico research lies in assisting biologists to identify disease-related microsatellite biomarkers and employ appropriate model organisms for further biomedical studies relying on microsatellite conservation information. Database http://ssrtc.cs.ntou.edu.tw is for academic use.

8-Alkylcoumarins from the fruits of Cnidium monnieri protect against hydrogen peroxide induced oxidative stress damage.

Three new 8-alkylcoumarins, 7-O-methylphellodenol-B (1), 7-methoxy-8-(3-methyl- 2,3-epoxy-1-oxobutyl)chromen-2-one (2), and 3'-O-methylvaginol (3), together with seven known compounds (4-10) were isolated from the fruits of Cnidium monnieri. Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. All the isolates were evaluated the cytoprotective activity by MTS cell proliferation assay and the results showed that all the three new 8-alkylcoumarins exhibited cytoprotective effect on Neuro-2a neuroblastoma cells injured by hydrogen peroxide.

Myricetin attenuates lipopolysaccharide-stimulated activation of mouse bone marrow-derived dendritic cells through suppression of IKK/NF-κB and MAPK signalling pathways.

BACKGROUND: Myricetin is a naturally occurring flavonoid that is found in many fruits, vegetables, teas and medicinal herbs. It has been demonstrated to have anti-inflammatory properties, but, to date, no studies have described the immunomodulatory effects of myricetin on the functions of dendritic cells (DCs). The aim of this study was to evaluate the potential for myricetin to modulate lipopolysaccharide (LPS)-stimulated activation of mouse bone marrow-derived DCs. RESULTS: Our experimental data showed that treatment with myricetin up to 10 µg mL(-1) does not cause cytotoxicity in cells. Myricetin significantly decreased the secretion of tumour necrosis factor-α, interleukin-6 and interleukin-12p70 by LPS-stimulated DCs. The expression of LPS-induced major histocompatibility class II, CD40 and CD86 on DCs was also inhibited by myricetin, and the endocytic and migratory capacity of LPS-stimulated DCs was blocked by myricentin. In addition, LPS-stimulated DC-elicited allogeneic T-cell proliferation was reduced by myricetin. Moreover, our results confirmed that myricetin attenuates the responses of LPS-stimulated activation of DCs via suppression of IκB kinase/nuclear factor-κB and mitogen-activated protein kinase-dependent pathways. CONCLUSION: Myricetin has novel immunopharmacological activity, and modulation of DCs by myricetin may be an attractive strategy for the treatment of inflammatory and autoimmune disorders, and for transplantation.

Ethylene glycol induces calcium oxalate crystal deposition in Malpighian tubules: a Drosophila model for nephrolithiasis/urolithiasis.

Several animal species are used to study calcium oxalate urolithiasis; however, an ideal model has yet to be identified. We used Drosophila as a model organism and fed the flies lithogenic agents such as ethylene glycol, hydroxyl-L-proline, and sodium oxalate. At different times, the Malpighian tubules, the kidney equivalent of insects, were dissected and a polarized light microscope used to highlight the birefringent crystals. Scanning electron microscopy and energy-dispersive X-ray spectroscopy confirmed that the crystal composition was predominately calcium oxalate. Furthermore, administration of potassium citrate successfully reduced the quantity of and modulated the integrity of the ethylene glycol-induced crystals. Thus, the Drosophila model of bio-mineralization produces crystals in the urinary system through many lithogenic agents, permits observation of crystal formation, and is amenable to genetic manipulation. This model may mimic the etiology and clinical manifestations of calcium oxalate stone formation and aid in identification of the genetic basis of this disease.

Inhibition of the interactions between eosinophil cationic protein and airway epithelial cells by traditional Chinese herbs.

BACKGROUND: The eosinophil cationic protein (ECP) is cytotoxic to bacteria, viruses, parasites and mammalian cells. Cells are damaged via processes of pore formation, permeability alteration and membrane leaking. Some clinical studies indicate that ECP gathers in the bronchial tract of asthma sufferers, damages bronchial and airway epithelial cells, and leads to in breathing tract inflammation; therefore, prevention of the cytotoxicity caused by ECP may serve as an approach to treat airway inflammation. To achieve the purpose, reduction of the ECP-cell interactions is rational. In this work, the Chinese herbal combinative network was generated to predict and identify the functional herbs from the pools of prescriptions. It was useful to select the node herbs and to demonstrate the relative binding ability between ECP and Beas-2B cells with or withour herb treatments. RESULTS: Eighty three Chinese herbs and prescriptions were tested and five effective herbs and six prescription candidates were selected. On the basis of effective single-herbal drugs and prescriptions, a combinative network was generated. We found that a single herb, Gan-cao, served as a node connecting five prescriptions. In addition, Sheng-di-huang, Dang-guei and Mu-tong also appeared in five, four and three kinds of prescriptions, respectively. The extracts of these three herbs indeed effectively inhibited the interactions between ECP and Beas-2B cells. According to the Chinese herbal combinative network, eight of the effective herbal extracts showed inhibitory effects for ECP internalizing into Beas-2B cells. The major components of Gang-cao and Sheng-di-huang, glycyrrhizic acid and verbascose, respectively, reduced the binding affinity between ECP and cells effectively. CONCLUSIONS: Since these Chinese herbs reduced the binding affinity between ECP and cells and inhibited subsequent ECP entrance into cells, they were potential for mitigating the airway inflammation symptoms. Additionally, we mentioned a new concept to study the Chinese herbs using combinative network in the field of systems biology. The functional single herbs could be identified from the set of prescriptions.

Aloe-emodin is an interferon-inducing agent with antiviral activity against Japanese encephalitis virus and enterovirus 71.

In this study, aloe-emodin was identified as a potential interferon (IFN)-inducer by screening compounds from Chinese herbal medicine. Aloe-emodin showed low cytotoxicity to human HL-CZ promonocyte cells and TE-671 medulloblastoma cells and significantly activated interferon-stimulated response element (ISRE) and gamma-activated sequence (GAS)-driven cis-reporting systems. Moreover, aloe-emodin upregulated expression of IFN-stimulated genes such as dsRNA-activated protein kinase and 2',5'-oligoisoadenylate synthase. Aloe-emodin resulted in significant activation of nitric oxide production. The antiviral activity of aloe-emodin against Japanese encephalitis virus (JEV) and enterovirus 71 (EV71) was evaluated using dose- and time-dependent plaque reduction assays in HL-CZ cells and TE-671 cells. The 50% inhibitory concentration (IC(50)) of aloe-emodin ranged from 0.50microg/mL to 1.51microg/mL for JEV and from 0.14microg/mL to 0.52microg/mL for EV71. Aloe-emodin showed clearly potent virus inhibitory abilities and achieved high therapeutic indices, in particular for HL-CZ cells. Therefore, the study demonstrated dose- and time-dependent actions of aloe-emodin on the inhibition of JEV and EV71 replication via IFN signalling responses.

Interferon antagonist function of Japanese encephalitis virus NS4A and its interaction with DEAD-box RNA helicase DDX42.

The interferon (IFN) antagonists of Japanese encephalitis virus (JEV) proteins contribute to the JE pathogenesis. Most flavivirus non-structural (NS) proteins correlate with virus-induced inflammation and immune escape. NS4A proteins of West Nile virus and dengue type 2 virus have been demonstrated to inhibit IFN signaling. In this study, JEV NS4A without the C-terminal 2K domain has been demonstrated to partially block activation of an IFN-stimulated response element (ISRE)-based cis-reporter by IFN-alpha/beta. In addition, JEV NS4A significantly inhibited the phosphorylation levels of STAT1 and STAT2, but not TYK2 in the IFN-treated cells. Moreover, the N-terminus of a RNA helicase DDX42 protein identified using a phage display human brain cDNA library have been demonstrated to specifically bind to JEV NS4A in vitro using a co-immunoprecipitation assay. The interaction between JEV NS4A and RNA helicase DDX42 showed partial co-localization in human medulloblastoma TE-671 cells by confocal microscopy. Importantly, the expression of N-terminal DDX42 is able to overcome JEV-induced antagonism of IFN responses. Therefore, these results show that JEV NS4A without the C-terminal 2K domain is associated with modulation of the IFN response and the interaction of JEV NS4A with RNA helicase DDX42 could be important for JE pathogenesis.

Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer.

The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.

Estrogen receptor alpha polymorphism is associated with pelvic organ prolapse risk.

Estrogen and estrogen receptors are known to play important roles in the pathophysiology of pelvic organ prolapse (POP). We investigated whether estrogen receptor alpha (ERalpha) gene polymorphisms were associated with POP risk by conducting a case-control association study in 88 women with POP and 153 women without POP. Genotypes of the ERalpha (ESR1) gene polymorphisms (rs17847075, rs2207647, rs2234693, rs3798577, and rs2228480) were determined by polymerase chain reaction, followed by restriction fragment length polymorphism analysis. There was significant difference between women with and those without POP in the distribution of the ESR1 rs2228480 genotypes evaluated. By using multivariable logistic regression, age and ESR1 rs2228480 genotype GA were significantly associated with POP risk. Although the sample size of women with POP studied is small, the present study shows that ERalpha genotype may be associated with POP risk.

Mass spectroscopic characteristics of low molecular weight proteins extracted from calcium oxalate stones: preliminary study.

It is believed that boundary compositions of matrix proteins might play a role in stone formation; however, few proteomic studies concerning matrix proteins in urinary stones have been conducted. In this study, we extracted low molecular weight proteins from calcium oxalate stones and measured their characteristic patterns by mass spectroscopy. A total of 10 stones were surgically removed from patients with urolithiasis. Proteins were extracted from the stones and identified by one-dimensional electrophoresis (sodium dodecyl sulfate buffer [SDS]-polyacrylamide gel electrophoresis [SDS-PAGE]). After in-gel digest, samples were analyzed by the surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) technique. The peptide sequences were analyzed from the data of mass spectroscopy. Proteins were identified from Database Search (SwissProt Protein Database; Swiss Institute of Bioinformatics; http://www.expasy.org/sprot) on a MASCOT server (Matrix Science Ltd.; http://www.matrixscience.com). A total of three bands of proteins (27, 18, and 14 kDa) were identified from SDS-PAGE in each stone sample. A database search (SwissProt) on a MASCOT server revealed that the most frequently seen proteins from band 1 (27 kDa) were leukocyte elastase precursor, cathepsin G precursor, azurocidin precursor, and myeloblastin precursor (EC 3.4.21.76) (leukocyte proteinase 3); band 2 (18 kDa) comprised calgranulin B, eosinophil cationic protein precursor, and lysozyme C precursor; band 3 (14 kDa) showed neutrophil defensin 3 precursor, calgranulin A, calgranulin C, and histone H4. The modifications and deamidations found from the mass pattern of these proteins may provide information for the study of matrix proteins. Various lower molecular weight proteins can be extracted from calcium oxalate stones. The characteristic patterns and their functions of those proteins should be further tested to investigate their roles in stone formation.

Insect NMDA receptors mediate juvenile hormone biosynthesis.

In vertebrates, the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) appears to play a role in neuronal development, synaptic plasticity, memory formation, and pituitary activity. However, functional NMDAR have not yet been characterized in insects. We have now demonstrated immunohistochemically glutamatergic nerve terminals in the corpora allata of an adult female cockroach, Diploptera punctata. Cockroach corpus allatum (CA) cells, exposed to NMDA in vitro, exhibited elevated cytosolic [Ca(2+)], but not in culture medium nominally free of calcium or containing NMDAR-specific channel blockers: MK-801 and Mg(2+). Sensitivity of cockroach corpora allata to NMDA changed cyclically during the ovarian cycle. Highly active glands of 4-day-old mated females, exposed to 3 microM NMDA, produced 70% more juvenile hormone (JH) in vitro, but the relatively inactive glands of 8-day-old mated females showed little response to the agonist. The stimulatory effect of NMDA was eliminated by augmenting the culture medium with MK-801, conantokin, or high Mg(2+). Having obtained substantive evidence of functioning NMDAR in insect corpora allata, we used reverse transcription PCR to demonstrate two mRNA transcripts, DNMDAR1 and DNMDAR2, in the ring gland and brain of last-instar Drosophila melanogaster. Immunohistochemical labeling, using mouse monoclonal antibody against rat NMDAR1, showed that only one of the three types of endocrine cells in the ring gland, CA cells, expressed rat NMDAR1-like immunoreactive protein. This antibody also labeled two brain neurons in the lateral protocerebrum, one neuron per brain hemisphere. Finally, we used the same primers for DNMDAR1 to demonstrate a fragment of putative NMDA receptor in the corpora allata of Diploptera punctata. Our results suggest that the NMDAR has a role in regulating JH synthesis and that ionotropic-subtype glutamate receptors became specialized early in animal evolution.