Receptor cDNA Research Articles

Single-cell Analysis of β2-Adrenergic Receptor Dynamics by Quantitative Fluorescence Microscopy.

G protein-coupled receptors (GPCRs) represent the largest family of surface proteins and are involved in the regulation of key physiological processes. GPCRs are characterized by seven transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. Cellular response of these receptors to their ligands is largely determined by their surface expression and postactivation behavior including expression, desensitization and resensitization. To develop a quantitative fluorescence Microscopy assay to study β2- Adrenergic receptor expression and desensitization. β2-Adrenergic receptor cDNA was engineered to put an HA tag at the extracellular N-terminus and GFP Tag at the intracellular C-terminus. GFP fluorescence serves as a measure of total cellular expression; whereas staining with CY3 conjugated anti-HA antibodies without permeabilizing the cells represents the surface expression of β2-AR. The images are quantified and amount of CY3 (surface) and GFP (total) fluorescence for each cell determined using image processing software. The method is sensitive and allows for the simultaneous measurement of surface and total expression of β2-AR. A highly accurate method is described for measuring β2-AR surface and total expression based on single-cell quantitative immunofluorescence. The method can be used to determine agonist-induced desensitization and resensitization process as well as receptor kinetics like endocytosis and exocytosis of β2-Adrenergic receptor and can be applied to essentially any other GPCR.

Pharmacological Characteristics of Porcine Orexin 2 Receptor and Mutants.

Orexin receptors (OXRs) play a critical regulatory role in central control of food intake, maintenance of sleeping states, energy metabolism, and neuroendocrine homeostasis. However, most previous studies have focused on the sleep-promoting functions of OXRs in human beings, while their potential value in enhancing food intake for livestock breeding has not been fully exploited. In this study, we successfully cloned porcine orexin 2 receptor (pOX2R) complementary DNA and constructed four pOX2R mutants (P10S, P11T, V308I, and T401I) by site-directed mutagenesis, and their functional expressions were further confirmed through Western blotting analysis. Pharmacological characteristics of pOX2R and their mutants were further investigated. These results showed that the P10S, P11T, and T401I mutants had decreased cAMP signaling with orexin A, whereas only the P11T mutant decreased under the stimulation of orexin B. Besides, only P10S displayed a decreased calcium release in response to both orexin ligands. Importantly, these mutants exhibited decreased phosphorylation levels of ERK1/2, p38, and CREB to some degree compared with wild-type pOX2R. Collectively, these findings highlight the critical role of these mutations in pOX2R signaling and expand our understanding of molecular and pharmacological characterization of pOX2R.

Molecular characterization and expression of arginine vasotocin V1a2 receptor in Atlantic croaker brain: Potential mechanisms of its downregulation by PCB77.

The arginine vasotocin (AVT)-V1a receptor mediates critical reproductive behaviors of the nonapeptide vasotocin in the teleost brain. In this study, we report the molecular characterization of the AVT-V1a2 receptor and its messenger RNA (mRNA) and protein expressions in the Atlantic croaker brain after exposure to the planar polychlorinated biphenyl congener 3,3',4,4'-tetrachlorobiphenyl (PCB77). The full-length sequence of croaker AVT-V1a2 receptor complementary DNA (cDNA) is highly homologous to other teleost AVT-V1a2 receptor cDNAs. Double-labeled immunohistochemistry showed coexpression of AVT-V1a2 receptor and gonadotropin-releasing hormone-I (GnRH-I, a neuropeptide that regulates gonadotropin secretion) in hypothalamic neurons, thereby providing the anatomical basis for possible AVT modulation of croaker reproduction through alterations in GnRH-I secretion. AVT-V1a2 receptor mRNA and protein levels as well as GnRH-I mRNA levels were markedly decreased in hypothalamic tissues of croaker exposed to PCB77 (dose: 2 and 8 µg/g body weight for 4 weeks) compared with levels in untreated (control) fish. In contrast, hypothalamic cytochrome P450 1A (CYP1A, a monooxygenase enzyme) and interleukin-1β (IL-1β, a cytokine indicator of inflammation and response to neuronal damage) mRNA levels, and plasma protein carbonyl (PC, an indicator of reactive oxygen species) contents, important biomarkers of neural stress, were increased in PCB77-exposed fish compared with controls. Collectively, these results suggest that the downregulation of hypothalamic AVT-V1a2 receptor and GnRH-I transcripts due to PCB77 exposure is associated with induction of CYP1A, cellular inflammation and oxidative stress in Atlantic croaker, a marine teleost that inhabits estuaries along the US Atlantic coast and the Gulf of Mexico that are often contaminated with persistent organic pollutants such as PCBs.

Pig endothelial protein C receptor is functionally compatible with the human protein C pathway.

Endothelial protein C receptor (EPCR) plays an anticoagulant and anti-inflammatory role by promoting the activation of protein C by thrombin bound to thrombomodulin (TBM). Incompatibility between pig TBM and human/primate thrombin is thought to contribute to dysregulated coagulation in pig-to-primate organ xenografts, and expression of human TBM (hTBM) in pigs has shown benefit in preclinical models. However, it is not known whether there are incompatibilities-or molecular barriers-between endogenous pig EPCR (pEPCR) and transgenically expressed human TBM. To clone and express pEPCR, and determine its function in the human protein C pathway in vitro. Pig endothelial protein C receptor cDNA was generated from pig lung RNA by RT-PCR. Primate COS-7 transfectants expressing various combinations of human and pig TBM and EPCR were incubated with human thrombin and human protein C, and tested for TBM cofactor activity. The predicted protein sequence of pEPCR shared 72.3% amino acid sequence identity with hEPCR, and residues critical for protein C binding were conserved. COS-7 cells transfected with hEPCR, pEPCR or vector showed minimal TBM cofactor activity (0.13±0.04, 0.13±0.02 and 0.14±0.06 U, respectively). The cofactor activity of hTBM-transfected cells (1.18±0.29 U) was 8-fold higher than vector-transfected cells (P=.004) and further increased 4-fold and 3-fold by co-transfection with hEPCR (5.01±1.12 U, P=.004) or pEPCR (3.73±0.65 U, P=.003), respectively. Our data show that pEPCR is largely compatible with the human TBM/thrombin complex, when expressed on COS-7 cells in vitro, promoting the activation of human protein C. These findings suggest that endogenous pEPCR will enhance the activity of transgenic hTBM in the xenograft setting.

Herceptin: A First Assault on Oncogenes that Launched a Revolution

This year's Lasker Clinical Research Award honors H. Michael Shepard, Dennis J. Slamon, and Axel Ullrich for their invention of Herceptin, the first monoclonal antibody that blocks a cancer-causing protein, and for its development as a life-saving therapy for women with breast cancer.

Mammalian Taste Cells Express Functional Olfactory Receptors.

The peripheral taste and olfactory systems in mammals are separate and independent sensory systems. In the current model of chemosensation, gustatory, and olfactory receptors are genetically divergent families expressed in anatomically distinct locations that project to disparate downstream targets. Although information from the 2 sensory systems merges to form the perception of flavor, the first cross talk is thought to occur centrally, in the insular cortex. Recent studies have shown that gustatory and olfactory receptors are expressed throughout the body and serve as chemical sensors in multiple tissues. Olfactory receptor cDNA has been detected in the tongue, yet the presence of physiologically functional olfactory receptors in taste cells has not yet been demonstrated. Here we report that olfactory receptors are functionally expressed in taste papillae. We found expression of olfactory receptors in the taste papillae of green fluorescent protein-expressing transgenic mice and, using immunocytochemistry and real-time quantitative polymerase chain reaction experiments, the presence of olfactory signal transduction molecules and olfactory receptors in cultured human fungiform taste papilla (HBO) cells. Both HBO cells and mouse taste papilla cells responded to odorants. Knockdown of adenylyl cyclase mRNA by specific small inhibitory RNA and pharmacological block of adenylyl cyclase eliminated these responses, leading us to hypothesize that the gustatory system may receive olfactory information in the periphery. These results provide the first direct evidence of the presence of functional olfactory receptors in mammalian taste cells. Our results also demonstrate that the initial integration of gustatory and olfactory information may occur as early as the taste receptor cells.

Molecular Cloning and Expression Analysis of 5-hydroxytryptamine Receptor 7 in Ant Polyrhachis vicina Roger (Hymenoptera: Formicidae).

Serotonin (5-hydroxytryptamine [5-HT]) is a monoamine neurotransmitter that plays an important role in regulating a variety of physiological and behavioral activities. In this study, the 5-HT7 receptor gene was cloned from the ant Polyrhachis vicina Roger (1863). The complete Pv5-HT7 receptor cDNA is 3054 bp, including a 5′-untranslated region (UTR) of 790 bp, a 3′-UTR of 752 bp and an open reading frame of 1512 bp encoding polypeptide of 503 amino acids. Hydrophobic analysis suggests that seven trans-membrane domains are the major sequence characteristic of the Pv5-HT7 receptor. In addition, the Pv5-HT7 receptor has three potential N-glycosylation sites, a palmitoylation site, three protein kinase A phosphorylation sites, and four protein kinase C phosphorylation sites. Phylogenetic analysis revealed that the deduced Pv5-HT7 receptor sequence shared a high homology with 5-HT7 receptor sequences of other species, such as a 78% similarity with the Am5-HT7 receptor (Apis mellifera). Real-time quantitative PCR (qRT-PCR) results showed that the expression level of the Pv5-HT7 receptor was low in the eggs and 1th-4th larval stages, but it was increased in the pupae stage and reached its peak in the adult workers. Western blot results showed that the highest protein expression was in the male body, head, and thorax. These results suggest that the Pv5-HT7 receptor may have specific functions in regulating the development of P. vicina, especially in adult formation and caste differentiation, feeding and caring behaviors of workers in the nest, and in the development of motor organs and mating behaviors in males.

Abstract A039: FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1

Abstract The outcome of cancer patients has recently been improved by utilizing novel immunotherapies that inhibit the regulatory immune system. Regulatory T-cells (Treg) contribute not only to suppression of excessive immune responses in the steady state but also to reduction of antitumor immune responses in cancer patients. We have shown that down-regulation of the Helios transcription factor undermines CD4 Treg stability inducing these cells to convert into effector cells that enhance the antitumor immune response. Helios is also the canonical transcription factor of CD8 Treg and essential for CD8 Treg function. CD8 Treg are Qa-1 (HLA-E in human)–restricted CD8 T-cells that mainly regulate Qa-1+ immune cells. The peptide FAM49B(p190-198) is a Qa-1 binding peptide derived from ubiquitously expressed FAM49B. Although FAM49B(p190-198) is regularly degenerated in the endoplasmic reticulum normal cells, we found that FAM49B(p190-198) is stably presented in some tumor cells. Here, we investigate the potential contribution of FAM49B(p190-198)/Qa-1 specific CD8 Treg to cancer.FAM49B(p190-198)/Qa-1 specific CD8 Treg were detected by FAM49B(p190-198)/Qa-1 tetramer after immunization with FAM49B(p190-198) peptide–loaded dendritic cells in CD8 Treg (i.e., CD44+CD122+Ly49+ CD8 T-cells). T-cell receptor cDNAs were amplified from the sorted single cells and inserted into retrovirus expression vectors for transduction in 58α–β– hybridoma cells and bone marrow cells. EL4-Qa-1 KO cells and EL4-FAM49B KO cells were generated using CRISPR/Cas9 technology. These tools allowed validation of the specificity of the TCRs and their Qa-1–restriction. For in vivo studies, TCR retrogenic mice were developed by transferring TCR-transduced Rag2–/– bone marrow hematopoietic stem cells into lethally irradiated CD45.1 mice. Retrogenic T-cells were prepared by CD8 enrichment of retrogenic mouse splenocytes and in vitro culture for 3 days in the presence of IL15c. For in vivo studies, we use an adoptive transfer model of retrogenic T-cells for EL4 tumor bearing mice. Rag2–/–γC–/– mice were inoculated with 0.5 x 10^6 EL4 WT-cells and 0.5 x 10^6 EL4 Qa-1 KO cells on each lateral flank, then intravenously injected with retrogenic T-cells (on day 13 and 17). FAM49B(p190-198)/Qa-1 specific CD8 T-cells preferentially use TCR Vα3.2 (TRAV9N-3) and Vβ5.1/5.2 (TRBV12-1/12-2) chains independent of their expression of Ly49+. We tested 17 pairs obtained from Ly49+ cells using the in vitro system and found that 11 pairs bound strongly to Fam49b(p190-198)/Qa-1 tetramer while 8 out of the 11 pairs exhibited antigen-specific activation. Interestingly, one TCR pair (No.8) responded to WT EL4 cells without antigen peptides, which was abolished in coculture wells with Qa-1 KO EL4 cells and FAM49B KO EL4 cells. This TCR hybridoma was also activated by stimulated WT B6 splenocytes but not by Qa-1 KO splenocytes suggesting its regulatory potential. Adoptive transfer of TCR No.8-expressing retrogenic T-cells delayed EL4 WT tumor growth but not EL4 Qa-1 KO tumor growth when inoculated in the same hosts. Tumor-infiltrating CD8 T-cells displayed a more activated phenotype in WT tumors compared to KO tumors, indicating that retrogenic T-cells recognize and target EL4 cells in a Qa-1-dependent manner. Citation Format: Hidetoshi Nakagawa, Hye-Jung Kim, Harvey Cantor. FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A039.

Genetically modified hematopoietic stem/progenitor cells that produce IL-10–secreting regulatory T cells

Random amino acid copolymers used in the treatment of multiple sclerosis in man or experimental autoimmune encephalomyelitis (EAE) in mice [poly(Y,E,A,K)n, known as Copaxone, and poly(Y,F,A,K)n] function at least in part by generation of IL-10-secreting regulatory T cells that mediate bystander immunosuppression. The mechanism through which these copolymers induce Tregs is unknown. To investigate this question, four previously described Vα3.2 Vβ14 T cell receptor (TCR) cDNAs, the dominant clonotype generated in splenocytes after immunization of SJL mice, that differed only in their CDR3 sequences were utilized to generate retrogenic mice. The high-level production of IL-10 as well as IL-5 and small amounts of the related cytokines IL-4 and IL-13 by CD4+ T cells isolated from the splenocytes of these mice strongly suggests that the TCR itself encodes information for specific cytokine secretion. The proliferation and production of IL-10 by these Tregs was costimulated by activation of glucocorticoid-induced TNF receptor (GITR) (expressed at high levels by these cells) through its ligand GITRL. A mechanism for generation of cells with this specificity is proposed. Moreover, retrogenic mice expressing these Tregs were protected from induction of EAE by the appropriate autoantigen.

Quantitative analysis of the T cell receptor repertoire.

The T cell receptor repertoire provides a window into the cellular adaptive immune response. In the context of cancer, determining the repertoire within a tumor can give important insights into the evolution of the T cell anti-cancer response, and has the potential to identify specific personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing and analyzing T cell receptors which is economical, robust, sensitive and versatile. The key experimental step is the ligation of a single stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. We describe a detailed protocol describing this method to create libraries of T cell receptors from in vitro T cell cultures, blood or tissue samples. We combine this with a computational pipeline, which incorporates sample multiplexing, T cell receptor annotation and error correction to provide accurate counts of individual T cell receptor sequences within samples. The integrated experimental and computational pipeline should be of value to researchers interested in documenting and understanding the T cell immune response to cancer, and in manipulating it for therapeutic purposes.

Abacavir, nevirapine, and ritonavir modulate intracellular calcium levels without affecting GHRH-mediated growth hormone secretion in somatotropic cells in vitro

Growth Hormone (GH) deficiency is frequent in HIV-infected patients treated with antiretroviral therapy. We treated GH3 cells with antiretrovirals (nevirapine, ritonavir or abacavir sulfate; 100 pM-1 mM range), after transfection with human growth hormone releasing hormone (GHRH) receptor cDNA. Cells viability, intracellular cAMP, phosphorylation of CREB and calcium increase, GH production and secretion were evaluated both in basal condition and after GHRH, using MTT, bioluminescence resonance energy transfer, western blotting and ELISA.Antiretroviral treatment did not affect GHRH 50% effective dose (EC50) calculated for 30-min intracellular cAMP increase (Mann-Whitney's U test; p ≥ 0.05; n = 4) nor 15-min CREB phosphorylation. The kinetics of GHRH-mediated, rapid intracellular calcium increase was perturbed by pre-incubation with drugs, while GHRH failed to induce the ion increase in ritonavir pre-treated cells (ANOVA; p < 0.05; n = 3). Antiretrovirals did not impact 24-h intracellular and extracellular GH levels (ANOVA; p ≥ 0.05; n = 3).We demonstrated the association between antiretrovirals and intracellular calcium increase, without consequences on somatotrope cells viability and GH synthesis. Overall, these results suggest that antiretrovirals may not directly impact on GH axis in HIV-infected patients.

An Economical, Quantitative, and Robust Protocol for High-Throughput T Cell Receptor Sequencing from Tumor or Blood.

The T cell receptor repertoire provides a window to the cellular adaptive immune response within a tumor, and has the potential to identify specific and personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing, and analyzing T cell receptors which is economical, robust, sensitive, and versatile. The key experimental step is the ligation of a single-stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. This method has been applied to the analysis of unfractionated human tumor lysates, subpopulations of tumor-infiltrating lymphocytes, and peripheral blood samples from patients with a variety of solid tumors.

Molecular cloning and functional expression of the 5-HT7 receptor in Chinese mitten crab (Eriocheir sinensis).

Serotonin (5-HT) regulates numerous physiological functions and processes, such as light adaptation, food intake and ovarian maturation, and plays the role through 5-HT receptors. To our knowledge, this is the first study to isolate and characterize the serotonin receptor 7 (5-HT7 receptor) cDNA encoded in Eriocheir sinensis, an economically important aquaculture species in China, by performing rapid-amplification of cDNA ends. The full-length of 5-HT7 receptor gene cDNA is 2328 bp and encodes a polypeptide with 590 amino acids that are highly homologous with other crustaceans 5-HT7 receptor genes. Analysis of the deduced amino acid sequence of the 5-HT7, including 7 transmembrane domains and some common features of G protein-coupled receptors (GPCRs), indicated that 5-HT7 receptor was a member of GPCRs family. A gene expression analysis of the 5-HT7 receptor by RT-PCR revealed that the 5-HT7 receptor transcripts were widely distributed in various tissues, in which high expression levels were observed in the cranial ganglia, thoracic ganglia and intestines. Further study about the effects of photoperiods on the 5-HT7 expression in the tissues showed that a significantly increasing expression of the 5-HT7 receptor was observed in the thoracic ganglia induced by constant light. In addition, in the eyestalks, the expression levels of 5-HT7 mRNA in constant darkness and constant light were lower than control treatment. Then, the expression levels of the 5-HT7 receptor in three feeding statuses displayed that there were significantly increasing expressions in the hepatopancreas and intestines after feeding, compared with before feeding and during the feeding period. Finally, the 5-HT7 mRNA expression levels in stage III and stage IV were higher than the levels in stage I of ovarian development. Our experimental results showed that the 5-HT7 receptor structurally belongs to GPCRs, and the thoracic ganglia and eyestalks are the important tissues of the 5-HT7 receptor for light adaptation. The 5-HT7 receptor may also be involved in the physiological regulation of the hepatopancreas and intestines after ingestion in E. sinensis. In addition, the 5-HT7 receptor is involved in the process of ovarian maturation. The study provided a foundation for further research of light adaptation, digestive functions and ovarian maturation of the 5-HT7 receptor in Decapoda.

Identification of molt-inhibiting hormone and ecdysteroid receptor cDNA sequences in Gammarus pulex, and variations after endocrine disruptor exposures

In amphipods, growth, development and reproduction are mediated by the molt, which is a hormonally controlled process and which, therefore, could be impacted by endocrine disruption compounds (EDC). The molt process is controlled by both X-organ (XO) and Y-organ (YO) through a variety of hormones and receptors including the molt-inhibiting hormone (MIH) and the ecdysteroid receptor (EcR). However, although many studies were devoted to characterize MIH and EcR in crustaceans, only few works evaluated their variations under EDCs exposures. Consequently, the present work aimed to characterize MIH and EcR genes of the amphipod Gammarus pulex, as well as to study their relative expression variations after exposure to four EDCs, proved in vertebrates: ethinylestradiol (estrogen), 4-hydroxytamoxifen (anti-estrogen), 17α-methyltestosterone (androgen) and cyproterone acetate (anti-androgen).PCR amplification allowed to obtain 204 bp length and 255 bp length fragments, encoding for partial sequences of 68 amino acids and 85 amino acids, which correspond to EcR and MIH, respectively, and which are highly conserved in crustacean species. Results highlighted MIH and EcR expressions mainly in G. pulex head, which is the localization of XO and YO. Moreover, irrespective of the EDC exposure, increases of MIH and EcR relative expressions were observed, as it was observed after the exposure to 20-hydroxyecdysone (20HE), the natural molt hormone, used as positive control. Therefore, it appeared that tested EDCs behaved like 20HE, suggesting that their effects could occur through the ecdysteroids pathways, and so impact the molt process of G. pulex on the long term. Finally, the present study is a first step in the possibility of using MIH and EcR relative expressions as biomarkers of exposure for EDCs risk assessment. However additional studies must first be carried out to better characterize and understand their variations, and also better predicted consequences for the exposed amphipods.

Understanding Angiotensin II Type 1 Receptor Signaling in Vascular Pathophysiology.

Angiotensin II is the most important endocrine ligand in the renin–angiotensin system (RAS), contributing to the development of several cardiovascular diseases including hypertension.1 Angiotensin II mediates its signal transduction and functions via the angiotensin II receptors.2 Historically, the presence of 2 subtypes of angiotensin II receptors were pharmacologically recognized based on the sensitivity to the first orally active nonpeptide angiotensin II receptor antagonist, losartan. The losartan-sensitive receptor was termed AT1 (angiotensin II receptor type 1) receptor. It was assumed to be a heterotrimeric GPCR (G protein–coupled receptor) because it generates inositol triphosphate and diacylglycerol, leading to intracellular Ca2+ elevation and protein kinase C activation, respectively. Most known physiological and pathophysiological functions of angiotensin II, including stimulation of vasoconstriction and salt and water reabsorption, are mediated through the AT1 receptor. The losartan-insensitive receptor was termed AT2 receptor, whereas its G protein coupling remains unclear.1,3,4 In 1991, 2 research groups in the United States independently isolated cDNA (termed AGTR1) encoding the mammalian AT1 receptor.5,6 Subsequently, rat AT2 receptor cDNA (AGTR2) was cloned in 1993.7,8 This pioneer work revealed complete amino acid sequences of the angiotensin II receptor subtypes belonging to the 7-transmembrane GPCR superfamily. In the early 1990s, several studies reported that AT1 receptor elicits tyrosine phosphorylation of multiple proteins and activation of MAPK (mitogen-activated protein kinase; eg, p42/p44 MAPK)/ERK (extracellular signal–regulated kinase; eg, ERK1/2) in various cell types including vascular smooth muscle cells (VSMC). The early 1990s also saw the establishment of the concept that angiotensin II via the AT1 receptor has a direct action on cardiac myocytes, fibroblasts, and VSMCs causing hypertrophic and fibrotic cardiovascular remodeling.9,10 The cardiovascular remodeling caused by angiotensin II seemed to be …

Cloning and functional characterization of the DA2 receptor gene in Chinese mitten crab (Eriocheir sinensis).

Dopamine (DA) plays a modulatory role in numerous physiological processes such as light adaptation and food intake, and exerts these functions through DA receptors (DARs). This study presents, for the first time, isolation and characterization of the dopamine receptor 2(DA2 receptor) cDNA from the intestinal tissue of Eriocheir sinensis, an economically important freshwater aquaculture species in China. The DA2 receptor cDNA sequence, which was obtained by rapid amplification of cDNA ends, is 2369bp long, encode peptide of 589 amino acid, and is highly homologous to related sequences in crustaceans. Analysis of the deduced amino acid sequence and the structure of the DA2 indicated that this receptor is a member of the family of G protein-coupled receptors (GPCRs), as it contains seven transmembrane domains and other common signatures of GPCRs. RT-PCR showed that the expression of the DA2 receptor gene was distributed in various tissues, and high expression levels were observed in the cranial ganglia and the thoracic ganglia. Further study of the effect of photoperiod on DA2 expression showed that constant darkness induced a significant increase in DA2 expression in the cranial ganglia. Finally, analysis of DA2 receptor expression under different feeding statuses showed that there was significantly greater expression in the hepatopancreas and intestines after feeding than before feeding, but there were no differences in expression between the before feeding and during feeding periods in either tissue. Our results indicate that the DA2 receptor structurally belongs to the family of G protein-coupled receptors, and that the cranial ganglia are the main tissues in which the DA2 receptor participates in light adaptation during dark hours. In addition, the DA2 receptor in E. sinensis may be involved in the physiological regulation of the hepatopancreas and digestive tract after the ingestion of food. This study provides a foundation for further exploration of the light adaptation and digestive functions of the DA2 receptor in decapods.

Molecular characterization and expression profiling of ryanodine receptor gene in the pink stem borer, Sesamia inferens (Walker)

The susceptibilities of three field populations of pink stem borer (PSB), Sesamia inferens (walker) to diamide insecticides, chlorantraniliprole and flubendiamide, were evaluated in this study. The results showed that these PSB field populations were still sensitive to the two diamide insecticides after many years of exposure. To further understand PSB and diamide insecticide, the full-length ryanodine receptor (RyR) cDNA (named as SiRyR), the molecular target of diamide insecticides was cloned from PSB and characterized. The SiRyR gene contains an open reading frame of 15,420 nucleotides, encoding 5140 amino acid residues, which shares 77% to 98% sequence identity with RyR homologous of other insects. All hallmarks of RyR proteins are conserved in the SiRyR protein, including the conserved C-terminal domain with the consensus calcium-biding EF-hands (calcium-binding motif), the six transmembrane domains, as well as mannosyltransferase, IP3R and RyR (pfam02815) (MIR) domains. Real-time qPCR analysis revealed that the highest mRNA expression levels of SiRyR were observed in pupa and adults, especially in males. SiRyR was expressed at the highest level in thorax, and the lowest level in wing. The full genetic characterization of SiRyR could provide useful information for future functional expression studies and for discovery of new insecticides with selective insecticidal activity.

Vitellogenin and vitellogenin receptor gene expression profiles in Spodoptera exigua are related to host plant suitability.

The beet armyworm Spodoptera exigua, a worldwide phytophagous pest, causes considerable economic agricultural losses. Understanding the relationship between its fecundity and the host plant is a basic and important component of early forecasting of beet armyworm outbreaks. However, little is known about the molecular mechanism by which distinct hosts affect S. exigua fecundity. In this study, key life-history parameters of S. exigua reared on distinct hosts were investigated; the host plants could be ranked as lettuce > shallot > tomato > celery in their order of suitability. Full-length S. exigua vitellogenin receptor (SeVgR) cDNA was cloned, and sex-, stage- and tissue-specific expression characteristics were assessed. Spodoptera exigua vitellogenin (SeVg) and SeVgR expression levels were markedly modulated by host nutrients (P < 0.05). SeVg and SeVgR expression levels were significantly higher in S. exigua reared on lettuce, the most preferred and most nutritive host, than in those reared on tomato and celery. Interestingly, significant linear regression correlations were found between SeVg and SeVgR expression levels and key S. exigua life-history parameters, especially life span, pupa weight, and female fecundity (P < 0.01). Host plant type and suitability could affect the expression pattern of SeVg and SeVgR, which influenced S. exigua fecundity. Vg and VgR have the potential to be used as molecular markers of S. exigua fecundity and for forecasting outbreaks of S. exigua on different hosts. © 2017 Society of Chemical Industry.

Ariadne merione ecdysone receptor (AmEcR) protein: An in silico approach for comparison of agonist and antagonist compounds

Ecdysteroid signal transduction plays a major role in insect metamorphosis, 20-hydroxyecdysone (20E) binds to the nuclear receptor composed of the ecdysone receptor ligand binding domine (EcR-LBD) and triggers the developmental transitions. Ariadne merione ecdysone receptor (AmEcR) cDNA was amplified and partially sequenced of about 553 bp, which encodes a polypeptide of 184 amino acids (aa). The theoretical molecular weight (MW), isoelectric point (pI) and aliphatic index of the deduced AmEcR protein were predicted using BIOEDIT (v7.2.5) to be 21.192 kDa, 9.31 and 101.739 respectively. Identified ecdysone receptor gene of A. merione showed maximum similarity with Precis coenia gene. In this research, we have employed ligand-receptor engineering technique to screen a specific compound which plays antagonist role and assist to formulate an insect specific pesticide. The EcR protein 3D structure of AmEcR modeled using Schrödinger maestro and virtual screening was performed using 5554 molecules from Zinc database, where ZINC20031812 showed highest glide score of −6.257 and Etoxazole chosen on literature basis and showed best glide score −6.671. We have compared the antagonist with agonist (20E) by molecular dynamics (MD) simulation. Root Mean Square Deviation (RMSD) value of agonist and antagonist indicates the binding were stable in water with a range of distance from 2.3 to 2.6 Å, 1.8 to 2.3 Å and 1.9 to 2.3 Å with a variation over the time scale of 1 ps. Since Etoxazole and ZINC20031812 are antagonists, computationally they were more stable than 20E.

The short neuropeptide F modulates olfactory sensitivity of Bactrocera dorsalis upon starvation

The insect short neuropeptide F (sNPF) family has been shown to modulate diverse physiological processes, such as feeding, appetitive olfactory behavior, locomotion, sleep homeostasis and hormone release. In this study, we identified the sNPF (BdsNPF) and its receptor (BdsNPFR) in an important agricultural pest, the oriental fruit fly Bactrocera dorsalis (Hendel). Afterwards, the receptor cDNA was functionally expressed in Chinese hamster ovary cell lines. Activation of BdsNPFR by sNPF peptides caused an increase in intracellular calcium ions, with a 50% effective concentration values at the nanomolar level. As indicated by qPCR, the BdsNPF and BdsNPFR transcripts were mainly detected in the central nervous system and antennae, and they showed significantly starvation-induced expression patterns. Furthermore, we found that the starved flies had an increased electroantennogram response compared to the normally fed flies. However, this enhanced olfactory sensitivity was reversed when we decreased the expression of BdsNPF by double-stranded RNA injection in adults. We concluded that sNPF plays an important role in modulating the olfactory sensitivity of B. dorsalis upon starvation. Our results will facilitate the understanding of the regulation of early olfactory processing in B. dorsalis.