Functional analysis of the odorant receptor coreceptor in odor detection in Grapholita molesta (lepidoptera: Tortricidae).

The olfactory system must detect and discriminate various semiochemicals in the environment. In response to such diversity, insects have evolved a family of odorant-gated ion channels composed of a common receptor (coreceptor, Orco) and a ligand-binding tuning odorant receptor (OR) that confers odour specificity. This study aims to examine the expression pattern of Orco gene of Grapholita molesta (GmolOrco) and to elucidate the role of GmolOrco in detecting G. molesta sex pheromone and green leaf volatiles by using gene silencing via RNA interference (RNAi) coupled antennal electrophysiological (EAG). Multiple sequence alignment showed that GmolOrco shared high sequence similarities with the Orco ortholog of lepidopterans. The results of real-time quantitative PCR detection demonstrated that GmolOrco was predominantly expressed in adult antennae and had the highest expression quantity in adult period among the different developmental stages. Compared with the noninjected controls, GmolOrco expression in GmolOrcodouble-stranded RNA (dsRNA)-injected males was reduced to 39.92% and that in females was reduced to 40.43%. EAG assays showed that the responses of GmolOrco-dsRNA injected males to sex pheromones (Z)-8-dodecenyl acetate (Z8-12:OAc) and (Z)-8-dodecenyl alcohol (Z8-12:OH) were significantly reduced, and the GmolOrco-dsRNA-injected female to green leaf volatile (Z)-3-hexenyl acetate also significantly declined. We inferred that Orco-mediated olfaction was different in male and female G. molesta adults and was mainly involved in recognizing the sex pheromones released by female moths.

[Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin].

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

Degradation of sex pheromone and plant volatile components by an antennal glutathione S-transferase in the oriental fruit moth,Grapholita molesta Busck (Lepidoptera: Tortricidae).

Insect antennae have a primary function of perceiving and discerning odorant molecules including sex pheromones and host plant volatiles. The assumption that genes highly expressed in the antennae may have an olfactory-related role associated with signal transduction. Here, one delta subfamily glutathione S-transferase (GST) gene (GmolGSTD1) was obtained from an antennal transcriptome of Grapholita molesta. Quantitative real-time polymerase chain reaction results revealed that GmolGSTD1 was mainly expressed in antennae and the expression levels were significantly higher in female antennae than in male antennae. The recombinant GmolGSTD1 (rGmolGSTD1) showed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The pH range for optimal rGmolGSTD1 enzyme activity was 6.0-6.5, and rGmolGSTD1 enzyme activity had maximal peaks at 35-40°C. Spectrophotometric analysis indicated that insecticides had weak inhibitory effects on the activity of rGmolGSTD1 with the inhibitory rates of 28.82% for chlorpyrifos, 22.27% for lambda-cyhalothrin, 18.07% for bifenthrin, 20.42% for acetamiprid, 17.57% for thiamethoxam, 25.67% for metaflumizone, 27.43% for abamectin, and 7.24% for chlorbenzuron. rGmolGSTD1 exhibited high degradation activity to the sex pheromone component (Z)-8-dodecenyl alcohol and the host plant volatile butyl hexanoate with the degradation efficiency of 75.01% and 48.54%, respectively. We speculate that GmolGSTD1 works in inactivating odorant molecules and maintaining sensitivity to olfactory communication of G. molesta.

Molecular and functional characterization of three odorant binding proteins from the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricide).

Odorant binding proteins (OBPs) act in recognizing odor molecules and their most well-studied functions are transporting odors across the sensillum lymph to olfactory receptor neurons within the insect antennal sensillum. The adults of Grapholita molesta highly depend on olfactory cues in locating host plants and selecting oviposition sites, in which OBPs play an important role in perceiving and recognizing host plant volatiles. Exploring the physiological function of OBPs could facilitate our understanding of their importance in insects' chemical communication. In this study, three OBP genes were cloned and named GmolOBP4, GmolOBP5, and GmolOBP10. Quantitative real-time PCR results indicated that GmolOBP4 and GmolOBP10 were predominantly expressed in adult antennae and GmolOBP5 was expressed in multiple tissues, including head, legs, and wings in addition to antennae. The binding affinities of the three recombinant GmolOBPs (rGmolOBPs) with four sex pheromone components and twenty-nine host plant volatiles were measured using 1-N-Phenyl-naphthylamine as a fluorescence probe. The three rGmolOBPs exhibited specific binding properties to potential ligands, GmolOBP4 and GmolOBP10 bound to minor sex pheromone components, such as (Z)-8-dodecenyl alcohol and dodecanol, respectively. rGmolOBP4 showed intermediate binding ability with hexanal, benzyl alcohol, and pear ester, rGmolOBP5 had a weak affinity for benzaldehyde, pear ester and, methyl jasmonate, and rGmolOBP10 showed strong binding capacity toward hexanol, decanol, and α-ocimene. We speculate that the GmolOBP4 and GmolOBP10 have dual functions in perception and recognition of host plant volatiles and sex pheromone components, while GmolOBP5 may serve other function(s).

Broadening of the sum-frequency phase-matching bandwidth by temperature gradient in MgO:PPLN.

We demonstrate bandwidth broadening in cascaded MgO-doped periodically poled lithium niobate (MgO:PPLN) crystals (Λ=10.3 µm) using the temperature-gradient technique. Up to 2.8 nm bandwidth at 600 nm spectral region is achieved using two 50 mm long cascaded MgO:PPLN crystals via sum frequency generation. This technique combines the merits of high conversion efficiency attributed to cascaded nonlinearity and the reconfigurability of temperature-gradient-induced chirp for broadening of the input wavelength acceptance range.

CLONING, EXPRESSION, AND FUNCTIONAL ANALYSIS OF THREE ODORANT-BINDING PROTEINS OF THE ORIENTAL FRUIT MOTH, Grapholita molesta (BUSCK) (LEPIDOPTERA: TORTRICIDAE).

Odorant-binding proteins (OBPs) act in insect olfactory processes. OBPs are expressed in the olfactory organs and serve in binding and transport of hydrophobic odorants through the sensillum lymph to olfactory receptor neurons within the antennal sensilla. In this study, three OBP genes were cloned from the antennal transcriptome database of Grapholita molesta via reverse-transcription PCR. Recombinant GmolOBPs (rGmolOBPs) were expressed in a prokaryotic expression system and enriched via Ni ion affinity chromatography. The binding properties of the three rGmolOBPs to four sex pheromones and 30 host-plant volatiles were investigated in fluorescence ligand-binding assays. The results demonstrated that rGmolOBP8, rGmolOBP11, and rGmolOBP15 exhibited high binding affinities with the major sex pheromone components (E)-8-dodecenyl acetate, (Z)-8-dodecenyl alcohol, and dodecanol. The volatiles emitted from peach and pear, decanal, butyl hexanoate, and α-ocimene, also showed binding affinities to rGmolOBP8 and rGmolOBP11. Hexanal, heptanal, and α-pinene showed strong binding affinities to rGmolOBP15. Results of the electrophysiological recording experiments and previous behavior bioassays indicated that adult insects had strong electroantennogram and behavioral responses toward butyl hexanoate, hexanal, and heptanal. We infer that the GmolOBP8 and GmolOBP11 have dual functions in perception and recognition of host-plant volatiles and sex pheromones, while GmolOBP15 was mainly involved in plant volatile odorants' perception.

[Correction of Fiber Throughput Variation Due to the Focal Ratio Degradation].

The focal ratio degradation (FRD) of optical fiber is one of major sources causing light loss in multi-fiber astronomical instruments. Meanwhile, the sky subtraction is crucial to multi-fiber spectra reduction, especially for the objects which are as faint as the sky background, not to mention for those even fainter ones. To improve the accuracy of sky subtraction, it is necessary to normalize the throughput among object fibers and sky sampling fibers. The rotation and twist during mounting and rotating could change the FRD of individual fibers, which means the variation of the transmission throughput among fibers. We investigate such throughput variation among LAMOST fibers and its correlation with the intensity of sky emission lines on all wavelength coverage in this paper. On the basis of this work, we present an approach to correcting the varied fiber throughput by measuring the intensity of the sky emission lines as the secondary throughput correction. This approach has been applied to LAMOST 2D Pipeline.

Involvement of calcium-sensing receptors in hypoxia-induced vascular remodeling and pulmonary hypertension by promoting phenotypic modulation of small pulmonary arteries.

Phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) plays an important role during hypoxia-induced vascular remodeling and pulmonary hypertension (PAH). We had previously shown that calcium-sensing receptor (CaSR) is expressed in rat PASMCs. However, little is known about the role of CaSR in phenotypic modulation of PASMCs in hypoxia-induced PAH as well as the underlying mechanisms. In this study, we investigated whether CaSR induces the proliferation of PASMCs in small pulmonary arteries from both rats and human with PAH. PAH was induced by exposing rats to hypoxia for 7-21 days. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVI), the percentage of medial wall thickness to the external diameter (WT %), and cross-sectional total vessel wall area to the total area (WA %) of small pulmonary arteries were determined by hematoxylin and eosin (HE), masson trichrome and Weigert's staining. The protein expressions of matrix metalloproteinase (MMP)-2 and MMP-9, the tissue inhibitors of metalloproteinase (TIMP)-3, CaSR, proliferating cell nuclear antigen (PCNA), phosphorylated extracellular signal-regulated kinase (p-ERK), and smooth muscle cell (SMC) phenotype marker proteins in rat small pulmonary arteries, including calponin, SMα-actin (SMAα), and osteopontin (OPN), were analyzed by immunohistochemistry and Western blotting, respectively. In addition, immunohistochemistry was applied to paraffin-embedded human tissues from lungs of normal human and PAH patients with chronic heart failure (PAH/CHF). Compared with the control group, mPAP, RVI, WT % and WA % in PAH rats were gradually increased with the prolonged hypoxia. At the same time, the expressions of CaSR, MMP-2, MMP-9, TIMP-3, PCNA, OPN, and p-ERK were markedly increased, while the expressions of SMAα and calponin were significantly reduced in lung tissues or small pulmonary arteries of PAH rats. Neomycin (an agonist of CaSR) enhanced but NPS2390 (an antagonist of CaSR) weakened these hypoxic effects. We further found that the expression change of CaSR, PCNA, and SMC phenotypic marker proteins in PAH/CHF lungs was similar to those in PAH rats. Our data suggest that CaSR is involved in the pulmonary vascular remodeling and PAH by promoting phenotypic modulation of small pulmonary arteries.

Sequence and phylogenetic analysis of surface protein genes of emerging H9N2 influenza viruses isolated from poultry in two geographical regions of China.

Subtype H9N2 avian influenza viruses (AIVs) circulating in China have aroused increasing concerns for their impact on poultry and risk to public health. The present study was an attempt to elucidate the phylogenetic relationship of H9N2 AIVs in two geographically distinct regions of China where vaccination is routinely practiced. A total of 18 emerging H9N2 isolates were identified and genetically characterized. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes confirmed that the isolates belonged to the Y280 lineage. Based on the HA genes, the isolates were subdivided into two subgroups. The viruses from Zhejiang Province were clustered together in Group I, while the isolates from Guangdong Province were clustered together in Group II. Antigenic characterization showed that the tested viruses were antigenically different when compared to the current used vaccine strain. It was notable that 14 out of total 18 isolates had an amino acid exchange (Q→L) at position 216 (226 by H3 Numbering) in the receptor-binding site, which indicated that the virus had potential affinity of binding to human like receptor. These results suggest that the emerging viruses have potential risk to public health than previously thought. Therefore, continuous surveillance studies of H9N2 influenza virus are very important to the prognosis and control of future influenza pandemics.

Parsimonious model for blood glucose level monitoring in type 2 diabetes patients.

To establish the parsimonious model for blood glucose monitoring in patients with type 2 diabetes receiving oral hypoglycemic agent treatment. One hundred and fifty-nine adult Chinese type 2 diabetes patients were randomized to receive rapid-acting or sustained-release gliclazide therapy for 12 weeks. Their blood glucose levels were measured at 10 time points in a 24 h period before and after treatment, and the 24 h mean blood glucose levels were measured. Contribution of blood glucose levels to the mean blood glucose level and HbA1c was assessed by multiple regression analysis. The correlation coefficients of blood glucose level measured at 10 time points to the daily MBG were 0.58-0.74 and 0.59-0.79, respectively, before and after treatment (P<0.0001). The multiple stepwise regression analysis showed that the blood glucose levels measured at 6 of the 10 time points could explain 95% and 97% of the changes in MBG before and after treatment. The three blood glucose levels, which were measured at fasting, 2 h after breakfast and before dinner, of the 10 time points could explain 84% and 86% of the changes in MBG before and after treatment, but could only explain 36% and 26% of the changes in HbA1c before and after treatment, and they had a poorer correlation with the HbA1c than with the 24 h MBG. The blood glucose levels measured at fasting, 2 h after breakfast and before dinner truly reflected the change 24 h blood glucose level, suggesting that they are appropriate for the self-monitoring of blood glucose levels in diabetes patients receiving oral anti-diabetes therapy.

Antennal Transcriptome Evaluation and Analysis for Odorant-Binding Proteins, Chemosensory Proteins, and Suitable Reference Genes in the Leaf Beetle Pest Diorhabda rybakowi Weise (Coleoptera: Chrysomelidae).

Diorhabda rybakowi Weise is one of the dominant pests feeding on Nitraria spp., a pioneer plant used for windbreaking and sand fixation purposes, and poses a threat to local livestock and ecosystems. To clarify the key olfactory genes of D. rybakowi and provide a theoretical basis for attractant and repellent development, the optimal reference genes under two different conditions (tissue and sex) were identified, and the bioinformatics and characterization of the tissue expression profiles of two categories of soluble olfactory proteins (OBPs and CSPs) were investigated. The results showed that the best reference genes were RPL13a and RPS18 for comparison among tissues, and RPL19 and RPS18 for comparison between sexes. Strong expressions of DrybOBP3, DrybOBP6, DrybOBP7, DrybOBP10, DrybOBP11, DrybCSP2, and DrybCSP5 were found in antennae, the most important olfactory organ for D. rybakowi. These findings not only provide a basis for further in-depth research on the olfactory molecular mechanisms of host-specialized pests but also provide a theoretical basis for the future development of new chemical attractants or repellents using volatiles to control D. rybakowi.

[Role of MEK1/ERK1, 2 pathways in the calcium-sensing receptor mediation of hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells].

OBJECTIVE: To explore the cell signal transduction pathway of calcium-sensing receptor (CaSR) mediated hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs). METHODS: The expressions of proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated protein kinase 1, 2 (ERK1, 2) were analyzed by Western blot. Cell proliferation was tested by a 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Cell cycle and proliferation index (PI) were analyzed by flow cytometry. RESULTS: Hypoxia significantly increased the expression of PCNA (0.528 ± 0.028), p-ERK1, 2 (1.12 ± 0.05, 0.91 ± 0.06), BrdU incorporation (143.3 ± 4.2) and cell proliferation index (12.5 ± 0.9) (all P < 0.05, versus control group, 0.243 ± 0.025, 0.47 ± 0.03, 0.40 ± 0.03, 100.0 ± 5.4, 7.5 ± 1.2). Gadolinium chloride (GdCl3, a CaSR agonist) amplified the effect of hypoxia (0.770 ± 0.039, 1.50 ± 0.06, 1.61 ± 0.05, 187.4 ± 3.9, 19.8 ± 0.6, all P < 0.05). PD98059 (a MEK1 inhibitor) decreased the up-regulation of PCNA expression, BrdU incorporation and the increase of cell proliferation index induced by hypoxia and GdCl3 in PASMCs (0.441 ± 0.020, 0.71 ± 0.07, 0.72 ± 0.06, 115.5 ± 4.0, 9.3 ± 1.1, all P < 0.05). CONCLUSION: Calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through ERK1, 2 pathways.

Functional disparity of four pheromone-binding proteins from the plum fruit moth Grapholita funebrana Treitscheke in detection of sex pheromone components.

Grapholita funebrana, also known as the plum fruit moth, is an oligophagous pest species that causes enormous economic losses of the fruits of Rosaceae. An eco-friendly method for the control of G. funebrana besides chemical control has not yet been developed. The sex pheromone communication system plays an important role in moth courtship and mating, in which pheromone-binding proteins (PBPs) are critical. In this research, we identified four PBPs, namely, GfunPBP1.1, GfunPBP1.2, GfunPBP2, and GfunPBP3, from the antennae of G. funebrana. The results of real-time quantitative PCR (RT-qPCR) showed that all four GfunPBPs were overwhelmingly expressed in the antennae and that GfunPBP1.2 and GfunPBP2 showed male-biased expression patterns, whereas GfunPBP1.1 and GfunPBP3 were equally expressed between sexes. The results of ligand-binding assays illustrated that although all four recombinant GfunPBPs (rGfunPBPs) had binding activity with the tested sex pheromone compounds, their preferred ligands were significantly different. rGfunPBP2 had the strongest binding affinity to Z8-12:Ac and Z8-12:OH; rGfunPBP1.1 preferred to bind Z8-14:Ac, Z10-14:Ac, and 12:OH more than to the other three GfunPBPs; and rGfunPBP1.2 exhibited stronger binding affinity to E8-12:Ac than to the other rGfunPBPs. Molecular docking results demonstrated that hydrophobic forces, especially van der Waals forces and hydrogen bonds, were the most important forces that maintained GfunPBP-pheromone ligand complexes. This study will improve our understanding of the sex pheromone recognition mechanisms of G. funebrana and promote the development of novel strategies for controlling G. funebrana.

'Candidatus Phytoplasma ziziphi' Changes the Metabolite Composition of Jujube Tree Leaves and Affects the Feeding Behavior of Its Insect Vector Hishimonus hamatus Kuoh.

Hishimonus hamatus Kuoh is a leafhopper species native to China that feeds on Chinese jujube leaves. This leafhopper species has been verified to transmit jujube witches' broom (JWB) disease, caused by phytoplasma, a fatal plant pathogen, which belongs to the phytoplasma subgroup 16SrV-B. The transmission of JWB phytoplasma largely relies on the feeding behavior of piercing-sucking leafhoppers. However, the specific mechanisms behind how and why the infection of JWB influences the feeding behavior of these leafhoppers are not fully understood. To address this, a study was conducted to compare the feeding patterns of H. hamatus when feeding JWB-infested jujube leaves to healthy leaves using the electrical penetration graph (EPG) technique. Then, a widely targeted metabolome analysis was performed to identify differences in the metabolite composition of JWB-infected jujube leaves and that of healthy jujube leaves. The results of EPG analyses revealed that when feeding on JWB-infected jujube leaves, H. hamatus exhibited an increased frequency of phloem ingestion and spent longer in the phloem feeding phase compared to when feeding on healthy leaves. In addition, the results of metabolomic analyses showed that JWB-infected leaves accumulated higher levels of small-molecular carbohydrates, free amino acids, and free fatty acids, as well as lower levels of lignans, coumarins and triterpenoids compared to healthy leaves. The above results indicated that the H. hamatus preferentially fed on the phloem of infected leaves, which seems to be linked to the transmission of the JWB phytoplasma. The results of metabolomic analyses partially imply that the chemical compounds might play a role in making the infected leaves more attractive to H. hamatus for feeding.

Increased expression of calcium-sensing receptors in atherosclerosis confers hypersensitivity to acute myocardial infarction in rats.

Acute myocardial infarction (AMI) is a leading cause of death worldwide. Most cases of AMI result from coronary atherosclerosis (AS). The pathogenic mechanisms underlying AS lesions and AMI are incompletely understood. Calcium-sensing receptors (CaSR) belong to a family of G-protein-coupled receptors. We previously discovered that CaSR was expressed in the heart tissue of adult rats. CaSR may contribute to AMI in AS. We initially established a rat model of AS by injection of vitamin D(3) and feeding with a high-fat diet. Isoproterenol (ISO) was used to induce AMI. The MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH), cardiac troponin T (cTnT), tetrazolium chloride staining, and cardiac function parameters were selected as indicators of myocardial damage or necrosis. Cardiac apoptosis was analyzed by transferase dUTP nick-end labeling (TUNEL) assay. Expression of CaSR, Bcl-2, Bax, caspase-3, p-ERK1/2, p-JNK, and p-p38 were determined by Western blot analysis. Compared with the control group, levels of cTnT, CK-MB, and LDH; number of TUNEL-positive cells; and expression of CaSR, Bax, caspase-3, p-ERK1/2, p-JNK and p-p38, were significantly increased, whereas cardiac function and expression of Bcl-2 were decreased markedly in isoproterenol (ISO)-treated group (C/ISO) and AS groups. These changes were significant in the AS/ISO group than in the C/ISO group or AS group. The upregulation of CaSR during AS formation renders hypersensitivity to AMI. Activation of the pro-apoptotic mitochondria pathway and JNK-p38 MAPK pathway triggered by increased expression of CaSR may be one of molecular mechanisms underlying AMI in AS.

Involvement of calcium-sensing receptor in oxLDL-induced MMP-2 production in vascular smooth muscle cells via PI3K/Akt pathway.

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs) and up-regulated in atherosclerotic lesion by various stimuli, such as oxidized low-density lipoprotein (oxLDL). Calcium-sensing receptor (CaSR) is also expressed in VSMCs, but it remains unclear whether CaSR is associated with overproduction of MMP-2 in VSMCs. In this study, the expression of MMP-2 was detected by real-time PCR and Western blot analysis, and the gelatinolytic activity of MMP-2 was measured using gelatin zymography. Our results showed that oxLDL enhanced MMP-2 expression and activity in rat aortic VSMCs in a time- and dose-dependent manner. In addition, CaSR expression was up-regulated by oxLDL. Manipulating CaSR function in these cells by NPS2390 (an antagonist of CaSR) or GdCl(3) (an agonist of CaSR) affected the oxLDL-induced MMP-2 production. In VSMCs, oxLDL stimulated the rapid activation of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, as determined by Western blot analysis. Phosphorylation of Akt and MMP-2 production stimulated by oxLDL were attenuated by LY294002 (a specific inhibitor of PI3K). Activation of Akt was suppressed by NPS2390 but enhanced by GdCl(3). In contrast, oxLDL had no stimulatory effect on the phosphorylation of JNK, and pretreatment with SP600125 (an inhibitor of JNK) produced no significant effect on oxLDL-induced MMP-2 production. These results suggest that CaSR mediates oxLDL-induced MMP-2 production in VSMCs via PI3K/Akt signal pathway.

Glucotoxicity and α cell dysfunction: involvement of the PI3K/Akt pathway in glucose-induced insulin resistance in rat islets and clonal αTC1-6 cells.

AIM/HYPOTHESIS: The objective of this study was to assess how long-term exposure to high glucose affects the α cell function and whether the increased glucagon secretion is mediated via insulin resistance. MATERIALS AND METHODS: We established a ß cell-depleted rat model to obtain pure primary α cells. Furthermore, isolated rat islets and TC1-6 cells (a clonal α cell line) were exposed to high glucose (25 or 30 mmol/L) and low glucose (5.5 mmol/L) for up to 5 days to evaluate the influence of chronic glucose toxicity on glucagon secretion and glucagon gene expression. Moreover, we added insulin and/or Wortmannin to examine if the inhibitory effect of insulin on glucagon secretion was impaired by high glucose via the phosphatidylinositol 3 kinase/PKB protein kinase B pathway. RESULTS: Both glucagon secretion and glucagon gene expression were increased in response to 5 days exposure to high glucose. While a moderate insulin concentration slightly inhibits glucagon secretion from rat islets and α TC1-6 cells at high glucose, a pronounced increase in glucagon secretion was observed at low glucose. We found that the insulin-mediated activity of the phosphatidylinositol 3 kinase/PKB protein kinase B pathway in the α cell was markedly impaired by chronic exposure to high glucose. CONCLUSION: The hypersecretion of glucagon induced by glucotoxicity may be secondary to insulin resistance of the α cell induced by impaired activity of the insulin signaling pathway.

Different Binding Affinities of Three General Odorant-Binding Proteins in Grapholita funebrana (Treitscheke) (Lepidoptera: Tortricidae) to Sex Pheromones, Host Plant Volatiles, and Insecticides.

Insect general odorant-binding proteins (GOBPs) play irreplaceable roles in filtering, binding, and transporting host odorants to olfactory receptors. Grapholita funebrana (Treitscheke) (Lepidoptera: Tortricidae), an economically important pest of fruit crops, uses fruit volatiles as cues to locate host plants. However, the functions of GOBPs in G. funebrana are still unknown. Three GOBP genes, namely, GfunGOBP1, GfunGOBP2, and GfunGOBP3, were cloned, and their expression profiles in different tissues were detected by the method of real-time quantitative PCR (RT-qPCR). The binding properties of recombinant GfunGOBPs (rGfunGOBPs) to various ligands were investigated via fluorescence binding assays. The three GfunGOBPs were mainly expressed in the antennae of both male and female moths. All these three rGfunGOBPs could bind to sex pheromones, while having varying affinities toward these pheromones. The three rGfunGOBPs also displayed a wide range of ligand-binding spectrums with tested host odorants. The rGfunGOBP1, rGfunGOBP2, and rGfunGOBP3 bound to 34, 33, and 30 out of the 41 tested odorants, respectively. Three rGfunGOBPs had overlapping binding activities to ß-myrcene, (-)-α-phellandrene, and ethyl isovalerate with the Ki less than 3.0 µM. The rGfunGOBP1 and rGfunGOBP3 could selectively bind to several insecticides, whereas rGfunGOBP2 could not. Three rGfunGOBPs had the dual functions of selectively binding to sex pheromones and host odorants. Moreover, the rGfunGOBP1 and rGfunGOBP3 can also serve as 'signal proteins' and bind to different insecticides. This study contributed to elucidating the potential molecular mechanism of the olfaction for G. funebrana, and thereby promotes the development of effective botanical attractants or pheromone synergists to control G. funebrana.

The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells.

BACKGROUND: The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown. METHODS: The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca2+]i) was detected by a laser-scanning confocal microscope. RESULTS: The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca2+]o (extracellular calcium concentration) or Gd3+ (an agonist of CaSR) induced an increase of [Ca2+]i and PAs constriction in a concentration-dependent manner. In addition, the above-mentioned effects of Ca2+ and Gd3+ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP3 receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase). CONCLUSIONS: CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca2+]i through G-PLC-IP3 pathway.

Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes.

BACKGROUND: Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D2 receptors are expressed in cardiac tissues. However, the roles of dopamine D2 receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D2 receptors agonist (bromocriptine) and antagonist (haloperidol) on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury. METHODS: Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic) buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium. RESULTS: Treatment of the cardiomyocytes with 10 µM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca2+]i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 µM) had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions. CONCLUSIONS: These data suggest that activation of dopamine D2 receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.