Effect of RMT1-10 on the immunological characteristics of dendritic cells cultured in vitro and corneal transplantation in vivo.

OBJECTIVE: Corneal allograft rejection is an immunological hypersensitive reaction caused by the antigenicity of the donor cornea. This study aimed to explore the effects of RMT1-10 on the prevention of corneal graft rejection by modifying immunological characteristics of dendritic cells (DCs). MATERIALS AND METHODS: DCs and CD4+T cells were sorted using flow cytometry and used for in vitro mixed lymphocyte culture. The cultured cells were prepared for the characterization of the DC cell phenotypes using the markers CD11c, CD80, MHC II, CD54, and TIM-4. Cytokine concentrations of IL-4, IL-12, and IL-10 of supernatants were measured by the enzyme-linked immunosorbent assay. CD4+T cells were examined by flow cytometry for apoptosis and proliferation. We also investigated the effect of RMT1-10 in the prevention and treatment of high-risk corneal graft rejection using a mouse model of corneal transplantation. RESULTS: DCs were identified as the CD11c+MHC-II-expressing subset. RMT1-10 suppressed the expression of CD11c, CD80, MHC II, CD54, and TIM-4 of DCs using the blockade of TIM-1 signaling. Moreover, TIM-1 blockade inhibited the production of IL-12 and IL-10 in a mixed lymphocyte culture system. However, a TIM-1 blockade had no effect on the apoptosis of CD4+T cells. RMT1-10 suppressed DC maturation, inhibiting the proliferation of CD4+T cells. CONCLUSIONS: RMT1-10 significantly improved the survival rate of the corneal allografts in mice compared with saline-injected controls. This clinical improvement from RMT1-10 occurred through the inhibition of CD4+T cell proliferation. Moreover, RMT1-10 induced antigen-specific detection of receptor immune tolerance. The cross-linking of TIM-1 on CD4+T cells with the agonist mAb provided a costimulatory inhibition signal for T cell activation or proliferation.

Protective Effect of Edaravone Against Aß25-35-Induced Mitochondrial Oxidative Damage in SH-SY5Y Cells.

Amyloid-ß (Aß)-induced oxidative stress plays an important role in the pathogenesis of Alzheimer's disease (AD). Recent studies show that Aß accumulation may lead to mitochondrial oxidative damage. In the present study, we investigated the protective effect of edaravone on mitochondrial damage in SH-SY5Y cells treated with Aß25-35. SH-SY5Y cells were pre-treated with 20, 40 or 80 µM edaravone before treatment with 25 µM Aß25-35. After 24h cell culture, cellular apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), ATP levels and mitochondrial morphology were evaluated. SH-SY5Y cells exposed to Aß25-35 had high levels of apoptosis and ROS; loss of ΔΨm, decreased ATP levels and presence of mitochondrial swelling. However, these effects were significantly inhibited by edaravone pre-treatment. These results indicate that edaravone prevents mitochondria oxidative damage caused by Aß in SH-SY5Y cells, which suggests that it may have potential clinical application in AD therapy.

Functional analysis of female-biased odorant binding protein 6 for volatile and nonvolatile host compounds in Adelphocoris lineolatus (Goeze).

The polyphagous mirid bug Adelphocoris lineolatus relies heavily on olfactory cues to track suitable host plants. Thus, a better understanding of the molecular basis of its olfactory detection could contribute to the development of effective pest management strategies. In the present study, we report the expression profile of the odorant binding protein gene A. lineolatus odorant binding protein 6 (AlinOBP6). Quantitative real-time PCR experiments suggest that AlinOBP6 is female adult antennae-biased. Cellular immunolocalization analyses show that AlinOBP6 is highly expressed in the lymph of both multiporous sensilla basiconica and uniporous sensilla chaetica. A ligand binding analysis showed that recombinant AlinOBP6 not only bound tightly to host plant volatile compounds but also to nonvolatile compounds. Homology modelling and molecular docking analyses confirmed these unusual ligand binding profiles and revealed that the amino acid residues involved in the recognition of volatile and nonvolatile compounds are distinct. The results of our study are the first to suggest that an antenna- and female-biased OBP in an hemipteran insect is expressed in both olfactory and gustatory sensilla as a mechanism to respond to volatile and nonvolatile host compounds. These findings warrant further research into the molecular mechanisms of chemosensation for mirid bugs in responsive to host plant location.

Effects of tumor necrosis factor and E-selectin on coronary artery flow.

OBJECTIVE: The aim of the study is to investigate the correlation between tumor necrosis factor (TNF-α), E-selectin and coronary artery flow following myocardial ischemia-reperfusion model (IR) in Yorkshire pigs. MATERIALS AND METHODS: Establishment of IR model in pigs. Following the injury model, Experiment group was administrated intravenously Shenfu injection solution (SFI, 1 mL/kg). The control group received the same amount of saline. After 30 min of blood reflux, thrombolysis in myocardial infarction frame count (TFC) was recorded following surgery. TNF-α, E-selectin expression was determined by ELISA in the venous sheath, coronary sinus, artery sinus, and proximal segment of the coronary artery. RESULTS: After the blood reflowing, TFC in both groups were upregulated, and TFC increased more than the control group. The difference is statistically significant (p<0.05) at the time of 30 min. TNF-α, E-selectin expression increased after IR. After reperfusion, TNF-α, E-selectin levels further increased and the myocardial injury was aggravated. SFI inhibited inflammation in the experimental group. TNF-α, E-selectin levels at coronary sinus, artery sinus, and distal segment of coronary artery after surgery was positively correlated with TIMI in the experimental group (p<0.05). TNF-α, E-selectin levels significantly increased after reperfusion (p<0.05). CONCLUSIONS: The result demonstrated that TNF-α, E-selectin levels were positively correlated with coronary artery reflow only in the experimental group but not in the control group.

Correlation study between interleukin-6 levels and coronary reflow.

OBJECTIVE: To analyze the correlation between local interleukin-6 (IL-6) levels in different parts of blood vessel and the record of Thrombolysis in Myocardial Infarction (TIMI) frame count (TFC) after myocardial ischemia-reperfusion (IR) model. MATERIALS AND METHODS: Establishment of IR model in Yorkshire pigs, the pigs were divided into two groups (n=6). Experiment group pigs were administrated with Shenfu injection (SF) intravenously (1 mL/kg), control group was given saline injection. The blood reflowed after 30 min. TIMI was recorded to evaluate the coronary blood flow and myocardial perfusion. IL-6 levels in venous sheath, coronary sinus, artery sinus, and proximal coronary artery were determined by ELISA. RESULTS: The records of TIMI in experimental group were higher than that in control group. The difference was statistically significant (p < 0.05). The level of IL-6 increased obviously compared with control group after reperfusion (p < 0.05). Shenfu injection reduced the level of IL-6. IL-6 level at the coronary sinus was positively correlated with TIMI in experimental group (p = 0.03, R2 = 0.97) but not in control group. CONCLUSIONS: IL-6 levels were significantly increased after reperfusion, which aggravated myocardial injury. IL-6 may be associated with coronary reflow, but further study is needed.

Gene structure and expression characteristic of a novel odorant receptor gene cluster in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae).

Odorant receptors (ORs) expressed in the antennae of parasitoid wasps are responsible for detection of various lipophilic airborne molecules. In the present study, 107 novel OR genes were identified from Microplitis mediator antennal transcriptome data. Phylogenetic analysis of the set of OR genes from M. mediator and Microplitis demolitor revealed that M. mediator OR (MmedOR) genes can be classified into different subfamilies, and the majority of MmedORs in each subfamily shared high sequence identities and clear orthologous relationships to M. demolitor ORs. Within a subfamily, six MmedOR genes, MmedOR98, 124, 125, 126, 131 and 155, shared a similar gene structure and were tightly linked in the genome. To evaluate whether the clustered MmedOR genes share common regulatory features, the transcription profile and expression characteristics of the six closely related OR genes were investigated in M. mediator. Rapid amplification of cDNA ends-PCR experiments revealed that the OR genes within the cluster were transcribed as single mRNAs, and a bicistronic mRNA for two adjacent genes (MmedOR124 and MmedOR98) was also detected in female antennae by reverse transcription PCR. In situ hybridization experiments indicated that each OR gene within the cluster was expressed in a different number of cells. Moreover, there was no co-expression of the two highly related OR genes, MmedOR124 and MmedOR98, which appeared to be individually expressed in a distinct population of neurons. Overall, there were distinct expression profiles of closely related MmedOR genes from the same cluster in M. mediator. These data provide a basic understanding of the olfactory coding in parasitoid wasps.

Identification and characterization of the distinct expression profiles of candidate chemosensory membrane proteins in the antennal transcriptome of Adelphocoris lineolatus (Goeze).

Chemosensory membrane proteins, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs) and sensory neurone membrane proteins (SNMPs), are supposed to be crucial macromolecules in the insect olfactory signal transduction pathway. The alfalfa plant bug Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) is highly attracted to high-nitrogen or flowering plants and destroys many important agricultural crops. We assembled the antennal transcriptome of A. lineolatus using Illumina sequencing technology and identified a total of 108 transcripts encoding chemosensory membrane proteins (88 ORs, 12 IRs, four GRs and four SNMPs), amongst which 90 candidates appeared to be full length. Subsequently, both semiquantitative reverse transcription PCR and quantitative real-time PCR experiments were performed to investigate their tissue- and sex-biased expression profiles. The results showed that nearly all of the 108 candidate chemosensory membrane protein genes were largely expressed in adult antennae, and some genes additionally displayed significant differences in the expression levels between sexes. The results of our phylogenetic analysis and the detailed tissue- and sex-biased expression characteristics given here provide an important foundation for further understanding of the complex chemoreception system of the alfalfa plant bug and other Hemiptera species, which also could help us use chemosensory membrane proteins as targets to manipulate insect olfactory behaviour and broaden the applications of available tools for insect pest control.

The antenna-specific odorant-binding protein AlinOBP13 of the alfalfa plant bug Adelphocoris lineolatus is expressed specifically in basiconic sensilla and has high binding affinity to terpenoids.

Odorant-binding proteins (OBPs) are crucial in the olfactory pathway of insects. In the present study, the antenna-enriched OBP AlinOBP13 was investigated because of its potential contribution to the peripheral olfactory perception in the alfalfa plant bug Adelphocoris lineolatus. The results of quantitative reverse transcriptase-PCR showed that the transcript level of AlinOBP13 was higher in the adult stage than in the nymph stages. The transcript levels of AlinOBP13 in the male and female antennae significantly increased after 4 and 8 h of starvation, respectively. Fine ultrastructures of different types of chemosensilla in both female and male antennae were investigated using transmission electron microscopy and immunocytochemical labelling. The results revealed that the anti-AlinOBP13 antiserum strongly and specifically labelled short basiconic sensilla; this antiserum was restricted to the inner lumen and the cavities below the sensillum base of the sensilla. By contrast, multiporous sensilla trichodea, medium long sensilla basiconica, and aporous sensilla chaetica were not labelled. The present study is the first to report an OBP showing specific expression in the short basiconic sensilla of a member of the Hemipteran species. The results of a fluorescence displacement binding assay indicated that recombinant AlinOBP13 showed a more specific binding preference to terpenoids than to sex pheromones and other classes of chemicals. This binding ability was dramatically affected by pH; higher binding affinities were displayed at pH 10.0 than at pH 7.4 and 5.0. In addition, the results of dose-dependent electroantennogram recordings from the antennae showed that both female and male adult bugs responded to the terpenoids tested, suggesting an apparent physiological relevance of AlinOBP13 in A. lineolatus chemoreception. The results of this study suggest that AlinOBP13 functions as a specific carrier of terpenoids and provide insights into the mechanism of A. lineolatus in response to green volatiles.

Phosphatidylinositol 3 kinase/protein kinase B is responsible for the protection of paeoniflorin upon H2O2-induced neural progenitor cell injury.

Promoting neural stem/progenitor cell (NSC/NPC) survival in the pro-apoptotic environment is critical to stem cell replacement for neurodegenerative disease therapy. Paeoniflorin (PF), one of the principal bioactive components in Paeoniae Radix, has been used widely in central nervous system (CNS) diseases treatment and serves as an antioxidant to protect neurons against oxidative stress. The present study investigated the protective effects of PF on NPC injury induced by hydrogen peroxide (H2O2). After challenge with 200 µM H2O2 for 2h, loss of cell viability and excessive apoptotic cell death were observed in cultured NPC, PF treatment conferred protective effects against the loss of cellular viability in a concentration-dependent manner. PF pretreatment also inhibited NPC apoptosis induced by H2O2 by reversing the decreased level of Procaspase-3 and balancing Bcl-2 and Bax expression. Furthermore, PF-mediated NPC protection was associated with an increase in phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt-1) phosphorylation in a time- and concentration-dependent manner. Selective inhibition of PI3K using LY294002 abolished PF-mediated phosphorylation of Akt-1 and NPC protection upon oxidative stress. These data suggest that PF-mediated NPC protection on H2O2 injury is reliant on the activation of the PI3K/Akt-1 pathway, giving insight to an essential role of PF in NPC protection.

Specific involvement of two amino acid residues in cis-nerolidol binding to odorant-binding protein 5 AlinOBP5 in the alfalfa plant bug, Adelphocoris lineolatus (Goeze).

Olfaction plays an important role in insects' survival and reproduction. Odorant-binding proteins (OBPs) are considered to be one of the crucial proteins in the insect olfactory pathway. In this study, an antenna-specific OBP of the alfalfa plant bug, Adelphocoris lineolatus AlinOBP5, was expressed and purified in vitro. The binding affinities of AlinOBP5 with sex pheromone analogues of the Miridae and cotton volatiles were investigated by fluorescence competitive binding assays. The binding sites of AlinOBP5 were predicted by three-dimensional structure modelling and molecular docking, and site-directed mutagenesis. AlinOBP5 could not effectively bind with sex pheromone analogues of Miridae but showed high binding abilities with specific cotton volatiles, such as cis-nerolidol, ethyl laurate, ß-ionone, ß-caryophyllene, 2,3-dimethylbenzoic acid and (E)-farnesol. The strongest binding affinity was to cis-nerolidol, suggesting a role of AlinOBP5 in general odorant chemoreception. Based on the relatively strong binding affinity and the reported physiological activity of cis-nerolidol in other insects, we chose cis-nerolidol for further homology modelling and ligand docking. The results of molecular simulation and site-directed mutagenesis indicated that two amino acids, Lys74 and Pro121, in the protein binding pocket are the key amino acids involved in the binding of cis-nerolidol. The Lys74 residue may participate in specific recognition of ligands, and the Pro121 residue plays a crucial role in ligand binding and release by changing the binding pocket environment and stabilizing the conformation of the C-terminus of AlinOBP5.

Return migration of Helicoverpa armigera (Lepidoptera: Noctuidae) during autumn in northern China.

The autumn migration of Helicoverpa armigera (Hübner) was observed with radar and two types of light-trap at Langfang, Hebei province, China in 2001 and 2002. The sudden increase in the proportion of H. armigera moths in the searchlight trap indicated migration into the area and catches increased 10-fold during the second half of the night due to the landing of migrants before dawn. The moths' migratory flights took place at up to 2000 m above the ground, and moths flew differentially at times, and heights, when favourable (i.e. northerly) winds occurred. This facilitated the maximum displacement of moths towards the south during these 'return' migrations. The moths flew over the radar site at consistently high densities through the night, and the resulting flight durations of c. 10 h, at displacement speeds of 30-33 km h-1, would allow moths emerging in the far northeast of China (i.e. Liaoning and Jilin provinces and the Inner Mongolia autonomous region) to migrate into northern China (Hebei, Shandong and Henan provinces). The association of the seasonal migratory movements of H. armigera with crops in northern China is briefly discussed.

Radar observations of the autumn migration of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) and other moths in northern China.

The autumn return migration of the beet armyworm Spodoptera exigua Hübner and other insects in northern China was observed with radar and with a simultaneously-operated searchlight trap and ground light-trap at a site in Langfang, near Beijing. The searchlight trap was found to be an efficient tool for trapping migrants and, operated alongside a ground light-trap, could distinguish migrant from locally-flying species. It was confirmed that S. exigua and some other species were high-altitude nocturnal windborne migrants during September and October in northern China. Maximum density of moths typically occurred below 500 m, and strong layering was often observed at about 200 m above ground level in airflows that would carry the moths towards the south. Descent of S. exigua in the vicinity of the radar site in late September was often associated with rain.

Cloning, expression and immunocytochemical localization of a general odorant-binding protein gene from Helicoverpa armigera (Hübner).

A cDNA clone coding for general odorant-binding protein2 was isolated from the antenna of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. Results of sequencing and structural analyses showed that the full-length of GOBP2Harm was 636 bp, possessing 162 amino acid residues and a signal peptide of 21 amino acids. Its predicted molecular weight and isoelectric point were 18.2 kDa and 5.21, respectively. This deduced amino acid sequence shared some common structural features with odorant-binding proteins from several moth species, including the six conserved cysteine motif, typical of insect's OBPs. Northern blot showed that GOBP2Harm is specifically expressed in the antenna of Helicoverpa armigera at similar levels in both sexes. In order to obtain sufficient GOBP2 for further determining its biochemical and physiological properties, a bacterical expression vector of GOBP2 was constructed and successfully expressed. The protein was obtained mainly as insoluble inclusion bodies, that, however, could be solubilized and refolded. The rGOBP2 was purified by affinity chromatography and gel filtration. The rGOBP2 was shown to cross-react with an anti-GOBP antiserum from Antheraea polyphemus. Finally, polyclonal antibodies against GOBP2Harm were used to mark the distribution of the protein in olfactory sensilla and were tested by immuno-electron microscopy. In the male, GOBP2Harm is mainly expressed in sensilla basiconica, while in the female, it is equally expressed in sensilla basiconica and in sensilla trichodea.