Volatile Dimethyl Disulfide from Guava Plants Regulate Developmental Performance of Asian Citrus Psyllid through Activation of Defense Responses in Neighboring Orange Plants.

Intercropping with guava (Psidium guajava L.) can assist with the management of Asian citrus psyllid (ACP, Diaphorina citri Kuwayama), the insect vector of the huanglongbing pathogen, in citrus orchards. Sulfur volatiles have a repellent activity and physiological effects, as well as being important components of guava volatiles. In this study, we tested whether the sulfur volatiles emitted by guava plants play a role in plant-plant communications and trigger anti-herbivore activities against ACP in sweet orange plants (Citrus sinensis L. Osbeck). Real-time determination using a proton-transfer-reaction mass spectrometer (PTR-MS) showed that guava plants continuously release methanethiol, dimethyl sulfide (DMS), and dimethyl disulfide (DMDS), and the contents increased rapidly after mechanical damage. The exposure of orange plants to DMDS resulted in the suppression of the developmental performance of ACP. The differential elevation of salicylic acid (SA) levels; the expression of phenylalanine ammonia lyase (PAL), salicylate-O-methyl transferase (SMT), and pathogenesis-related (PR1) genes; the activities of defense-related enzymes PAL, polyphenol oxidase (PPO), and peroxidase (POD); and the total polyphenol content were observed in DMDS-exposed orange plants. The emission of volatiles including myrcene, nonanal, decanal, and methyl salicylate (MeSA) was increased. In addition, phenylpropanoid and flavonoid biosynthesis, and aromatic amino acid (such as phenylalanine, tyrosine, and tryptophan) metabolic pathways were induced. Altogether, our results indicated that DMDS from guava plants can activate defense responses in eavesdropping orange plants and boost their herbivore resistance to ACP, which suggests the possibility of using DMDS as a novel approach for the management of ACP in citrus orchards.

Burn-Induced Impairment of Ileal Muscle Contractility Is Associated with Increased Extracellular Matrix Components.

INTRODUCTION: Severe burns lead to marked impairment of gastrointestinal motility, such as delayed gastric emptying and small and large intestinal ileus. However, the cellular mechanism of these pathologic changes remains largely unknown. METHODS: Male Sprague Dawley rats approximately 3 months old and weighing 300-350 g were randomized to either a 60% total body surface area full-thickness scald burn or sham procedure and were sacrificed 24 h after the procedure. Gastric emptying, gastric antrum contractility ileal smooth muscle contractility, and colonic contractility were measured. Muscularis externa was isolated from the ileal segment to prepare smooth muscle protein extracts for Western blot analysis. RESULTS: Compared with sham controls, the baseline rhythmic contractile activities of the antral, ileal, and colonic smooth muscle strips were impaired in the burned rats. Simultaneously, our data showed that ileal muscularis ECM proteins fibronectin and laminin were significantly up-regulated in burned rats compared with sham rats. TGF-ß signaling is an important stimulating factor for ECM protein expression. Our results revealed that TGF-ß signaling was activated in the ileal muscle of burned rats evidenced by the activation of Smad2/3 expression and phosphorylation. In addition, the total and phosphorylated AKT, which is an important downstream factor of ECM signaling in smooth muscle cells, was also up-regulated in burned rats' ileal muscle. Notably, these changes were not seen in the colonic or gastric tissues. CONCLUSION: Deposition of fibrosis-related proteins after severe burn is contributors to decreased small intestinal motility.

Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat.

Electroacupuncture has been shown to effectively reduce chronic pain in patients with nerve injury. The underlying mechanisms are not well understood. Accumulated evidence suggests that purinergic P2X3 receptors (P2X3Rs) in dorsal root ganglion neurons play a major role in mediating chronic pain associated with nerve injury. The aim of this study is to determine if electroacupuncture stimulation alters P2X3R activity in dorsal root ganglia to produce analgesia under neuropathic pain condition. Peripheral neuropathy was produced by ligation of the left lumbar 5 (L5) spinal nerve in rats. Low-frequency (2 Hz) electrical stimulation was applied to ipsilateral ST36 and BL60 acupoints in rats. The P2X3R agonist (α,ß-meATP)-induced flinch responses were reduced after electroacupuncture treatment. Western analyses showed that P2X3R expression was upregulated in nerve-uninjured lumbar 4 (L4) dorsal root ganglion neurons ipsilateral to the spinal nerve ligation. Electroacupuncture-stimulation reversed the upregulation. In nerve-injured L5 dorsal root ganglia, P2X3R expression was substantially reduced. Electroacupuncture had no effect on the reduction. We also determined the injury state of P2X3R expressing dorsal root ganglion neurons using the neuronal injury marker, activating transcription factor 3 (ATF3). Immunohistochemical assay showed that in L4 dorsal root ganglia, almost all P2X3Rs were expressed in uninjured (ATF3-) neurons. Spinal nerve ligation increased the expression of P2X3Rs. Electroacupuncture reduced the increase in P2X3R expression without affecting the percentage of ATF + neurons. In ipsilateral L5 dorsal root ganglion neurons, spinal nerve ligation reduced the percentage of P2X3R + neurons and markedly increased the percentage of ATF3 + cells. Almost all of P2X3Rs were expressed in damaged (ATF3+) neurons. Electroacupuncture had no effect on spinal nerve ligation-induced changes in the percentage of P2X3R or percentage of ATF3 + cells in L5 dorsal root ganglia. These observations led us to conclude that electroacupuncture effectively reduces injury-induced chronic pain by selectively reducing the expression of P2X3Rs in nerve-uninjured L4 dorsal root ganglion neurons.

Toxicity and biochemical effects of itol A on the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

Itol A, a novel isoryanodane diterpene derived from Itoa orientalis Hemsl., has potent activities against insect pests. This study was conducted to determine the contact toxicity and biochemical effects of itol A on the Nilaparvata lugens. After macropterous females of N. lugens were exposed to itol A from 0.5 to 24 h, the mortality and poisoning symptoms were measured. Effects of itol A on the major enzymes activity and oxidative stress level were assessed in dose-response (with LD10-LD70 at 24 h) and time-course (with LD50 at 0.5-24 h) experiments for the potential toxicity mechanisms. Based on the results, the mortality of N. lugens showed significant dose- and time-dependent effects, with the 24-h LD50 value was 0.58 µg/insect. The symptoms of excitation, convulsion and paralysis were also observed. However, acetylcholinesterases (AChE) activity was not altered after itol A treatment compared to control. Na+/K+-ATPases, Ca2+-ATPases, Ca2+/Mg2+-ATPases, glutathione S-transferases (GSTs), cytochrome P450 monooxygenases (P450s), superoxide dismutases (SOD) and catalases (CAT) activities were significantly reduced in dose-response and time-course experiments. While acid phosphatases (ACP) and glutathione peroxidases (GPX) activities were significantly increased. We further revealed that itol A exposure resulted in the decrease of GSH/GSSG (reduced to oxidized glutathione) ratio and the increase of hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels in both experiments. The results indicated that the inhibition of Na+/K+-ATPases, Ca2+-ATPases, Ca2+/Mg2+-ATPases, GSTs, P450s, SOD and CAT activities and the induction of oxidative stress was one of the potential biochemical mechanisms of itol A against N. lugens.

Inflammation induces Epac-protein kinase C alpha and epsilon signaling in TRPV1-mediated hyperalgesia.

The exchange proteins activated by cAMP (Epacs) have been shown to play important roles in producing inflammation-induced nociception. Transient receptor potential vanilloid type 1 (TRPV1) is a major receptor processing thermal and chemosensitive nociceptive information. The role of Epacs in modulating the activity of TRPV1 has yet to be determined. Studying the effect of complete Freund adjuvant (CFA)-induced inflammation on capsaicin-activated TRPV1 nociceptive responses in dorsal root ganglia (DRG), we found that CFA produced a large increase in capsaicin-induced responses. The increase was inhibited by Epac1 and Epac2 antagonists. Thus, activation of Epacs is critical in producing enhancement in TRPV1-mediated responses under inflammatory conditions. In addition, the inflammation-induced enhancement of TRPV1 responses was blocked by PKCα and PKCε inhibitors, suggesting the essential roles of these PKCs in enhancing TRPV1 responses. To determine the mechanism underlying the Epac actions on TRPV1, we studied the effects of the Epac activator, 8-(4-chlorophenylthio)-2-O-methyl-cAMP (CPT), on capsaicin-induced nociceptive behavioral responses, capsaicin-activated currents, expression and membrane trafficking of PKC and TRPV1 in DRG. CPT was found to enhance capsaicin-induced nociception and ionic currents. The enhancement was inhibited by PKCα and PKCε inhibitors. In addition, CPT increased the expression of phosphorylated PKCα (pPKCα) and membrane TRPV1 expression in DRG. Studying the colocalization of TRPV1 and pPKCα or pPKCε in DRG slices prepared from CFA-treated rats, we found that pPKCα or pPKCε expressed with TRPV1 in different-sized neurons to exert differential influences on TRPV1 activity. Thus, Epac-PKC signaling is critically important in producing inflammation-induced potentiation of TRPV1 functions.

Epac and Nociceptor Sensitization.

Abstract: Primary sensory neurons are responsible for transmitting sensory information from the peripheral to the central nervous system. Their responses to incoming stimulation become greatly enhanced and prolonged following inflammation, giving rise to exaggerated nociceptive responses and chronic pain. The inflammatory mediator, prostaglandin E2 (PGE2), released from the inflamed tissue surrounding the terminals of sensory neurons contributes to the abnormal pain responses. PGE2 acts on G protein-coupled EP receptors to activate adenylyl cyclase, which catalyzes the conversion of adenosine triphosphate to cyclic adenosine 3',5'-monophosphate (cAMP). Under normal conditions, cAMP activates primarily protein kinase A. After inflammation, cAMP also activates the exchange proteins activated by cAMP (Epacs) to produce exaggerated PGE2-mediated hyperalgesia. The role of cAMP-Epac signaling in the generation of hypersensitivity is the topic of this review.

EXPRESS: F-actin links Epac-PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation.

Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund's adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund's adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.

Epac-protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation.

Sensitization of purinergic P2X3 receptors (P2X3Rs) is a major mechanism contributing to injury-induced exaggerated pain responses. We showed in a previous study that cyclic adenosine monophosphate (cAMP)-dependent guanine nucleotide exchange factor 1 (Epac1) in rat sensory dorsal root ganglia (DRGs) is upregulated after inflammatory injury, and it plays a critical role in P2X3R sensitization by activating protein kinase C epsilon (PKCε) inside the cells. protein kinase C epsilon has been established as the major PKC isoform mediating injury-induced hyperalgesic responses. On the other hand, the role of PKCα in receptor sensitization was seldom considered. Here, we studied the participation of PKCα in Epac signaling in P2X3R-mediated hyperalgesia. The expression of both Epac1 and Epac2 and the level of cAMP in DRGs are greatly enhanced after complete Freund adjuvant (CFA)-induced inflammation. The expression of phosphorylated PKCα is also upregulated. Complete Freund adjuvant (CFA)-induced P2X3R-mediated hyperalgesia is not only blocked by Epac antagonists but also by the classical PKC isoform inhibitors, Go6976, and PKCα-siRNA. These CFA effects are mimicked by the application of the Epac agonist, 8-(4-chlorophenylthio)-2 -O-methyl-cAMP (CPT), in control rats, further confirming the involvement of Epacs. Because the application of Go6976 prior to CPT still reduces CPT-induced hyperalgesia, PKCα is downstream of Epacs to mediate the enhancement of P2X3R responses in DRGs. The pattern of translocation of PKCα inside DRG neurons in response to CPT or CFA stimulation is distinct from that of PKCε. Thus, in contrast to prevalent view, PKCα also plays an essential role in producing complex inflammation-induced receptor-mediated hyperalgesia.

Identification of dysregulated pathways associated with pancreatic cancer by survival analysis.

In order to identify the dysregulated pathways associated with pancreatic cancer, the fourth leading cause of cancer mortality in the United States, tumor and non-tumor samples were systematically analyzed in the present study. Initially, dysregulated genes in pancreatic cancer were identified using paired t-test. Subsequently, dysregulated biological pathways involved in the development of pancreatic cancer were identified by enrichment analysis. Finally, individual survival analysis of the significantly dysregulated functions was conducted at the pathway level. Our results indicated that the pathway named ̔Pathways in cancer was significantly correlated with survival time. In addition, the mean survival time of individual and genetic variation demonstrated a significantly negative correlation, that is, the lower the genetic variation, the longer the survival time. Furthermore, detailed analysis of genes on the pathway named ̔Pathways in cancer denoted that this pathway involved multiple cancer hallmark signals and several dysregulated cancer genes, including tumor protein p53, myelocytomatosis, Kirsten rat sarcoma, phosphatidylinositol 3-kinase, v-raf murine sarcoma viral oncogene homolog B1 and cyclin-dependent kinase inhibitor 2A. According to the DrugBank database, certain oncogenes have been validated to be the targets of drugs, including Sorafenib, Trastuzumab, Imatinib and Paclitaxel or were under investigation. An improved understanding of the pathophysiology of pancreatic cancer has been achieved based on our results and the present study aimed to provide guidance for the development of drugs to treat pancreatic cancer.

Evaluation of in vivo antioxidant and immunity enhancing activities of sodium aescinate injection liquid.

Oxidative stress is involved in the development and progression of disease. Because sodium aescinate has been reported to have immunity enhancing and antioxidative effects, we investigated its activity by employing a hepatocellular carcinoma (HCC) mouse model. Sixty BALB/c mice were randomly divided into four groups, including a 1.4 mg/kg treated group (n = 15), a 2.8 mg/kg treated group (n = 15), an untreated hepatocellular carcinoma control group (n = 15) and a normal control group (n = 15). After H22 cells were cultured for one week, we collected 2 × 106 cells and injected them subcutaneously as 0.2 mL cell suspensions in sterile saline into the right shoulder region of every mouse. The animals were monitored for changes in activity, physical condition and body weight during the experiment. The next day after injection of H22 cells, animals in these test groups received one intraperitoneal injection of drug or physiological saline for 13 days. Results showed that in the sodium aescinate injection liquid (SAIL)-treated HCC mice, serum interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), Gamma-glutamyltransferase (γ-GT), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) levels were significantly decreased compared with normal control mice. In addition, treatment with sodium aescinate injection liquid significantly decreased blood and liver malondialdehyde (MDA) levels, increased glutathione (GSH) levels, and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)] activities in a dose-dependent manner. We conclude that sodium aescinate injection liquid can decrease oxidative injury and enhance immunity functions in HCC mice.

Neuronal soma-satellite glial cell interactions in sensory ganglia and the participation of purinergic receptors.

It has been known for some time that the somata of neurons in sensory ganglia respond to electrical or chemical stimulation and release transmitters in a Ca2+-dependent manner. The function of the somatic release has not been well delineated. A unique characteristic of the ganglia is that each neuronal soma is tightly enwrapped by satellite glial cells (SGCs). The somatic membrane of a sensory neuron rarely makes synaptic contact with another neuron. As a result, the influence of somatic release on the activity of adjacent neurons is likely to be indirect and/or slow. Recent studies of neuron-SGC interactions have demonstrated that ATP released from the somata of dorsal root ganglion neurons activates SGCs. They in turn exert complex excitatory and inhibitory modulation of neuronal activity. Thus, SGCs are actively involved in the processing of afferent information. In this review, we summarize our understanding of bidirectional communication between neuronal somata and SGCs in sensory ganglia and its possible role in afferent signaling under normal and injurious conditions. The participation of purinergic receptors is emphasized because of their dominant roles in the communication.

A critical role of the cAMP sensor Epac in switching protein kinase signalling in prostaglandin E2-induced potentiation of P2X3 receptor currents in inflamed rats.

Sensitization of purinergic P2X receptors is one of the mechanisms responsible for exaggerated pain responses to inflammatory injuries. Prostaglandin E2 (PGE2), produced by inflamed tissues, is known to contribute to abnormal pain states. In a previous study, we showed that PGE2 increases fast inactivating ATP currents that are mediated by homomeric P2X3 receptors in dorsal root ganglion (DRG) neurons isolated from normal rats. Protein kinase A (PKA) is the signalling pathway used by PGE2. Little is known about the action of PGE2 on ATP currents after inflammation, although the information is crucial for understanding the mechanisms underlying inflammation-induced sensitization of P2X receptors. We therefore studied the effects of PGE2 on P2X3 receptor-mediated ATP currents in DRG neurons dissociated from complete Freund's adjuvant (CFA)-induced inflamed rats. We found that PGE2 produces a large increase in ATP currents. PKCepsilon, in addition to PKA, becomes involved in the modulatory action of PGE2. Thus, PGE2 signalling switches from a solely PKA-dependent pathway under normal conditions to both PKA- and PKC-dependent pathways after inflammation. Studying the mechanisms underlying the switch, we demonstrated that cAMP-responsive guanine nucleotide exchange factor 1 (Epac1) is up-regulated after inflammation. The Epac agonist CPT-OMe mimics the potentiating effect of PGE2 and occludes the PKC-mediated PGE2 action on ATP currents. These results suggest that Epac plays a critical role in P2X3 sensitization by activation of de novo PKC-dependent signalling of PGE2 after inflammation and would be a useful therapeutic target for pain therapies.

Remote nerve injection of mu opioid receptor adeno-associated viral vector increases antinociception of intrathecal morphine.

UNLABELLED: We have shown previously that using recombinant adeno-associated viral vector (rAAV) to up-regulate mu opioid receptors (muORs) in dorsal root ganglia (DRGs) increases the potency of subcutaneous morphine. Here we report an improved method of introducing rAAV-muOR viral vectors into DRGs. Instead of injecting the rAAV-muOR gene directly into DRGs as shown before, the vector was introduced into the sciatic nerve of rats. Changes in muOR expression and antinociceptive effects of intrathecal morphine in rAAV-muOR rats were examined. Immunocytochemical studies showed that the transduced muORs were expressed in all types (ie, small, medium, and large) of DRG neurons. The expression of muORs in DRG neurons, quantified by Western blotting, was increased by 1.7-fold 4 weeks after the sciatic nerve injection. The up-regulation persisted for more than 6 months. The effects of intrathecal morphine on paw withdrawal latencies to heat were studied in rats inflamed with complete Freund's adjuvant. Compared with rats injected with rAAV containing the enhanced green fluorescent protein gene (rAAV-EGFP), the antinociceptive potency of intrathecal morphine in rAAV-muOR rats was significantly increased, and the effective dose (ED50) for morphine was 5.4-fold lower (rAAV-muOR: ED50 = 0.84 microg, confidence interval, 0.70-0.99 microg; rAAV-EGFP: ED50 = 4.50 microg, confidence interval, 3.55-5.86 microg). With minimum tissue damage and a large persistent increase in the opioid potency, remote nerve injection of rAAV-muOR to up-regulate muORs could be a useful therapeutic strategy for the treatment of chronic pain. PERSPECTIVE: Injection of adeno-associated viral vector containing the muOR gene into the sciatic nerve produces a significant up-regulation of muORs in DRGs for up to 6 months without producing any immune responses in the injected animals. This results in a 5.4-fold increase in the potency of intrathecal morphine.

Efficiencies of transgene expression in nociceptive neurons through different routes of delivery of adeno-associated viral vectors.

Transferring therapeutic genes into the nociceptive system, including dorsal root ganglia (DRGs) and the spinal cord, is potentially a powerful approach for the treatment of chronic pain in humans. Adeno-associated viral vectors (AAVs) are particularly useful in delivering foreign genes to targeted tissues because they seldom induce immune responses or produce cytotoxicity. To determine the efficiency of transgene expression and the best route(s) of delivery, a recombinant AAV type 2 vector containing the enhanced green fluorescent protein (EGFP) gene driven by the neuron-specific enolase (NSE) promoter (rAAV-EGFP) was constructed. We injected the vector into subcutaneous tissue, sciatic nerve, DRGs, and subarachnoid space, and examined EGFP expression in the DRG, spinal cord, and nerve fibers. Both sciatic nerve and DRG injection led to strong EGFP expression in a large number of DRG neurons. The expression persisted for more than 6-8 months. We then delivered the mu-opioid receptor (muOR) gene into DRGs through direct DRG or sciatic nerve injection of rAAV-muOR and found a significant increase in morphine efficacy. These results suggest that delivering therapeutic genes to DRGs by the rAAV-NSE vector is a valid strategy for treatment of chronic pain.

Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat.

Pluripotent or multipotent stem cells isolated from human embryos or adult central nervous system (CNS) may provide new neurons to ameliorate neural disorders. A major obstacle, however, is that the majority of such cells do not differentiate into neurons when grafted into non-neurogenic areas of the adult CNS. Here we report a new in vitro priming procedure that generates a nearly pure population of neurons from fetal human neural stem cells (hNSCs) transplanted into adult rat CNS. Furthermore, the grafted cells differentiated by acquiring a cholinergic phenotype in a region-specific manner. This technology may advance stem cell-based therapy to replace lost neurons in neural injury or neurodegenerative disorders.

Gabapentin actions on N-methyl-D-aspartate receptor channels are protein kinase C-dependent.

Gabapentin (Neurontin) (GBP) is a widely prescribed analgesic used in treating pain patients with peripheral nerve injuries, diabetic neuropathy and cancer. To understand the mechanism of its action, we used the whole-cell patch recording technique to study the effects of GBP on N-methyl-D-aspartate (NMDA)-evoked currents in single dorsal horn neurons isolated from normal rats and from rats with inflammation induced by the injection of complete Freund adjuvant (CFA) to the hindpaw. We found that GBP enhanced NMDA currents in normal neurons only when protein kinase C (PKC) was added to these cells. The enhancement resulted from an increase in the affinity of glycine for NMDA receptors by GBP. In contrast, in neurons isolated from CFA-treated rats, GBP enhanced NMDA responses without any PKC treatment. Since endogenous PKC in inflamed tissue is elevated, these results suggest that GBP exerts its effects only on those cells affected by inflammatory injuries. Thus, the effects of GBP on NMDA receptors are plastic; they depend on the phosphorylation states of cells or receptors. These observations point to a new strategy for drug design. A chemical whose action depends on the state of cells would maximize its effectiveness while keeping its side-effects to a minimum.