LncRNA AWPPH accelerates the progression of non-small cell lung cancer by sponging miRNA-204 to upregulate CDK6.

OBJECTIVE: This study aims to uncover the function of long non-coding RNA (lncRNA) AWPPH in the progression of non-small cell lung cancer (NSCLC) and the potential mechanism. PATIENTS AND METHODS: AWPPH and microRNA (miRNA-204) levels in NSCLC tissues and adjacent normal tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Kaplan-Meier curves were introduced for assessing overall survival in NSCLC patients expressing high or low level of AWPPH. Potential correlation between expression levels of AWPPH and miRNA-204 in NSCLC tissues was analyzed by Spearman correlation test. Through Dual-Luciferase reporter gene assay, the interaction among AWPPH, miRNA-204, and CDK6 was identified. Potential impacts of AWPPH/miRNA-204/CDK6 regulatory loop on mediating proliferative, migratory, and invasive capacities of A549 cells were evaluated through cell counting kit-8 (CCK-8) and transwell assay. RESULTS: Upregulated AWPPH and downregulated miRNA-204 were determined in NSCLC tissues. AWPPH level was negatively correlated to overall survival in NSCLC patients and miRNA-204 level in NSCLC tissues. Silence of AWPPH attenuated proliferative, migratory, and invasive capacities in A549 cells. MiRNA-204 was the downstream gene of AWPPH. Knockdown of miRNA-204 reversed the decreased viability, migratory, and invasive rates in A549 cells with AWPPH knockdown. In addition, CDK6 was the target gene of miRNA-204. Overexpression of miRNA-204 downregulated CDK6 level in A549 cells. The attenuated proliferative, migratory, and invasive capacities in A549 cells overexpressing miRNA-204 were reversed after CDK6 overexpression. CONCLUSIONS: LncRNA AWPPH serves as the miRNA-204 sponge to upregulate CDK6 level, thus aggravating the progression of NSCLC.

rPSGL-1-Ig, a recombinant PSGL-1-Ig fusion protein, ameliorates LPS-induced acute lung injury in mice by inhibiting neutrophil migration.

The binding of selectin to P—selectin glycoprotein ligand—1 (PSGL—1) mediates the tethering and rolling of leukocytes on the endothelium during leukocyte migration and inflammation. Recombinant human PSGL—1—Ig fusion protein (rPSGL—1—Ig) is a widely used selectin inhibitor that prevents neutrophil entry into inflamed or reperfused tissues. We hypothesized that rPSGL—1—Ig could be used to as a drug for the treatment of acute lung injury (ALI). We induced murine ALI by injecting mice with lipopolysaccharide (LPS) and then treated the mice with rPSGL—1—Ig. We determined the lung injury index, wet/dry ratio, and inflammatory cytokine level in differentially treated mice. The symptoms of LPS—induced lung injury were alleviated by rPSGL—1—Ig treatment. The histopathological index of LPS—induced lung injury improved after rPSGL—1—Ig treatment. rPSGL—1—Ig treatment also reduced the recruitment of inflammatory cells, including neutrophils, into the lung, as well as reducing the level of inflammatory cytokines. These data suggest that rPSGL—1—Ig protein has a therapeutic effect on LPS—induced lung injury.

Down-regulation of MAO-B activity and imidazoline receptors in rat brain following chronic treatment of morphine.

AIM: To study the regulation of monoamine oxidase-B (MAO-B) activity and imidazoline receptors (I-R) during long term treatment of morphine. METHODS: MAO-B activity was detected by high performance liquid chromatography; I-R was detected by [3H]idazoxan binding test. RESULTS: Idazoxan and morphine inhibited whole brain homogenate MAO-B activity in a dose-dependent manner, while agmatine, an endogenous imidazoline ligand, didn't affect the activity of MAO-B, and it had no effect on the inhibition of MAO-B activity by idazoxan or morphine. MAO-B activity of rats decreased markedly in all five brain regions detected (cerebral cortex, hippocampus, thalamus, cerebellum, and striatum) after chronic administration of morphine for 16 d (P < 0.01). Acute challenge with naloxone or idazoxan did not influence MAO-B activity in morphine chronically treated rats. Although agmatine itself did not affect MAO-B activity, co-administration of agmatine with morphine could reverse the effect of morphine on MAO-B activity. Chronic administration of morphine significantly decreased the density of [3H]idazoxan binding sites and increased the binding affinity in cerebral cortex and cerebellum (P < 0.05 or P < 0.01). CONCLUSION: MAO-B activity was relevant to the abstinent syndrome of morphine dependent rats, but not related to the effect of agmatine on morphine analgesia; influence of agmatine on the pharmacological effects of morphine was based on its activation of imidazoline receptors.

Structural basis of Smad1 activation by receptor kinase phosphorylation.

Phosphorylation of Smad1 at the conserved carboxyl terminal SVS sequence activates BMP signaling. Here we report the crystal structure of the Smad1 MH2 domain in a conformation that reveals the structural effects of phosphorylation and a molecular mechanism for activation. Within a trimeric subunit assembly, the SVS sequence docks near two putative phosphoserine binding pockets of the neighboring molecule, in a position ready to interact upon phosphorylation. The MH2 domain undergoes concerted conformational changes upon activation, which signal Smad1 dissociation from the receptor kinase for subsequent heteromeric assembly with Smad4. Biochemical and modeling studies reveal unique favorable interactions within the Smad1/Smad4 heteromeric interface, providing a structural basis for their association in signaling.

Influence of idazoxan on analgesia, tolerance, and physical dependence of morphine in mice and rats in vivo.

AIM: To study the influence of idazoxan (Ida), an antagonist of imidazoline receptors (I-R), on analgesia, tolerance, and physical dependence of morphine. METHODS: The effects of Ida on pain threshold and morphine analgesia were observed in mouse acetic acid writhing test and 55 degrees C hot plate test. The effect of Ida on morphine tolerance and physical dependence were observed in mouse tolerant model and in mice and rat models. RESULTS: Ida (3-9 mg/kg) significantly decreased the pain threshold by 120% in acetic acid writhing test and by 39% in 55 degrees C hot plate test of mice, respectively. It inhibited the analgesic effect of morphine in a dose-dependent manner. Ida promoted the development of tolerance to morphine in mice and induced the abstinence syndrome in morphine-dependent mice and rats similar to naloxone. CONCLUSION: I-R and its endogenous ligand agmatine might participate in the pain threshold and influence morphine analgesia as well as negatively regulate tolerance to and physical dependence on morphine.

Effects of agmatine on tolerance to and substance dependence on morphine in mice.

AIM: To study the effects of agmatine on tolerance to and dependence on morphine. METHODS: Inhibitory effects of agmatine on tolerance to and substance dependence on morphine were observed in mouse tolerant models and in mouse jumping test, respectively. RESULTS: Agmatine 0.125-2.5 mg.kg-1 prevented the development of tolerant to morphine in a dose-dependent manner. Pretreatment of mice with morphine induced an over 3-fold increase in analgesic ED50 (20.1, 14.4-28.0 mg.kg-1) than those with normal saline (6.3, 5.1-7.8 mg.kg-1). Pretreatment of mice with both of agmatine and morphine made morphine loss the ability to induce tolerance. Withdrawal jumps and loss in body weight induced by naloxone in morphine-dependent mice were prevented by agmatine (2.5-10 mg.kg-1) in a dose-dependent manner. ED50 of naloxone (21.4, 18.4-24 mg.kg-1) required to precipitate withdrawal jumps in mice pretreated with both agmatine and morphine was 8 times higher than that with morphine alone (2.5, 2.1-2.8 mg.kg-1). These effects of agmatine were blocked by idazoxan. CONCLUSION: Agmatine prevented tolerance to and substance dependence on morphine in mice by activation of imidazoline receptors.

Correlation between inhibitions of morphine withdrawal and nitric-oxide synthase by agmatine.

AIM: To study correlation between inhibitions of naloxone-precipitated withdrawal jumps and nitric-oxide synthase (NOS) activity by agmatine. METHODS: NOS activities in mouse brain were measured by determination of concentration of [3H]citrulline, the product of [3H]arginine. RESULTS: Agmatine inhibited NOS activity in naive and morphine-dependent mouse cerebellum, forebrain, and thalamus in substrate-competitive manner in vitro. Naloxone induced withdrawal jumps and an increase in NOS activity in cerebellum, forebrain, and thalamus of abstinent mice. Pretreatment of mice with morphine plus agmatine inhibited the effect of naloxone to precipitate withdrawal jumps and increase in NOS activity. The effect of agmatine was blocked by idazoxan. CONCLUSION: The inhibitory effect of agmatine on naloxone-precipitated withdrawal jumps is related to its inhibition of NOS activity by substrate competitive manner and activation of imidazoline receptors.

Analgesic effect of agmatine and its enhancement on morphine analgesia in mice and rats.

AIM: To study the effect of agmatine on pain and morphine analgesia. METHODS: The effect of agmatine on pain was observed in mouse heat radiant tail-flick test, mouse acetic acid writhing test, and rat 4% saline test. Its enhancing effect on analgesia of morphine and clonidine was assessed in rat and mouse heat radiant tail-flick tests. RESULTS: Agmatine did not significantly prolong tail-flick latency of mice, but reduced the number of acetic acid-induced writhing of mice and inhibited writhing responses to saline completely. It potentiated the analgesic effects of morphine and clonidine in dose-dependent manner and decreased the analgesic ED50 of morphine and clonidine by more than 75% in mouse heat radiant tail-flick test. These effects of agmatine were antagonized by idazoxan. CONCLUSION: Agmatine has weak analgesic effects and potentiates morphine and clonidine analgesia by activation of imidazoline receptors.

Methadone-induced desensitization of the delta-opioid receptor is mediated by uncoupling of receptor from G protein.

Chronic exposure of neuroblastoma x glioma (NG108-15) hybrid cells and rat mu-receptor-transfected Chinese hamster ovary (CHO) cells to 10 microM morphine resulted in a compensatory and antagonist-precipitated increase in cAMP accumulation. However, incubation of these cells with 10 microM methadone during chronic exposure to morphine substantially prevented the actions of morphine. Chronic methadone treatment caused a pronounced reduction in agonist-stimulated binding of [35S]GTPgammaS to G proteins, but it did not produce significant down-regulation of delta-opioid receptors, whereas chronic morphine treatment failed to induce either uncoupling of delta-opioid receptors from G proteins or down-regulation of delta-opioid receptors. In contrast to chronic treatment with morphine alone, treatment of cells with morphine and methadone simultaneously resulted in a significant decrease in agonist-stimulated binding of [35S]GTPgammaS to G proteins. The action of methadone-mediated uncoupling of the receptor from the G protein was blocked by the nonselective protein kinase inhibitor [1-(5-isoqinolinesulfony)-2-methylpiprazine](H7), but not by the specific protein kinase C inhibitor, chelerythrine. The data demonstrate that methadone desensitizes the delta-opioid receptor by uncoupling the receptor from the G protein. In this way, methadone antagonizes the morphine-mediated adaptive sensitization and overshoot of adenylate cyclase. The functional desensitization of opioid receptors by methadone may explain why methadone is effective in the treatment of morphine dependence.

The difference between methadone and morphine in regulation of delta-opioid receptors underlies the antagonistic effect of methadone on morphine-mediated cellular actions.

To investigate the cellular and molecular basis for using methadone in substitution therapy for morphine addiction, the difference between methadone and morphine in causing desensitization of delta-opioid receptors was examined, and the effects of methadone pretreatment on opiate-induced inhibition of forskolin-stimulated cAMP accumulation was studied. Methadone substantially attenuated the ability of [D-Ala2,D-Leu5]enkephalin (DADLE), morphine and methadone to inhibit forskolin-stimulated cAMP accumulation. Methadone was able to block the morphine-induced compensatory increase in intracellular cAMP levels and naloxone-precipitated cAMP overshoot after chronic exposure to morphine. The protein kinase inhibitor (1-5-isoquinolinesulfony)-2-methylpiperazine) (H7) could significantly block the chronic methadone treatment-induced loss of the ability of DADLE to inhibit adenylate cyclase. The protein kinase inhibitor chelerythrine was able to block the acute methadone treatment-induced loss of the ability of DADLE to inhibit adenylate cyclase. In contrast, morphine did not cause a substantial desensitization of the delta-opioid receptor. These results indicate that methadone is different from morphine in its regulation of the delta-opioid receptor. In addition, these results also indicate that the mechanisms of delta-opioid receptor desensitization induced by acute and chronic methadone treatment are different.

Functional implications of structural differences between variants A and B of bovine beta-lactoglobulin.

The structure of the trigonal crystal form of bovine beta-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 A and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014-14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61-67), there are small changes in main-chain conformation, whereas the substitution V118A on beta-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.

Effects of opioid receptor agonists on cAMP second messenger system.

AIM: To study the mechanism underlying the difference in physical dependence potential of morphine (Mor), methadone (Met), buprenorphine (Bup), etorphine (Eto), and dihydroetorphine (DHE). METHODS: Adenylate cyclase of NG108-15 cells were used for studying the effects of different opiates on cAMP second messenger system. RESULTS: Bup, DHE, and Eto were distinct from Mor in naloxone-precipitated rebound response of cAMP in NG108-15 cells chronically treated with these opiates. Naloxone given to NG108-15 cells treated with Mor for 24 h produced marked rebound response of adenylate cyclase. While no such rebound response was detected when the cells were treated with Bup, DHE, and Eto for 24 h. The naloxone-induced rebound response of cAMP in chronic Met-treated NG108-15 cells was also lower than that in chronic Mor-treated NG108-15 cells. Following a prolonged exposure to Bup, DHE, and Eto for 72 h, the naloxone-induced rebound response of cAMP in these cells was still markedly lower than that in Mor-treated cells. The substitution of Mor with Bup, Met, DHE, and Eto inhibited naloxone-induced rebound response of cAMP in chronic Mor-treated NG108-15 cells. CONCLUSION: There were distinct differences among these opiates in regulating cAMP second messenger system, which was related to their physical dependence potential.

Influence of agmatine in adaptation of cAMP signal transduction system of opiate receptors.

AIM: To observe attenuative effects of agmatine on opiate desensitization and substance dependence. METHODS: Guanosine 5'-O-(3-[35S] thiotriphosphate) ([35S]GTTP) binding and cellular cyclic AMP (cAMP) level were determined by radioligand binding assay and radioimmunoassay in NG108-15 cells, respectively. RESULTS: Agmatine increased stimulative action of opioids on [35S]GTTP binding by about 35% and inhibitory effects of opioids on cellular cAMP concentration by about 114.3% in NG108-15 cells pretreated with opioids. On the other hand, it also inhibited cAMP over-shooting by 214.9% of morphine substance dependent cells precipitated by naloxone compared with that of control. These effects of agmatine were antagonized by idazoxan in a concentration-dependent manner. CONCLUSION: Agmatine reversed the formative process of adaptation in cAMP signal transduction cascade.

Effects of low-pH treatment on cAMP second messenger system regulated by different opioid agonists.

AIM: To study the mechanism of opioid agonists in regulation of cAMP second messenger system. METHODS: Low-pH treatment was used to deplete the stimulatory G protein (Gs) function. The effects of some opiates on adenylate cyclase were compared between control and low-pH treatment membranes. RESULTS: In contrast to dehydroetorphine (DHE), etorphine (Eto), morphine (Mor) and methadone (Met) substantially increased the inhibitory effects on adenylate cyclase in membranes prepared from naive and chronic Mor- or Met-treated NG108-15 cells by low-pH treatment. In contrast to Mor, DHE and Eto did not result in significant decrease in the inhibitory effects on adenylate cyclase in membranes from the cells treated chronically with DHE or Eto. Marked rebound of adenylate cyclase was also not observed in membranes from chronic DHE or Eto-treated cells when precipitated with naloxone. Low-pH treatment eliminated naloxone-induced rebound of adenylate cyclase in chronic Mor-treated cells. CONCLUSION: The difference in opiate-induced functional adaptive alteration of Gs is at least one biochemical mechanism of developing opiate tolerance and dependence.

12-Bromododecanoic acid binds inside the calyx of bovine beta-lactoglobulin.

The X-ray structure of bovine beta-lactoglobulin with the ligand 12-bromododecanoic acid as a model for fatty acids has been determined at a resolution of 2.23 A in the trigonal lattice Z form. The ligand binds inside the calyx, resolving a long-standing controversy as to where fatty-acid like ligands bind. The carboxylate head group lies at the surface of the molecule, and the lid to the calyx is open at the pH of crystallization (pH 7.3), consistent with the conformation observed in ligand-free bovine beta-lactoglobulin in lattice Z at pH 7.1 and pH 8.2.

Structural basis of the Tanford transition of bovine beta-lactoglobulin.

The structures of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods at a resolution of 2.56, 2. 24, and 2.49 A, respectively. The corresponding values for R (Rfree) are 0.192 (0.240), 0.234 (0.279), and 0.232 (0.277). The C and N termini as well as two disulfide bonds are clearly defined in these models. The glutamate side chain of residue 89 is buried at pH 6.2 and becomes exposed at pH 7.1 and 8.2. This conformational change, involving the loop 85-90, provides a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.

Agmatine inhibited tolerance to and dependence on morphine in guinea pig ileum in vitro.

AIM: To observe effect of agmatine (Agm) on tolerance to and substance dependence on morphine (Mor) in guinea pig ileum longitudinal muscle (GPILM). METHODS: The experiment was performed in electric field stimulation (EFS) test in vitro. RESULTS: Mor inhibited twitch contractions of GPILM induced by EFS [IC50 = 140 (107-182) nmol.L-1]. Incubation of GPILM with Mor 270 nmol.L-1 for 8 h evoked a 37-fold increase in IC50 of Mor (tolerance) and a contractile response to naloxone (Nal, substance dependence). When the preparations were coincubated with Mor + Nal and Mor + Agm, Mor lost the ability to induce tolerance and inhibited the contractile responses of the preparations to Nal by 90% and 75%, respectively. These effects of Agm could be almost completely antagonized by idazoxan. CONCLUSION: Agm prevented the development of tolerance to and substance dependence on Mor in GPILM in vitro by activation of imidazoline receptors.

Ultrastructural changes in rat locus coeruleus induced by chronic opioids.

The locus coeruleus (LC) is a major noradrenergic nucleus in the brain. The activity of the LC neurons is chronically regulated by opioids. So far, very little is known about the morphological changes induced by chronic treatment with opioids. In the present study, the effects of chronic treatment with morphine and dihydroetorphine, a new narcotic analgesic with lower physical dependence potential than morphine, were investigated on the ultrastructure of the rat LC. Rats received saline or increasing doses of morphine or dihydroetorphine for 5 days by twice daily subcutaneous injections. Withdrawal was precipitated in half of the opioid-treated rats by a single intraperitoneal injection of naloxone 4 h after the last injections of opioids. The ultrastructure of the LC was examined by electron microscopy. Results showed that chronic morphine treatment induced a marked injury to the LC neurons. The primary changes in the cell body were the indentation of nuclei, the fragmentation and degranulation of rough endoplasmic reticulum, as well as the disaggregation of polyribosomes. Myelinoid bodies were seen in the processes. An accumulation of presynaptic vesicles was observed in some of the terminals which formed synaptic junctions with the LC neurons as compared to the normal controls. Naloxone-precipitated withdrawal from morphine did not stop the morphine-induced injury on the LC neurons except that less accumulation of presynaptic vesicles occurred. Chronic dihydroetorphine treatment only induced a slight change in the ultrastructure of the LC neurons. These results indicate that the LC neurons are more vulnerable to chronic treatment with morphine than to that with dihydroetorphine.

Anxiogenic effect of naltrexone in social interaction test in rats.

AIM: To study the anxiogenic effect of naltrexone (Nal) on the emotional state of rats. METHODS: The duration of active interaction was measured in the social interaction test in rats. RESULTS: Without influence on the locomotor activity, Nal (0.1-50 mg.kg-1) dose- and time-dependently decreased the duration of active interaction, which was antagonized by morphine (5 mg.kg-1) or fenclonine (Fen, 150 mg.kg-1 x 3 d) and was enhanced by 5-hydroxytryptophan (5-HTP, 50 mg.kg-1). CONCLUSION: Nal produced anxiety via its blockade of opioid receptors; central opioidergic neurons were involved in the regulation of anxiety through their tonic inhibitions in serotonergic neurons.