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.

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.

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.

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.

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.

Dihydroetorphine is a mu-receptor-selective ligand.

The selectivity to opioid receptors of dihydroetorphine, a potent analgesic with only mild physical dependence, was investigated using radioligand binding assay and its analgesic activity in mice determined. The relative affinity ratio of dihydroetorphine to mu-, delta- and kappa- opioid receptors was 333:1:1. The analgesic effect of intracerebro-ventricular injection in mice could be antagonized by the mu-antagonist beta-funaltrexamine but could not be antagonized by delta- and kappa-selective antagonists naltrindole and norbinaltorphimine. We conclude that dihydroetorphine is a selective ligand for the mu-opioid receptor.

[Effects of 7-methoxy-4'-hydroxyl-3'-diethylaminomethylisoflavone (MHDF) on hemodynamics and aorta in rats].

MHDF is a synthetic isoflavone derivative with anti-ischemic effect. In this paper, the effects of MHDF on in vivo hemodynamics and in vitro contraction of rat aorta were reported. MHDF (3-12.8 mg/kg) was found to decrease +/- dP/dtmax, Vmax, Vpm and LVSP, prolong T-dP/dtmax of left ventricle and slow the HR in rats. It was also shown to inhibit the changes of heart performance induced by CaCl2 in rats. In isolated rat aorta, MHDF caused a noncompetitive antagonism of noradrenaline-and CaCl2-induced contraction with the pD2' of 3.11 +/- 0.21 and 3.73 +/- 0.07. The high K(+)-evoked contraction with IC50 of 17.6 mu mol/L was also found to be inhibited. The results suggest that the effect of MHDF on vascular muscle is different from that of an alpha-blocker and may be attributable to inhibition of extracellular calcium influx or intracellular calcium release.

[Effect of dihydroetorphine on contraction response of guinea pig ileum, mouse, and rabbit vas deferens induced by electric field stimulation in vitro].

Dihydroetorphine (DHE) inhibited contraction response of isolated guinea pig ileum and mouse vas deferens induced by electric field stimulation with IC50 values of (4.4 +/- 4.3) x 10(-16) mol.L-1 and (1.1 +/- 1.4) x 10(-18) mol.L-1 respectively. These effects of DHE were antagonized by naloxone (Nal). On guinea pig ileum, the antagonistic effect of Nal against DHE was similar to those against morphine and etorphine. However, on mouse vas deferens, the antagonistic effect of Nal against DHE was more potent than those against morphine and etorphine. Besides, DHE showed antagonistic effect on k-receptors in isolated rabbit vas deferens.

[Inhibitory properties of cycloguanide phenylsulfone on acetylcholinesterase].

Mouse brain homogenates, mouse RBC, immobilized enzyme of pig brain, and human RBC were chosen as source of AChE, AChE activities were determined by colorimetric and gasometric methods. Cycloguanide phenylsulfone (CGP) exerted a moderate inhibitory effect on AChE. The pI50 (negative logarithm of molar concentration causing 50% inhibition of AChE) towards AChE in mouse RBC and brain were 5.75 and 5.50, respectively. The binding potency to AChE was very loose. The AChE inhibition was easily reversed by washing. It showed that CGP belonged to the contra-competitive AChE inhibitor.

[Binding of [3H] dihydroetorphine to opioid receptors in rat brain membrane].

AIM: To study the binding characteristics of dihydroetorphine (DHE) to opioid receptors. METHODS: The binding of [3H] DHE to rat brain membrane opioid receptors was observed by radioligand binding study. RESULTS: Saturation study showed the binding of [3H] DHE to rat brain membrane presented a single high-affinity class of binding sites with Kd = 0.19 nmol.L-1 and Bmax = 115 pmol/g protein. Kinetics study showed the association rate of [3H] DHE to opioid receptor was very rapid and dissociation very slow. NaCl 100 mmol.L-1 + guanosine triphosphate (GTP) 50 mumol.L-1 inhibited the binding of [3H] DHE and made Kd increase to 7.87 nmol.L-1, without effect on Bmax. Competitive inhibition of agonists and antagonists indicated that the affinity of [3H]DHE to mu opioid receptor was higher than to delta and kappa opioid receptors. CONCLUSION: DHE is a selective agonist of mu opioid receptors.

Influences of locus coeruleus lesions and reserpine treatment on opioid physical dependence in rats.

AIM: To study the role of locus coeruleus (LC) noradrenergic neurons in the opioid dependence. METHODS: Chemical lesion of LC was produced by 6-hydroxydopamine (4 micrograms in 1 microL). Reserpine (Res) was used to deplete central noradrenaline. Composite scores of naloxone (4 mg.kg-1, ip) precipitated abstinence syndromes were calculated and the magnitude of weight loss was determined in chronic morphine (Mor)- or dihydroetorphine (DHE)-treated rats. RESULTS: Lesions of LC made Mor, but not DHE, abstinence syndrome more serious. Multiple doses of Res (0.5 mg.kg-1.d-1 x 3 d) increased scores of Mor and DHE abstinence syndrome, made worse the loss of body weight in chronic Mor-, but not DHE-, treated rats. A single dose of Res (0.5 mg.kg-1) speeded up the development of physical dependence on Mor. CONCLUSION: The LC noradrenergic neurons play a role in suppression of some withdrawal symptoms.

[Effects of 7-methoxy-4'-hydroxyl-3'-diethylaminomethylisoflavone on heart atrium and ventricular papillary muscles of guinea pig].

In isolated guinea pig right atria, 7-methoxy-4'-hydroxyl-3'-diethylaminomethylisoflavone (MHDF), a new synthetic isoflavone produced noncompetitive antagonisms to isoproterenol- and histamine-induced positive chronotropic actions with pD'2 values of 5.04 +/- 0.10 and 4.90 +/- 0.18, respectively. MHDF inhibited the positive chronotropic response to CaCl2. In isolated left atria, the negative inotropic action of MHDF increased as the frequency increased. In papillary muscles, MHDF 3 mumol/L reduced the contractile force, while Vmax was decreased and APD and ERP were prolonged. These results indicated that the mechanism of MHDF on myocardium is related to inhibition of Ca2+ influx, Na+ influx and K+ efflux, not by blocking beta or H2 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.

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.

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.

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.