BACKGROUND: It has been considered that Cestrum nocturnum L. (CNL) has the effects of antiarrhythmia, local anesthesia and central inhibition.OBJECTIVE: To investigate the analgesic effect of CNL extract on mice,so as to find new drugs for clinical treatment of pain.DESIGN: A randomized control observation.SETTING: Center of Modern Education and Department of Pharmacology,Gannan Medical College.MATERIALS: The experiments were carried out in the laboratory of scientific research center, Gannan Medical College between March and April in 2005. ① A total of 150 healthy adult Kunming mice were used in four independent experiments. ② Drugs: CNL extract was provided by the Department of Phytochemistry, Shenyang Pharmaceutical University (batch number: 2002080901), morphine hydrochloride injection by Shenyang No.1Pharmaceutical Factory (batch number: 000305), and naloxone hydrochloride injection by Yanqiao (Hunan) Pharmaceutical Co. Ltd., (batch number:20021109).METHODS: ① Effects of CNL extract on writhing times induced by acetic acid: Forty female mice were randomly divided into four groups with10 mice in each, and they were treated with intraperitoneal injections of 0.02 mL/g saline, 0.10 and 0.20 mg/g CNL extract and 0.10 mg/g aminophenazone respectively. The intraperineal injection of 6 g/L glacial acetic acid was given after 15 minutes. The writhing times of mice within 15 minutes were observed and recorded in each group. ② Effects of CNL extract on the pain induced by hot pla in mice: Forty female mice were randomly divided into four groups with 10 mice in each, and they were treated with intraperineal injections of 0.02 mL/g saline, 0. 10 and 0.20 mg/g CNL extract and 0.10 mg/g morphine respectively. The pain responses were detected at 15, 30 and 60 minutes after administration. ③ The antagonistic effect of naloxone on morphine and CNL extract to the pain induced by hot plate in mice: Thirty female mice were randomly divided into three groups ith 10 mice in each group, and they were given intraperitoneal injections of 0.02 mL/g saline, naloxone 0.004 mg/g +morphine 0.01 mg/g and naloxone 0.004 mg/g+CNL extract 0.01 mg/g respectively. The pain responses were detected at 15, 30 and 60 minutes after administration respectively. ④ Effects of CNL extract on electrostimulation induced pain in mice: Forty mice were randomly divided into four groups with 10 mice in ach group, and they were administrated with intraperineal injections of 0.02 mL/g saline, 0.10 and 0.20 mg/g CNL extract and 1 g/L morphine respectively. Repeated electrostimulations were given at 20, 35, 50 and 70minutes after administration, and the pain responses were detected by means of electrostimulation.MAIN OUTCOME MEASURES: ① Writhing times; ② Time for the pain response induced by hot plate; ③ Analgesic rate induced by electrostimulation.RESULTS: Totally 150 healthy adult Kunming mice were used in the four independent experiments, and all were involved in the analysis of results. ①Writhing times in the mice: 0.10 and 0.20 mg/g CNL extracts and 0.10 mg/g aminophenazone had very significant analgesic effects on writhing induced byacetic acid in mice, and the writhing times after administration were all fewer than those in the saline group (20.2±10.8, 14.5±7.6, 7.6±4.5,50.6±15.5, P ＜ 0.01), and the analgesic effects of CNL extract were dosedependently. ② Time for the pain response induced by hot plate: 0.10 and 0.20 mg/g CNL extracts had significant analgesic effects on the pain in duced by hot plate, and the time for pain sensation at 15, 30 and 60 minutes after administration were all longer than those in the saline group (P ＜ 0.05 or P ＜ 0.01), and the analgesic effect was dose-dependently. The times for pain sensation at each time point after administration in the naloxone 0.004 mg/g+CNL extract 0.01 mg/g group were all longer than those in the saline group, but those were close between the naloxone 0.004 mg/g+morphine 0.01 mg/g group and the saline group. ③ Analgesic rate induced by electrostimulation in the mice: The analgesic rates at20, 35, 50 and 70minutes after administration in the CNL extract 0.10 and 0.20 mg/g groups were all higher than those in the saline group (P ＜ 0.01).CONCLUSION: Our data suggested that CNL extract has obvious analgesic effect, and the analgesic intensity is dose-dependently. Naloxone, an opiate receptor antagonist, can antagonize the analgesic effect of morphine,but cannot antagonize that of CNL extract on mice with pain induced by hot plate, which indicates that CNL extract exert its analgesic role not through binding with opiate receptor.

Objective To investigate the incidence rate, onset time and electrophysiological characteristics of rapid eye movement sleep behavior disorder (RBD) and the relationship between RBD and synucleinopathies as well as the electrophysiological diagnostic criteria of RBD in Parkinson' s disease (PD) and multiple system atrophy (MSA). Methods Sleep survey and night video-polysomnography (NPSG)were used to study sleep disturbance of PD and MSA. (1) Subjective sleep assessments: All subjects,including 66 PD patients, 65 age and sex matched healthy controls and 30 MSA patients, completed the sleep questionnaires, and the RBD incidence rate and onset time were got. (2) Objective sleep assessments: 8 PD patients, 13 MSA patients, and 15 age and sex matched healthy controls underwent video-NPSG recording on two consecutive nights. Sleep architect were analyzed. The NPSG characteristics of RBD accompany with PD and MSA were analyzed, and the electrophysiological diagnostic varameters of it were determined. Results Patients with PD or MSA had a higher prevalence of RBD. RBD was found in 59. 1% (39/66) PD patients and 86. 6% (26/30) MSA patients, among those, 46. 2% ( 18/39 ) and 84.6% (22/26) had the waking symptoms of MSA and PD. The main NPSG characteristics of RBD of PD or MSA were chin REM without atonia (RWA) and increased movement. Conclusions The relatively higher RBD prevalence in MSA and PD patients indicates that RBD has close relationship with PD and MSA.Part of patients with RBD preceding neurology disease indicates that RBD may be the early marker of PD and MSA. The main NPSG characteristics of RBD accompany with PD and MSA are chin RWA and the motor manifestations. RWA and phasic EMG activity density are supposed to be the NPSG diagnostic parameters.

Objective To describe the clinical spectrum,especially sleep disorder in three patients diagnosed with Morvan syndrome.Methods Three consecutive patients were identified with Morvan syndrome in the Department of Neurology, Peking Union Medical College Hospital between December 2014 and March 2016.The character in three cases has been studied from several aspects such as clinical presentation, imaging, polysomnography (PSG), cerebrospinal fluid and serum.Results Serum test showed serum contactin-associated protein 2 (CASPR2)antibodies strongly positive (+++) and leucine-rich glioma inactivated protein 1 antibodies positive (+) in three patients.Neuropsychiatric features, neuromyotonia, neuropathic pain, dysautonomia, agrypnia excitata presented in all three patients.The agrypnia excitata was characterized by severe insomnia, excessive motor activity during the night.Agrypnia excitata was diagnosed in three patients according to their history.PSG was finished in case 2 and case 3.PSG in one patient (case 2) documented severe insomnia (sleep efficiency was 59%), lack of cyclic sleep organization with a predominance of stage 1 non-rapid eye movement sleep episodes intermixed with brief rapid eye movement, and a marked reduction of spindles and delta sleep;PSG in another patient (case 3) revealed complete absence of recognizable sleep.Sleep disorders and other symptoms resolved completely or almost completely in two patients (case 1,case 2) who received immunotherapy.Case 3 died from sudden cardiac death before immunotherapy.Conclusions Morvan syndrome usually is associated with high-titer CASPR2 antibodies in serum.Agrypnia excitata is cardinal manifestation of Morvan syndrome in association with a spectrum of neurologic presentations.Early immunotherapy could provide a favorable outcome.

Objective To cultivate human leukemia cells (HL-60) which were transiently transfected either a miR-126 mimic or inhibitor,and then to characterize the proliferation and apoptotic behavior of the transfected leukemia cells.Methods The leukemia cell line was developed and RT-PCR was performed to evaluate miR-126 expression levels.An instantaneous plasmid tmnsfection technique was used to transfect cells with either the miR-126 mimic or inhibitor.CCK-8,FCM,and clone formation tests were performed to analyze the proliferative and apoptotic behaviors of the leukemia cells.Results Proliferation was significantly decreased in cells transfected with the miR-126 mimic for 0,24,48,and 72 hours (P ＜ 0.05).Specifically,the G1,S,and G2 phases were significantly inhibited (P ＜ 0.05),the late and early apoptosis (UR+LR) rate increased (P ＜ 0.05),and the average rate of colony formation was also significantly decreased (P ＜ 0.05).Additionally,proliferation was significantly increased in cells transfected with the miR-126 inhibitor for 0,24,48,and 72 hours (P ＜ 0.05).Specifically,the G1,S,and G2 phases were increased (P ＜ 0.05),the UR + LR decreased significantly (P ＜ 0.05),and the average rate of colony formation was significantly increased (P ＜0.05).Conclusion In HL-60 cells,miR-126 can inhibit proliferation and promote apoptosis;thus,miR-126 may play an important role in the occurrence and development of leukemia as a tumor-suppressor miRNA.

OBJECTIVETo study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma.

METHODSMelanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression.

RESULTSIn melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector.

CONCLUSIONSBased on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Humans ; MAP Kinase Signaling System ; Melanoma ; genetics ; metabolism ; Mutation ; Phenotype ; Protein-Serine-Threonine Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Proto-Oncogene Proteins B-raf ; metabolism ; Signal Transduction ; Transfection

Objective: To investigate the function and mechanism of zinc finger protein 750 (ZNF750) in esophageal squamous cell carcinoma (ESCC). Methods: Xenograft in nude mice was applied to detect the tumorigenesis of ZNF750-depleted ESCC cells. Western blot was performed to observe the expression of downstream target protein of ZNF750 in ESCC cell lines and xenograft tumor tissues in which ZNF750 was knocked down. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay was used to determine the proliferation of ZNF750 stably depleted cells after restoration of its target protein. Results: The tumor weight of blank control, negative control and ZNF750 knockdown groups was 137±26 mg, 161±31 mg and 463±89 mg, respectively, with a statistically significant difference (P<0.01). The expressions of Kruppel-like factor 4 (KLF4) in ZNF750-depleted ESCC cells and its derived tumor tissue xenograft in nude mice were significantly down-regulated. Restoration of KLF4 in ZNF750 stably depleted cells significantly inhibited the cell proliferation (P<0.01). Conclusions ZNF750 may be a new tumor suppressor in the tumorigenesis of ESCC, and the inhibition of cell proliferation induced by ZNF750 may be partially through the regulation of KLF4 expression.