PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates.

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrate that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate, a peptide containing the phosphorylated NSF-pY83 site, specify PTP-MEG2 substrate selectivity, and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface reveals that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites. Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies thus provide mechanistic insights in complex exocytosis processes.

Real-time endocytosis imaging as a rapid assay of ligand-GPCR binding in single cells.

Most G protein-coupled receptors (GPCRs) do not generate membrane currents in response to ligand-receptor binding (LRB). Here, we describe a novel technique using endocytosis as a bioassay that can detect activation of a GPCR in a way analogous to patch-clamp recording of an ion channel in a living cell. The confocal imaging technique, termed FM endocytosis imaging (FEI), can record ligand-GPCR binding with high temporal (second) and spatial (micrometer) resolution. LRB leads to internalization of an endocytic vesicle, which can be labeled by a styryl FM dye and visualized as a fluorescent spot. Distinct from the green fluorescence protein-labeling method, FEI can detect LRB endocytosis mediated by essentially any receptors (GPCRs or receptors of tyrosine kinase) in a native cell/cell line. Three modified versions of FEI permit promising applications in functional GPCR studies and drug screening in living cells: 1) LRB can be recorded in "real time" (time scale of seconds); 2) internalized vesicles mediated by different GPCRs can be discriminated by different colors; and 3) a high throughput method can screen ligands of a specific GPCR.

[Conserved motifs in voltage sensing proteins].

This paper was aimed to study conserved motifs of voltage sensing proteins (VSPs) and establish a voltage sensing model. All VSPs were collected from the Uniprot database using a comprehensive keyword search followed by manual curation, and the results indicated that there are only two types of known VSPs, voltage gated ion channels and voltage dependent phosphatases. All the VSPs have a common domain of four helical transmembrane segments (TMS, S1-S4), which constitute the voltage sensing module of the VSPs. The S1 segment was shown to be responsible for membrane targeting and insertion of these proteins, while S2-S4 segments, which can sense membrane potential, for protein properties. Conserved motifs/residues and their functional significance of each TMS were identified using profile-to-profile sequence alignments. Conserved motifs in these four segments are strikingly similar for all VSPs, especially, the conserved motif [RK]-X(2)-R-X(2)-R-X(2)-[RK] was presented in all the S4 segments, with positively charged arginine (R) alternating with two hydrophobic or uncharged residues. Movement of these arginines across the membrane electric field is the core mechanism by which the VSPs detect changes in membrane potential. The negatively charged aspartate (D) in the S3 segment is universally conserved in all the VSPs, suggesting that the aspartate residue may be involved in voltage sensing properties of VSPs as well as the electrostatic interactions with the positively charged residues in the S4 segment, which may enhance the thermodynamic stability of the S4 segments in plasma membrane.

[Characteristics and associated factors of long-term retention for methadone maintenance treatment patients].

OBJECTIVE: To describe the characteristics and factors associated with long-term retention for methadone maintenance treatment (MMT) patients. METHODS: This study was conducted in eight MMT clinics located in Sichuan, Yunnan, Guangxi, Guizhou and Zhejiang provinces. Five hundred and thirty-nine MMT patients who enrolled in MMT clinics in 2004 and retained in treatment by June 2010 were selected as study subjects. Chi-square tests were used to compare the demographics and drug abuse history at enrollment and treatment characteristics during the follow-up period between continuous treatment patients and discontinuous treatment patients. RESULTS: Of the 539 patients, 110 (20.4%) were continuous treatment patients whereas 429 (79.6%) were discontinuous treatment patients. Of these 429 discontinuous treatment patients, 84.1% (361/429) had 2-4 treatment episodes whereas 15.9% (68/429) had 5 or more episodes during follow-up period. When continuous treatment patients were compared with discontinuous treatment patients, living with family members or friends (88.2% (97/110), 78.5% (337/429)), age of first drug use under 25 (61.8% (68/110), 71.3% (306/429)), low urine morphine positive test results (67.3% (74/110), 38.2% (164/429)) and living within 5 kilometers of the MMT clinic (72.7% (80/110), 61.3% (263/429)) were positively associated with higher possibility of continuous treat retention (P < 0.05). Demographics and drug abuse characteristics at enrollment, including gender, age, employment status, family relationship, injection, needle sharing, criminal behavior, contacts with drug users, MMT daily dosage and family members receiving MMT were not significantly associated with treatment retention (P > 0.05). CONCLUSION: Illicit drug use during the treatment and longer distance travelling to MMT clinic might have negative impact on patients' continuous treatment retention. Mobile MMT vehicles and expanded MMT service sites could be introduced to improve compliance of treatment retention of MMT patients.

Two distinct vesicle pools for depolarization-induced exocytosis in somata of dorsal root ganglion neurons.

The somata of dorsal root ganglion (DRG) neurons release neurotransmitters and neuropeptides. In addition to the conventional Ca2+-dependent secretion (CDS), Ca2+-independent but voltage-dependent secretion (CIVDS) also occurs in the somata of DRG neurons. Electrical stimulation induces both CDS and CIVDS, which differ in size and are coupled with different types of endocytosis contributed by CIVDS and CDS, respectively. However, it is unclear whether they use a common vesicle pool, so we investigated the relationship between the vesicle pools of CDS and CIVDS. Membrane capacitance recording and photolysis of a caged-Ca2+ compound showed that, in low external Ca2+ solutions, the depolarization-induced exocytosis contained two (fast and slow) phases, which were contributed by CIVDS and CDS, respectively. Depletion of the CDS readily releasable pool using photolysis did not affect the CIVDS. When the CIVDS and CDS vesicle pools were depleted by electrical stimulation, the pools had different sizes. Their kinetics of exocytosis-coupled endocytosis were also different. Thus, CIVDS and CDS used different vesicle pools in DRG neurons.

Co-transfection and tandem transfection of HEK293A cells for overexpression and RNAi experiments.

pIRES2-EGFP was employed and a non-target shRNA expressing plasmid was constructed to simulate overexpression and RNAi (RNA interference) experiments. Transfection of pIRES2-EGFP into HEK293A cells by cationic lipids VigoFect demonstrated that transfection efficiency increased in a dose-dependent manner with amount of DNA plasmid used, and optimal transfection time and cell density should be identified to reach a compromise of higher transfection efficiency and lower toxicity. Co-transfection experiments indicated that the two co-transfected plasmids were equivalently delivered into the same cells, and the co-transfection efficiency was rarely affected by cell density and proportion of the two plasmids. However, plasmid-receipted cells seemed indisposed to accept plasmid again during the second transfection, and very low co-transfection efficiency was observed in tandem transfection.

[Determination of supermolecular inclusion constants of hydroxylpropyl-beta-cylcodextrin with imperatorin and isoimperatorin by phase solubility method].

OBJECTIVE: To study the supermolecular interaction between different concentrations of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and imperatorin (IM) and isoimperatorin (ISO) using phase solubility analysis. METHODS: Different concentrations of hydroxypropyl-beta-cyclodextrin were prepared, added overdose of imperatorin or isoimperatorin, made a phase solubility diagram. RESULTS: The relationship of phase solubility was approximatively linear and the model of inclusion compound was AL and the inclusion ratio of IM, ISO versus HP-beta-CD was 1: 1. The inclusion constants were 214.4, 587.2 L/mol respectively. CONCLUSIONS: Supermolecular inclusion of HP-beta-CD of imperatorin/isoimperatorin could evidently increase the solubility of imperatorin and isoimperatorin, the enhancing effect of isoimperatorin solubility is better than that of imperatorin.

The use of solid lipid nanoparticles to target a lipophilic molecule to the liver after intravenous administration to mice.

Taspine solid lipid nanoparticles (Ta-SLN) and taspine solid lipid nanoparticles modified by galactoside (Ta-G2SLN) were prepared by the film evaporation-extrusion method. The nanoparticles were spherical or near-spherical particles with smooth surface, small size and high encapsulation efficiency. Ta-G2SLN and Ta-SLN showed significant inhibition on 7721 cell growth. Intravenous injection of either Ta-SLN or Ta-G2SLN resulted in a higher plasma and liver concentration and a longer retention time in mice compared with the administration of Ta. These results suggested that SLN tended to be preferentially delivered to the liver and Ta-G2SLN may further enhance liver targeting.

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells.

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-ß-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide-Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.

RNAi-based knockdown of multidrug resistance-associated protein 1 is sufficient to reverse multidrug resistance of human lung cells.

Up-regulation of multidrug resistance-associated protein 1 (MRP1) is regarded as one of the main causes for multidrug resistance (MDR) of tumor cells, leading to failure of chemotherapy-based treatment for a multitude of cancers. However, whether silencing the overexpressed MRP1 is sufficient to reverse MDR has yet to be validated. This study demonstrated that RNAi-based knockdown of MRP1 reversed the increased efflux ability and MDR efficiently. Two different short haipin RNAs (shRNAs) targeting MRP1 were designed and inserted into pSilence- 2.1-neo. The shRNA recombinant plasmids were transfected into cis-dichlorodiamineplatinum-resistant A549 lung (A549/DDP) cells, and then shRNA expressing cell clones were collected and maintained. Real time PCR and immunofluorescence staining for MRP1 revealed a high silent efficiency of these two shRNAs. Functionally, shRNA-expressing cells showed increased rhodamine 123 retention in A549/DDP cells, indicating reduced efflux ability of tumor cells in the absence of MRP1. Consistently, MRP1-silent cells exhibited decreased resistance to 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and DDP, suggesting reversal of MDR in these tumor cells. Specifically, MRP1 knockdown increased the DDP-induced apoptosis of A549/DDP cells by increased trapping of their cell cycling in the G2 stage. Taken together, this study demonstrated that RNAi- based silencing of MRP1 is sufficient to reverse MDR in tumor cells, shedding light on possible novel clinical treatment of cancers.

[Factors associated with overdose-caused mortality of HIV-positive patients who were on methadone maintenance treatment program].

OBJECTIVE: To study the factors that associated with the mortality of overdose on methadone maintenance treatment(MMT)among HIV-positive patients. METHODS: A 1 : 1 matched case-control design was used to identify the relationship between factors related to demography, drug use, characteristics of treatment and the mortality of overdose. 110 HIV-positive patients who died of drug overdose from March 2004 to September 2012 were defined as cases. Controls were another 110 patients who were still alive and paired with the cases, according to the same gender, similar date of MMT initiation and from the same clinics. RESULTS: Multivariate conditional logistic regression analyses indicated that risk factors as sharing needles before enrolled in the MMT program(OR = 5.19, 95% CI:1.39-19.33), HIV infection because of injecting drug-use (OR = 3.08, 95% CI: 1.16-8.21), and off from the treatment before the end point of the program(OR = 2.54, 95% CI: 1.23-5.23)were associated with mortality caused by overdose. Higher adherence(OR = 0.31, 95%CI: 0.10-0.95)appeared to be associated with lower mortality when compared with the control group. CONCLUSION: In order to reduce the mortality rate, comprehensive intervention could be introduced to improve the compliance of retention on MMT among patients. Intervention efforts should be focused on those patients who shared needle/syringes.

[Current situation related to antiretroviral therapy and related influential factors on HIV infected injection drug users in the methadone maintenance treatment clinics].

OBJECTIVE: To find out the current coverage of antiretroviral therapy (ART) among HIV positive subjects and to identify the major influential factors associated with the participation in ART among them. METHODS: 291 HIV positive subjects from 6 methadone maintenance treatment (MMT) clinics in Guangxi and Yunnan province were surveyed by questionnaires. RESULTS: 217 males (74.6%) and 74 females (25.4%) were under investigation, with the average age of 38.4 +/- 5.9. Most of them received less than senior high school education, married and unemployed. Results from the single factor logistic regression analysis showed that: working status, living alone, self-reported history of drinking alcohol in the last month, negative attitude towards MMT among family members,poor self-reported compliance to MMT in the last month,lack of incentives in the MMT clinics, reluctance on disclosure of their own HIV status, good self-perception on their health status, lack of communication on ART related topics among family members in the last 6 months, lack of correct attitude and knowledge on ART etc. appeared as the main factors that influencing the participation in ART program among the patients. Data from the multivariate logistic regression analysis showed that factors as: living alone, unwilling to tell others about the status of HIV infection, poor self-perception on HIV infection, lack of discussion of ART related topics within family members in the last 6 months and poor awareness towards ART among the family members etc., were associated with the low participation rate of ART. Conclusion Strengthening the publicity and education programs on HIV positive patients and their family members at the MMT clinics seemed to be effective in extending the ART coverage. Attention should also be paid to increase the family support to the patients.

[Evaluation on the long-term effectiveness among the first set eight methadone maintenance treatment clinics in China].

OBJECTIVE: To evaluate the long-term effectiveness of the first set 8 methadone maintenance treatment (MMT) clinics in China. METHODS: Repeated cross-sectional surveys were conducted on the first month after the enrollment and 5 years later, among drug users who received MMT, using a standard questionnaire. Data on demographic characteristics, HIV-related high-risk behaviors, criminal records associated with drug use and related family/social functions were collected and analyzed. RESULTS: There were 252 and 195 participants being interviewed at the baseline and the 5-year surveys, respectively. Of them, 66 participants were involved in both surveys. There was no significant differences on factors as ethnicity, level of education, working status, marital status and living status (P > 0.05) between the baseline and the 5-year surveys. Compared with data from the baseline survey, participants' behavior on drug abuse (100.0% vs. 24.1%, P < 0.001), needle sharing behavior (19.4% vs. 0.0%, P < 0.001), and exchanging sex for drugs (34.5% vs. 0.0%, P < 0.001) had significant decreases at the 5-year survey. Rates on condom use (10.6% vs. 25.0%, P = 0.004), and having jobs (27.8% vs. 47.7%, P < 0.001) had been improved significantly, while self-reported criminal cases related to drug use (15.1% vs. 1.5%, P < 0.001) and the contacts with drug users (88.9% vs. 31.3%, P < 0.001) had been significantly reduced after joining the MMT program. CONCLUSION: MMT could play an active role in reducing the HIV-related high-risk behaviors, criminal cases associated with drug abuse as well as enhancing the family and social functions of the MMT patients. Providing high quality service to the MMT clinic and trying to keep the drug users stick to the program remain difficult. It was also important to improve the training programs for staff working at the MMT clinics.

Validation and application by HPLC for simultaneous determination of vitexin-2″-O-glucoside, vitexin-2″-O-rhamnoside, rutin, vitexin, and hyperoside.

A simple, precise, and rapid high-performance liquid chromatographic method was developed and validated for the simultaneous determination of vitexin-2″-O-glucoside, vitexin-2″-O-rhamnoside, rutin, vitexin, and hyperoside. The HPLC separation was performed using a Shim-pack VP-ODS C18 column (250 mm×4.6 mm i.d., 5 µm) with the isocratic mobile phase consisting of tetrahydrofuran/ acetonitrile/0.05% phosphoric acid solution (20:3:77, v/v/v), and the flow rate was set at 1.0 mL/min. UV detection was carried out at a wavelength of 360 nm and the whole analysis took 25 min. The method was linear in the range of 4.12-206.00 µg/mL for vitexin-2″-O-glucoside, 4.05-202.50 µg/mL for vitexin-2″-O-rhamnoside, 1.64-82.00 µg/mL for rutin, 1.74-87.00 µg/mL for vitexin, and 1.41-70.60 µg/mL for hyperoside with the correlation coefficient for each analyte more than 0.998. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.6 and 2 ng for vitexin-2″-O-glucoside, 0.6 and 2 ng for vitexin-2″-O-rhamnoside, 0.3 and 1 ng for rutin, 1 and 3 ng for vitexin, and 0.5 and 2 ng for hyperoside, respectively. Intra- and inter-day precision and accuracy (RSD) were less than 3%. The developed HPLC method was successfully applied to the analysis of five flavonoids in hawthorn leaves, hawthorn fruits, and the preparations containing hawthorn leaves or fruits.

[Risk factors which were associated with heroin use during the methadone maintenance treatment among 1301 patients in 9 cities of China.].

OBJECTIVE: To determine the proportion of heroin use among patients who were involved in community-based methadone maintenance treatment (MMT) program and to identify the risk factors associated with heroin use. METHODS: This study was conducted in 9 MMT clinics within 3 provinces. Thirteen hundred and one patients who met the study criteria were selected from each of the five groups with different dosages of methadone users. An administrative questionnaire was applied to explore the demographics, drug abuse-related behaviors and MMT services received by the clients, etc. The prevalence of depression and anxiety among the clients were also collected by SAS and SDS. Urine samples were collected as a biological marker to indicate if heroin had been used. RESULTS: Of the 1301 patients, 76.2% were males. The mean age was (34.6 +/- 6.5) years while 71.7% had an education level of primary school or below. The average daily dosage of methadone was (48.1 +/- 29.4) mg and self-satisfied evaluation score on treatment was 8.6. On average, 27.7% urine samples showed positive opiate evidence. Marital status, employment status, treatment retention, self-satisfied evaluation score on dosage and dropout history were found to be significantly associated with heroin use, while gender, education level and dosage had no significant association with heroin use. It seemed that risk factors that associated with heroin use were different from areas to areas. CONCLUSION: High quality MMT clinic services, high self-satisfied score, longer treatment retention and low dropout rate seemed to have the effects of reducing the risk of ongoing heroin abuse under the methadone maintenance treatment program.

[Evaluation of first 8 pilot methadone maintenance treatment clinics in China].

OBJECTIVE: To evaluate the effectiveness of the first eight pilot methadone maintenance treatment (MMT) clinics in China. METHODS: A questionnaire survey of the clients at the 8 pilot MMT clinics was performed at entry, 6 month and 12 month follow-up. Drug using behaviors, drug related crime behaviors, and relationships in families were compared among at entry, 6 and 12 months follow-up. RESULTS: There were 585, 609 and 468 clients involved at baseline, 6 month and 12 month follow-up surveys, respectively. At entry, 6 month and 12 month follow-up, the proportion of clients whose injection of drugs reduced from 69.1% to 8.9% and 8.8%, and the frequency of injection in the past month had reduced from 90 times per month to 2 times per month. Employment rate increased from 22.9% to 43.2% and 40.6%, and self-reported criminal behaviors reduced from 20.7% to 3.6% and 3.8%. At 12 month follow-up, 65.8% of clients reported a healthy family relationship, increased from 46.8% at entry, 95.9% of clients reported that they were satisfied with the MMT service. CONCLUSION: Pilot MMT program reduced drug use, drug injecting behaviors, drug related criminal behaviors, and improved relationship with family members. Therefore, MMT clinic should be considered as a platform for providing comprehensive services to drug users.