Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction.
addiction; heroin; hnRNPH; opioid receptor; SNP; splicing
So far, a number of case-control or cohort studies have been carried out to investigate the relationship between rs759853 polymorphism in the promoter of aldose reductase (AR) gene and the risk of diabetic nephropathy (DN). However, the results have generated considerable controversy. We performed this study to clarify the linkage between this gene mutation and the risk of DN.
A comprehensive literature search of electronic databases and a well-organized meta-analysis were conducted.
Twelve comparisons and 4,735 individuals from nine published case-control or cohort studies were included finally. From none to large heterogeneity was observed, therefore, both fixed and random models were used. Significant differences were found between AR rs759853 polymorphism and susceptibility of DN from both type 1 and type 2 diabetes in all genetic models (allele contrast, OR = 1.37, CI (1.18, 1.59), P < 0.0001; additive model, OR = 1.78, CI (1.25, 2.53), P = 0.01; recessive model OR = 1.33 CI (1.08, 1.63), P = 0.008; dominant model, OR = 1.52, CI (1.26, 1.84), P < 0.0001; codominance model OR = 1.30 (1.15, 1.47), P < 0.0001). In stratified meta-analyses for type 2 diabetes by ethnicity, the significant relationship was found in allele contrast and dominant model in Caucasians, and in allele contrast and codominance model in Asians. However, data do not support the linkage between this gene mutation and the progression of DN. There was no significant publication bias.
The evidence currently available shows that the AR rs759853 polymorphism may correlate with the susceptibility of DN. However, data do not support the association between this DNA variation and the progression of DN.
Aldose reductase; Diabetic nephropathy; Meta-analysis
In China, the Compulsory Detoxification Centres are the main response for people who use illegal drugs. Due to high relapse rates among people released from the Compulsory Detoxification Centres, it is likely that they may seek medical help, including Methadone Maintenance Treatment (MMT) services, at some point. Therefore, better understanding of the attitudes and beliefs of people in the Compulsory Detoxification Centres can help to provide more adequate response to opioid dependence.
In total, 329 detained heroin users and 112 active MMT clients were recruited from a local Compulsory Detoxification Centre and MMT clinic, respectively. The survey contained specific questions relating to attitudes and beliefs regarding MMT.
Participants at the Compulsory Detoxification Centre and the MMT clinic expressed different opinions, regarding positive and negative attitudes and beliefs towards MMT. In addition, participants from both sites hold certain negative attitudes and beliefs about methadone despite their acknowledgement of the positive effects of MMT. Finally, participants at the Compulsory Detoxification Centre and the MMT clinic reported distinctive treatment preferences, with the former preferring community-based treatment and the latter MMT.
Developing targeted education about MMT for people at the Compulsory Detoxification Centres could help improve access to accurate and evidence-based health and treatment information. The study may also help providers understand and adjust services needed for target population in the future.
Methadone; Compulsory detoxification centre; China
G-quadruplexes (G4s) are four-stranded DNA secondary structures, which are involved in a diverse range of biological processes. Although the anti-cancer potential of G4s in oncogene promoters has been thoroughly investigated, the functions of promoter G4s in non-cancer-related genes are not well understood. We have explored the possible regulatory roles of promoter G4s in cardiac function-related genes using both computational and a wide range of experimental approaches. According to our bioinformatics results, it was found that potential G4-forming sequences are particularly enriched in the transcription regulatory regions (TRRs) of cardiac function-related genes. Subsequently, the promoter of human cardiac troponin I (TnIc) was chosen as a model, and G4s found in this region were subjected to biophysical characterisations. The chromosome 19 specific minisatellite G4 sequence (MNSG4) and near transcription start site (TSS) G4 sequence (−80 G4) adopt anti-parallel and parallel structures respectively in 100 mM KCl, with stabilities comparable to those of oncogene G4s. It was also found that TnIc G4s act cooperatively as enhancers in gene expression regulation in HEK293 cells, when stabilised by a synthetic G4-binding ligand. This study provides the first evidence of the biological significance of promoter G4s in cardiac function-related genes. The feasibility of using a single ligand to target multiple G4s in a particular gene has also been discussed.
Methamphetamine (METH) use, especially in females, has become a growing public health concern in China. In this study, we aimed to characterize the factors that contributed to drug craving in female METH users under isolated compulsory detoxification. We characterized factors contributing to craving such as duration of detoxification, history of drug use and self-reported mood state.
Subjects (N=113) undergoing a 1- to 3-year METH detoxification program were recruited from the Zhejiang Compulsory Detoxification Center for Women. The Questionnaire of METH-use Urge (QMU) was used to evaluate the level of craving for METH. The Abbreviate Profile of Mood States (A-POMS) was applied as an assessment for the negative mood disturbances.
The participants were at a mean age of 25.2, primarily lowly educated and unemployed, and single. Smoking was the only route of METH administration at an average dose of 0.5 g/day, and 4 times/week. The reported craving level was positively correlated with the negative mood disturbances and the weekly dose of METH, but independent of the duration of detoxification. Furthermore, all five aspects of negative mood disturbances, including fatigue, bewilderment, anxiety, depression and hostility, were shown to positively correlate to the self-reported craving level after controlling for weekly dose of METH.
The data demonstrate a robust correlation between mood distress and craving for METH. Our results call for close evaluation of mood distress in treatment of METH users in China.
Methamphetamine; Addiction; Craving; Mood distress; Detoxification
Relapse to drug seeking was studied using a rodent model of reinstatement induced by exposure to drug-related cues. Here, we used intravenous drug self-administration procedures in rats to further investigate the beneficial effects of electroacupuncture (EA) on heroin-seeking behavior in a reinstatement model of relapse. We trained Sprague-Dawley rats to nose-poke for i.v. heroin either daily for 4 h or 25 infusions for 14 consecutive days. Then the rats were abstinent from heroin for two weeks. 2 Hz EA stimulation was conducted once daily for 14 days during heroin abstinence. We tested these animals for contextual and discrete cue-induced reinstatement of active responses. We also applied immunohistochemistry to detect Fos-positive nuclei in the nucleus accumbens (NACc) core and shell after reinstatement test. We found that active responses elicited by both contextual cues and discrete cues were high in the rats trained with heroin than in saline controls. EA treatment significantly reduced active responses elicited by discrete cues. EA stimulation attenuated Fos expression in the core but not the shell of the NACc. Altogether, these results highlight the therapeutic benefit of EA in preventing relapse to drug addiction.
To date there has been limited research on both the prevalence of overdose and drug user knowledge about overdose prevention and response methods in China. In addition, there has been no effort to integrate naloxone information and distribution into pre-release services for drug users detained in isolated compulsory detoxification facilities in China.
The authors conducted a survey of 279 heroin users in isolated compulsory detoxification centers in Ningbo, China in an attempt to evaluate the possibility of conducting prelease peer naloxone programs in Ningbo isolated compulsory detoxification centers. Respondents' demographic background, history of heroin overdoses, and attitudes/knowledge about overdose prevention and response were collected.
While drug users in Ningbo's compulsory detoxification centers have limited understandings of how to effectively respond to overdoses, they expressed concern about the possibility of overdose, interest in participating in overdose prevention and response programs, and a willingness to help their peers. In general, there was no significant difference in history and attitudes/knowledge of overdose between male and female participants.
Based on the findings of this research, our survey provides preliminary evidence that detained drug users have considerable interest in overdose prevention and response information and willingness to help peers. However, drug users in Ningbo isolated compulsory detoxification centers currently have limited understandings of effective ways of helping to prevent overdose deaths.
Overdose prevention; Opiate addiction; Naloxone; Peer education compulsory detoxification centers
Previous studies show that the acute administration of N-acetylcysteine inhibits the desire for cocaine in addicts and cocaine seeking in animals
Rats were trained to self-administer heroin and the reinstatement model of drug seeking was used to determine if chronic N-acetylcysteine treatment inhibited heroin seeking.
Daily N-acetylcysteine administration inhibited cue- and heroin-induced seeking. Moreover, repeated N-acetylcysteine administration during extinction training reduced extinction responding and inhibited cue- and heroin-induced reinstatement for up to 40 days after discontinuing daily N-acetylcysteine injection.
These data show that daily N-acetylcysteine inhibits heroin-induced reinstatement and produces an enduring reduction in cue- and heroin-induced drug seeking for over a month after the last injection of N-acetylcysteine. Both the inhibitory effect of N-acetylcysteine on the reinstatement of heroin-seeking and the ability of N-acetylcysteine to reduce extinction responding support clinical evaluation of repeated N-acetylcysteine administration to decreases in drug seeking in heroin addicts.
heroin; N-acetylcysteine; reinstatement; addiction; self-administration; extinction
Functional assays are commonly used to measure the antibodies of ADAMTS13 found in patients of thrombotic thrombocytopenic purpura (TTP). In this study we used an enzyme-linked immunoassay to analyze the ADAMTS13-binding IgG levels in six groups of individuals: normal, random hospitalized patients, acute TTP,TTP after receiving plasma therapy, TTP in remission, and other types of thrombotic microangiopathy (TMA). The results showed thatADAMTS13-binding IgG levels were elevated in 100% of the acute TTP group, 75% of the TTP group after receiving plasma therapy, and 40% of the remission group. Overall, the ADAMTS13-binding IgG levels correlated with the inhibitory activity levels against ADAMTS13 (r=−0.69,P<0.0001). The assay also detected elevated IgG binding levels in 5% – 15% of the normal, random, and other TMA control groups. Addition of purified ADAMTS13 protein to the plasma samples suppressed the IgG binding in each of the acute TTP patients, but in none of the non-TTP groups. Serial measurement in a patient that had two exacerbations of TTP within the first three weeks revealed that the ADAMTS13 activity levels remained < 0.1 U/ml during this period, and the ADAMTS13-binding IgG remained elevated, suggesting thatADAMTS13 analysis may provide valuable insight to the disease status during the course of therapy. Analysis of ADAMTS13-binding IgG is helpful for the diagnosis and management of TTP.
ADAMTS13; thrombotic thrombocytopenic purpura; antibody
ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motifs), a circulating multidomain zinc metalloprotease of the reprolysin subfamily, is critical for preventing von Wille-brand factor-platelet interaction under high shear stress conditions. A deficiency of the protease, due to mutations in the ADAMTS13 gene or the presence of antibodies that inhibit the activity of the protease, causes thrombotic thrombocytopenic purpura (TTP). Plasma therapy, the conventional therapy for TTP, may cause serious adverse reactions and is ineffective in some patients. In order to develop new strategies for improving the diagnosis and treatment of TTP, we produced a series of truncated ADAMTS13 proteins in mammalian cells and analyzed their binding with and suppression by the IgG derived from the TTP patients. The results revealed that truncation of the ADAMTS13 protein at its cysteine-rich region eliminated its recognition by the antibodies without abolishing its von Willebrand factor-cleaving activity. This raises the possibility that resistant ADAMTS13 variants may be exploited to circumvent inhibitory antibodies that cause TTP.
ADAMTS13 is a circulating zinc metalloprotease that cleaves the hemostatic glycoprotein von Willebrand factor (VWF) in a shear-dependent manner. Deficiency in ADAMTS13, owing to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura (TPP). Northern blot analysis has shown that ADAMTS13 is expressed primarily in the liver. By using real-time RT-PCR, we confirmed that in mice the liver had the highest level of the ADAMTS13 transcript. To identify the liver cell-type-specific origin of ADAMTS13, we used in situ hybridization techniques to investigate the pattern of ADAMTS13 expression in the liver; analyzed the ADAMTS13 proteolytic activity in the culture media of fractionated liver cells; and confirmed ADAMTS13 expression with RT-PCR analysis and cloning of the mouse ADAMTS13 gene. The results revealed that ADAMTS13 was expressed primarily in cell fractions enriched in hepatic stellate cells. The mouse ADAMTS13 cloned from primary hepatic stellate cells was similar to its human counterpart in digesting VWF and was susceptible to suppression by EDTA or the IgG inhibitors of patients with TTP. Since hepatic stellate cells are believed to play a major role in the development of hepatic fibrosis and cirrhosis, the identification of the liver cell-type expressing ADAMTS13 will have important implications for understanding pathophysiological mechanisms regulating ADAMTS13 expression.
ADAMTS13; hepatic stellate cell; von Willebrand factor; metalloprotease
In a patient with fatal thrombotic thrombocytopenic purpura, the inhibitory activity of antibodies against ADAMTS13 rapidly escalated to extremely high levels despite daily plasma exchange and corticosteroid therapy. This increase was found to be due to a combination of higher antibody concentration and potency. Furthermore, during her course of the disease, the percentage of IgG1 antibody progressively decreased while that of IgG2 antibody increased, suggesting Th1-type cytokine response. These changes suggest that the course of TTP may be exacerbated by complex immune reactions. Further characterization of the factors contributing to this exacerbation may have important pathogenetic and therapeutic implications.
thrombotic thrombocytopenic purpura; von Willebrand factor; ADAMTS13; somatic hypermutation; antibody class switch
The involvement of cholinergic transmission in heroin self-administration and the reinstatement of heroin-seeking was examined in rats trained to nose-poke for intravenous heroin. Systemic treatment with physostigmine, an inhibitor of acetylcholinesterase, modestly reduced the acquisition and rate of heroin self-administration, and this suppression of heroin intake was reversed by pretreatment with scopolamine but not by mecamylamine. Following 10–14 days of self-administration, rats were left in the home environment for 14 days. Subsequently, rats were evaluated for extinction of nose-pokes during the first hour after being returned to the self-administration apparatus. One hr later a conditioned stimulus (house light, light in the nose-poke hole, sound of the infusion pump) was presented to initiate cue-induced reinstatement. Physostigmine produced a dose-dependent inhibition of cue-induced reinstatement, but only the dose of 0.5 mg/kg significantly decreased nose-poke responding in the extinction test. Chronic treatment with physostigmine (0.1 mg/kg) did not impair performance during acquisition of heroin self-administration. However, during a subsequent reinstatement test conducted in the absence of physostigmine pretreatment, heroin seeking was significantly below that of rats chronically pretreated with saline. To evaluate brain regions mediating the effects of systemic drug treatment on reinstatement, physostigmine was microinjected into the nucleus accumbens (NAc) or ventral tegmental area (VTA). Microinjection of physostigmine into the NAc prior to presenting conditioned cues inhibited the reinstatement of heroin-seeking, without affecting extinction responding. In contrast, microinjection of physostigmine into the VTA augmented the reinstatement induced by conditioned cues and extinction responding. Inactivation of either NAc or VTA by microinjecting tetrodotoxin blocked both extinction responding and cue-induced reinstatement. These data demonstrate that cholinergic transmission influences heroin self-administration and reinstatement. Moreover, cue-induced reinstatement was inhibited by physostigmine in the NAc and potentiated by cholinergic stimulation in the VTA.
rat; craving; abstinence; acetylcholinesterase inhibitor; reinstatement; conditioning
Severe deficiency of ADAMTS13, a von Willebrand factor cleaving metalloprotease, causes thrombotic thrombocytopenic purpura. When analyzed with VWF multimers, but not with an abbreviated VWF peptide (VWF73) as the substrate, the plasma ADAMTS13 activity levels of mouse strains segregated in a high and a low group that differed by approximately 10 folds. Low ADAMTS13 activity was detected in mice containing two alleles of IAP-type retrotransposon sequence in the ADAMTS13 gene. Molecular cloning of mouse ADAMTS13 identified two truncated variants (IAP-a and IAP-b) in the low activity mice. Both the IAP variants lacked the two carboxyl terminus thrombospondin type 1 repeat (TSR) and CUB domains of full-length ADAMTS13. The IAP-b variant also had splicing abnormalities affecting the spacer domain sequence and had miniscule enzymatic activity. Compared to full-length ADAMTS13, the IAP-a variant was approximately one ninth as active in cleaving VWF multimers but was only slightly less active in cleaving VWF73 peptide. Recombinant human ADAMTS13 was also less effective in cleaving VWF multimers than VWF73 when the C-terminal TSR sequence was deleted. In summary, the carboxyl terminus TSR sequence is important for cleaving VWF multimers. Assay results should be interpreted with caution when peptide substrates are used for analysis of variant ADAMTS13 proteins.
ADAMTS13; IAP; Mouse; Retrotransposon; von Willebrand factor
The activity of ADAMTS13, the von Willebrand factor cleaving protease, is deficient in patients with thrombotic thrombocytopenic purpura (TTP). In the present study, the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting. Normal plasma (n = 20) revealed a single band at 190 kD under reducing conditions using a polyclonal antibody, and a single band at 150 kD under non-reducing conditions using a monoclonal antibody. ADAMTS13 was not detected in the plasma from patients with congenital TTP (n = 5) by either antibody, whereas patients with acquired TTP (n = 2) presented the normal phenotype. Following immunoadsorption of immunoglobulins, the ADAMTS13 band was removed from the plasma of the patients with acquired TTP, but not from that of normal individuals. This indicates that ADAMTS13 is complexed with immunoglobulin in these patients. The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis, impaired cellular secretion, or enhanced degradation in the circulation. This study differentiated between normal and TTP plasma, as well as between congenital and acquired TTP. This method may, therefore, be used as a complement in the diagnosis of TTP.
ADAMTS13; von Willebrand factor; Thrombotic thrombocytopenic purpura; Immunoblotting; Plasma; von Willebrand factor cleaving protease