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Immune-modulatory therapy, especially with immune-checkpoint inhibitors (ICIs), has reshaped cancer therapeutics. Immunotherapy is relatively a novel approach that can effectively delay the progression of aggressive tumors and inhibit tumor recurrence and metastasis in many different tumor types. In the past years, ICIs have shown a sustained response and promising long-term survival in patients with advanced hepatocellular carcinoma (HCC). Nevertheless, ICI therapy can unbalance the immune system and result in a wide range of immune-related adverse events (irAEs), which are generally manageable but occasionally lead to a fatal outcome. HCC generally develops in the context of liver cirrhosis which is typically caused by viral hepatitis and non-alcoholic steatohepatitis. These underlying diseases may cause symptoms that overlap with irAEs and lead to consequences such as late recognition, inadequate work-up, and inappropriate treatment. Owing to the growing use of immunotherapy in HCC, it is necessary for clinicians to strengthen their understanding of the frequency, clinical features, and management of irAEs. This review focuses on the common toxicities associated with ICI therapy in patients with HCC and summarizes therapeutic strategies that can be used to monitor and manage such toxicities.

Schizophrenia is a complex mental illness characterized by positive and negative symptoms. Antipsychotic medications are the main treatment for this condition; however, many patients have only a partial response. All currently available antipsychotics involve modulation of the dopamine system, although the underlying pathophysiology of this illness remains poorly understood. Increasing evidence suggests that inflammation plays a significant role in the development of schizophrenia. Thus, a medication that modulates both dopamine signaling and inflammation may be a promising treatment for schizophrenia. L-tetrahydropalmatine (l-THP) has modest affinity for dopamine receptors, acting as an antagonist at D1, D2, and D3 receptors. L-THP also has anti-inflammatory effects, decreasing levels of tumor necrosis factor-α and interleukin 6. We performed a 4-week, randomized, double-blind, placebo-controlled trial of oral l-THP (30 mg twice daily) as an adjunctive treatment in 63 adults with schizophrenia (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria) on stable antipsychotic medication. The primary psychiatric outcome measures were the presence of positive and negative symptoms. Plasma concentrations of 11 cytokines and C-reactive protein (CRP) were also measured. L-THP had no significant effect on any psychiatric measure but was well tolerated and associated with a significant decrease in extrapyramidal symptoms. L-THP significantly increased plasma concentrations, compared with placebo, of two inflammatory markers: soluble intracellular adhesion molecule 1 and CRP. These findings do not support the efficacy of L-THP as an add-on treatment for schizophrenia but suggest that L-THP may have previously unrecognized proinflammatory effects in people with this disease.

The etiology and pathophysiology underlying opioid tolerance and dependence are still unknown. Because mu opioid receptor (MOR) plays an essential role in opioid action, many vulnerability-related studies have focused on single nucleotide polymorphisms of MOR, particularly on A118G. In this study, we found that a single-point mutation at the MOR T394 phosphorylation site could be another important susceptive factor in the development of opioid tolerance and dependence in mice. T394A mutation, in which a threonine at 394 was replaced by an alanine, did not alter agonist binding to MOR and opioid analgesia, but resulted in loss of etorphine-induced MOR internalization in spinal dorsal horn neurons and opioid analgesic tolerance induced by either morphine or etorphine. In addition, this mutation also caused an increase in intravenous heroin self-administration and in nucleus accumbens dopamine response to heroin. These findings suggest that T394 phosphorylation following MOR activation causes MOR internalization and desensitization, which subsequently contributes to the development of tolerance in both opioid analgesia and opioid reward. Accordingly, T394A mutation blocks opioid tolerance and leads to an increase in brain dopamine response to opioids and in opioid-taking behavior. Thus, the T394 may serve as a new drug target for modulating opioid tolerance and the development of opioid abuse and addiction.SIGNIFICANCE STATEMENTThe mechanisms underlying opioid tolerance and susceptibility to opioid addiction remain unclear. The present studies demonstrate that a single-point mutation at the T394 phosphorylation site in the C-terminal of mu opioid receptor (MOR) results in loss of opioid tolerance and enhanced vulnerability to heroin self-administration. These findings suggest that modulation of the MOR-T394 phosphorylation or dephosphorylation status may have therapeutic potential in management of pain, opioid tolerance, and opioid abuse and addiction. Accordingly, MOR-T394 mutation or polymorphisms could be a risk factor in developing opioid abuse and addiction and therefore be used as a new biomarker in prediction and prevention of opioid abuse and addiction.

Cocaine use disorder (CUD) remains a significant public health challenge. l-Tetrahydropalmatine (l-THP), a well-tolerated and nonaddictive compound, shows promise for the management of CUD. Its pharmacologic profile includes blockade at dopamine and other monoamine receptors and attenuation of cocaine self-administration, reinstatement, and rewarding properties in rats. This study evaluated the safety of l-THP in human cocaine users and its influence on the safety and pharmacokinetics (PK) of cocaine. Twenty-four cocaine-using adult men were randomized to receive l-THP (30 mg twice a day orally) or placebo double-blind for 4 days, with an intranasal cocaine (40 mg) challenge on the fourth day. Safety and tolerability were evaluated using vital signs, ECG, clinical laboratory tests, and standardized self-report instruments. Peripheral venous blood was collected periodically and later assayed for l-THP and cocaine using highly sensitive and specific ultraperformance liquid chromatography-fluorescence detection (UPLC-FLD) methods. Twenty subjects completed the study, of whom 19 provided complete PK data. The short 3.5-day course of l-THP was safe and well tolerated and did not affect cocaine's PK or its acute cardiovascular effects. The cocaine AUC0→∞ was 211.5 and 261.4 h·ng/mL, and the Cmax was 83.3 and 104.5 ng/mL for the l-THP and placebo groups, respectively. In addition there were no significant differences in the number of side effects reported in each group (l-THP group 22 [48%], placebo group 24 [52%]) or vital signs including, heart rate, blood pressure, complete blood count, or ECG. These findings suggest that oral THP has promise for further development as a treatment for CUD.

Social isolation (SI) rearing has been demonstrated to induce behavioral abnormalities like anxiety, impulsivity, aggression, and learning and memory deficits which are relevant to core symptoms in patients with some certain neuropsychiatric disorders. But the underlying pathophysiological mechanisms remain unclear. Recent studies have revealed HINT1 has close relation with diverse neuropsychiatric diseases. In this present study, the SI rearing mice exhibited depression-like and aggressive behavior. Besides, HINT1 protein levels decreased in PFC but increased in HIP. Based on the data obtained, we concluded that HINT1 is involved in the behavioral abnormalities induced by social isolation and exerts distinct roles in different encephalic regions.

Previous studies have implicated a role of the histidine triad nucleotide-binding protein 1 (Hint1) in the pathogenesis of schizophrenia. Protein kinase C gamma (PKCγ) could be potentially involved in the Hint1-implicated pathogenesis since PKCγ was identified as a Hint1 interacting protein. Recently, a debate was brought forward from the understanding how Hint1 affects the expression and activity of PKCγ in the brain. In the present study, we use Hint1 knockout mice and biochemical analysis to define the effect of Hint1 on protein PKCγ. Our data reveal that Hint1-deficiency in mouse brains led to increased protein levels of PKCγ in the cortex and hippocampus, the striatum and thalamus and amygdala. Without stimulation, PKCγ protein in Hint1-deficient brain displayed a basal activity that was reflected by control-leveled phosphorylations of PKCγ T514 and T674 at its kinase domain. Upon psycho-stimulation, both sites of PKCγ T514 and T674 were activated in these brain structures via phosphorylation; however, the phosphorylation level at the site of PKCγ T674 apparently attenuated in Hint1-deficient mice compared to wild-type control. Thus, we conclude that Hint1 deficiency leads to an increased protein level of PKCγ in the brain and a compromised activation response of PKCγ upon stimulation. These findings suggest an inhibitory role of Hint1 on the protein PKCγ in the brain and an impaired PKCγ-mediated phosphorylation signal in Hint1-deficient neuron.

The histidine triad nucleotide binding protein1(HINT1),which belongs to the histidine triad(HIT) enzyme superfamily,exerts its enzymic activities as hydrolase or transferase. Its physiological functions are still unclear. HINT1 protein is expressed in various tissues and plays an important role in transcription and signal transduction. Earlier studies have identified HINT1 as a haplo-insufficient tumor suppressor. Other evidences indicate that HINT1 is involved in a wide variety of physiological processes,some of which are irrelevant with its basic enzymic activities. Investigations recently suggest that HINT1 is closely related to many peripheral and central nervous system diseases,and plays a vital role in some of neuropsychiatric diseases such as inherited peripheral neuropathies,schizophrenia,mood disorder,drug addiction,and Down's syndrome. In this review,the role of HINT1 in above-mentioned neuropsychiatric disorders was summarised,and the research findings of HINT1 in each of the above diseases were summarized and analyzed,in order to provide some guidance for further research on this protein.三联组氨酸核苷结合蛋白1(HINT1)是三联组氨酸(HIT)酶家族成员,作为核苷水解酶或转移酶起作用,其生物学功能尚不清楚。HINT1蛋白可在多种组织中表达,在转录和信号转导中发挥重要作用。早期研究已经明确HINT1是单倍剂量不足的肿瘤抑制因子;也有研究表明HINT1广泛参与一系列生理过程,且部分功能与其基本的酶活性无关;新近研究表明这一蛋白与诸多外周、中枢神经系统疾病存在密切关系,在遗传性周围神经病、精神分裂症、心境障碍、药物成瘾、唐氏综合征等神经精神疾病中起作用。本文针对HINT1蛋白在以上不同神经精神疾病中的作用做了详细综述,分析归纳了HINT1在上述每一种疾病中的研究发现,以期对今后该蛋白的深入研究起到一定的指导作用。.

Currently, there are no FDA approved medications for treatment of cocaine addiction underscoring the dire need to develop such a product. There is an accumulating body of evidence that l-tetrahydropalmatine (l-THP), a non-selective dopamine antagonist, can be used for the treatment of cocaine addiction. Indeed, the FDA recently approved its usage in a Phase I study in cocaine abusers and it was indispensable to develop a simple and sensitive method for the simultaneous determination of l-THP and cocaine in human plasma. We developed a UPLC-FLD method for quantitation of these molecules using an ACQUITY BEH C18 column (2.1 mm × 50mm, 1.7 μm) and a mobile phase that consisted of 10mM ammonium phosphate (pH=4.75), methanol, and acetonitrile (v:v:v, 78:16:6). Venlafaxine was used as the internal standard while hexane was used for the liquid-liquid extraction. The flow rate was 0.4 mL/min with fluorescence detection using an excitation wavelength of 230 nm and emission detection wavelength of 315 nm. This method was selective, linear and sensitive with a lower limit of quantification of 2.5 ng/mL for both cocaine and l-THP. The intra-day precision of cocaine and l-THP was

In the early stage of deep-water exploration, the research about subtle trap prediction is mostly confined to technologies such as seismic attribute, due to which ambiguity and uncertainty in the results can occur. Therefore, in this paper, a new process of identifying deep-water reservoirs is proposed. First, strata slice technology is adopted to guide the analysis of seismic facies and preliminary recognition of the distribution and scale of geologic bodies; then based on the seismic facies analysis, the reservoir is depicted in detail with the application of seismic phase-controlled nonlinear random inversion; and finally, the comprehensive analysis of various features like strata slices, seismic facies, and seismic inversion are used to predict the favorability of the reservoir. The results show that the application of this method not only successfully predicts the distribution of the reservoir but also provides a reliable basis for future well drilling.

A synthetic monoketone analog of curcumin, termed 3, 5-bis (2-flurobenzylidene) piperidin-4-one (EF24), has been reported to inhibit the growth of a variety of cancer cells both in vitro and in vivo. However, whether EF24 has anticancer effects on cholangiocarcinoma (CCA) cells and the mechanisms remain to be investigated. The aim of our study was to evaluate the molecular mechanisms underlying the anticancer effects of EF24 on CCA tumor growth and metastasis. Cell proliferation, apoptosis, migration, invasion, tumorigenesis and metastasis were examined. EF24 exhibited time- and dose-dependent inhibitory effects on HuCCT-1, TFK-1 and HuH28 human CCA cell lines. EF24 inhibited CCA cell proliferation, migration, and induced G2/M phase arrest. EF24 induced cell apoptosis along with negative regulation of NF-κB- X-linked inhibitor of apoptosis protein (XIAP) signaling pathway. XIAP inhibition by lentivirus mediated RNA interference enhanced EF24-induced apoptosis, while XIAP overexpression reduced it in CCA cells. In vivo, EF24 significantly suppressed the growth of CCA tumor xenografts and tumor metastasis while displaying low toxicity levels. Our findings indicate that EF24 is a potent antitumor agent that inhibits tumor growth and metastasis by inhibiting NF-κB dependent signaling pathways. EF24 may represent a novel approach for CCA treatment.

Cocaine use disorder (CUD) remains a significant public health challenge. Levo-tetrahydropalmatine (L-THP), a well-tolerated and non-addictive compound, shows promise for the management of CUD. Its pharmacologic profile includes blockade at dopamine and other monoamine receptors and attenuation of cocaine self-administration, reinstatement, and rewarding properties in rats. This study evaluated the safety of L-THP in human cocaine users and its influence on the safety and pharmacokinetics (PK) of cocaine. Twenty-four cocaine-using adult men were randomized to receive L-THP (30 mg BID orally) or placebo double-blind for 4 days, with an intranasal cocaine (40 mg) challenge on the fourth day. Safety and tolerability were evaluated using vital signs, EKG, clinical laboratory tests, and standardized self-report instruments. Peripheral venous blood was collected periodically and later assayed for L-THP and cocaine using highly sensitive and specific ultra-performance liquid chromatography-fluorescence detection (UPLC-FLD) methods. Twenty subjects completed the study, of whom 19 provided complete PK data. The short 3.5-day course of L-THP was safe and well tolerated and did not affect cocaine’s PK or its acute cardiovascular effects. The cocaine AUC0→∞ was 211.5 and 261.4 h*ng/ml, and the Cmax was 83.3 and 104.5 ng/ml for the L-THP and placebo groups respectively. In addition there were no significant difference in the number of side effects reported in each group (L-THP group: 22 [48%], Placebo group: 24 [52%]), or vital signs including, heart rate, blood pressure, complete blood count or EKG. These findings suggest that oral THP has promise for further development as a treatment for CUD.

The three parameters of P-wave velocity, S-wave velocity, and density have remarkable differences in conventional prestack inversion accuracy, so study of the consistency inversion of the “three parameters” is very important. In this paper, we present a new inversion algorithm and approach based on the in-depth analysis of the causes in their accuracy differences. With this new method, the inversion accuracy of the three parameters is improved synchronously by reasonable approximations and mutual constraint among the parameters. Theoretical model calculations and actual data applications with this method indicate that the three elastic parameters all have high inversion accuracy and maintain consistency, which also coincides with the theoretical model and actual data. This method has good application prospects.

UNLABELLED Photodynamic therapy (PDT) is an effective local cancer treatment when aphotosensitizer is administered and the tumor is irradiated with light. We examined the effect of PDT using HMME as the photosensitizer, and the 630nm diode laser on human cholangiocarcinoma cell line QBC939. Cell viability was determined by MTT assay. The percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Two methods were used for the determination of apoptosis: terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay and laser scanning confocal microscope detection. The procaspase-3 and cytochrome cwere measured by western blot. In vitro PDT showed excellent cytotoxicity that was afunction of laser energy and drug concentration to the QBC939 cell lines. PDT-mediated cell death occurred predominantly by apoptosis in vitro. Furthermore, this treatment initiates early cytochrome crelease, followed by late procaspase-3 activation. Our study demonstrates that PDT using HMME and the diode laser induces apoptosis that is mediated by cytochrome crelease and caspase activation in human cholangiocarcinoma cell lines. It is expected that this therapy would be clinically useful for the treatment of patients with cholangiocarcinoma. KEYWORDS Hematoporphyrin monomethyl ether, photodynamic therapy, apoptosis, cholangiocarcinoma.

A general strategy for the design of dual labeled peptides was developed, and derivatives of the delta opioid receptor (DOR) selective antagonist TIPP (Tyr-Tic-Phe-PheOH) containing both an affinity label and biotin were prepared by solid-phase synthesis. Tyr-Tic-Phe-Phe(p-X)-Asp-NH(CH2CH2O)2-CH2CH2NH-biotin (where X = N=C=S or NHCOCH2Br) exhibit nanomolar DOR affinity. The ability to detect receptors labeled with these peptides following solubilization and SDS-PAGE demonstrate the applicability of this design approach for dual labeled peptide derivatives.

The purpose of this study was to determine whether ultrasound exposure combined with microbubble destruction could be used to enhance non-viral gene delivery in human pancreatic carcinoma cells (PANC-1).The study was performed with four experimental groups: Group P, plasmid alone; Group P+M, plasmid and microbubbles; Group P+U, plasmid and ultrasound; Group P+U+M, plasmid with ultrasound and microbubbles. Plasmid DNA encoding enhanced green fluorescent protein (pEGFP) was gently mixed with commercially available ultrasound microbubble contrast agents (SonoVue; Bracco Diagnostics Inc, Milan, Italy) in Group P+M and Group P+U+M. The different combinations of DNA and DNA plus microbubbles were added to cultured PANC-1 cells under different conditions. Transfection efficiency and cell viability were assessed by FACS analysis (Becton Dickinson, San Jose, CA, USA), confocal laser scanning microscopy, and trypan blue staining.The results demonstrated that microbubbles with ultrasound exposure could significantly enhance the reporter gene expression as compared with other groups (Group P+U+M, 21.4%+/-3.16%; Group P, 2.9%+/-0.45%; Group P+M, 3.1%+/-0.51%; Group P+U, 6.1%+/-1.27%; P0.01). No statistically significant difference was observed in the PANC-1 cell viability between Group P+U+M and other groups (P0.05).Our in-vitro findings suggest that ultrasound-mediated microbubble destruction has the potential to promote efficient gene transfer into PANC-1 cells without significant cell death. This non-invasive gene transfer method may be a useful tool for safe clinical gene therapy of pancreatic cancer in the future.

This study was to characterize the effects of a point-mutant at C-terminal of mu opioid receptor (MOR), namely MORT394A, in chronic opioid-induced cellular responses. After 18 h of exposure to [D-Ala 2 , N-Me-Phe 4 , Gly-ol 5 ] enkephalin (DAMGO), adenylyl cyclase (AC) superactivation, a hallmark for the cellular adaptive response after chronic opioid stimulation, was observed in the cells expressing wild-type receptor, but was totally abolished in the cells expressing MOR T394A. Receptor phosphorylation was also attenuated in cells with MOR T394A after prolonged preexposure to agonist. Furthermore, MAP kinase kinase-1 (MKKI) overexpression was able to rescue AC superactivation in cells with MOR T394A, but showed no effect in the wild-type MOR-expressing cells. These results indicated that the amino acid T394 at C-terminus of MOR played a critical role in chronic agonist-induced AC superactivation and receptor phosphorylation.

Protein kinase C interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) is a member of the histidine triad protein family. Although this protein is widely expressed in the mammalian brain including mesocorticolimbic and mesostriatal regions, its physiological function in CNS remains unknown. Recent microarray studies reported decreased mRNA expression of PKCI/HINT1 in the frontal cortex of individuals with schizophrenia, suggesting the possible involvement of this protein in the pathophysiology of the disease. In view of the documented link between dopamine (DA) transmission and schizophrenia, the present study used behavioral and neurochemical approaches to examine the influence of constitutive PKCI/HINT1 deletion upon: (i) basal and amphetamine (AMPH)-evoked locomotor activity; (ii) DA dynamics in the dorsal striatum, and (iii) postsynaptic DA receptor function. PKCI/HINT1(-/-) (KO) mice displayed lower spontaneous locomotion relative to wild-type (WT) controls. Acute AMPH administration significantly increased locomotor activity in WT mice; nonetheless, the effect was enhanced in KO mice. Quantitative microdialysis studies revealed no alteration in basal DA dynamics in the striatum or nucleus accumbens of KO mice. The ability of acute AMPH to increase DA levels was unaltered indicating that function in presynaptic DA neurotransmission in these regions do not underlie the behavioral phenotype of KO mice. In contrast to WT mice, systemic administration of the direct-acting DA receptor agonist apomorphine (10 mg/kg) significantly increased locomotor activity in KO mice suggesting that postsynaptic DA function is altered in these animals. These results demonstrate an important role of PKCI/HINT1 in modulating the behavioral response to AMPH. Furthermore, they indicate that the absence of this protein may be associated with dysregulation of postsynaptic DA transmission.

The magnocellular medial preoptic nucleus plays a crucial role in the regulation of male sexual behavior in Syrian hamsters. Histological and behavioral studies suggest that the magnocellular medial preoptic nucleus regulates male mating behavior by integrating chemosensory and hormonal signals. The present study is the first to systematically identify the afferent connections of the magnocellular medial preoptic nucleus by tracing the uptake of cholera toxin B from deposits in the magnocellular medial preoptic nucleus of adult male Syrian hamsters. Our findings indicate that the magnocellular medial preoptic nucleus receives 1) chemosensory input from areas in the main and accessory olfactory pathways including the posterior medial bed nucleus of the stria terminalis, anterior medial, anterior cortical and posterior cortical nuclei of the amygdala; 2) input from steroid responsive structures such as the posterior medial nucleus of the amygdala, bed nucleus of the stria terminalis, lateral septum, anteroventral periventricular nucleus, medial preoptic nucleus, ventromedial nucleus of the hypothalamus and arcuate nucleus; 3) input from structures in the brainstem such as the subparafascicular thalamic nucleus, peripeduncular nucleus and the premamillary nucleus in the hypothalamus that carry sensory information from the genitalia. The major afferent input to the magnocellular medial preoptic nucleus was confirmed by injecting anterograde tracer biotinylated dextran amine into the anterior medical nucleus of the amygdala, the posterodorsal part of the medial nucleus of the amygdala, the posteromedial part of the bed nucleus of the stria terminalis and the posterointermediate part of the bed nucleus of the stria terminalis. Our results support the hypothesis that the magnocellular medial preoptic nucleus is part of the chemosensory pathway that receives chemosensory and hormonal input to regulate mating behavior and suggest that the magnocellular medial preoptic nucleus may utilize information from the genitalia to regulate male mating behavior.

A growing body of literature indicates that chronic morphine exposure alters the expression and function of cytoskeletal proteins in addition to the well established interactions between mu opioid receptors and G proteins. In the present study, we hypothesized that chronic morphine alters the expression and functional effects of G alpha12, a G protein that regulates downstream cytoskeletal proteins via its control of RhoA. Our results showed that chronic morphine treatment decreased the expression of G alpha i2 (64%) and G alpha i3 (60%), had no effect of G alpha o, and increased G alpha12 (66%) expression in Chinese hamster ovary (CHO) cells expressing the cloned human mu opioid receptors (hMOR-CHO cells) but not in cells expressing a mutant mu opioid receptor that do not develop morphine tolerance and dependence (T394A-CHO cells). Morphine treatment had no significant effect on PAR-1 thrombin receptor-activated G protein activity, as measured by thrombin-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate binding. Chronic morphine treatment significantly enhanced thrombin-stimulated RhoA activity and thrombin-stimulated expression of alpha-actinin, a cytoskeletal anchoring protein, in hMOR-CHO cells. Proteomic analysis of two-dimensional gel spots prepared from hMOR-CHO cells showed that morphine treatment affected the expression of a number of proteins associated with morphological changes. Up-regulation of G alpha12 and alpha-actinin by chronic morphine was also observed in mouse brain. Viewed collectively, these findings indicate, for the first time, that chronic morphine enhances the G alpha12-associated signaling system, which is involved in regulating cellular morphology and growth, supporting other findings that chronic morphine may alter cellular morphology, in addition to cellular function.

The human mu-opioid receptor (HmuOR) is a G-protein coupled receptor that mediates analgesia, euphoria and other important central and peripheral neurological functions. In this study, we found in a yeast two-hybrid screen that a protein kinase C-interacting protein (PKCI) specifically interacts with the C terminus of HmuOR. The interaction of PKCI with HmuOR was recapitulated in Chinese hamster ovary cells that express the full-length HmuOR and PKCI proteins. The affinity of HmuOR for an opioid ligand and its ability to mediate the activation of a G-protein were not altered by their interaction. However, the association of PKCI with HmuOR reduced agonist-induced inhibition of adenylyl cyclase and suppressed HmuOR desensitization partially at the G protein level and completely at the adenylyl cyclase level. Furthermore, PMA-induced, but not DAMGO-induced, HmuOR phosphorylation was partially inhibited by the coexpression of PKCI, suggesting that PKCI exerts a selective regulatory effect on HmuOR signaling. This effect was specific to the mu-opioid receptor because delta-opioid receptor desensitization was unaffected by PKCI. In addition, behavioral studies revealed that both basal and morphine-induced analgesia were significantly enhanced in the mutant mice that lacked expression of PKCI gene, and these mice developed a greater extent of tolerance to morphine analgesia. Taken together, these results suggest that PKCI functions as a negative regulator in HmuOR desensitization, phosphorylation, and in mediating morphine analgesia.

Sugar amino acids (SAAs) bearing an amine group on the anomeric position are a challenging class of SAAs to synthesize due to the inherent instability of glycosylamines. We developed a novel synthetic strategy towards both furanoid and pyranoid δ-SAAs of this type, based on a Curtius rearrangement. The latter reaction, which is known to proceed with retention of configuration, was performed on carboxylic acids derived from the oxidation of glycosidic primary hydroxyls. Leu-enkephalin analogs were prepared by replacing the Gly-Gly moiety in the parent Leu-enkephalin pentapeptide with the furanoid and pyranoid δ-SAA. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Histidine triad nucleotide-binding protein 1 (HINT1) is a member of a superfamily of histidine triad proteins named by the conserved nucleotide-binding motif histidine-x-histidine-x-histidine-xx, in which x represents hydrophobic amino acid. HINT1 is implicated in pathological progress of many human diseases including cancer and schizophrenia; however, little is known about the essential role and pathological consequences of HINT1 in cellular physiology and diseases. Therefore, we summarize the structure, distribution, and physiological function of HINT1 in cells and tissues as well as the correlation between HINT1 and human diseases.

By metabolically labeling tissue slices from striatum and thalamus with [32P]orthophosphoric acid and immunoprecipitating the receptor with mu receptor-specific antiserum, we found that the endogenous mu receptor in the brain tissue did undergo phosphorylation. The phosphorylation occurred at basal level (no drug treatment) and was enhanced with DAMGO-treatment. The enhancement of the phosphorylation was blocked by naloxone. Morphine stimulation also increased the phosphorylation, but the amount of enhancement was less than that caused by DAMGO-treatment. Mu receptor phosphorylation in the thalamus was much greater than the striatum, while no phosphorylation of the mu receptor in the cerebellum was detected, even with DAMGO treatment. The extent of mu receptor phosphorylation identified in the thalamus, striatum and cerebellum is consistent with the previous studies of mu receptor distribution. The time course and dose-response studies demonstrated that mu receptor phosphorylation was a rapid event, exhibited a positive dose-dependent response, and was similar to that observed in the cloned mu receptor in CHO cells. Furthermore, we correlated the change of mu receptor phosphorylation with the desensitization of the mu receptor function, specifically, inhibition of adenylyl cyclase activity in the thalamus of morphine-tolerant rats. We found that in the thalamus of rats chronically treated with morphine, the enhancement of mu receptor phosphorylation in basal and DAMGO-treated samples paralleled the desensitization of DAMGO-mediated inhibition of adenylyl cyclase. Our results suggest that mu receptor phosphorylation in vivo may play an important role in the modulation of mu receptor function following both acute exposure to morphine and during the development of morphine tolerance.

Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.

HINT1 is a mouse histidine triad nucleotide binding protein. Here we report the assignments for the backbone nitrogen, carbon and proton NMR signals.

The μ opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [d-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation. We have previously shown that the Thr394 in the carboxyl tail is essential for agonist-induced early desensitization, presumably by serving as a primary phosphorylation site for G protein-coupled receptor kinase. Using a T394A mutant receptor, we determined that Thr394 was also responsible for μ opioid receptor down-regulation. The T394A mutant receptor displayed 50% reduction of receptor down-regulation (14.8%) compared with wild type receptor (34%) upon 1 h of exposure to DAMGO. Agonist-induced T394A receptor down-regulation was unaffected by pertussis toxin treatment, indicating involvement of a mechanism independent of G protein function. Interestingly, pertussis toxin-insensitive T394A receptor down-regulation was completely inhibited by a tyrosine kinase inhibitor, genistein. Tyrosine kinase inhibition blocked wild type MOR down-regulation by 50%, and the genistein-resistant wild type MOR down-regulation was completely pertussis toxin-sensitive. Following DAMGO stimulation, MOR was shown to be phosphorylated at tyrosine residue(s), indicating that the receptor was a direct substrate for tyrosine kinase action. Mutagenesis of the four intracellular tyrosine residues resulted in complete inhibition of the G protein-insensitive MOR internalization. Therefore, agonist-induced MOR down-regulation appears to be mediated by two distinct cellular signal transduction pathways. One is G protein-dependent and GRK-dependent, which can be abolished by pertussis toxin treatment of wild type MOR or by mutagenesis of Thr394. The other novel pathway is G protein-independent but tyrosine kinase-dependent, blocked by genistein treatment, and one in which Thr394 has no regulatory role but phosphorylation of tyrosine residues appears essential.

Previous data obtained with the cloned rat mu opioid receptor demonstrated that the "super-potent" opiates, ohmefentanyl (RTI-4614-4) and its four enantiomers, differ in binding affinity, potency, efficacy, and intrinsic efficacy. Molecular modeling (Tang et al., 1996) of fentanyl derivatives binding to the mu receptor suggests that Asp147, Tyr148, Trp318, and His319 are important residues for binding. According to this model, Asp147 interacts with the positively charged opiate agonist to form potent electrostatic and hydrogen-bonding interactions. In this study, the role of weak electrostatic and hydrogen-bonding "pi-pi" interactions of the O atom of the carbonyl group and the phenyl ring structures of RTI-4614-4 and its four enantiomers with residues Tyr148, Trp318, and His319 were explored via site-directed mutagenesis. Tyr148 (in transmembrane helix 3 {TMH3}), Trp318 (TMH7), and His319 (TMH7) were individually replaced with phenylalanine or alanine. Receptors transiently expressed in COS-7 cells were labeled with [125I]IOXY according to published procedures. Mutation of Tyr148 to phenylalanine reduced the binding affinities of some mu-selective agonists (2-7 fold) but did not alter the affinities of DAMGO, naloxone, and the non-selective opiates etorphine and buprenorphine. In contrast, this mutation significantly increased the binding affinities (decreased the Kd values) of [D-Ala2,D-Leu5]enkephalin, IOXY, and dermorphin. Mutation of Trp318 decreased opioid receptor binding to almost undetectable levels. Substitution of alanine for His319 significantly reduced binding affinities for the opioid ligands tested (1.3- to 48-fold), but did not alter the affinities of naloxone and bremazocine. These results indicate the importance of Tyrl48 and His319 for the binding of fentanyl derivatives to the mu receptor. Functional studies using the mutant receptors will provide additional insight into the mechanism of action of RTI-4614-4 and its four enantiomers.

The orvinols are a class of potent opioids which have been extensively studied, yet little is known about the effects of introducing substituents into the 18- and 19-positions. The etheno bridge of thevinone was hydroxylated to give both the 18- and 19-hydroxyl substituted thevinols. After 3-O-demethylation to the corresponding orvinols, binding and GTPgammaS functional assays indicated that hydroxyl substitution at the 18- and 19-positions differentially affects the mu opioid efficacy of orvinols.

Many motor effects of opiates acting at mu-opioid receptors are thought to reflect functional interactions with dopaminergic inputs to the caudate-putamen nucleus. We examined the cellular and subcellular bases for this interaction in the rat caudate-putamen nucleus by dual immunocytochemical labelling for mu-opioid receptors and tyrosine hydroxylase, a marker mainly for dopamine in this region. mu-Opioid receptor-like immunoreactivity showed a patchy distribution by light microscopy. Within the patches, electron microscopy revealed that immunogold labelling for mu-opioid receptors was mainly distributed along extrasynaptic plasma membranes of medium spiny neurons. In contrast, immunoperoxidase labelling for tyrosine hydroxylase was exclusively located in axons and axon terminals without detectable mu-opioid receptor-like immunoreactivity. Forty-six percent of the total mu-opioid receptor-labelled neuronal profiles (n = 1441) were in contact with tyrosine hydroxylase-immunoreactive axons and terminals. These contacts were characterized by closely apposed parallel plasma membrane segments, without well-defined synaptic junctions, or with punctate symmetric specializations. From 639 noted appositions, over 90% were between mu-opioid receptor-labelled dendrites and/or dendritic spines and tyrosine hydroxylase-containing terminals. The dendritic spines containing mu-opioid receptor-like immunoreactivity often received asymmetric synapses characteristics of excitatory inputs from unlabelled terminals. Axon terminals containing mu-opioid receptor-like immunoreactivity formed asymmetric synapses with dendritic spines, or apposed tyrosine hydroxylase-labelled terminals. Our results suggest that, in striatal patch compartments, mu-agonists and dopamine dually modulate the activity of single spiny neurons mainly through changes in their postsynaptic responses to excitatory inputs. In addition, our findings implicate mu-opioid receptors and dopamine in the presynaptic regulation of excitatory neurotransmitter release within the striatal patch compartments.

Recent findings indicate that histidine triad nucleotide-binding protein 1 (HINT1) is implicated in the pathophysiology of certain psychiatric disorders and also exhibits tumor suppressor properties. However, the authentic functions of HINT1 in cellular physiology and especially its role in Ca2+signaling remain unclear. Here, we studied Ca2+signaling in cultured embryonic fibroblasts derived from wild-type control and HINT1 knockout (KO) mice. The resting cytosolic Ca2+level (measured with fura-2) was not altered in fibroblasts lacking HINT1. The stored Ca2+evaluated by measuring peak amplitude of ATP (10 μM)-induced Ca2+transients in Ca2+-free medium was significantly larger in HINT1 KO fibroblasts than in wild-type cells. Ca2+influx after external Ca2+restoration, likely via store- and receptor-operated channels (SOCs and ROCs, respectively), was greatly (by 2-fold) reduced in HINT1 KO fibroblasts. This correlated with a downregulated expression of Orai1 and stromal interacting molecule 1 (STIM1), essential components of store-operated Ca2+entry pathway. Expression of canonical transient receptor potential (TRPC)3 and TRPC6, which function as ROCs, was not altered in HINT1 KO fibroblasts. Immunoblots also revealed that Orai1 was downregulated by twofold in brain lysates of HINT1 KO mice compared with the wild-type littermates. Importantly, silencer RNA knockdown of HINT1 in Neuro-2A cells markedly downregulated Orai1 and STIM1 protein expression and significantly (by 2.5-fold) reduced ATP-induced Ca2+influx, while ATP-evoked Ca2+release was not changed. Thus the study demonstrates a novel function of HINT1 that involves the regulation of SOC-mediated Ca2+entry pathway (Orai1 and STIM1), essential for regulation of cellular Ca2+homeostasis.

To delineate the cellular sites for the motor effects of opiates acting at the mu opioid receptor (MOR) in the rat caudate-putamen nucleus, we examined the ultrastructural immunogold and immunoperoxidase labeling of an antipeptide antiserum specific for the MOR. We also combined these labeling methods to examine the subcellular relationship between the MOR and the endogenous opioid peptide, Leu5-enkephalin (LE). By light microscopy, MOR-labeling was seen in a heterogeneous patchy distribution. Electron microscopic analysis of these patches showed that more than 80% of the total neuronal profiles (n = 1,586) containing MOR-like immunoreactivity (MOR-IR) were dendrites and dendritic spines. The remaining labeled profiles included a few perikarya and many axon terminals. MOR-IR was predominantly localized to extrasynaptic plasma membranes of dendrites, and to both synaptic vesicles and plasma membranes in terminals. Ten percent of the total MOR-labeled terminals (n = 272) formed asymmetric synapses with unlabeled or MOR-labeled dendritic spines. Terminals containing LE-IR formed synapses, in almost equal proportions, on MOR-labeled dendrites and dendritic spines, while over 80% of the unlabeled terminals formed synapses on MOR-labeled dendritic spines. Moreover, colocalization of MOR- and LE-IR was often seen in both dendrites and terminals. These results indicate that in patch compartments of the caudate-putamen nucleus, the MOR is mainly involved in extrasynaptic modulation of spiny neurons, including those that contain LE. In addition, the findings provide a cellular basis for presynaptic opioid modulation of neurotransmitter release through MOR located on axon terminals.

Many of the analgesic effects of opiate drugs and of endogenous opioid ligands, such as Leu5-enkephalin (LE) are thought to be mediated in part by mu-opioid receptors (MOR) in the dorsal horn of the spinal cord. To establish the cellular sites for the spinally mediated analgesic effects of MOR activation and the potential anatomical substrates for interactions with LE, we examined the ultrastructural localization of MOR and LE immunoreactivities in the adult rat cervical spinal cord (C3-C5). Anti-MOR sera recognizing the carboxyl terminal domain of MOR was localized using immunoperoxidase and immunogold-silver methods. mu-opioid receptor-like immunoreactivity (MOR-LI) was observed mainly in the superficial layers of the dorsal horn. Electron microscopy of this region revealed that small unmyelinated axons and axon terminals constituted 48% (91/189) and 15% (28/189), respectively, while dendrites comprised 36% (68/189) of the total population of neuronal profiles containing the MOR. MOR-LI was localized mainly along extrasynaptic portions of the plasma membrane in both axons and dendrites. In sections dually labeled for MOR and LE, 21% (14/68) of the dendrites containing MOR-LI closely apposed or received synaptic contact from axon terminals exhibiting LE reaction product. The results provide the first ultrastructural evidence that within the dorsal horn of the spinal cord, LE, as well as exogenous opiates may alter both axonal release of neurotransmitters and postsynaptic responsiveness of target neurons to afferent input through activation of extrasynaptic MOR.

GABAA receptors link binding of GABA (gamma-aminobutyric acid) to inhibitory chloride flux in the brain. They are the site of action of several important classes of drugs, and have been implicated in animal models of epilepsy and in the actions of alcohol. We compare the sequence and expression of the beta 1, beta 2 and beta 3 subunits of GABAA receptors in two inbred strains of mice, DBA/2J and C57BL/6J, which differ markedly in seizure susceptibility and in a variety of behaviors related to alcohol. Only the beta 3 subunit had strain differences in cDNA nucleotide sequence, which did not affect amino acid sequence but which did create restriction fragment length polymorphisms (RFLPs) potentially useful in gene mapping. We have also tested mouse beta 1 and beta 2 subunits for internal alternative splicing, detecting none.

An herb derived compound, levo-tetrahydropalmatine (L-THP), attenuates self-administration of cocaine and opiates in rodents. Since L-THP mainly antagonizes dopamine D2 receptors (D2R) in the brain, it is likely to regulate other addictive behaviors as well. Here, we examined whether L-THP regulates ethanol drinking in C57BL/6J mice using a two-bottle choice drinking experiment. L-THP treated mice consumed less ethanol compared to vehicle-treated mice during the 15% ethanol drinking session while water consumption remained similar between each group. We then examined the molecular basis underlying the pharmacological effect of L-THP in mice. Our results indicated that a single injection of L-THP increased active phosphorylated forms of PKA, AKT and ERK in the caudate-putamen (CPu), but not in the nucleus accumbens (NAc), of alcohol naive mice. Interestingly, we found that systematic treatment with L-THP for 4 consecutive days while mice were drinking 15% ethanol increased pPKA levels in the CPu, but not in the NAc. In contrast to the effect of acute L-THP treatment, no differences were detected for pAKT or pERK in either striatal regions. Together, our findings suggest that reduction of ethanol drinking by L-THP treatment is possibly correlated with D2R-mediated PKA signaling in the CPu.

Liver resection has been established currently as an effective and standard treatment for patients suffering from both benign and malignant hepatobiliary diseases. Although substantial improvement in perioperative mortality rate and morbidity resulting from appropriate candidates selection, advanced surgical techniques and enhanced perioperative care, hepatectomy is still burdened by about 5% mortality rate and some lethal postoperative complications, especially postoperative liver insufficiency and failure. Various approaches have been advocated to minimize stress and insult on patients due to operative procedures. It becomes important to preserve remnant hepatic function as much as possible to improve the outcome of hepatectomy. Minimally invasive concept and fast track surgery are crucial breakthrough in the natural history of surgery and have been employed in liver resection. To safely and accurately perform hepatic resection, owing to our experiences with recent advances in surgical techniques and perioperative administration for liver resection, a novel strategy, "precise hepatectomy" originating from minimally invasive surgery has been developed, which includes precise preoperative planning, sophisticated intraoperative techniques and careful postoperative management. This strategy is characteristic by involvement of minimally invasive concept in overall therapy, from preoperative assessment to postoperative care, optimization of a series of advanced techniques and proper employment of surgical instruments in light of actual individual information. However, further prospective studies, especially randomized controlled trials in high volume centers, remain essential to compare the safety and therapeutic efficacies between precise hepatectomy and conventional surgical procedures.

Levo-tetrahydropalmatine (l-THP) is an active constituent of herbal preparations containing plant species of the genera Stephania and Corydalis and has been approved and used in China for a number of clinical indications under the drug name Rotundine. The pharmacological profile of l-THP, which includes antagonism of dopamine D1 and D2 receptors and actions at dopamine D3, α adrenergic and serotonin receptors, suggests that it may have utility for treating cocaine addiction. In this review, we provide an overview of the pharmacological properties of l-THP and the evidence supporting its development as an anti-addiction medication. The results of preclinical work demonstrating that l-THP attenuates cocaine’s reinforcing/rewarding effects and reinstatement in rat models of cocaine relapse are summarized, and the outcomes of studies demonstrating efficacy in human addicts are described. Finally, an overview of the safety profile of l-THP is provided and challenges associated with US FDA approval of l-THP are discussed.

To investigate roles of second extracellular loop sequences in peptide and nonpeptide ligand recognition by human opiate receptors, we have constructed a chimeric receptor in which this domain of the human mu opiate receptor has been replaced with that of the human kappa opiate receptor. The chimeric opiate receptor displays dramatically increased affinity for dynorphin peptides. Affinities for dynorphin A-(1-17), dynorphin A-(1-13), and alpha-neoendorphin increase by up to 250-fold when compared with the wild-type human mu opiate receptor. The chimera maintains recognition of the mu-selective ligands morphine and [D-Ala2,MePhe4,Gly-ol5]enkephalin and displays no significant changes in affinity for the kappa-selective small molecule ligand U50,488. The chimeric opiate receptor displays evidence for effective G-protein coupling; 100 nM dynorphin A-(1-17) is as effective as 100 nM morphine at inhibiting forskolin-stimulated adenyl cyclase activity through actions at the chimeric receptor. These data suggest that the putative second extracellular loop contributes substantially to the kappa receptor's selectivity in dynorphin ligand recognition.

The mu opiate receptor is a principal brain site for activities of morphine, other opiate drugs, and opioid peptides in modulating pain and altering mood. Recent cloning of cDNAs encoding rat and human mu receptors reveals charged amino acid residues within putative transmembrane domains (TMs) II, III, and VI, a substantial N-terminal extracellular domain, and a C-terminal intracellular domain. Deletion of 64 N-terminal amino acids produced little effect on receptor function (Wang, J.B., Imai, Y., Eppler, C.M., Gregor, P., Spivak, C.E., and Uhl, G.R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10230-10234). Further deletion of 33 C-terminal amino acids yielded a receptor at which morphine, but not the substituted enkephalin DAMGO ([D-Ala2,MePhe4,Glyol5]enkephalin), inhibited adenylate cyclase. Alanine substitution for each charged TM residue in the N-terminally deleted receptor reduced affinities for morphine, DAMGO, and the opiate antagonist naloxone. Replacement of TM II Asp114 with asparagine or glutamic acid increased mu receptor affinity for naloxone. TM II and TM III glutamic acid substitutions for Asp114 and Asp147 reduced agonist binding affinities but allowed full inhibition of adenylate cyclase at high agonist concentrations. TM VI histidine substitution with alanine yielded a receptor that produced almost twice the cyclase inhibition displayed by the wild type receptor in parallel transient expression assays. These findings underscore the importance of charged residues in TM II, III, and VI for different receptor functions and the modest involvement of extensive portions of N- and C-terminal receptor domains in these processes.