Heterogeneity of cannabinoid ligand-induced modulations in intracellular Ca2+ signals of mouse pancreatic acinar cells in vitro.

We recently reported that a CB2R agonist, GW405833 (GW), reduced both the ACh-induced Ca2+ oscillations and the L-arginine-induced Ca2+ signal enhancement in mouse pancreatic acinar cells, suggesting that GW-induced inhibition may prevent the pathogenesis of acute pancreatitis. In this study, we aim to evaluate the effects of other cannabinoid ligands on Ca2+ signaling in acinar cells. Patch-clamp whole-cell recordings were applied to measure ACh-induced intracellular Ca2+ oscillations in pancreatic acinar cells acutely dissociated from wild-type (WT), CB1R knockout (KO), and CB2R KO mice, and the pharmacological effects of various cannabinoid ligands on the Ca2+ oscillations were examined. We found that all the 8 CB2R agonists tested inhibited ACh-induced Ca2+ oscillations. Among them, GW, JWH133, and GP1a caused potent inhibition with IC50 values of 5.0, 6.7, and 1.2 µmol/L, respectively. In CB2R KO mice or in the presence of a CB2R antagonist (AM630), the inhibitory effects of these 3 CB2R agonists were abolished, suggesting that they acted through the CB2Rs. The CB1R agonist ACEA also induced inhibition of Ca2+ oscillations that existed in CB1R KO mice and in the presence of a CB1R antagonist (AM251), suggesting a non-CB1R effect. In WT, CB1R KO, and CB2R KO mice, a nonselective CBR agonist, WIN55,212-2, inhibited Ca2+ oscillations, which was not mediated by CB1Rs or CB2Rs. The endogenous cannabinoid substance, 2-arachidonoylglycerol (2-AG), did not show an inhibitory effect on Ca2+ oscillations. In conclusion, CB2R agonists play critical roles in modulating Ca2+ signals in mouse pancreatic acinar cells, while other cannabinoid ligands modulate Ca2+ oscillations in a heterogeneous manner through a CB receptor or non-CB-receptor mechanism.

Pharmacological and functional comparisons of α6/α3ß2ß3-nAChRs and α4ß2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6*-nAChRs remain poorly understood because of the lack of selective agonists for α6*-nAChRs and the challenging heterologous expression of functional α6*-nAChRs in mammalian cell lines. In particular, the α6 subunit is commonly co-expressed with α4*-nAChRs in the midbrain, which masks α6*-nAChR (without α4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of α6*-nAChRs and compared these properties with those of α4ß2 nAChRs expressed in the same cell line. Heterologously expressed human α6/α3 chimeric subunits (α6 N-terminal domain joined with α3 trans-membrane domains and intracellular loops) with ß2 and ß3 subunits in the human SH-EP1 cell line (α6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed α6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with α4ß2-nAChRs. Pharmacologically, α6*-nAChR was highly sensitive to the α6 subunit-selective antagonist α-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-ß-erythroidine. Nicotine and acetylcholine were found to be full agonists for α6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed α6*-nAChRs exhibited pharmacology and function distinct from those of α4ß2-nAChRs, suggesting that α6*-nAChRs may mediate different cholinergic signals. Our α6*-nAChR expression system can be used as an excellent cell model for future investigations of α6*-nAChR function and pharmacology.

Hippocampal synaptic and neural network deficits in young mice carrying the human APOE4 gene.

INTRODUCTION: Apolipoprotein E4 (APOE4) is a major genetic risk factor for late-onset sporadic Alzheimer disease. Emerging evidence demonstrates a hippocampus-associated learning and memory deficit in aged APOE4 human carriers and also in aged mice carrying human APOE4 gene. This suggests that either exogenous APOE4 or endogenous APOE4 alters the cognitive profile and hippocampal structure and function. However, little is known regarding how Apoe4 modulates hippocampal dendritic morphology, synaptic function, and neural network activity in young mice. AIM: In this study, we compared hippocampal dendritic and spine morphology and synaptic function of young (4 months) mice with transgenic expression of the human APOE4 and APOE3 genes. METHODS: Hippocampal dendritic and spine morphology and synaptic function were assessed by neuronal imaging and electrophysiological approaches. RESULTS: Morphology results showed that shortened dendritic length and reduced spine density occurred at hippocampal CA1 neurons in Apoe4 mice compared to Apoe3 mice. Electrophysiological results demonstrated that in the hippocampal CA3-CA1 synapses of young Apoe4 mice, basic synaptic transmission, and paired-pulse facilitation were enhanced but long-term potentiation and carbachol-induced hippocampal theta oscillations were impaired compared to young Apoe3 mice. However, both Apoe genotypes responded similarly to persistent stimulations (4, 10, and 40 Hz for 4 seconds). CONCLUSION: Our results suggest significant alterations in hippocampal dendritic structure and synaptic function in Apoe4 mice, even at an early age.

Brain cannabinoid receptor 2: expression, function and modulation.

Cannabis sativa (marijuana) is a fibrous flowering plant that produces an abundant variety of molecules, some with psychoactive effects. At least 4% of the world's adult population uses cannabis annually, making it one of the most frequently used illicit drugs in the world. The psychoactive effects of cannabis are mediated primarily through cannabinoid receptor (CBR) subtypes. The prevailing view is that CB1Rs are mainly expressed in the central neurons, whereas CB2Rs are predominantly expressed in peripheral immune cells. However, this traditional view has been challenged by emerging strong evidence that shows CB2Rs are moderately expressed and function in specific brain areas. New evidence has demonstrated that brain CB2Rs modulate animal drug-seeking behaviors, suggesting that these receptors may exist in brain regions that regulate drug addiction. Recently, we further confirmed that functional CB2Rs are expressed in mouse ventral tegmental area (VTA) dopamine (DA) neurons and that the activation of VTA CB2Rs reduces neuronal excitability and cocaine-seeking behavior. In addition, CB2R-mediated modulation of hippocampal CA3 neuronal excitability and network synchronization has been reported. Here, we briefly summarize recent lines of evidence showing how CB2Rs modulate function and pathophysiology in the CNS.

Clinical analysis for the application of endoscopic ultrasonography in the diagnosis of patients with a retroperitoneal space-occupying lesion.

OBJECTIVE: To report our experience and to evaluate the application of endoscopic ultrasonography (EUS) in the qualitative diagnosis of retroperitoneal space-occupying lesions. METHODS: Twenty-six patients with retroperitoneal space-occupying lesions confirmed by CT or MRI were studied. All the patients underwent endoscopic ultrasonography guided fine needle aspiration (EUS-FNA) at the Department of Gastroenterology, Xiangyang Central Hospital, Hubei Province, China from August 2009 to August 2011. Different parameters were evaluated, such as complications of EUS-FNA, the ratio of definite pathological diagnosis, and the pathologic types of all the specimens. RESULTS: Of the 26 patients, 24 had definite pathological diagnosis; the ratio of histodiagnosis was 92.3%. There were no complications such as hemorrhage, infection, or injury to the abdominal viscera in the process of EUS-FNA. Eight patients had benign tumors, with a ratio of 33.3%, and 16 patients had malignant tumors, with a ratio of 66.7%. Two patients had no definite pathological diagnosis because of the shortness of tissue sample. Eight patients did not undergo operation due to the diagnosis of benign tumors. CONCLUSION: The EUS-FNA has the advantage of lower complications, and higher diagnostic ratio, which is valuable in the qualitative diagnosis of retroperitoneal space-occupying lesions.

The relationship among human papilloma virus infection, survivin, and p53 gene in lung squamous carcinoma tissue.

OBJECTIVE: To study the relationship between the infection of human papillomavirus (HPV) type 16, type 18, the expression of survivin, and the mutation of p53 gene in lung squamous carcinoma tissue for the research of pathogenesis of lung carcinoma. METHODS: This study was carried out at the Laboratory of Molecular Biology, Xiangfan Central Hospital of Hubei Province, China from September 2008 to May 2010. Forty-five specimens of lung squamous carcinoma tissue confirmed by histopathology were the excisional specimens taken by the Thoracic Surgery of Xiangfan Central Hospital. Normal tissue, closely adjacent to the fresh carcinoma specimens, was used as the control group for p53 gene mutation analysis. Sixteen surgical excisional specimens of benign lung disease were used as a control group of non-carcinomatous diseases. Human papillomavirus DNA were detected by polymerase chain reaction (PCR), and we used the PCR-single-strand conformation polymorphism-ethidium bromide (PCR-SSCP-EB) method to detect the mutations of the p53 gene. The expression of the survivin gene was detected by immunohistochemistry methods. RESULTS: Approximately 68.9% of 45 lung squamous carcinoma tissue had p53 gene mutations. The mutation rate of exon 5-8 p53 were 15.6%, 17.8%, 15.6% and 20%. Approximately 42.2% of lung squamous cell carcinoma samples were shown to be positive for HPV DNA expression and 62.2% were positive for survivin expression. There was an inverse correlation between the presence of HPV infections and mutations of p53 gene; and the mutations of p53 gene and expression of survivin had a positive relationship. CONCLUSION: Mutation of p53 gene and HPV infection may facilitate each other in the generation of lung squamous cell carcinoma. Abnormal expression of the survivin gene may take part in the onset and progression of lung squamous cell carcinoma.

Construction and expression of anti-CD62P/anti-GPIIb-IIIa diabody.

OBJECTIVE: To construct 3 expression plasmids for the targeted therapy of thrombosis: single chain variable fragment (scFv) of monoclonal antibody (mAb) 7E3 that can identify and bind platelet glycoprotein GPIIb-IIIa complex, scFv of mAb WAPS12.2 that can identify and bind P selectin (CD62P), a diabody that can identify and bind GPIIb-IIIa and CD62P simultaneously, and to investigate whether the vectors can express correctly. METHODS: This study was carried out at the Laboratory of Hunan Yuantai Biological Technology Co. Ltd, Hubei, China from September 2007 to May 2008. Total RNA of mAb 7E3 cells and WAPS12.2 cells were obtained. Reverse transcriptase polymerase chain reaction (PCR) was carried out to obtain the genes of variable regions of light and heavy chains of 7E3 and WAPS12.2. Target genes were named 7E3VL, 7E3VH, CD62PVL, and CD62PVH. The 7E3VL-7E3VH and CD62PVL-CD62PVH were obtained by PCR and connected with pET-22b(+). Products were named pET-scFv7E3 and pET-scFvCD62P. The 7E3VL-CD62PVH and CD62PVL-7E3VH were obtained by using overlap PCR and were ligated to pET-22b(+). The products were named pET-ED1 and pET-ED2. The PCR was performed by taking pET-ED2 as a template to obtain the complete operon gene and was ligated to pET-ED1. The product was named pET-7ECD. RESULTS: The identification by restriction endonuclease cleavage and DNA sequencing confirmed that the construction of these expression plasmids was successful. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed that these plasmids expressed correctly. CONCLUSION: The expression plasmids pET-scFv7E3, pET-scFvCD62P, and pET-7ECD were constructed and expressed successfully, and laid a good foundation for further research on target-oriented thrombolytic agents.

Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate.

AIM: To construct a new target-oriented conjugate of humanized carcinoembryonic antigen (CEA) specific single chain variable fragment (scFv) and mitomycin (MMC) against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells. METHODS: The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamHI and EcoRI in its upstream and downstream. PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamHI and EcoRI, and connected to an expression vector which also has the restriction enzyme cleavage sites BamHI and EcoR. Expression of the reaction was induced by isopropy-beta-D-thiogalactoside (IPTG). Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay (ELISA). The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry (FCM). RESULTS: Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) confirmed that this vector correctly expressed the fusion protein. ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent manner. CONCLUSION: The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.

Construction of adeno-associated virus coexpression system for human angiopoietin-1 and VEGF gene.

BACKGROUND: Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165(VEGF165) gene in adeno-associated virus (AAV) gene delivery system. METHODS: Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site HindIII, and the downstream of angiopoietin 1 contained restriction enzyme site BamHI. The upstream of VEGF165 contained restriction enzyme site BglII, and the downstream of VEGF165 contained restriction enzyme site BamHI. Using the multiple cloning sites (MCS) in plasmid pZero++ such as BamHI, BglII, HindIII, NotI, XhoI, XbaI, SalI, BspHI, KspI and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS. RESULTS: DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion, which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietin1 and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes. CONCLUSION: Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.