December 1965

Massachusetts Turf and Lawn Grass Council Better Turf Through Research and Educatio

Cognitive impairment induced by delta9-tetrahydrocannabinol occurs through heteromers between cannabinoid CB1 and serotonin 5-HT2A receptors

Delta-9-tetrahydrocannabinol (THC), the main psychoactive compound of marijuana, induces numerous undesirable effects, including memory impairments, anxiety, and dependence. Conversely, THC also has potentially therapeutic effects, including analgesia, muscle relaxation, and neuroprotection. However, the mechanisms that dissociate these responses are still not known. Using mice lacking the serotonin receptor 5-HT2A, we revealed that the analgesic and amnesic effects of THC are independent of each other: while amnesia induced by THC disappears in the mutant mice, THC can still promote analgesia in these animals. In subsequent molecular studies, we showed that in specific brain regions involved in memory formation, the receptors for THC and the 5-HT2A receptors work together by physically interacting with each other. Experimentally interfering with this interaction prevented the memory deficits induced by THC, but not its analgesic properties. Our results highlight a novel mechanism by which the beneficial analgesic properties of THC can be dissociated from its cognitive side effects

Characterization of Chromosomal Instability in Murine Colitis-Associated Colorectal Cancer

Patients suffering from ulcerative colitis (UC) bear an increased risk for colorectal cancer. Due to the sparsity of colitis-associated cancer (CAC) and the long duration between UC initiation and overt carcinoma, elucidating mechanisms of inflammation-associated carcinogenesis in the gut is particularly challenging. Adequate murine models are thus highly desirable. For human CACs a high frequency of chromosomal instability (CIN) reflected by aneuploidy could be shown, exceeding that of sporadic carcinomas. The aim of this study was to analyze mouse models of CAC with regard to CIN. Additionally, protein expression of p53, beta-catenin and Ki67 was measured to further characterize murine tumor development in comparison to UC-associated carcinogenesis in men.The AOM/DSS model (n = 23) and IL-10(-/-) mice (n = 8) were applied to monitor malignancy development via endoscopy and to analyze premalignant and malignant stages of CACs. CIN was assessed using DNA-image cytometry. Protein expression of p53, beta-catenin and Ki67 was evaluated by immunohistochemistry. The degree of inflammation was analyzed by histology and paralleled to local interferon-γ release.CIN was detected in 81.25% of all murine CACs induced by AOM/DSS, while all carcinomas that arose in IL-10(-/-) mice were chromosomally stable. Beta-catenin expression was strongly membranous in IL-10(-/-) mice, while 87.50% of AOM/DSS-induced tumors showed cytoplasmatic and/or nuclear translocation of beta-catenin. p53 expression was high in both models and Ki67 staining revealed higher proliferation of IL-10(-/-)-induced CACs.AOM/DSS-colitis, but not IL-10(-/-) mice, could provide a powerful murine model to mechanistically investigate CIN in colitis-associated carcinogenesis

Selective inhibitors of cardiac ADPR cyclase as novel anti-arrhythmic compounds

ADP-ribosyl cyclases (ADPRCs) catalyse the conversion of nicotinamide adenine dinucleotide to cyclic adenosine diphosphoribose (cADPR) which is a second messenger involved in Ca2+ mobilisation from intracellular stores. Via its interaction with the ryanodine receptor Ca2+ channel in the heart, cADPR may exert arrhythmogenic activity. To test this hypothesis, we have studied the effect of novel cardiac ADPRC inhibitors in vitro and in vivo in models of ventricular arrhythmias. Using a high-throughput screening approach on cardiac sarcoplasmic reticulum membranes isolated from pig and rat and nicotinamide hypoxanthine dinuleotide as a surrogate substrate, we have identified potent and selective inhibitors of an intracellular, membrane-bound cardiac ADPRC that are different from the two known mammalian ADPRCs, CD38 and CD157/Bst1. We show that two structurally distinct cardiac ADPRC inhibitors, SAN2589 and SAN4825, prevent the formation of spontaneous action potentials in guinea pig papillary muscle in vitro and that compound SAN4825 is active in vivo in delaying ventricular fibrillation and cardiac arrest in a guinea pig model of Ca2+ overload-induced arrhythmia. Inhibition of cardiac ADPRC prevents Ca2+ overload-induced spontaneous depolarizations and ventricular fibrillation and may thus provide a novel therapeutic principle for the treatment of cardiac arrhythmias

Aberrant Cyclization Affords a C-6 Modified Cyclic Adenosine 5′-Diphosphoribose Analogue with Biological Activity in Jurkat T Cells

*S Supporting Information ABSTRACT: Two nicotinamide adenine dinucleotide (NAD +) analogues modified at the 6 position of the purine ring were synthesized, and their substrate properties toward Aplysia californica ADP-ribosyl cyclase were investigated. 6-N-Methyl NAD + (6-N-methyl nicotinamide adenosine 5′-dinucleotide 10) hydrolyzes to give the linear 6-N-methyl ADPR (adenosine 5′-diphosphoribose, 11), whereas 6-thio NHD + (nicotinamide 6-mercaptopurine 5′-dinucleotide, 17) generates a cyclic dinucleotide. Surprisingly, NMR correlation spectra confirm this compound to be the N1 cyclic product 6-thio N1-cIDPR (6-thio cyclic inosine 5′-diphosphoribose, 3), although the corresponding 6-oxo analogue is well-known to cyclize at N7. In Jurkat T cells, unlike the parent cyclic inosine 5′-diphosphoribose N1-cIDPR 2, 6-thio N1-cIDPR antagonizes both cADPR- and N1cIDPR-induced Ca 2+ release but possesses weak agonist activity at higher concentration. 3 is thus identified as the first C-6 modified cADPR (cyclic adenosine 5′-diphosphoribose) analogue antagonist; it represents the first example of a fluorescent N1cyclized cADPR analogue and is a new pharmacological tool for intervention in the cADPR pathway of cellular signaling

Activation Status of Wnt/ß-Catenin Signaling in Normal and Neoplastic Breast Tissues: Relationship to HER2/neu Expression in Human and Mouse

Wnt/ß-catenin signaling is strongly implicated in neoplasia, but the role of this pathway in human breast cancer has been controversial. Here, we examined Wnt/ß-catenin pathway activation as a function of breast cancer progression, and tested for a relationship with HER2/neu expression, using a human tissue microarray comprising benign breast tissues, ductal carcinoma in situ (DCIS), and invasive carcinomas. Cores were scored for membranous ß-catenin, a key functional component of adherens junctions, and for nucleocytoplasmic ß-catenin, a hallmark of Wnt/ß-catenin pathway activation. Only 82% of benign samples exhibited membrane-associated ß-catenin, indicating a finite frequency of false-negative staining. The frequency of membrane positivity was similar in DCIS samples, but was significantly reduced in carcinomas (45%, P<0.001), consistent with loss of adherens junctions during acquisition of invasiveness. Negative membrane status in cancers correlated with higher grade (P = 0.04) and estrogen receptor-negative status (P = 0.03), both indices of poor prognosis. Unexpectedly, a substantial frequency of nucleocytoplasmic ß-catenin was observed in benign breast tissues (36%), similar to that in carcinomas (35%). Positive-staining basal nuclei observed in benign breast may identify putative stem cells. An increased frequency of nucleocytoplasmic ß-catenin was observed in DCIS tumors (56%), suggesting that pathway activation may be an early event in human breast neoplasia. A correlation was observed between HER2/neu expression and nucleocytoplasmic ß-catenin in node-positive carcinomas (P = 0.02). Furthermore, cytoplasmic ß-catenin was detected in HER2/neu-induced mouse mammary tumors. The Axin2NLSlacZ mouse strain, a previously validated reporter of mammary Wnt/ß-catenin signaling, was utilized to define in vivo transcriptional consequences of HER2/neu-induced ß-catenin accumulation. Discrete hyperplastic foci observed in mammary glands from bigenic MMTV/neu, Axin2NLSlacZ mice, highlighted by robust ß-catenin/TCF signaling, likely represent the earliest stage of mammary intraepithelial neoplasia in MMTV/neu mice. Our study thus provides provocative evidence for Wnt/ß-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia

High Resolution Genome-Wide Analysis of Chromosomal Alterations in Burkitt's Lymphoma

Additional chromosomal abnormalities are currently detected in Burkitt's lymphoma. They play major roles in the progression of BL and in prognosis. The genes involved remain elusive. A whole-genome oligonucleotide array CGH analysis correlated with karyotype and FISH was performed in a set of 27 Burkitt's lymphoma-derived cell lines and primary tumors. More than half of the 145 CNAs<2 Mb were mapped to Mendelian CNVs, including GSTT1, glutathione s-transferase and BIRC6, an anti-apoptotic protein, possibly predisposing to some cancers. Somatic cell line-specific CNVs localized to the IG locus were consistently observed with the 244 K aCGH platform. Among 136 CNAs >2 Mb, gains were found in 1q (12/27), 13q (7/27), 7q (6/27), 8q(4/27), 2p (3/27), 11q (2/27) and 15q (2/27). Losses were found in 3p (5/27), 4p (4/27), 4q (4/27), 9p (4/27), 13q (4/27), 6p (3/27), 17p (3/27), 6q (2/27),11pterp13 (2/27) and 14q12q21.3 (2/27). Twenty one minimal critical regions (MCR), (range 0.04–71.36 Mb), were delineated in tumors and cell lines. Three MCRs were localized to 1q. The proximal one was mapped to 1q21.1q25.2 with a 6.3 Mb amplicon (1q21.1q21.3) harboring BCA2 and PIAS3. In the other 2 MCRs, 1q32.1 and 1q44, MDM4 and AKT3 appeared as possible drivers of these gains respectively. The 13q31.3q32.1 <89.58–96.81> MCR contained an amplicon and ABCC4 might be the driver of this amplicon. The 40 Kb 2p16.1 <60.96–61> MCR was the smallest gained MCR and specifically encompassed the REL oncogene which is already implicated in B cell lymphomas. The most frequently deleted MCR was 3p14.1 <60.43–60.53> that removed the fifth exon of FHIT. Further investigations which combined gene expression and functional studies are essential to understand the lymphomagenesis mechanism and for the development of more effective, targeted therapeutic strategies

Molecular Characterization of a Novel Intracellular ADP-Ribosyl Cyclase

Background. ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates. Methodology/Principal Findings. Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1) is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained. Conclusions/Significance. Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized