A novel, rapidly acquired and persistent spatial memory task that induces immediate early gene expression

<p>Abstract</p> <p>Background</p> <p>The Morris water maze task is a hippocampus-dependent learning and memory test that typically takes between 3 days to 2 weeks of training. This task is used to assess spatial learning and induces the expression of genes known to be crucial to learning and memory in the hippocampus. A major caveat in the protocol is the prolonged duration of training, and difficulty of assessing the time during training in which animals have learned the task. We introduce here a condensed version of the task that like traditional water maze tasks, creates lasting hippocampus-dependent spatial cognitive maps and elicits gene expression following learning.</p> <p>Methods</p> <p>This paradigm was designed for rats to quickly acquire a hippocampus-dependent spatial cognitive map and retain this memory for at least 24 hours. To accomplish this, we interspersed visible and hidden training trials, delivering them in a massed fashion so training takes a maximum of 15 minutes. Learning was assessed based on latencies to the platform during each training trial, as well as time spent in the goal quadrant during probe testing 30 minutes and 24 hours after training. Normal rats were compared to two impaired cohorts (rats with fimbria-fornix lesions and rats administered NMDA receptor antagonist (CPP)). To quantitate hippocampal expression of known learning genes, real-time polymerase chain reaction (RT-PCR) was performed on hippocampal cDNA.</p> <p>Results</p> <p>We show that massed training using alternating visible and hidden training trials generates robust short-term working and long-term reference memories in rats. Like the traditional Morris water maze paradigm, this task requires proper hippocampal function, as rats with fimbria-fornix lesions and rats administered CPP fail to learn the spatial component of the task. Furthermore, training in this paradigm elicits hippocampal expression of genes upregulated following learning in a variety of spatial tasks: <it>homer1a, cfos </it>and <it>zif268</it>.</p> <p>Conclusions</p> <p>We introduce here a condensed version of the Morris water maze, which is like a traditional water maze paradigm, in that it is hippocampus-dependent, and elicits hippocampal expression of learning genes. However, this task is administered in 15 minutes and induces spatial memory for at least 24 hours.</p

Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR) in neuromuscular junction and cardiac intercalated discs

BACKGROUND: The Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown. RESULTS: Using antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart. CONCLUSION: The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus

Ultrasound increases plasmid-mediated gene transfer to dystrophic muscles without collateral damage

Studies have shown that ultrasound, used either alone or in combination with microbubble contrast agents, can increase cell membrane permeability to plasmid DNA. Because ultrasound is a non-painful and well-established tool in clinical medicine, its potential to enhance DNA uptake into the muscles of patients with muscular dystrophy is conceptually attractive. Therefore, we evaluated the ability of ultrasound pulses (1 MHz; 1.5 W/cm2) to increase exogenous (LacZ) gene expression in normal wild-type and dystrophic Dmd(mdx/mdx) mice after plasmid DNA injection into muscle. We also ascertained whether co-injection of lipid-encapsulated perfluoropropane microbubbles (Definity) or pretreatment with hyaluronidase could further increase the level of gene transfer to ultrasound-treated muscles. The use of ultrasound did not increase transfection efficiency in normal mice. In contrast, dystrophic mice demonstrated an increase in the number of transfected fibers (threefold) as well as the amount of LacZ protein (22-fold) after ultrasound exposure, provided that Definity was also co-injected with the DNA. Pretreatment of muscles with hyaluronidase before ultrasound exposure was not effective in augmenting the level of gene transfer. Under the optimal conditions for dystrophic muscle transfection (ultrasound + Definity), there was no associated increase in muscle damage. Hence ultrasound may provide a safe and effective method for enhancing gene transfer to dystrophic muscles, thereby increasing the prospects for therapeutic application of naked DNA in muscular dystrophy patients.Peer reviewed: YesNRC publication: N

Purification and Characterization of Membrane Proteins: Beef Heart Mitochondrial Succinate Dehydrogenase

Beef heart succinate dehydrogenase (SDH) was solubilized from mitochondrial membranes by a medium containing 4% Triton X-100/4M urea ipH8.6). It was subsequently purified to homogeneity via ion-exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on BioGel A-O.Sm. The pure SDH preparation had a specific activity of 14 - 15.5u moles succinateoxidized/min/mg biuret protein at 38°C in the ferricyanide reductase assay. The pure SDH was composed of two polypeptide subunits (30k and 70k) present at a molar ratio of 1. The N-terminal amino acid residue of the 70k subunit was found to be isoleucine whereas the 30k subunit may have a blocked N-terminal amino acid. Analytical polyacrylamide gel electrophoresis demonstrated that the pure SDH preparation (detergent-free) was not present in an aggregated form. The Triton-free protein had a hydrodynamic molecular weight of 105,000 daltons and a partial specific volume of 0.74ml/g as determined by sedimentation equilibrium ultracentrifugation. […]La déhydrogénase de succinate du muscle cardiaque bovin a été solubilisée des membranes de la mitochondrie dans un milieu contenant 4% Triton X-100/4H urée (pH8.6). L'enzyme fut purifiée à l'état homogène par la chromatographie a échange d'ions (DEAE Sepharose CL-6B) et la chromatographie sur gel (BioGel A-0.5m). La préparation ainsi purifiée avait une activité spécifique de 14 - 15.5u moles succinate oxydée/min/mg protéine a 38°C dans l'essai de la réductase de ferricyanure. La protéine purifiée était composée de deux sous-unités polypeptidiques (30k et 70k) qui étaient présents dans un rapport de 1 à 1. L'acide aminé N-terminal du sous-unité de 70k se trouve à être l'isoleucine tandis que celui du 30k est bloqué. Aucune aggrégation était apparente quand la molécule (sans détergent) fut examinée par électrophorèse sur gel de polyacrylamide. L'ultra centrifugation à équilibre révéla que la protéine (ne contenant aucun détergent) avait un poids moléculaire hydrodynamique de 105,000 daltons et un volume spécifique partiel de 0.74ml/g. […

The Coxsackievirus and Adenovirus Receptor (CAR) undergoes ectodomain shedding and regulated intramembrane proteolysis (RIP).

The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates' ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP

Suicide gene therapy with an adenovirus expressing the fusion gene CD::UPRT in human glioblastomas: different sensitivities correlate with p53 status

DA - 20041230IS - 1099-498XLA - engPT - Journal ArticleSB - IMNRC publication: Ye

Confirmation of the area of ECD cleavage on CAR in HEK 293 cells.

<p>(A) A schematic showing putative ADAM10 cleavage sites on CAR’s extracellular domain (arrows), as obtained from <i>in vitro</i> peptide digestion and mass spectrometry (Figure S4). (B) HEK 293 stable cell populations were generated to express wild-type CAR, the mutant MLRLAAAA or the mutant Δ221-232. The MLRLAAAA mutant did not shed into conditioned media of HEK 293 cells, while the Δ221-232 mutant shed its ECD similarly to wild-type CAR. Cell surface biotinylation experiments revealed that wild-type CAR and the two mutants are expressed similarly on the cell surface. Note that the low level of endogenous CAR in HEK 293 was detected after enrichment of cell surface biotinylated proteins (mock lane). Western blotting was performed using the anti-CAR N-terminus antibody 2240.</p