AB9485 Sigma-AldrichAnti-Somatostatin Receptor Type 1 Antibody

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABAA receptors (GABAARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Hetα1KO) of the human epilepsy gene, the GABAAR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Hetα1KO on the expression and physiology of GABAARs in the mouse cortex. We found that Hetα1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABAARs. Cortices partially compensated for Hetα1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Hetα1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Hetα1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Hetα1KO reduced base-line GABAAR endocytosis, an effect that probably contributes to the observed changes in GABAAR expression. These findings demonstrate that Hetα1KO exerts two principle disinhibitory effects on cortical GABAAR-mediated inhibitory neurotransmission: 1) a modest reduction of GABAAR number and 2) a partial compensation with GABAAR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABAARs.

Oxidant stress, among other effectors, is implicated in the sequel of myocardial reperfusion injury. It is generally accepted that maintaining the balance between oxidant and antioxidant signalling within the cell provides protection against reperfusion damage. The cardioprotective strategy of postconditioning (PC) reduces reperfusion injury through complex mechanisms; however, the contribution of the antioxidant system has not been fully investigated. In this study, isolated rat hearts were subjected to PC after 30 min global ischaemia, and then to 5 min (IR5) or 60 min of reperfusion (IR60). Postconditioning significantly increased the left ventricular developed pressure and the double product (heart rate × left ventricular developed pressure) for both early (PC5) and prolonged reperfusion (PC60, PC before 60 min of reperfusion). Necrotic tissue diminished to 10.8% in PC60 hearts, compared with 49% of infarct size measured in IR60 hearts (P less than 0.05 versus IR60). Also, protein carbonylation and malondialdehyde levels decreased and were correlated with a significant augmentation in CuZn superoxide dismutase activity (P less than 0.05, PC60 versus IR60) and increased glutathione redox state (GSH:GSSG ratio; P less than 0.05, PC60 versus IR60). Diethylthiocarbamate, a non-selective superoxide dismutase inhibitor, significantly diminished the protection afforded by PC when administered throughout the protocol. However, administration of this inhibitor only during reperfusion had no effect on PC-induced cardioprotection. These results indicate that non-enzymatic antioxidants account for the protective effect of PC, modifying the oxidant stress caused by ischaemic reperfusion in rats. The contribution of CuZn superoxide dismutase activity in the observed cardioprotective effect is less clear, and could be relevant if acting in concert with other PC-activated mechanisms.

Aging promotes accumulation of reactive oxygen/nitrogen species (ROS/RNS) in cardiomyocytes, which leads to contractile dysfunction and cardiac abnormalities. These changes may contribute to increased cardiovascular disease in the elderly. Inducible antioxidant pathways are regulated by nuclear erythroid 2 p45-related factor 2 (Nrf2) through antioxidant response cis-elements (AREs) and are impaired in the aging heart. Whereas acute exercise stress (AES) activates Nrf2 signaling and promotes myocardial antioxidant function in young mice (~2 months), aging mouse (greater than 23 months) hearts exhibit significant oxidative stress as compared to those of the young. The purpose of this study was to investigate age-dependent regulation of Nrf2-antioxidant mechanisms and redox homeostasis in mouse hearts and the impact of exercise. Old mice were highly susceptible to oxidative stress following high endurance exercise stress (EES), but demonstrated increased adaptive redox homeostasis after moderate exercise training (MET; 10m/min, for 45 min/day) for ~6 weeks. Following EES, transcription and protein levels for most of the ARE-antioxidants were increased in young mice but their induction was blunted in aging mice. In contrast, 6-weeks of chronic MET promoted nuclear levels of Nrf2 along with its target antioxidants in the aging heart to near normal levels as seen in young mice. These observations suggest that enhancing Nrf2 function and endogenous cytoprotective mechanisms by MET, may combat age-induced ROS/RNS and protect the myocardium from oxidative stress diseases.

Antiprogestins have been largely utilized in reproductive medicine, yet their repositioning for oncologic use is rapidly emerging. In this study we investigated the molecular mediators of the anti-ovarian cancer activity of the structurally related antiprogestins RU-38486, ORG-31710 and CDB-2914. We studied the responses of wt p53 OV2008 and p53 null SK-OV-3 cells to varying doses of RU-38486, ORG-31710 and CDB-2914. The steroids inhibited the growth of both cell lines with a potency of RU-38486 greater than ORG-31710 greater than CDB-2914, and were cytostatic at lower doses but lethal at higher concentrations. Antiprogestin-induced lethality associated with morphological features of apoptosis, hypodiploid DNA content, DNA fragmentation, and cleavage of executer caspase substrate PARP. Cell death ensued despite RU-38486 caused transient up-regulation of anti-apoptotic Bcl-2, ORG-31710 induced transient up-regulation of inhibitor of apoptosis XIAP, and CDB-2914 up-regulated both XIAP and Bcl-2. The antiprogestins induced accumulation of Cdk inhibitors p21(cip1) and p27(kip1) and increased association of p21(cip1) and p27(kip1) with Cdk-2. They also promoted nuclear localization of p21(cip1) and p27(kip1), reduced the nuclear abundances of Cdk-2 and cyclin E, and blocked the activity of Cdk-2 in both nucleus and cytoplasm. The cytotoxic potency of the antiprogestins correlated with the magnitude of the inhibition of Cdk-2 activity, ranging from G1 cell cycle arrest towards cell death. Our results suggest that, as a consequence of their cytostatic and lethal effects, antiprogestin steroids of well-known contraceptive properties emerge as attractive new agents to be repositioned for ovarian cancer therapeutics.

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

The prion protein gene PRNP directs the synthesis of one of the most intensively studied mammalian proteins, the prion protein (PrP). Yet the physiological function of PrP has remained elusive and has created controversies in the literature. We found a downstream alternative translation initiation AUG codon surrounded by an optimal Kozak sequence in the +3 reading frame of PRNP. The corresponding alternative open reading frame encodes a polypeptide termed alternative prion protein (AltPrP) with a completely different amino acid sequence from PrP. We introduced a hemagglutinin (HA) tag in frame with AltPrP in PrP cDNAs from different species to test the expression of this novel polypeptide using anti-HA antibodies. AltPrP is constitutively coexpressed with human, bovine, sheep, and deer PrP. AltPrP is localized at the mitochondria and is up-regulated by endoplasmic reticulum stress and proteasomal inhibition. Generation of anti-AltPrP antibodies allowed us to test for endogenous expression of AltPrP in wild-type human cells expressing PrP. By transfecting cells with siRNA against PrP mRNA, we repressed expression of both PrP and AltPrP, confirming endogenous expression of AltPrP from PRNP. AltPrP was also detected in human brain homogenate, primary neurons, and peripheral blood mononuclear cells. These results demonstrate an unexpected function for PRNP, which, in addition to plasma membrane-anchored PrP, also encodes a second polypeptide termed AltPrP.-Vanderperre, B., Staskevicius, A. B., Tremblay, G., McCoy, M., O\'Neill, M. A., Cashman, N. R., Roucou, X. An overlapping reading frame in the PRNP gene encodes a novel polypeptide distinct from the prion protein.

The formation and plasticity of dendritic spines and synapses, which are poorly understood on a molecular level, are critical for cognitive functions, such as learning and memory. The adaptor protein containing a PH domain, PTB domain, and leucine zipper motif (APPL1) is emerging as a critical regulator of various cellular processes in non-neuronal cells, but its function in the nervous system is not well understood. Here, we show that APPL1 localizes to dendritic spines and synapses and regulates the development of these structures in hippocampal neurons. Knockdown of endogenous APPL1 using siRNA led to a significant decrease in the number of spines as well as synapses and this defect could be rescued by expression of siRNA-resistant APPL1. Expression of exogenous APPL1 increased the spine and synaptic density and the amount of surface GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Deletion of the C-terminal phosphotyrosine binding domain of APPL1, which binds the serine/threonine kinase Akt, resulted in a significant decrease in the spine and synaptic density, suggesting a role for Akt in regulating the development of these structures. Consistent with this, knockdown of Akt with siRNA or expression of dominant negative Akt led to a dramatic decrease in spine and synapse formation. In addition, APPL1 increased the amount of active Akt in spines and synapses and the effects of APPL1 on these structures were dependent on Akt, indicating that Akt is an effector of APPL1 in the regulation of these processes. Moreover, APPL1 signaling modulates spine and synapse formation through p21-activated kinase (PAK). Thus, our results indicate that APPL1 signaling through Akt and PAK is critical for spine and synaptic development and point to a role for APPL1 and its effectors in regulating cognitive function.

Background: The degradation of many nuclear receptors is controlled by ligand-binding and mediated by the ubiquitin-proteasome pathway. However, the mechanisms implicated in thyroid hormone receptor (TR) degradation remain unclear. Our objective was to define the kinetics, mechanisms, and sub-cellular fractions involved in TRs degradation. Methods: We used pulse-chase analyses, time-course experiments carried out in presence of cycloheximide (to inhibit new protein synthesis), and biochemical fractionation with Western blot analyses to determine the kinetics of the degradation of the TRβ isoform, TRβ1, in transiently transfected QBI-HEK 293A cells. Results: We observed that TRβ1 degradation is mediated by the proteasome pathway. Also, the kinetics of TRβ1 degradation is atypical due to the co-existence of more than one TRβ1 population, located in different cellular compartments and having different stability profiles. Moreover, TRβ1 degradation was unaffected by a mutation in its putative PEST motif, which confers turnover of other proteins. Conclusion: Our findings introduce novel evidence suggesting that stable and unstable forms of TRβ1, which might have distinct functions, co-exist in cells.

Several mammalian forkhead transcription factors have been shown to impact on cell cycle regulation and are themselves linked to cell cycle control systems. Here we have investigated the little studied mammalian forkhead transcription factor FOXK2 and demonstrate that it is subject to control by cell cycle-regulated protein kinases. FOXK2 exhibits a periodic rise in its phosphorylation levels during the cell cycle, with hyperphosphorylation occurring in mitotic cells. Hyperphosphorylation occurs in a cyclin-dependent kinase (CDK)·cyclin-dependent manner with CDK1·cyclin B as the major kinase complex, although CDK2 and cyclin A also appear to be important. We have mapped two CDK phosphorylation sites, serines 368 and 423, which play a role in defining FOXK2 function through regulating its stability and its activity as a transcriptional repressor protein. These two CDK sites appear vital for FOXK2 function because expression of a mutant lacking these sites cannot be tolerated and causes apoptosis.