Alpha1 Subunit Research Articles

Altered expression of laminin alpha1 in aganglionic colon of endothelin receptor-B null mouse model of Hirschsprung's disease.

Laminin, an extracellular matrix molecule, is essential for normal development of the nervous system. The alpha1 subunit of laminin-1 (LAMA1) has been reported to promote neurites and outgrowth and is expressed only during embryogenesis. Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize Enteric neural crest-derived cell (ENCC)s with a green fluorescent protein, Venus. We designed this study to investigate the expression of LAMA1 using Sox10-VENUS mice gut. We harvested the gut on days 13.5 (E13.5) and 15.5 (E15.5) of gestation. Sox10-VENUS+/Ednrb -/- mice (n = 8) were compared with Sox10-VENUS+/Ednrb +/+ mice (n = 8) as controls. Gene expression of LAMA1 was analysed by real-time RT-PCR. Fluorescent immunohistochemistry was performed to assess protein distribution. The relative mRNA expression levels of LAMA1 were significantly increased in HD in the proximal and distal colon on E15.5 compared to controls (p < 0.05), whereas there were no significant differences on E13.5. LAMA1 was expressed in the serosa, submucosa and basal lamina in the gut, and was markedly increased in the proximal and distal colon of HD on E15.5. Altered LAMA1 expression in the aganglionic region may contribute to impaired ENCC migration, resulting in HD. These data could help in understanding the pathophysiologic interactions between LAMA1 and ENCC migration.

Lateral septum stimulation disinhibits dopaminergic neurons in the antero-ventral region of the ventral tegmental area: Role of GABA-A alpha 1 receptors

The mechanisms commanding the activity of dopaminergic neurons of the ventral tegmental area (VTA) and the location of these neurons are relevant for the coding and expression of motivated behavior associated to reward-related signals. Anatomical evidence shows that several brain regions modulate VTA dopaminergic neurons activity via multiple mechanisms. However, there is still scarce knowledge of how the lateral septum (LS) modulates VTA activity. We performed in-vivo dual-probe microdialysis to measure VTA dopamine, glutamate and GABA extracellular levels after LS stimulation in the presence or absence of GABAergic antagonists. Anterograde tracing and immunohistochemical analysis was used to reveal the anatomical relationship between LS and VTA. LS stimulation significantly increased dopamine and GABA, but not glutamate, VTA extracellular levels. Intra VTA infusion of bicuculline, GABA-A receptor antagonist, inhibited the increase of dopamine but not of GABA VTA levels induced by LS stimulation. Intra VTA infusion of indiplon, selective positive allosteric modulator of GABA-A receptors containing alpha1 subunit, significantly increases VTA dopamine extracellular levels induced by LS. Combined c-Fos and tyrosine hydroxylase immunohistochemistry, revealed that LS stimulation increases the activity of dopaminergic neurons in the antero-ventral region of the VTA. Consistently, anterograde tracing with biotinylated dextran amine revealed the existence of fibers arising from the LS to the antero-ventral region of the VTA. Taken together, our results suggest that LS modulates dopaminergic activity in the antero-ventral region of VTA by inhibiting GABAergic interneurons bearing GABA-A receptors containing alpha1 subunit.

P 76 Maturation deficits in striatal interneurons in a phenotypic model of dystonia

GABAergic disinhibition has been suggested to play a critical role in the pathophysiology of several basal ganglia disorders, including dystonia, a common incurable movement disorder characterized by involuntary muscle contractions resulting in abnormal movements and/or postures. Previous studies have shown a deficit of striatal GABAergic interneurons (IN) in the dtsz mutant hamster, one of the few phenotypic animal models of dystonia. The resulting lack of inhibition of striatal projection neurons is likely involved in development of dystonic episodes which is corroborated by antidystonic effects of GABA potentiating drugs in the animal model and in dystonia patients. However, mechanisms underlying this deficit in striatal IN are largely unknown. In the present study, we investigated the migration and maturation of striatal IN during postnatal development (18 days of age) and at age of highest severity of dystonia (33 days of age) in the dtsz hamster by immunohistochemistry and real time PCR. In line with previous findings, the density of GAD67 positive IN and the level of parvalbumin mRNA, a marker for fast spiking GABAergic IN, were lower in mutant hamsters than in controls. However, an unaltered density of Nkx2.1 labeled cells and Nkx2.1 mRNA level indicated that the migration of GABAergic IN into the striatum was not retarded. KCC2 cation/chloride transporter and cytosolic carboanhydrase VII mRNA expression, used as markers for maturation of striatal GABAergic signaling, as well as expression of BDNF were unaltered. However, we found a reduced number of IN expressing the alpha1 subunit of the GABAA-receptor (37.5%) in dtsz hamsters at an age of 33 days, but not after spontaneous remission of dystonia at an age of 90 days. Since IN shift expression from alpha2 to alpha1 subunits during postnatal maturation, this result together with a decreased parvalbumin mRNA expression suggest a delayed maturation of striatal GABAergic IN in this animal model, which might underlie abnormal neuronal activity and striatal plasticity. The potential link between alterations in GABAA receptor subunits and GABAergic disinhibition in dystonia deserves further attention in research on the pathophysiology and therapeutic targets.

Bovine lineage specification revealed by single-cell gene expression analysis from zygote to blastocyst.

Preimplantation embryos undergo zygotic genome activation and lineage specification resulting in three distinct cell types in the late blastocyst. The molecular mechanisms underlying this progress are largely unknown in bovines. Here, we sought to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the bovine blastocyst. Using a quantitative microfluidics approach in single cells, we analyzed mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency in parallel in 384 individual cells from bovine preimplantation embryos. The developmental transitions can be distinguished by distinctive gene expression profiles and we identified NOTCH1, expressed in early developmental stages, while T-box 3 (TBX3) and fibroblast growth factor receptor 4 (FGFR4), expressed in late developmental stages. Three lineages can be segregated in bovine expanded blastocysts based on the expression patterns of lineage-specific genes such as disabled homolog 2 (DAB2), caudal type homeobox 2 (CDX2), ATPase H+/K+ transporting non-gastric alpha2 subunit (ATP12A), keratin 8 (KRT8), and transcription factor AP-2 alpha (TFAP2A) for trophectoderm; GATA binding protein 6 (GATA6) and goosecoid homeobox (GSC) for primitive endoderm; and Nanog homeobox (NANOG), teratocarcinoma-derived growth factor 1 (TDGF1), and PR/SET domain 14 (PRDM14) for epiblast. Moreover, some lineage-specific genes were coexpressed in blastomeres from the morula. The commitment to trophectoderm and inner cell mass lineages in bovines occurs later than in the mouse, and KRT8 might be an earlier marker for bovine trophectoderm cells. We determined that TDGF1 and PRDM14 might play pivotal roles in the primitive endoderm and epiblast specification of bovine blastocysts. Our results shed light on early cell fate determination in bovine preimplantation embryos and offer theoretical support for deriving bovine embryonic stem cells.

Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization.

Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

Balancing the autonomic nervous system to reduce inflammation in rheumatoid arthritis.

Imbalance in the autonomic nervous system (ANS) has been observed in many established chronic autoimmune diseases, including rheumatoid arthritis (RA), which is a prototypic immune-mediated inflammatory disease (IMID). We recently discovered that autonomic dysfunction precedes and predicts arthritis development in subjects at risk of developing seropositive RA. In addition, RA patients with relatively high vagus nerve tone (higher parasympathetic parameters, measured by heart rate variability) respond better to antirheumatic therapies. Together, these data suggest that the ANS may control inflammation in humans. This notion is supported by experimental studies in animal models of RA. We have found that stimulation of the so-called cholinergic anti-inflammatory pathway by efferent electrical vagus nerve stimulation (VNS) or pharmacological activation of the alpha7 subunit of nicotinic acetylcholine receptors (α7nAChR) improves clinical signs and symptoms of arthritis, reduces cytokine production and protects against progressive joint destruction. Conversely, increased arthritis activity was observed in alpha7nAChR knockout mice. These studies together with previous work in animal models of sepsis and other forms of inflammation provided the rationale for an experimental clinical trial in patients with RA. We could for the first time show that an implantable vagus nerve stimulator inhibits peripheral blood cytokine production in humans. VNS significantly inhibited TNF and IL-6 production and improved RA disease severity, even in some patients with therapy-resistant disease. This work strongly supports further studies using a bioelectronic approach to treat RA and other IMIDs.

A mutation in GABRB3 associated with Dravet syndrome.

Dravet syndrome is a rare and severe type of epilepsy in infants. Approximately, 70-80% of patients with Dravet syndrome have mutations in SCN1A, the gene encoding the alpha-1 subunit of the sodium channel, while some simplex patients have variants in one of several other genes, including but not limited to GABRA1, SCN2A, STXBP1, GABRG2, and SCN1B. In this study, we performed exome sequencing in six patients with SCN1A-negative Dravet syndrome to identify other genes related to this disorder. In one affected individual, we detected a novel de novo heterozygous missense variant, c.695G>A, p.(Arg232Gln), in GABRB3, the gene encoding the β3-subunit of the gamma-aminobutyric acid type A (GABAA) receptor, which mediates inhibitory signaling within the central nervous system. In summary, the data in this study identify GABRB3 as a candidate gene for Dravet syndrome.

A Proteomic Approach Suggests Unbalanced Proteasome Functioning Induced by the Growth-Promoting Bacterium Kosakonia radicincitans in Arabidopsis.

Endophytic plant growth-promoting bacteria have significant impact on the plant physiology and understanding this interaction at the molecular level is of particular interest to support crop productivity and sustainable production systems. We used a proteomics approach to investigate the molecular mechanisms underlying plant growth promotion in the interaction of Kosakonia radicincitans DSM 16656 with Arabidopsis thaliana. Four weeks after the inoculation, the proteome of roots from inoculated and control plants was compared using two-dimensional gel electrophoresis and differentially abundant protein spots were identified by liquid chromatography tandem mass spectrometry. Twelve protein spots were responsive to the inoculation, with the majority of them being related to cellular stress reactions. The protein expression of 20S proteasome alpha-3 subunit was increased by the presence of K. radicincitans. Determination of proteasome activity and immuno blotting analysis for ubiquitinated proteins revealed that endophytic colonization interferes with ubiquitin-dependent protein degradation. Inoculation of rpn12a, defective in a 26S proteasome regulatory particle, enhanced the growth-promoting effect. This indicates that the plant proteasome, besides being a known target for plant pathogenic bacteria, is involved in the establishment of beneficial interactions of microorganisms with plants.

Expression of interleukin-13 and its receptors in mycosis fungoides

Objective To measure the expression of interleukin-13 (IL-13) and its receptors in mycosis fungoides (MF) lesions, and to investigate their clinical significance. Methods A total of 34 paraffin-embedded specimens of MF, which was confirmed by clinical and histopathological features, immunophenotyping and/or T-cell receptor gene rearrangements, were collected from Hangzhou Third People's Hospital between January 2010 and March 2016. According to the tumor-node-metastasis (TNM) staging system, 5 patients were at stageⅠA, 9 at stageⅠB, 17 at stageⅡA, and 3 at stageⅡB. Ten normal skin tissue specimens served as controls. Immunohistochemical study was conducted to measure the expression of IL-13, IL-13Rα1 and IL-13Rα2. Results IL-13, IL-13Rα1 and IL-13Rα2 were all expressed in atypical lymphoid cells and epidermotropic lymphoid cells in MF lesions at various stages. IL-13Rα2 was highly expressed in all the MF lesions. None of IL-13 and its receptors were expressed in normal skin tissues and lymphocytes. The expression rates of IL-13 and its receptors in MF lesions increased along with the progression of MF. Additionally, the expression rates of IL-13 (10.00% ± 3.14%) , IL-13Rα1 (21.43% ± 6.88%) and IL-13Rα2 (31.14% ± 6.38%) significantly decreased in MF lesions at stage Ⅰ compared with those at stageⅡ (27.50% ± 11.00%, 39.45% ± 9.43%, 44.40% ± 11.15%, respectively, all P 0.05) . Conclusion IL-13 and its receptors, especially IL-13Rα2, may be expected to serve as biomarkers for early diagnosis of MF and prediction of its biological behaviors. Key words: Mycosis fungoides; Cell transformation, neoplastic; Interleukin-13; Interleukin-13 receptor alpha1 subunit; Interleukin-13 receptor alpha2 subunit

Test-retest reproducibility of quantitative binding measures of [11C]Ro15-4513, a PET ligand for GABAA receptors containing alpha5 subunits

IntroductionAlteration of γ-aminobutyric acid “A” (GABAA) receptor-mediated neurotransmission has been associated with various neurological and psychiatric disorders. [11C]Ro15-4513 is a PET ligand with high affinity for α5-subunit-containing GABAA receptors, which are highly expressed in limbic regions of the human brain (Sur et al., 1998). We quantified the test-retest reproducibility of measures of [11C]Ro15-4513 binding derived from six different quantification methods (12 variants). MethodsFive healthy males (median age 40 years, range 38–49 years) had a 90-min PET scan on two occasions (median interval 12 days, range 11–30 days), after injection of a median dose of 441 MegaBequerels of [11C]Ro15-4513. Metabolite-corrected arterial plasma input functions (parent plasma input functions, ppIFs) were generated for all scans.We quantified regional binding using six methods (12 variants), some of which were region-based (applied to the average time-activity curve within a region) and others were voxel-based: 1) Models requiring arterial ppIFs – regional reversible compartmental models with one and two tissue compartments (2kbv and 4kbv); 2) Regional and voxelwise Logan’s graphical analyses (Logan et al., 1990), which required arterial ppIFs; 3) Model-free regional and voxelwise (exponential) spectral analyses (SA; (Cunningham and Jones, 1993)), which also required arterial ppIFs; 4) methods not requiring arterial ppIFs – voxelwise standardised uptake values (Kenney et al., 1941), and regional and voxelwise simplified reference tissue models (SRTM/SRTM2) using brainstem or alternatively cerebellum as pseudo-reference regions (Lammertsma and Hume, 1996; Gunn et al., 1997).To compare the variants, we sampled the mean values of the outcome parameters within six bilateral, non-reference grey matter regions-of-interest. Reliability was quantified in terms of median absolute percentage test-retest differences (MA-TDs; preferentially low) and between-subject coefficient of variation (BS-CV, preferentially high), both compounded by the intraclass correlation coefficient (ICC). These measures were compared between variants, with particular interest in the hippocampus. ResultsTwo of the six methods (5/12 variants) yielded reproducible data (i.e. MA-TD <10%): regional SRTMs and voxelwise SRTM2s, both using either the brainstem or the cerebellum; and voxelwise SA. However, the SRTMs using the brainstem yielded a lower median BS-CV (7% for regional, 7% voxelwise) than the other variants (8–11%), resulting in lower ICCs. The median ICCs across six regions were 0.89 (interquartile range 0.75–0.90) for voxelwise SA, 0.71 (0.64–0.84) for regional SRTM-cerebellum and 0.83 (0.70–0.86) for voxelwise SRTM-cerebellum. The ICCs for the hippocampus were 0.89 for voxelwise SA, 0.95 for regional SRTM-cerebellum and 0.93 for voxelwise SRTM-cerebellum. ConclusionQuantification of [11C]Ro15-4513 binding shows very good to excellent reproducibility with SRTM and with voxelwise SA which, however, requires an arterial ppIF. Quantification in the α5 subunit-rich hippocampus is particularly reliable. The very low expression of the α5 in the cerebellum (Fritschy and Mohler, 1995; Veronese et al., 2016) and the substantial α1 subunit density in this region may hamper the application of reference tissue methods.

AMPK modulatory activity of olive-tree leaves phenolic compounds: Bioassay-guided isolation on adipocyte model and in silico approach.

ScopeOlive-tree polyphenols have demonstrated potential for the management of obesity-related pathologies. We aimed to explore the capacity of Olive-tree leaves extract to modulate triglyceride accumulation and AMP-activated protein kinase activity (AMPK) on a hypertrophic adipocyte model.MethodsIntracellular triglycerides and AMPK activity were measured on the hypertrophic 3T3-L1 adipocyte model by AdipoRed and immunofluorescence microscopy, respectively. Reverse phase high performance liquid chromatography coupled to time-of-flight mass detection with electrospray ionization (RP-HPLC-ESI-TOF/MS) was used for the fractionation of the extract and the identification of the compounds. In-silico molecular docking of the AMPK alpha-2, beta and gamma subunits with the identified compounds was performed.ResultsOlive-tree leaves extract decreased the intracellular lipid accumulation through AMPK-dependent mechanisms in hypertrophic adipocytes. Secoiridoids, cinnamic acids, phenylethanoids and phenylpropanoids, flavonoids and lignans were the candidates predicted to account for this effect. Molecular docking revealed that some compounds may be AMPK-gamma modulators. The modulatory effects of compounds over the alpha and beta AMPK subunits appear to be less probable.ConclusionsOlive-tree leaves polyphenols modulate AMPK activity, which may become a therapeutic aid in the management of obesity-associated disturbances. The natural occurrence of these compounds may have important nutritional implications for the design of functional ingredients.

Protective Effect of Resveratrol on the Brain in a Rat Model of Epilepsy.

Accumulating evidence has suggested resveratrol as a promising drug candidate for the treatment of epilepsy. To validate this, we tested the protective effect of resveratrol on a kainic acid (KA)-induced epilepsy model in rats and investigated the underlying mechanism. We found that acute resveratrol application partially inhibited evoked epileptiform discharges in the hippocampal CA1 region. During acute, silent and chronic phases of epilepsy, the expression of hippocampal kainate glutamate receptor (GluK2) and the GABAA receptor alpha1 subunit (GABAAR-alpha1) was up-regulated and down-regulated, respectively. Resveratrol reversed these effects and induced an antiepileptic effect. Furthermore, in the chronic phase, resveratrol treatment inhibited the KA-induced increased glutamate/GABA ratio in the hippocampus. The antiepileptic effects of resveratrol may be partially attributed to the reduction of glutamate-induced excitotoxicity and the enhancement in GABAergic inhibition.

Identification of differentially-expressed of Olfactomedin-related proteins 4 and COL11A1 in Iranian patients with intestinal gastric cancer.

Due to limited information on these genes and to a better understanding of common biomarkers associated with cancer of the digestive tract routes, we aim to evaluated expression level of Olfactomedin4 (OLFM4) and (pro)collagen11A1/COL11A1 genes in people with gastric cancer in Iran. Gastric cancer is one of the main cause of cancer death. The early prognosis of gastric cancer is still a matter of debate. Human olfactomedin4 (OLFM4) is a glycoprotein that generally known as the antiapoptotic protein. (pro) collagen11A1/COL11A1 codes for the alpha-1 subunit of type XI collagen which exists in extracellular minor fibrillar collagen. In most cases, OLFM4 and COL11A1 are found to be up-regulated in many types of human cancers including gastric cancer. 35 tissue samples were collected including 25 sample of patients with intestinal gastric cancer and 10 healthy controls. Expression level of OLFM4 and COL11A1 genes identified by using RGQ software. For analysis of real time-PCR products, Rotor-Gene Q series software was used. Our finding showed that expression level of OLFM4 was significantly upregulated and COL11A1 did not show any significant difference in expression level in Iranian population with gastric cancer samples compared with those in normal samples. The results recommend that expression profiling of OLFM4 can be used for diagnosis of gastric cancer, and OLFM4 seems to be used as a biomarker for the diagnosis of gastric cancer. Regarding to our result, unlike some studies, COL11A1 did not show any significant difference between normal and tumor tissue which could explain ethological role in distribution of gastric cancer.

TNF-alpha stimulation increases dental pulp stem cell migration in vitro through integrin alpha-6 subunit upregulation

ObjectiveThe dissemination of stem cells into tissues requiring inflammatory and reparative response is fundamentally dependent upon their chemotactic migration. Expression of TNF-α is up regulated in inflamed pulps. Dental pulp cells are also known to express integrin α6 subunit. Expression of integrin subunit α6 has been linked to the acquisition of migratory potential in a wide variety of cell types in both pathological and physiological capacities. Therefore, in this study we examined the effects of a pleiotropic cytokine TNF-α on the migration of hDPSCs and investigated its relationship with expression of integrin α6 in hDPSCs during chemotactic migration. DesignhDPSC cultures were established. Protein expression profile of α6 integrin subunit was determined. Effect of exogenous TNF-α (50ng/mL) on hDPSCs’ migration potential was evaluated by transwell inserts and in vitro scratch assay. Upregulation/downregulation of TNF-α mediated migration was assayed in presence/absence of integrin α6 respectively. To suppress integrin α6 expression, cells were transfected with integrin α6 siRNA and then cell migration and cytoskeletal changes were evaluated. ResultsOur results showed significant increase of hDPSCs’ migration after stimulation with TNF-α. By knockdown of integrin α6, which is upregulated by TNF-α, we observed a decrease in the TNF-α directed chemotaxis of hDPSCs. ConclusionIn this study, we show that activation of integrin α6 brought about by TNF-α led to an increase in migratory activity in DPSCs in vitro thus describing a novel association between a cytokine TNF-α and α6 chain of an adhesion receptor integrin in regulating migration of hDPSCs.

Altered postnatal maturation of striatal GABAergic interneurons in a phenotypic animal model of dystonia

GABAergic disinhibition has been suggested to play a critical role in the pathophysiology of several basal ganglia disorders, including dystonia, a common movement disorder. Previous studies have shown a deficit of striatal GABAergic interneurons (IN) in the dtsz mutant hamster, one of the few phenotypic animal models of dystonia. However, mechanisms underlying this deficit are largely unknown. In the present study, we investigated the migration and maturation of striatal IN during postnatal development (18days of age) and at age of highest severity of dystonia (33days of age) in this hamster model. In line with previous findings, the density of GAD67-positive IN and the level of parvalbumin mRNA, a marker for fast spiking GABAergic IN, were lower in the dtsz mutant than in control hamsters. However, an unaltered density of Nkx2.1 labeled cells and Nkx2.1 mRNA level suggested that the migration of GABAergic IN into the striatum was not retarded. Therefore, different factors that indicate maturation of GABAergic IN were determined. While mRNA of the KCC2 cation/chloride transporters and the cytosolic carboanhydrase VII, used as markers for the so called GABA switch, as well as BDNF were unaltered, we found a reduced number of IN expressing the alpha1 subunit of the GABAA-receptor (37.5%) in dtsz hamsters at an age of 33days, but not after spontaneous remission of dystonia at an age of 90days. Since IN shift expression from alpha2 to alpha1 subunits during postnatal maturation, this result together with a decreased parvalbumin mRNA expression suggest a delayed maturation of striatal GABAergic IN in this animal model, which might underlie abnormal neuronal activity and striatal plasticity.

Abstract P268: The Effects of a Slow Pressor Dose of Angiotensin II on Sodium Transporters Expression in the Kidney Cortex are Independent of PPAR-alpha

A slow pressor dose of Angiotensin II (Ang II) has been shown to increase the expression of sodium transporters in the proximal tubules (NHE3), TALH (NKCC2) and distal nephrons (NCC) of Sprague Dawley rats before an increase in blood pressure. Peroxisome Proliferator Activated Receptor - alpha (PPAR-alpha) has been shown to be involved in pressure natriuresis through changes in sodium transport via ameloride and thiazide-dependent mechanisms. We hypothesized that the changes in expression of the sodium transporters during Ang II hypertension were dependent upon PPAR-alpha expression. To address this hypothesis, we treated wild-type (WT) and PPAR-alpha knockout (KO) mice with a slow pressor dose of Ang II (400 ng/kg/min) for 12 days. Mean arterial pressure (MAP) was measured by radiotelemetry. Control MAP was not different between WT (110 ± 8 mmHg) and PPAR-alpha KO mice (112 ± 12 mmHg). On day 12 of Ang II, MAP for PPAR-alpha KO (156 ± 16) mice was significantly higher than WT (138 ± 11 mmHg) mice. The expression of NHE3, NHERF1, NKA-α1 subunit, NKCC2, and NCC was detected in kidney cortical homogenates by western blotting. Kidneys were homogenized and 25 μg of supernatant proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose paper, and blotted against antibodies to NHE3, NHERF1, NKA α1 subunit, NKCC2, and NCC. The slow pressor dose of Ang II decreased the expression of NHE3 in WT + Ang II (0.14 ± 0.02 ODU) and PPAR-alpha KO + Ang II (0.10 ± 0.02 ODU), when compared to WT (2.61 ± 0.93 ODU) and PPAR-alpha KO (2.20 ± 0.58 ODU) controls. Ang II-treatment also decreased NKCC2 in both WT (0.31 ± 0.10 ODU) and PPAR-alpha KO (0.22 ± 0.03 ODU). Ang II hypertension caused similar decreases in NCC and NHERF1 expression in WT and PPAR-α KO mice. NKA alpha1 subunit expression was increased during Ang II hypertension in both WT (1.06 ± 0.26 ODU) and PPAR-α KO (1.64 ± 0.26 ODU) mice. Our results suggest that the effects of a slow pressor dose of Ang II on expression of sodium transporters are independent of PPAR-alpha expression. Future studies are needed to determine the effects of decreasing NHE3, NKCC2, NCC and NHERF1 expression in the kidney on urinary salt excretion during a slow pressor dose of Ang II.

The Nicotinic α6-Subunit Selective Antagonist bPiDI Reduces Alcohol Self-Administration in Alcohol-Preferring Rats.

Cigarettes and alcohol are the most abused substances in the world and are commonly co-abused. Nicotine primarily acts in the brain on nicotinic acetylcholine receptors (nAChR), which are also a target for alcohol. The alpha6 subunit of nAChR is expressed almost exclusively in the brain reward system and may modulate the rewarding properties of alcohol and nicotine. Recently, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI) was synthesized as a selective, brain penetrant α6 subunit antagonist that reduces nicotine self-administration. The current study aimed to examine the effects of bPiDI on alcohol self-administration in inbred alcohol-preferring (iP) rats. Adult, male iP rats were trained to self-administer alcohol or sucrose. Once stable responding was achieved, rats were injected with bPiDI (1, 3mg/kg, i.p.) and tested for self-administration under fixed and progressive ratio schedules of reinforcement. They subsequently underwent extinction, in which no rewards or cues were presented in the operant chambers. Then, they were injected with bPiDI prior to testing for cue-induced reinstatement of reward seeking. bPiDI (3mg/kg) significantly reduced alcohol self-administration in both fixed and progressive ratios without any effects on sucrose self-administration or locomotor activity. In contrast, bPiDI (3mg/kg) did not inhibit cue-induced reinstatement of either alcohol or sucrose seeking. The results support the involvement of α6 containing nAChR in reinforcing effects of alcohol, but not relapse to alcohol-seeking, without any impact on responding for a natural reward or general activity. bPiDI may be a potential lead molecule for a therapeutic strategy to limit nicotine and alcohol consumption.

Paracrine nitric oxide induces expression of cardiac sarcomeric proteins in adult progenitor cells through soluble guanylyl cyclase/cyclic-guanosine monophosphate and Wnt/β-catenin inhibition

Cardiac progenitor cells (CPC) from adult hearts can differentiate to several cell types composing the myocardium but the underlying molecular pathways are poorly characterized. We examined the role of paracrine nitric oxide (NO) in the specification of CPC to the cardiac lineage, particularly through its inhibition of the canonical Wnt/β-catenin pathway, a critical step preceding cardiac differentiation. Sca1 + CPC from adult mouse hearts were isolated by magnetic-activated cell sorting and clonally expanded. Pharmacologic NO donors increased their expression of cardiac myocyte-specific sarcomeric proteins in a concentration and time-dependent manner. The optimal time window for NO efficacy coincided with up-regulation of CPC expression of Gucy1a3 (coding the alpha1 subunit of guanylyl cyclase). The effect of paracrine NO was reproduced in vitro upon co-culture of CPC with cardiac myocytes expressing a transgenic NOS3 (endothelial nitric oxide synthase) and in vivo upon injection of CPC in infarcted hearts from cardiac-specific NOS3 transgenic mice. In mono- and co-cultures, this effect was abrogated upon inhibition of soluble guanylyl cyclase or nitric oxide synthase, and was lost in CPC genetically deficient in Gucy1a3. Mechanistically, NO inhibits the constitutive activity of the canonical Wnt/β-catenin in CPC and in cell reporter assays in a guanylyl cyclase-dependent fashion. This was paralleled with decreased expression of β-catenin and down-regulation of Wnt target genes in CPC and abrogated in CPC with a stabilized, non-inhibitable β-catenin. Exogenous or paracrine sources of NO promote the specification towards the myocyte lineage and expression of cardiac sarcomeric proteins of adult CPC. This is contingent upon the expression and activity of the alpha1 subunit of guanylyl cyclase in CPC that is necessary for NO-mediated inhibition of the canonical Wnt/β-catenin pathway.

Dravet Syndrome: Diagnosis and Long-Term Course.

Dravet syndrome is one of the most severe epilepsy syndromes of early childhood, and it comes with very high morbidity and mortality. The typical presentation is characterized by hemiclonic or generalized clonic seizures triggered by fever during the first year of life, followed by myoclonic, absence, focal and generalized tonic-clonic seizures. Non-convulsive status epilepticus and epileptic encephalopathy are common. Development is normal in the first year of life, but most individuals eventually suffer from intellectual impairment. Dravet syndrome is associated with mutations in the sodium channel alpha1 subunit gene (SCN1A) in 70-80% of individuals. SCN1A mutation results in inhibition of the GABAergic inhibitory interneurons, leading to excessive neuronal excitation. The "interneuron hypothesis" is the current most accepted pathophysiological mechanism of Dravet syndrome. The mortality rate is increased significantly in Dravet syndrome. Ataxia, a characteristic crouched gait and Parkinson's symptoms may develop in some individuals. It is likely that Dravet syndrome is underdiagnosed in adults with treatment-resistant epilepsy. Early diagnosis is important to avoid anti-seizure medications that exacerbate seizures.

Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample.

Variation in genes coding for nicotinic acetylcholine receptor (nAChR) subunits affect cognitive processes and may contribute to the genetic architecture of neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs) in the CHRNA4 gene that codes for the alpha4 subunit of alpha4/beta2-containing receptors have previously been implicated in aspects of (mostly visual) attention and smoking-related behavioral measures. Here we investigated the effects of six synonymous but functional CHRNA4 exon 5 SNPs on the N100 event-related potential (ERP), an electrophysiological endophenotype elicited by a standard auditory oddball. A total of N = 1,705 subjects randomly selected from the general population were studied with electroencephalography (EEG) as part of the German Multicenter Study on nicotine addiction. Two of the six variants, rs1044396 and neighboring rs1044397, were significantly associated with N100 amplitude. This effect was pronounced in females where we also observed an effect on reaction time. Sequencing of the complete exon 5 region in the population sample excluded the existence of additional/functional variants that may be responsible for the observed effects. This is the first large-scale population-based study investigation the effects of CHRNA4 SNPs on brain activity measures related to stimulus processing and attention. Our results provide further evidence that common synonymous CHRNA4 exon 5 SNPs affect cognitive processes and suggest that they also play a role in the auditory system. As N100 amplitude reduction is considered a schizophrenia-related endophenotype the SNPs studied here may also be associated with schizophrenia outcome measures.