Modulation of NLRP3 inflammasomes activation contributes to improved survival and function of mesenchymal stromal cell spheroids.

Mesenchymal stem cells (MSCs) are ubiquitous multipotent cells that exhibit significant therapeutic potentials in a variety of disorders. Nevertheless, their clinical efficacy is limited owing to poor survival, low rate of engraftment, and impaired potency upon transplantation. Spheroidal three-dimensional (3D) culture of MSCs (MSC3D) has been proven to better preserve their in vivo functional properties. However, the molecular mechanisms underlying the improvement in MSC function by spheroid formation are not clearly understood. NLRP3 inflammasomes, a key component of the innate immune system, have recently been shown to play a role in cell fate decision of MSCs. The present study examined the role of NLRP3 inflammasomes in the survival and potency of MSC spheroids. We found that MSC3D led to decreased activation of NLRP3 inflammasomes through alleviation of ER stress in an autophagy-dependent manner. Importantly, downregulation of NLRP3 inflammasomes signaling critically contributes to the enhanced survival rate in MSC3D through modulation of pyroptosis and apoptosis. The critical role of NLRP3 inflammasome suppression in the enhanced therapeutic efficacy of MSC spheroids was further confirmed in an in vivo mouse model of DSS-induced colitis. These findings suggest that 3D culture confers survival and functional advantages to MSCs by suppressing NLRP3 inflammasome activation.

An Efficient Ionic Liquid-Mediated Extraction and Enrichment of Isoimperatorin from Ostericum koreanum (Max.) Kitagawa.

Ionic liquids (ILs) have attracted significant interest because of their desirable properties. These characteristics have improved their application to overcome the shortcomings of conventional separation techniques for phytochemicals. In this study, several ILs were investigated for their capacity to extract isoimperatorin, a bioactive furanocoumarin, from the roots of Ostericum koreanum. Herein, 1-Butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4]) was selected as a promising IL for separating isoimperatorin. A central composite design was applied to optimize the extraction conditions. Under the optimal conditions, the yield of isoimperatorin reached 97.17 ± 1.84%. Additionally, the recovery of isoimperatorin from the [Bmim][BF4] solution was successfully achieved (87.73 ± 2.37%) by crystallization using water as an antisolvent. The purity of the isoimperatorin was greatly enhanced, from 0.26 ± 0.28% in the raw material to 26.94 ± 1.26% in the product, in a one-step crystallization process. Namely, an enhancement of approximately 103-folds was reached. The developed approach overcomes the shortcomings of conventional separation methods applied for gaining isoimperatorin by significantly reducing the laboriousness of the process and the consumption of volatile organic solvents. Moreover, the simplicity and effectiveness of the method are assumed to be valuable for producing isoimperatorin-enriched products and for promoting its purification. This work also confirms the efficiency of ILs as a promising material for the separation of phytochemicals.

Effect of high-fat diet on cognitive impairment in triple-transgenic mice model of Alzheimer's disease.

High-fat diet (HFD)-induced obesity is a risk factor for cognitive impairment in Alzheimer's disease (AD). It has been reported that two typical neuropathological markers of AD, ß-amyloid (Aß) peptide and hyperphosphorylated protein tau can cause neuronal apoptosis via oxidative stress, which ultimately leads to cognitive dysfunction. In this study, we tried to explore the molecular pathway underlying memory impairment in young AD transgenic mice model in response to HFD. We maintained non-transgenic control mice (non-Tg) and triple transgenic AD (3xTg-AD) mice aged 8 weeks on either normal diet (ND) containing 10% fat or HFD (60% fat) for 16 weeks. Cognitive functions were evaluated by Morris water maze and Y-maze tests. Behavioral tests showed a significant memory impairment in 3xTg-AD mice fed with HFD. HFD did not alter the levels of Aß and phospho-tau protein in the cortical region regardless of groups. However, 3xTg-AD mice fed with HFD exhibited increased neuronal oxidative stress and apoptosis as assessed by augmentation of lipid peroxidation, activation of caspase-3 and elevated ratio of Bax/Bcl-2. Furthermore, HFD markedly reduced the activation of redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2) by suppressing its up-stream regulatory protein kinase B/Akt as well as down-stream targets such as heme oxygenase-1 and manganese superoxide dismutase in these mice. Our findings suggest that HFD may accelerate cognitive impairment by enhancing oxidative stress and aggravating neuronal apoptosis via inactivation of Nrf2 signaling pathway.

Critical Role of AMPK/FoxO3A Axis in Globular Adiponectin-Induced Cell Cycle Arrest and Apoptosis in Cancer Cells.

Adiponectin predominantly secreted from adipose tissue has exhibited potent anti-proliferative properties in cancer cells via modulating cell cycle and apoptosis. FoxO3A, a Forkhead box O member of the transcription factor, plays a critical role in modulating expression of genes involved in cell death and/or survival. In this study, we investigated the role of FoxO3A signaling in anti-cancer activities of adiponectin. Herein, we have shown that treatment with globular adiponectin (gAcrp) increases p27 but decreases cyclinD1 expression in human hepatoma (HepG2) and breast (MCF-7) cancer cells. Gene ablation of FoxO3A prevented gAcrp-induced increase in p27 and decreased in cyclin D1 expression, and further ameliorated cell cycle arrest by gAcrp, indicating a critical role of FoxO3A in gAcrp-induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase-3/7 activation and increased Fas ligand (FasL) expression in both HepG2 and MCF-7 cells. Transfection with FoxO3A siRNA inhibited gAcrp-induced caspase-3/7 activation and FasL expression, suggesting that FoxO3A signaling also plays an important role in gAcrp-induced apoptosis of cancer cells. We also found that gene silencing of AMPK prevented gAcrp-induced nuclear translocation of FoxO3A in HepG2 and MCF-7 cells. In addition, suppression of AMPK also blocked gAcrp-induced cell cycle arrest and further attenuated gAcrp-induced caspase-3/7 activation, indicating that AMPK signaling plays a pivotal role in both gAcrp-induced cell cycle arrest and apoptosis via acting as an upstream signaling of FoxO3A. Taken together, our findings demonstrated that AMPK/FoxO3A axis plays a cardinal role in anti-proliferative effect of adiponectin in cancer cells.

Anti-tumor activity of WK88-1, a novel geldanamycin derivative, in gefitinib-resistant non-small cell lung cancers with Met amplification.

Although epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been introduced for the treatment of non-small cell lung cancer (NSCLC), the emergence of secondary T790M mutation in EGFR or amplification of the Met proto-oncogene restrain the clinical success of EGFR-TKIs. Since heat shock protein-90 (Hsp90) stabilizes various oncoproteins including EGFR and c-Met, the inhibition of Hsp90 activity appears as a rational strategy to develop anticancer drugs. Despite preclinical efficacy of geldanamycin-anasamycin (GA)-derivatives containing benzoquinone moiety as Hsp90 inhibitors, the hepatotoxicity of these GA-derivatives restricts their therapeutic benefit. We have prepared WK-88 series of GA-derivatives, which lack the benzoquinone moiety. In this study, we have examined the anticancer effects of WK88-1 in Met-amplified- and gefitinib-resistant (HCC827GR) NSCLC cells and its parental HCC827 cells. Treatment with WK88-1 reduced the cell viability in both HCC827 and HCC827GR cells, which was associated with marked decrease in the constitutive expression of Hsp90 client proteins, such as EGFR, ErbB2, ErbB3, Met and Akt. Moreover, WK88-1 attenuated phosphorylation of these Hsp90 client proteins and reduced the anchorage-independent growth of HCC827GR cells. Administration of WK88-1 did not cause hepatotoxicity in animals and significantly reduced the growth of HCC827GR cells xenograft tumors in nude mice. Our study provides evidence that ErbB3 might be a client for Hsp90 in Met-amplified NSCLCs. In conclusion, we demonstrate that inhibition of Hsp90 dampens the activation of EGFR- or c-Met-mediated survival of Met-amplified NSCLCs and that WK88-1 as a Hsp90 inhibitor alleviates gefitinib resistance in HCC827GR cells.

Effects of Corticosterone on Beta-Amyloid-Induced Cell Death in SH-SY5Y Cells.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuronal cell death and memory impairment. Corticosterone (CORT) is a glucocorticoid hormone produced by the hypothalamic-pituitary-adrenal axis in response to a stressful condition. Excessive stress and high CORT levels are known to cause neurotoxicity and aggravate various diseases, whereas mild stress and low CORT levels exert beneficial actions under pathophysiological conditions. However, the effects of mild stress on AD have not been clearly elucidated yet. In this study, the effects of low (3 and 30 nM) CORT concentration on Aß25-35-induced neurotoxicity in SH-SY5Y cells and underlying molecular mechanisms have been investigated. Cytotoxicity caused by Aß25-35 was significantly inhibited by the low concentration of CORT treatment in the cells. Furthermore, CORT pretreatment significantly reduced Aß25-35-mediated pro-apoptotic signals, such as increased Bim/Bcl-2 ratio and caspase-3 cleavage. Moreover, low concentration of CORT treatment inhibited the Aß25-35-induced cyclooxygenase-2 and pro-inflammatory cytokine expressions, including tumor necrosis factor-α and interleukin-1ß. Aß25-35 resulted in intracellular accumulation of reactive oxygen species and lipid peroxidation, which were effectively reduced by the low CORT concentration. As a molecular mechanism, low CORT concentration activated the nuclear factor-erythroid 2-related factor 2, a redox-sensitive transcription factor mediating cellular defense and upregulating the expression of antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase, glutamylcysteine synthetase, and manganese superoxide dismutase. These findings suggest that low CORT concentration exerts protective actions against Aß25-35-induced neurotoxicity and might be used to treat and/or prevent AD.

Quantitation and pharmacokinetics of 1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene (U0126) in rat plasma by liquid chromatography-tandem mass spectrometry.

A simple, robust, and rapid LC-MS/MS method was developed for the quantitation of U0126 and validated in rat plasma. Plasma samples (20 µL) were deproteinized using 200 µL ACN containing 30 ng/mL of chlorpropamide, internal standard. Chromatographic separation performed on an Agilent Poroshell 120 EC-C(18) column (4.6 × 50 mm, 2.7 µm particle size) with an isocratic mobile phase consisting of a 70:30 v/v mixture of ACN and 0.1% aqueous formic acid. Each sample was run at 0.6 mL/min for a total run time of 2 min per sample. Detection and quantification were performed using a mass spectrometer in selected reaction-monitoring mode with positive ESI at m/z 381 → 123.9 for U0126 and m/z 277 → 175 for the internal standard. The standard curve was linear over a concentration range of 20-5000 ng/mL with correlation coefficients greater than 0.9965. Precision, both intra- and interday, was less than 10.1% with an accuracy of 90.7-99.4%. No matrix effects were observed. U0126 in rat plasma degraded approximately 41.3% after 3-h storage at room temperature. To prevent degradation, sample handling should be on an ice bath and all solutions kept at 4°C. This method was successfully applied to a pharmacokinetic study of U0126 at various doses in rats.

α-Pinene Attenuates Methamphetamine-Induced Conditioned Place Preference in C57BL/6 Mice.

Methamphetamine (METH) is a powerful neurotoxic psychostimulant affecting dopamine transporter (DAT) activity and leading to continuous excess extracellular dopamine levels. Despite recent advances in the knowledge on neurobiological mechanisms underlying METH abuse, there are few effective pharmacotherapies to prevent METH abuse leading to brain damage and neuropsychiatric deficits. α-Pinene (APN) is one of the major monoterpenes derived from pine essential oils and has diverse biological properties including anti-nociceptive, anti-anxiolytic, antioxidant, and anti-inflammatory actions. In the present study, we investigated the therapeutic potential of APN in a METH abuse mice model. METH (1 mg/kg/day, i.p.) was injected into C57BL/6 mice for four alternative days, and a conditioned place preference (CPP) test was performed. The METH-administered group exhibited increased sensitivity to place preference and significantly decreased levels of dopamine-related markers such as dopamine 2 receptor (D2R) and tyrosine hydroxylase in the striatum of the mice. Moreover, METH caused apoptotic cell death by induction of inflammation and oxidative stress. Conversely, APN treatment (3 and 10 mg/kg, i.p.) significantly reduced METH-mediated place preference and restored the levels of D2R and tyrosine hydroxylase in the striatum. APN increased the anti-apoptotic Bcl-2 to pro-apoptotic Bax ratio and decreased the expression of inflammatory protein Iba-1. METH-induced lipid peroxidation was effectively mitigated by APN by up-regulation of antioxidant enzymes such as manganese-superoxide dismutase and glutamylcysteine synthase via activation of nuclear factor-erythroid 2-related factor 2. These results suggest that APN may have protective potential and be considered as a promising therapeutic agent for METH-induced drug addiction and neuronal damage.

Dopaminergic neuroprotective effects of inosine in MPTP-induced parkinsonian mice via brain-derived neurotrophic factor upregulation.

Parkinson's disease (PD) is the second most common neurodegenerative disease. However, no curative or modifying therapy is known. Inosine is a purine nucleoside that increases brain-derived neurotrophic factor (BDNF) expression in the brain through adenosine receptors. Herein, we investigated the neuroprotective effects of inosine and elucidated the mechanisms underlying its pharmacological action. Inosine rescued SH-SY5Y neuroblastoma cells from MPP+ injury in a dose-dependent manner. Inosine protection correlated with BDNF expression and the activation of its downstream signaling cascade, as the TrkB receptor inhibitor, K252a and siRNA against the BDNF gene remarkably reduced the protective effects of inosine. Blocking the A1 or A2A adenosine receptors diminished BDNF induction and the rescuing effect of inosine, indicating a critical role of adenosine A1 and A2A receptors in inosine-related BDNF elevation. We assessed whether the compound could protect dopaminergic neurons from MPTP-induced neuronal injury. Beam-walking and challenge beam tests revealed that inosine pretreatment for 3 weeks reduced the MPTP-induced motor function impairment. Inosine ameliorated dopaminergic neuronal loss and MPTP-mediated astrocytic and microglial activation in the substantia nigra and striatum. Inosine ameliorated the depletion of striatal dopamine and its metabolite following MPTP injection. BDNF upregulation and the activation of its downstream signaling pathway seemingly correlate with the neuroprotective effects of inosine. To our knowledge, this is the first study to demonstrate the neuroprotective effects of inosine against MPTP neurotoxicity via BDNF upregulation. These findings highlight the therapeutic potential of inosine in dopaminergic neurodegeneration in PD brains.

MS-5, a Naphthalene Derivative, Induces Apoptosis in Human Pancreatic Cancer BxPC-3 Cells by Modulating Reactive Oxygen Species.

Pancreatic cancer is one of the most fatal cancers with a poor prognosis. Standard chemotherapies have proven largely ineffective because of their toxicity and the development of resistance. Therefore, there is an urgent need to develop novel therapies. In this study, we investigated the antitumor activity of MS-5, a naphthalene derivative, on BxPC-3, a human pancreatic cancer cell line. We observed that MS-5 was cytotoxic to BxPC-3 cells, as well as inhibited the growth of cells in a concentration- and time- dependent manner. Flow cytometry analysis revealed that the percentage of annexin V-positive cells increased after MS-5 treatment. We also observed cleavage of caspases and poly (ADP-ribose) polymerase, and downregulation of Bcl-xL protein. Flow cytometry analysis of intracellular levels of reactive oxygen species (ROS) and mitochondrial superoxide suggested that MS-5 induced the generation of mitochondrial superoxide while lowering the overall intracellular ROS levels. Thus, MS-5 may be potential candidate for pancreatic cancer treatment.

Caspase Cleavage of Receptor Tyrosine Kinases in the Dependence Receptor Family.

Dependence receptors are a group of receptor proteins with shared characteristics of transducing two different signals within cells. They can transduce a positive signal of survival and differentiation in the presence of ligands. On the other hand, dependence receptors can transduce an apoptosis signal in the absence of ligands. The function of these receptors depends on the availability of their ligands. Several receptor tyrosine kinases (RTKs) have been reported as dependence receptors. When cells undergo apoptosis by dependence receptors, the intracellular domain of some RTKs is cleaved by the caspases. Among the RTKs that belong to dependence receptors, we focused on eight RTKs (RET, HER2, MET, ALK, TrkC, EphA4, EphB3, and c-KIT) that are cleaved by caspases. In this review, we describe the features of the receptors, their cleavage sites, and the fate of the cleaved products, as well as recent implications on them being used as potential therapeutics for cancer treatment.

CO-Induced TTP Activation Alleviates Cellular Senescence and Age-Dependent Hepatic Steatosis via Downregulation of PAI-1.

Aging can increase the risk of various hepatic diseases, especially non-alcoholic fatty liver disease (NAFLD). Although the mechanisms underlying the pathogenesis of age-related disorders such as NAFLD remain incompletely understood, recent studies have implicated the accumulation of senescent cells as a contributing factor. Here, we show that tristetraprolin (TTP) deficiency accelerates NAFLD during aging by enhancing the senescence-associated secretory phenotype (SASP) as well as several hallmarks of senescence. The sequestration of plasminogen activator inhibitor (PAI)-1, a mediator of cellular senescence, in stress granules, (SGs) inhibits cellular senescence. In our previous report, we have shown that carbon monoxide (CO), a small gaseous mediator, can induce the assembly of SGs via an integrated stress response. Here, we show that CO treatment promotes the assembly of SGs which can sequester PAI-1, resulting in the inhibition of etoposide (ETO)-induced cellular senescence. Notably, CO-induced TTP activation enhances PAI-1 degradation, leading to protection against ETO-induced cellular senescence. CO-dependent Sirt1 activation promotes the inclusion of TTP into SGs, leading to decreased PAI-1 levels. Therefore, our findings highlight the importance of TTP as a therapeutic target in age-related NAFLD and offer a potential new strategy to reduce the detrimental effects of senescent cells in hepatic disorders.

Protective Role of Corticosterone against Hydrogen Peroxide-Induced Neuronal Cell Death in SH-SY5Y Cells.

Stress breaks body balance, which can cause diverse physiological disorders and worsen preexisting diseases. However, recent studies have reported that controllable stress and overcoming from stress reinforce resilience to resist against more intense stress afterwards. In this study, we investigated the protective effect of corticosterone (CORT), a representative stress hormone against hydrogen peroxide (H2O2)-induced neuronal cell death and its underlying molecular mechanism in SH-SY5Y cells, a human neuroblastoma cell line. The decreased cell viability by H2O2 was effectively restored by the pretreatment with low concentration of CORT (0.03 µM for 72 h) in the cells. H2O2-increased expression of apoptotic markers such as PUMA and Bim was decreased by CORT pretreatment. Furthermore, pretreatment of CORT attenuated H2O2-mediated oxidative damages by upregulation of antioxidant enzymes via activation of nuclear factor erythroid 2-related factor 2 (Nrf2). These findings suggest that low concentration of CORT with eustressed condition enhances intracellular self-defense against H2O2-mediated oxidative cell death, suggesting a role of low concentration of CORT as one of key molecules for resilience and neuronal cell survival.

Hydroxyzine Induces Cell Death in Triple-Negative Breast Cancer Cells via Mitochondrial Superoxide and Modulation of Jak2/STAT3 Signaling.

Treatment of triple-negative breast cancer (TNBC) has been limited due to the lack of molecular targets. In this study, we evaluated the cytotoxicity of hydroxyzine, a histamine H1 receptor antagonist in human triple-negative breast cancer BT-20 and HCC-70 cells. Hydroxyzine inhibited the growth of cells in dose- and time-dependent manners. The annexin V/propidium iodide double staining assay showed that hydroxyzine induced apoptosis. The hydroxyzine-induced apoptosis was accompanied down-regulation of cyclins and CDKs, as well as the generation of reactive oxygen species (ROS) without cell cycle arrest. The effect of hydroxyzine on the induction of ROS and apoptosis on TNBC cells was prevented by pre-treatment with ROS scavengers, N-acetyl cysteine or Mito-TEMPO, a mitochondria-targeted antioxidant, indicating that an increase in the generation of ROS mediated the apoptosis induced by hydroxyzine. Western blot analysis showed that hydroxyzine-induced apoptosis was through down-regulation of the phosphorylation of JAK2 and STAT3 by hydroxyzine treatment. In addition, hydroxyzine induced the phosphorylation of JNK and p38 MAPK. Our results indicate that hydroxyzine induced apoptosis via mitochondrial superoxide generation and the suppression of JAK2/STAT3 signaling.

Formulation of a Gastroretentive In Situ Oral Gel Containing Metformin HCl Based on DoE.

A gastroretentive in situ oral gel containing metformin hydrochloride (Met HCl) was prepared based on sodium alginate (Sod ALG), calcium carbonate, and hydroxyethylcellulose (HEC). The optimal composition of the formulation was explored based on the design of experiments (DoE). First, a 32 full factorial design was used for formulation E1 to determine proper composition of Sod ALG and calcium carbonate. Second, a circumscribed central composite design was employed to add HEC as a thickening agent (formulation E2). The dissolution rates at 15, 30, 60, 120, and 240 min were used as responses. Partial least squares regression analysis indicated the effect of each component in delaying the release of Met HCl in the oral gel formulation. The optimized formulation E2-08 consisting of 1.88% Sod ALG, 0.63% HEC, and 1.00% calcium carbonate and two more formulations, E2-10 and E2-12 conformed to USP monograph for extended release. Other physicochemical properties, including floating lag time and duration, viscosity, and pH, measured for each batch and FT-IR spectrometry analysis showed no unexpected interaction between Met HCl and excipients. The current study suggests the potential use of a gastroretentive in situ oral gel for Met HCl helping patient compliance. This study highlights that a systematic approach based on DoE allows the formulation optimization.

Protective Effect of Alpinia oxyphylla Fruit against tert-Butyl Hydroperoxide-Induced Toxicity in HepG2 Cells via Nrf2 Activation and Free Radical Scavenging and Its Active Molecules.

Alpinia oxyphylla Miq. (Zingiberaceae) extract exerts protective activity against tert-butyl hydroperoxide-induced toxicity in HepG2 cells, and the antioxidant response element (ARE) luciferase activity increased 6-fold at 30 µg/mL in HepG2 cells transiently transfected with ARE-luciferase. To identify active molecules, activity-guided isolation of the crude extract led to four sesquiterpenes (1, 2, 5, 6) and two diarylheptanoids (3 and 4) from an n-hexane extract and six sesquiterpenes (7-12) from an ethyl acetate extract. Chemical structures were elucidated by one-dimensional, two-dimensional nuclear magnetic resonance (1D-, 2D-NMR), and mass (MS) spectral data. Among the isolated compounds, eudesma-3,11-dien-2-one (2) promoted the nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and increased the promoter property of the ARE. Diarylheptanoids, yakuchinone A (3), and 5'-hydroxyl-yakuchinone A (4) showed radical scavenging activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assays. Furthermore, optimization of extraction solvents (ratios of water and ethanol) was performed by comparison of contents of active compounds, ARE-inducing activity, radical scavenging activity, and HepG2 cell protective activity. As a result, 75% ethanol was the best solvent for the extraction of A. oxyphylla fruit. This study demonstrated that A. oxyphylla exerted antioxidant effects via the Nrf2/HO-1 (heme oxygenase-1) pathway and radical scavenging along with active markers eudesma-3,11-dien-2-one (2) and yakuchinone A (3).

Reduced survival of motor neuron (SMN) protein in motor neuronal progenitors functions cell autonomously to cause spinal muscular atrophy in model mice expressing the human centromeric (SMN2) gene.

Spinal muscular atrophy (SMA) is a common (approximately 1:6400) autosomal recessive neuromuscular disorder caused by a paucity of the survival of motor neuron (SMN) protein. Although widely recognized to cause selective spinal motor neuron loss when deficient, the precise cellular site of action of the SMN protein in SMA remains unclear. In this study we sought to determine the consequences of selectively depleting SMN in the motor neurons of model mice. Depleting but not abolishing the protein in motor neuronal progenitors causes an SMA-like phenotype. Neuromuscular weakness in the model mice is accompanied by peripheral as well as central synaptic defects, electrophysiological abnormalities of the neuromuscular junctions, muscle atrophy, and motor neuron degeneration. However, the disease phenotype is more modest than that observed in mice expressing ubiquitously low levels of the SMN protein, and both symptoms as well as early electrophysiological abnormalities that are readily apparent in neonates were attenuated in an age-dependent manner. We conclude that selective knock-down of SMN in motor neurons is sufficient but may not be necessary to cause a disease phenotype and that targeting these cells will be a requirement of any effective therapeutic strategy. This realization is tempered by the relatively mild SMA phenotype in our model mice, one explanation for which is the presence of normal SMN levels in non-neuronal tissue that serves to modulate disease severity.

Effect on Chemical and Physical Properties of Soil Each Peat Moss, Elemental Sulfur, and Sulfur-Oxidizing Bacteria.

Peat moss is an organic substance corroded by sphagnum moss and has a pH of 3.0-4.0. Elemental sulfur is sulfated and oxidized by the action of bacteria to become sulfuric acid. These biological factors can alter the soil environment. Blueberries require soil with a pH of 4.5-5.2 and high organic matter content. In this experiment, we investigated whether different treatment rates of peat moss, elemental sulfur, and sulfur-oxidizing bacteria affect changes in soil pH, physicochemical properties, and electrical conductivity. We detected strong changes in soil pH as a reaction to the supply of peat moss, elemental sulfur, and sulfur-oxidizing bacteria. The pH of the soil when peat moss and elemental sulfur each were supplied was reduced. In addition, the pH decreased faster when elemental sulfur and sulfur-oxidizing bacteria were supplied together than elemental sulfur alone, satisfying an acidic soil environment suitable for blueberry cultivation. In this experiment, it is shown that peat moss, elemental sulfur, and sulfur-oxidizing bacteria are suitable for lowering soil pH. It was demonstrated that when elemental sulfur and sulfur-oxidizing bacteria were treated together, the pH decreased faster than when treated with peat moss. It could be economically beneficial to farmers to use elemental sulfur and sulfur-oxidizing bacteria, which are cheaper than peat moss, to reduce the pH of the soil.

Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common pediatric neuromuscular disorder caused by insufficient levels of the survival of motor neuron (SMN) protein. Studies involving SMA patients and animal models expressing the human SMN2 gene have yielded relatively little information about the earliest cellular consequences of reduced SMN protein. In this study, we have used severe- and mild-SMN2 expressing mouse models of SMA as well as material from human patients to understand the initial stages of neurodegeneration in the human disease. We show that the earliest structural defects appear distally and involve the neuromuscular synapse. Insufficient SMN protein arrests the post-natal development of the neuromuscular junction (NMJ), impairing the maturation of acetylcholine receptor (AChR) clusters into 'pretzels'. Pre-synaptic defects include poor terminal arborization and intermediate filament aggregates which may serve as a useful biomarker of the disease. These defects are reflected in functional deficits at the NMJ characterized by intermittent neurotransmission failures. We suggest that SMA might best be described as a NMJ synaptopathy and that one promising means of treating it could involve maintaining function at the NMJ.

Activation of microglial cells via protease-activated receptor 2 mediates neuronal cell death in cultured rat primary neuron.

The role of protease-activated receptor (PARs) in the regulation of microglial activation process is increasingly evident. In the present study, we have investigated the role of PAR-2, which can be activated by trypsin-like proteases, in microglial activation and neuronal cell death. In cultured rat primary microglia, activation of PAR-2 induced nitrite production by PKC- and MAPKs-dependent mechanism. Among the three members of MAPK pathway, ERK and JNK but not p38 mediated PAR-2-induced microglial activation. The down-stream regulator of PAR-2-PKC-MAPK pathway-induced microglial activation was NF-kappaB pathway. Besides nitrite, PAR-2 activation increased production of a variety of inflammatory mediators such as ROS and pro-inflammatory cytokines including TNF-alpha and IL-1beta. The addition of culture spent media from PAR-2 activated microglia induced neuronal cell death in primary rat cortical neuron cultures with apoptotic features such as increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive neurons, dissipation of mitochondrial membrane potential, increased expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, Bcl-X(L), and activation of caspase-3 in neurons. Interestingly, the increased production of cytoactive molecules as well as the neuronal cell death was normalized by PAR-2 or trypsin inhibitor or an NO synthase inhibitor, N(G)-nitro-l-arginine-methyl ester. Taken together, these results suggest that overt PAR-2 activation may induce microglial activation, which contributes to neuronal cell death.