AP132P Sigma-AldrichGoat Anti-Rabbit IgG Antibody, Peroxidase Conjugated

Alzheimer's disease (AD) is the most common form of dementia. The deposition of brain amyloid-β peptides (Aβ), which are cleaved from amyloid precursor protein (APP), is one of the pathological hallmarks of AD. Aβ-induced oxidative stress and neuroinflammation play important roles in the pathogenesis of AD. Antroquinonol, a ubiquinone derivative isolated from Antrodia camphorata, has been shown to reduce oxidative stress and inflammatory cytokines via activating the nuclear transcription factor erythroid-2-related factor 2 (Nrf2) pathway, which is downregulated in AD. Therefore, we examined whether antroquinonol could improve AD-like pathological and behavioral deficits in the APP transgenic mouse model. We found that antroquinonol was able to cross the blood-brain barrier and had no adverse effects via oral intake. Two months of antroquinonol consumption improved learning and memory in the Morris water maze test, reduced hippocampal Aβ levels, and reduced the degree of astrogliosis. These effects may be mediated through the increase of Nrf2 and the decrease of histone deacetylase 2 (HDAC2) levels. These findings suggest that antroquinonol could have beneficial effects on AD-like deficits in APP transgenic mouse.

The renal proximal tubule (PT) plays a major role in whole-body pH homeostasis by secreting H(+) into the tubule lumen. Previous work demonstrated that PTs respond to basolateral changes in [CO2] and [HCO3-] by appropriately altering H(+) secretion-responses blocked by the ErbB inhibitor PD168393, or by eliminating signaling through AT1 angiotensin receptors. In the present study, we analyze phosphorylation of three downstream targets of both ErbBs and AT1: phospholipase C-γ1 (PLC-γ1), extracellular-regulated kinase 1 (Erk1), and Erk2. We expose rabbit PT suspensions for 5 and 20 min to our control (Ctrl) condition (5% CO2, 22 mmol/L HCO3-, pH 7.40) or one of several conditions that mimic acid-base disturbances. We found that each disturbance produces characteristic phosphorylation patterns in the three enzymes. For example, respiratory acidosis (elevated [CO2], normal [HCO3-]) at 20 min decreases PLC-γ1 phosphorylation at tyrosine-783 (relative to Ctrl). Metabolic acidosis (normal [CO2], decreased [HCO3-]) for 5 min increases Erk1 phosphorylation (p-Erk1) but not p-Erk2, whereas metabolic alkalosis (normal [CO2], elevated [HCO3-]) for 5 min decreases p-Erk1 and p-Erk2. In the presence of CO2/HCO3-, PD168393 blocks only two of eight induced decreases in phosphorylation. In two cases in which disturbances have no remarkable effects on phosphorylation, PD168393 unmasks decreases and in two others, increases. These drug effects provide insight into the roles of PD168393-sensitive kinases. Our results indicate that PLC-γ1.pY783, p-Erk1, and p-Erk2 in the PT change in characteristic ways in response to acute acid-base disturbances, and thus presumably contribute to the transduction of acid-base signals.

Sann-Joong-Kuey-Jian-Tang (SJKJT), a traditional Chinese medicine, was previously reported to induce autophagy and inhibit the proliferation of the human HepG2 hepatocellular carcinoma cell line via an extrinsic pathway. In the present study, the effects of SJKJT-induced autophagy and the cytotoxic mechanisms mediating these effects were investigated in HepG2 cells. The cytotoxicity of SJKJT in the HepG2 cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that the half-maximal inhibitory concentration of SJKJT was 2.91 mg/ml at 24 h, 1.64 mg/ml at 48 h and 1.26 mg/ml at 72 h. The results of confocal fluorescence microscopy indicated that SJKJT resulted in the accumulation of green fluorescent protein-LC3 and vacuolation of the cytoplasm. Flow cytometric analysis revealed the accumulation of acidic vesicular organelles. Furthermore, western blot analysis, used to determine the expression levels of autophagy-associated proteins, demonstrated that the HepG2 cells treated with SJKJT exhibited LC3B-I/LC3B-II conversion, increased expression levels of Beclin, Atg-3 and Atg-5 and reduced expression levels of p62 and decreased signaling of the phosphoinositide-3 kinase/Akt/mammalian target of rapamycin and the p38 mitogen-activated protein kinase pathways. Taken together, these findings may assist in the development of novel chemotherapeutic agents for the treatment of malignant types of liver cancer.

Ethanol exposure to rodents during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces long-term potentiation and memory deficits. However, the molecular mechanisms underlying these deficits are still poorly understood.In the present study, we explored the potential role of epigenetic changes at cannabinoid type 1 (CB1R) exon1 and additional CB1R functions, which could promote memory deficits in animal models of fetal alcohol spectrum disorder.We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively. We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice. The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression. CB1R knockout mice exhibited neither ethanol-induced neurodegeneration nor inhibition of CREB phosphorylation or Arc expression. However, both neonatal and adult mice did exhibit enhanced CREB phosphorylation and Arc protein expression. P7 ethanol-treated adult mice exhibited impaired spatial and social recognition memory, which were prevented by the pharmacological blockade or deletion of CB1Rs at P7.Together, these findings suggest that P7 ethanol treatment induces CB1R expression through epigenetic modification of the CB1R gene, and that the enhanced CB1R function induces pCREB, Arc, spatial, and social memory deficits in adult mice.

The high mobility group (HMG) family transcription factor Sox9 is critical for induction and maintenance of neural stem cell pool in the central nervous system (CNS). In the spinal cord and retina, Sox9 is also the master regulator that defines glial fate choice by mediating the neurogenic-to-gliogenic fate switch. On the other hand, the genetic repertoire governing the maintenance and fate decision of neural progenitor pool in the cerebellum has remained elusive.By employing the Cre/loxP strategy, we specifically inactivated Sox9 in the mouse cerebellum. Unexpectedly, the self-renewal capacity and multipotency of neural progenitors at the cerebellar ventricular zone (VZ) were not perturbed upon Sox9 ablation. Instead, the mutants exhibited an increased number of VZ-derived neurons including Purkinje cells and GABAergic interneurons. Simultaneously, we observed continuous neurogenesis from Sox9-null VZ at late gestation, when normally neurogenesis ceases to occur and gives way for gliogenesis. Surprisingly, glial cell specification was not affected upon Sox9 ablation.Our findings suggest Sox9 may mediate the neurogenic-to-gliogenic fate switch in mouse cerebellum by modulating the termination of neurogenesis, and therefore indicate a functional discrepancy of Sox9 between the development of cerebellum and other major neural tissues.

The Neuregulin 1 transmembrane domain heterozygous mutant (Nrg1 TM HET) mouse is used to investigate the role of Nrg1 in brain function and schizophrenia-like behavioural phenotypes. However, the molecular alterations in brain Nrg1 expression that underpin the behavioural observations have been assumed, but not directly determined. Here we comprehensively characterise mRNA Nrg1 transcripts throughout development of the Nrg1 TM HET mouse. In addition, we investigate the regulation of high-frequency (gamma) electrophysiological oscillations in this mutant mouse to associate molecular changes in Nrg1 with a schizophrenia-relevant neurophysiological profile.Using exonic probes spanning the cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and intracellular domain encoding regions of Nrg1, mRNA levels were measured using qPCR in hippocampus and frontal cortex from male and female Nrg1 TM HET and wild type-like (WT) mice throughout development. We also performed electrophysiological recordings in adult mice and analysed gamma oscillatory at baseline, in responses to auditory stimuli and to ketamine.In both hippocampus and cortex, Nrg1 TM HET mice show significantly reduced expression of the exon encoding the transmembrane domain of Nrg1 compared with WT, but unaltered mRNA expression encoding the extracellular bioactive EGF-like and the cysteine-rich (type III) domains, and development-specific and region-specific reductions in the mRNA encoding the intracellular domain. Hippocampal Nrg1 protein expression was not altered, but NMDA receptor NR2B subunit phosphorylation was lower in Nrg1 TM HET mice. We identified elevated ongoing and reduced sensory-evoked gamma power in Nrg1 TM HET mice.We found no evidence to support the claim that the Nrg1 TM HET mouse represents a simple haploinsufficient model. Further research is required to explore the possibility that mutation results in a gain of Nrg1 function.

Endometriosis is an inflammatory disease of women of reproductive age featured by the presence of ectopic endometrium and is strongly related to infertility. Galectins, carbonhydrate-binding proteins, have been found to have pro- or anti-inflammatory roles in the reproductive tract and in pathological conditions concerning infertility. Galectin-1, which is expressed at endometrium and decidua, plays a major role in implantation and trophoblast invasion. Also, the neuropeptides, corticotropin releasing hormone (CRH) and urocortin (UCN) and their receptors are expressed in eutopic and ectopic endometrium showing a differential expression pattern in endometriotic women compared to healthy ones. The aim of this study was to examine the galectin-1 expression in endometriotic lesions and compare its expression in eutopic endometrium of endometriotic and healthy women. Furthermore, we examined the effect of CRH and UCN in galectin-1 expression in Ishikawa cell line and macrophages and investigated the implication of CRHR1 in these responses. Eutopic and ectopic endometrium specimens, Ishikawa cell line and mice macrophages were used. Immunohistochemistry and western blot analysis were performed in order to identify galectin-1 expression in ectopic and eutopic endometrium of women with and without endometriosis and the regulatory effect of CRH and UCN on galectin-1 expression. This study presents for the first time that galectin-1 is overexpressed in endometriotic lesions compared to eutopic endometrium of endometriotic women and is more abundantly expressed in eutopic endometrium of disease women compared to healthy ones. Furthermore, it is shown that CRH and UCN upregulate galectin-1 expression in Ishikawa cell line and macrophages and this effect is mediated through CRHR1. These results suggest that galectin-1 might play an important role in endometriosis pathology and infertility profile of women suffering from endometriosis by being at the same time regulated by CRH and UCN interfering in the immune disequilibrium which characterizes this pathological condition.

Store-operated Ca(2+) entry (SOCE) is activated following depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca(2+) pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca(2+) levels within the endoplasmic reticulum (ER) Ca(2+) reservoir, and a number of a Ca(2+)-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1-7) family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC) patients. SOCE was induced following pharmacological depletion of the ER Ca(2+) store, but not by InsP3-dependent Ca(2+) release. Metastatic RCC cells express Stim1-2, Orai1-3, and TRPC1-7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd(3+) and Pyr6, while it was inhibited by 100 µM Gd(3+), 2-APB, and carboxyamidotriazole (CAI). Neither Gd(3+) nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca(2+) signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors.

Metastasis associated with lung adenocarcinoma transcript-1 (MALAT1) is a functional long non-coding RNA (lncRNA), which is highly expressed in several tumours, including colorectal cancer (CRC). Its biological function and mechanism in the prognosis of human CRC is still largely under investigation.This study aimed to investigate the new effect mechanism of MALAT1 on the proliferation and migration of CRC cells in vitro and in vivo, and detect the expression of MALAT1, SFPQ (also known as PSF (PTB-associated splicing factor)), and PTBP2 (also known as PTB (polypyrimidine-tract-binding protein)) in CRC tumour tissues, followed by correlated analysis with clinicopathological parameters.We found that overexpression of MALAT1 could promote cell proliferation and migration in vitro, and promote tumour growth and metastasis in nude mice. The underlying mechanism was associated with tumour suppressor gene SFPQ and proto-oncogene PTBP2. In CRC, MALAT1 could bind to SFPQ, thus releasing PTBP2 from the SFPQ/PTBP2 complex. In turn, the increased SFPQ-detached PTBP2 promoted cell proliferation and migration. SFPQ critically mediated the regulatory effects of MALAT1. Moreover, in CRC tissues, MALAT1 and PTBP2 were overexpressed, both of which were associated closely with the invasion and metastasis of CRC. However, the SFPQ showed unchanged expression either in CRC tissues or adjacent normal tissues.Our findings implied that MALAT1 might be a potential predictor for tumour metastasis and prognosis. Furthermore, the interaction between MALAT1 and SFPQ could be a novel therapeutic target for CRC.

Oxidative stress is becoming increasingly linked to the pathogenesis of abdominal aortic aneurysms (AAAs). The antioxidant activity of flavonoids has attracted attention for their possible role in the prevention of cardiovascular diseases. The purpose of this study was to determine whether an antioxidant mechanism is involved in the aneurysm formation inhibitory effect afforded by quercetin. Male C57/BL6 mice received quercetin continuously from 2 weeks prior to and 6 weeks following the AAA induction with extraluminal CaCl2. Quercetin treatment decreased AAA incidence and inhibited the reactive oxygen species generation, nitrotyrosine formation and lipid peroxidation production in the aortic tissue during AAA development. In addition, quercetin‑treated mice exhibited significantly lower expression of the p47phox subunit of nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase, as well as coordinated downregulation of manganese‑superoxide dismutase activities and glutathione peroxidase (GPx)‑1 and GPx‑3 expression. Quercetin also blunted the expression of c‑Jun N‑terminal kinase (JNK) and phospho‑JNK and, in addition, diminished activation of the activator protein (AP)‑1 transcription factor. Gelatin zymography showed that quercetin eliminated matrix metalloproteinase (MMP)‑2 and MMP‑9 activation during AAA formation. In conclusion, the inhibitory effects of quercetin on oxidative stress and MMP activation, through modulation of JNK/AP‑1 signaling, may partly account for its benefit in CaCl2‑induced AAA.