Significance of medial preoptic area among the subcortical and cortical areas that are related to pain regulation in the rats with stress-induced hyperalgesia.

Psychophysical stresses frequently increase sensitivity and response to pain, which is termed stress-induced hyperalgesia (SIH). However, the mechanism remains unknown. The subcortical areas such as medial preoptic area (MPO), dorsomedial nucleus of the hypothalamus (DMH), basolateral (BLA) and central nuclei of the amygdala (CeA), and the cortical areas such as insular (IC) and anterior cingulate cortices (ACC) play an important role in pain control via the descending pain modulatory system. In the present study we examined the expression of phosphorylated -cAMP-response element binding protein (pCREB) and the acetylation of histone H3 in these subcortical and cortical areas after repeated restraint stress to reveal changes in the subcortical and cortical areas that affect the function of descending pain modulatory system in the rats with SIH. The repeated restraint stress for 3 weeks induced a decrease in mechanical threshold in the rat hindpaw, an increase in the expression of pCREB in the MPO and an increase in the acetylation of histone H3 in the MPO, BLA and IC. The MPO was the only area that showed an increase in both the expression of pCREB and the acetylation of histone H3 among these examined areas after the repeated restraint stress. Furthermore, the number of pCREB-IR or acetylated histone H3-IR cells in the MPO was negatively correlated with the mechanical threshold. Together, our data represent the importance of the MPO among the subcortical and cortical areas that control descending pain modulatory system under the condition of SIH.

Geniposide ameliorated fluoxetine-suppressed neurite outgrowth in Neuro2a neuroblastoma cells.

AIM: Fluoxetine (FXT), a selective serotonin reuptake inhibitor (SSRI), is one of the most common psychiatric medications clinically prescribed; while over-produced serotonin may suppress neurite development. The role of major iridoids like geniposide (GPS) and genipin (GNP) from Gardenia jasminoides Ellis fruit (family Rubiaceae) in ameliorating the anti-neurite outgrowth effect of FXT is poorly understood. In this study, the effects of these iridoids on FXT-suppressed neurite outgrowth in Neuro2a neuroblastoma cells were investigated. MAIN METHODS: Neuro2a cells were treated with FXT and GPS. The effect of GPS-FXT co-treatment on neurite outgrowth was observed using inverted phase-contrast microscope imaging system, while neurite outgrowth markers - microtubule-associated protein-2 (MAP2) and growth-associated protein 43 (GAP43) were analyzed using RT-PCR, Western blot and immunofluorescence staining. The transcription factor-cAMP response element binding (CREB), and signaling pathways - mitogen-activated protein kinase (MAPK) and protein kinase B/mammalian target of rapamycin (AKT/mTOR) were also analyzed with the help of Western blot. KEY FINDINGS: The results showed that FXT decreased the neurite outgrowth in Neuro2a cells and also downregulated gene and protein expression of MAP2 and GAP43. It also downregulated the protein expression of phosphorylated-CREB, MAPK, and AKT/mTOR signaling pathways. In contrast, GPS counteracted the effects of FXT. GPS-FXT co-treatment increased the percentage of neurite-bearing cells by 3.6-fold at 200 µM as compared to FXT treatment only. SIGNIFICANCE: This study has provided the possible molecular mechanism showing how FXT exerted its detrimental side-effects on the neurite differentiation, and via the same mechanism how GPS attenuated these side effects.

CREB signals as PBMC-based biomarkers of cognitive dysfunction: A novel perspective of the brain-immune axis.

To date, there is no reliable biomarker for the assessment or determination of cognitive dysfunction in Alzheimer's disease and related dementia. Such a biomarker would not only aid in diagnostics, but could also serve as a measure of therapeutic efficacy. It is widely acknowledged that the hallmarks of Alzheimer's disease, namely, amyloid deposits and neurofibrillary tangles, as well as their precursors and metabolites, are poorly correlated with cognitive function and disease stage and thus have low diagnostic or prognostic value. A lack of biomarkers is one of the major roadblocks in diagnosing the disease and in assessing the efficacy of potential therapies. The phosphorylation of cAMP Response Element Binding protein (pCREB) plays a major role in memory acquisition and consolidation. In the brain, CREB activation by phosphorylation at Ser133 and the recruitment of transcription cofactors such as CREB binding protein (CBP) is a critical step for the formation of memory. This set of processes is a prerequisite for the transcription of genes thought to be important for synaptic plasticity, such as Egr-1. Interestingly, recent work suggests that the expression of pCREB in peripheral blood mononuclear cells (PBMC) positively correlates with pCREB expression in the postmortem brain of Alzheimer's patients, suggesting not only that pCREB expression in PBMC might serve as a biomarker of cognitive dysfunction, but also that the dysfunction of CREB signaling may not be limited to the brain in AD, and that a link may exist between the regulation of CREB in the blood and in the brain. In this review we consider the evidence suggesting a correlation between the level of CREB signals in the brain and blood, the current knowledge about CREB in PBMC and its association with CREB in the brain, and the implications and mechanisms for a neuro-immune cross talk that may underlie this communication. This Review will discuss the possibility that peripheral dysregulation of CREB is an early event in AD pathogenesis, perhaps as a facet of immune system dysfunction, and that this impairment in peripheral CREB signaling modifies CREB signaling in the brain, thus exacerbating cognitive decline in AD. A more thorough understanding of systemic dysregulation of CREB in AD will facilitate the search for a biomarker of cognitive function in AD, and also aid in the understanding of the mechanisms underlying cognitive decline in AD.

Neuroprotective Effect of Astragalus Polysacharin on Streptozotocin (STZ)-Induced Diabetic Rats.

BACKGROUND In the recent years, there has been increasing interest in traditional Chinese medicine as a neuroprotective nutrient in the management of chronic neurodegenerative disease, such as diabetic cognitive decline. Astragalus polysacharin (APS), a Chinese herb extract, is a biologically active treatment for neurodegenerative diseases. Therefore, in the present study, we investigated the neuroprotective effects of APS (20 mg/kg) on diabetes-induced memory impairments in Sprague-Dawley (SD) rats and explored its underlying mechanisms of action. MATERIAL AND METHODS Thirty SD rats were randomly divided into a control group (CON group, n=10), a diabetic model (DM) group (n=10), and an APS group (n=10). We administered 55 mg/kg streptozotocin (STZ, Sigma) by intraperitoneal injection to induce a diabetic model. Food and water intake, body weight, and blood fasting plasma glucose (FPG) were measured. The Morris water maze test (MWM) was used to assess learning and memory ability, and we measured levels of N-methyl-D-aspartate receptor (NMDA), calcium/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element-binding protein (CREB) in the hippocampus. RESULTS APS (20 mg/kg) administration decreased the rats' fasting plasma glucose (FPG) levels and body weight. APS (20 mg/kg) administration improved the cognitive performance of diabetes-induced rats in the Morris water maze test. APS (20 mg/kg) administration reduced the number of dead cells in the CA1 region of the hippocampus. Furthermore, APS (20 mg/kg) administration obviously upregulated the phosphorylation levels CREB, NMDA, and CaMK II. CONCLUSIONS These results suggest that APS has the neuroprotective effects, and it may be a candidate for treatment of neurodegenerative diseases such as diabetic cognitive impairment.

Neuroprotective effect of tempeh against lipopolysaccharide-induced damage in BV-2 microglial cells.

Objectives: This study evaluated the bioactive composition of tempeh products and examined the effects of tempeh on BV-2 microglial cell cytotoxicity, neurotrophic effects, and expression of inflammatory genes.Methods: Tempeh products included soybean fermented by Rhizopus, soybean fermented through cocultivation with Rhizopus and Lactobacillus, and red bean fermented through cocultivation with Rhizopus and Lactobacillus (RT-C). We analyzed the bioactive contents of tempeh extracts and evaluated the effects of tempeh water extract on lipopolysaccharide (LPS)-treated BV-2 cells.Results: The results showed that RT-C water extract had the highest concentrations of γ-aminobutyric acid (GABA) and anthocyanin. The tempeh water extracts, especially RT-C, reduced the formation of LPS-induced reactive oxygen species, downregulated the levels of nitric oxide synthase and phospho-cyclic-AMP response element-binding protein, and upregulated the expression of brain-derived neurotrophic factor (BDNF).Discussion: Our data demonstrate that RT-C has the highest concentrations of GABA and anthocyanin, more effectively reduces oxidative stress and inflammation, and increases the expression of BDNF in LPS-induced BV-2 cells.

The protective effect of zeranol in cerebral ischemia reperfusion via p-CREB overexpression.

AIMS: Cerebral ischemia reperfusion (I/R) is a neurovascular disease leading to cerebral damage. It was found that postmenopausal women are liable to more dangerous effects than men at same age in stroke. The objective of this study is to investigate the neuroprotective effect of zeranol against cerebral ischemia reperfusion in ovariectomized rats. MAIN METHODS: 36 female wistar rats divided in to 3 groups: sham group, I/R group (where I/R was induced 7 weeks after ovariectomy), zeranol group (0.5 mg/kg every 3 days for 5 weeks before I/R). Cerebral ischemia reperfusion (I/R) was performed by bilateral common carotid artery occlusion then de-ligated to restore blood flow. After 24 h of reperfusion, rats performed cylinder test to evaluate behavioral dysfunction followed by decapitation. Brain tissues were collected for biochemical measures such as oxidative stress marker malondialdehyde, antioxidant markers reduced glutathione, inflammatory markers (interleukin-1 beta, tumor necrosis factor alpha, and inducible nitric oxide synthase), matrix metalloproteinase-9, adenosine triphosphate, brain derived neurotrophic factor, glucose transporter-3, phosphorylated c-AMP response element binding protein and finally nissl staining for histopathological examination. KEY FINDINGS: The zeranol administered group showed a reversal of neuronal damage caused by ischemia evidenced by the decrease in MDA, IL-1ß, TNF-α, and MMP-9 levels, increase GSH, and ATP levels, decrease expression of iNOS in both regions cortex and hippocampus, increase protein level of p-CREB, GLUT-3 and BDNF, increase number of intact neuron cells in both regions and attenuated histological changes in both cortex and hippocampus regions. SIGNIFICANCE: Zeranol has neuroprotective potential against cerebral ischemia reperfusion in ovariectomized rats.

Luteolin inhibits colorectal cancer cell epithelial-to-mesenchymal transition by suppressing CREB1 expression revealed by comparative proteomics study.

The cyclic AMP (cAMP) response element binding protein 1 (CREB1) is a promising target for cancer therapy. Here, we report that luteolin, a natural product, inhibits the expression of CREB1 at the transcriptional level and blocks epithelial-to-mesenchymal transition (EMT) of colorectal cancer cells. Treatment of colorectal cancer cells with luteolin induced mesenchymal-to-epithelial transition, reduced the expressions of mesenchymal markers and inhibited cell mobility in vitro. Through comparison of the proteomic profile of HCT-116 cells with and without luteolin treatment, we identified 366 differentially expressed proteins. Bioinformatics analysis revealed that downregulation of CREB1 plays a central role in this process. Immunoblot analysis verified that the protein levels of CREB1 and its downstream target genes were decreased in luteolin-treated cells. Moreover, forced expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells, suggesting the important role of CREB1 in this process. Furthermore, luciferase reporter assay and examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX) revealed that luteolin inhibits the expression of CREB1 at the transcriptional level. In summary, our results demonstrated that suppressing the expression of CREB1 is crucial in the mechanism-of-action of luteolin inhibiting EMT of colorectal cancer cells. SIGNIFICANCE: It is no doubt that understanding the mechanism-of-action of natural products at the molecular level is important for their translational application. Proteomics is a powerful platform to explore the effects of natural products on the cells. In this study, we compared the proteomic profile of HCT-116 colorectal cancer cells with and without luteolin treatment to investigate the mechanism-of-action of luteolin against colorectal cancer cells. Subsequent bioinformatics analysis revealed that CREB1 could be one of the main targets of luteolin against colorectal cancer cells. Downregulation of CREB1 by luteolin affects glucagon signaling pathway and cAMP signaling pathway. The proteomics findings were verified with mechanistic analyses. We first identified that luteolin decreased the mRNA and protein levels of CREB1 and its downstream target genes. We then found that luteolin inhibits CREB1 expression at the transcriptional level by real-time PCR and luciferase reporter assay which confirmed by examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX). Finally, we generated CREB1-overexpressing stable cell line and showed that ectopic expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells and restored the expression levels of CREB1 target genes in colorectal cancer cells, and thereby demonstrated the critical role of CREB1 in the mechanism-of-action of luteolin against colorectal cancer. In summary, we revealed a novel mechanism-of-action of luteolin against colorectal cancer cell by the combination of proteomics discovery and mechanistic analyses.

CCAR2 Is Required for Proliferation and Tumor Maintenance in Human Squamous Cell Carcinoma.

CCAR2 is a widely expressed protein involved in the regulation of a variety of transcriptional complexes. High expression of CCAR2 correlates with poor outcomes in many human tumor types such as squamous cell carcinoma (SCC). Paradoxically, loss of Ccar2 in the mouse results in an increased tumor burden, suggesting that CCAR2 may in fact function as a tumor suppressor. This tumor suppressor function is dependent on p53, a protein that is inactivated in the vast majority of SCC tumors, leaving the role of CCAR2 in p53-null tumors unclear. We sought to identify p53-independent CCAR2 functions in SCC and to examine its role in tumorigenesis. We found that CCAR2 is highly overexpressed in p53-deficient SCC cell lines compared with normal primary keratinocytes due to increased protein stability. We identify a role for CCAR2 in promoting the stability of the transcription factors RFX1 and CREB1, which are both required for proliferation. Finally, we show that CCAR2 is required for proliferation in vitro and in established SCC tumors in vivo. Our data suggest an important role for CCAR2 in maintaining cell cycle progression and promoting SCC tumorigenesis.

Nano-particle delivery of brain derived neurotrophic factor after focal cerebral ischemia reduces tissue injury and enhances behavioral recovery.

BACKGROUND: Low levels of brain-derived neurotrophic factor (BDNF) are linked to delayed neurological recovery, depression, and cognitive impairment following stroke. Supplementation with BDNF reverses these effects. Unfortunately, systemically administered BDNF in its native form has minimal therapeutic value due to its poor blood brain barrier permeability and short serum half-life. In this study, a novel nano-particle polyion complex formulation of BDNF (nano-BDNF) was administered to mice after experimental ischemic stroke. METHODS: Male C57BL/6J (8-10weeks) mice were randomly assigned to receive nano-BDNF, native-BDNF, or saline treatment after being subjected to 60min of reversible middle cerebral artery occlusion (MCAo). Mice received the first dose at 3 (early treatment), 6 (intermediate treatment), or 12h (delayed treatment) following stroke onset; a second dose was given in all cohorts at 24h after stroke onset. Post-stroke outcome was evaluated by behavioral, histological, and molecular analysis for 15days after stroke. RESULTS: Early and intermediate nano-BDNF treatment led to a significant reduction in cerebral tissue loss. Delayed treatment led to improved memory/cognition, reduced post-stroke depressive phenotypes, and maintained myelin basic protein and brain BDNF levels, but had no effect on tissue atrophy. CONCLUSIONS: The results indicate that administration of a novel nano-particle formulation of BDNF leads to both neuroprotective and neuro-restorative effects after stroke.

Chronic administration of aripiprazole activates GSK3ß-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats.

Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3ß-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3ß pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3ß signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, ß-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3ß signalling, Dvl-GSK3ß-ß-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects.

Regulation of cyclic adenosine monophosphate response element binding protein on renin expression in kidney via complex cyclic adenosine monophosphate response element-binding-protein-binding protein/P300 recruitment.

INTRODUCTION: Renin synthesis and release is the rate-limiting step in the renin-angiotensin system, because cyclic adenosine monophosphate (cAMP) has been identified as dominant pathway for renin gene expression, and cAMP response element-binding protein (CREB) is found in the human and mouse renin promoter. This study aimed to evaluate the role of CREB in expression of the renin gene. MATERIALS AND METHODS: We created conditional deletion of CREB in mice with low-sodium diet, specifically in renin cells of the kidney. To assess the effect of CREB on renin expression, immunostaining of renin was used in samples from wild-type mice and mice with gene knock-down of CREB. Cyclic AMP response element-binding-protein-binding protein (CBP) and p300 were measured in cultured renin cells of the mice, and RNA detection was done with real-time polymerase chain reaction. RESULTS: With low-sodium diet, renin was expressed along the whole wall of the afferent glomerular arterioles in wild-type mice, while there was no increase or even decrease in renin expression in CREB-specific deletion mice; RNA level of renin in cultured cells decreased by 50% with single knock-down of CREB, CBP, or p300, and decreased 70% with triple knock-down of CREB, CBP, and p300. CONCLUSIONS: This study found that CREB was important for renin synthesis and the role of CREB can be achieved through the recruitment of co-activators CBP and p300.

Acupuncture stimulation improves scopolamine-induced cognitive impairment via activation of cholinergic system and regulation of BDNF and CREB expressions in rats.

BACKGROUND: Acupuncture is an alternative therapy that is widely used to treat various neurodegenerative diseases and effectively improve cognitive and memory impairment. The aim of this study was to examine whether acupuncture stimulation at the Baihui (GV20) acupoint improves memory defects caused by scopolamine (SCO) administration in rats. We also investigated the effects of acupuncture stimulation at GV20 on the cholinergic system as well as the expression of brain-derived neurotrophic factor (BDNF) and cAMP-response element-binding protein (CREB) in the hippocampus. METHODS: SCO (2 mg/kg, i.p.) was administered to male rats once daily for 14 days. Acupuncture stimulation at GV20 was performed for 5 min before SCO injection. After inducing cognitive impairment via SCO administration, we conducted a passive avoidance test (PAT) and the Morris water maze (MWM) test to assess behavior. RESULTS: Acupuncture stimulation at GV20 improved memory impairment as measured by the PAT and reduced the escape latency for finding the platform in the MWM test. Acupuncture stimulation at GV20 significantly alleviated memory-associated decreases in the levels of choline acetyltransferase (ChAT), BDNF and CREB proteins in the hippocampus. Additionally, acupuncture stimulation at GV20 significantly restored the expression of choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT), BDNF and CREB mRNA in the hippocampus. These results demonstrate that acupuncture stimulation at GV20 exerts significant neuroprotective effects against SCO-induced neuronal impairment and memory dysfunction in rats. CONCLUSIONS: These findings suggest that acupuncture stimulation at GV20 might be useful in various neurodegenerative diseases to improve cognitive functioning via stimulating cholinergic enzyme activities and regulating BDNF and CREB expression in the brain.

Prognostic value of coexistence of abnormal expression of micro-RNA-200b and cyclic adenosine monophosphate-responsive element-binding protein 1 in human astrocytoma.

Our aim was to investigate the expression of micro-RNA-200b (miR-200b) and cAMP-responsive element-binding protein 1 (CREB-1) in astrocytoma and its efficacy for predicting outcome. Both miR-200b and CREB-1 messenger RNA expression was measured in 122 astrocytomas and 30 nonneoplastic brain specimens by quantitative real-time polymerase chain reaction. Expression of miR-200b was significantly lower in astrocytoma than in nonneoplastic brain (P < .001), whereas CREB-1 messenger RNA expression was significantly elevated in the tumors (P < .001). Both miR-200b down-regulation and CREB-1 up-regulation were significantly associated with advanced pathologic grade (P = .002 and P = .006, respectively). Low miR-200b expression correlated negatively with Karnofsky performance score (P = .03), and high CREB-1 expression correlated positively with mean tumor diameter (P = .03). By Kaplan-Meier analysis, low miR-200b, high CREB-1, and coexistence of abnormal miR-200b and CREB-1 expression (low miR-200b/high CREB-1) were predictive of shorter progression-free survival and overall survival in both grade III and grade IV astrocytoma. By multivariate analysis, only low miR-200b/high CREB-1 expression was an independent prognostic factor for poor prognosis in astrocytoma of advanced grade. Both miR-200b and CREB-1 may play important cooperative roles in the progression of human astrocytoma. The efficacy of miR-200b and CREB-1 together as a predictor of prognosis in astrocytoma patients is shown for the first time.

Detection of CREB phosphorylation via Zr (IV) ion mediated signal amplification.

Phosphorylation of protein plays a vital regulatory role in a variety of biological processes. We herein report a novel method to assay the level of phosphorylated cAMP-response element binding protein (CREB) via Zr(4+) mediated signal amplification using gold nanoparticle/DNA/methylene blue (GNP/DNA/MB) nanocomposites. In this method the probe DNA immobilized at a gold electrode surface can specifically and efficiently recognize the phosphorylated target protein CREB. Then Zr(4+) links the phosphorylated CREB with GNP/DNA/MB nanocomposites by coordinating the phosphate groups on both CREB and the nanocomposites. Since the nanocomposites can provide high sensitivity (limit of detection: 0.25 nM) for the detection, efficient and highly sensitive bioanalysis of the expression level of phosphorylated protein CREB in human placenta tissues has also been conducted in this work. Our method is reported which shows acceptable stability, reproducibility for assaying of the protein phosphorylation states in real biosamples under physiological and pathological conditions with great potential for clinical applications in future.

Multiple reaction monitoring mass spectrometry for the discovery and quantification of O-GlcNAc-modified proteins.

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification regulating proteins involved in a variety of cellular processes and diseases. Unfortunately, O-GlcNAc remains challenging to detect and quantify by shotgun mass spectrometry (MS) where it is time-consuming and tedious. Here, we investigate the potential of Multiple Reaction Monitoring Mass Spectrometry (MRM-MS), a targeted MS method, to detect and quantify native O-GlcNAc modified peptides without extensive labeling and enrichment. We report the ability of MRM-MS to detect a standard O-GlcNAcylated peptide and show that the method is robust to quantify the amount of O-GlcNAcylated peptide with a method detection limit of 3 fmol. In addition, when diluted by 100-fold in a trypsin-digested whole cell lysate, the O-GlcNAcylated peptide remains detectable. Next, we apply this strategy to study glycogen synthase kinase-3 beta (GSK-3ß), a kinase able to compete with O-GlcNAc transferase and modify identical site on proteins. We demonstrate that GSK-3ß is itself modified by O-GlcNAc in human embryonic stem cells (hESC). Indeed, by only using gel electrophoresis to grossly enrich GSK-3ß from whole cell lysate, we discover by MRM-MS a novel O-GlcNAcylated GSK-3ß peptide, bearing 3 potential O-GlcNAcylation sites. We confirm our finding by quantifying the increase of O-GlcNAcylation, following hESC treatment with an O-GlcNAc hydrolase inhibitor. This novel O-GlcNAcylation could potentially be involved in an autoinhibition mechanism. To the best of our knowledge, this is the first report utilizing MRM-MS to detect native O-GlcNAc modified peptides. This could potentially facilitate rapid discovery and quantification of new O-GlcNAcylated peptides/proteins.

Neuroprotective effect of estradiol-loaded poly(lactic-co-glycolic acid) nanoparticles on glutamate-induced excitotoxic neuronal death.

Different concentrations of estradiol (E2)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (E2-PLGA-NPs) were synthesized using the emulsion-diffusion method. Transmission electron microscopy results showed that the average particle size of E2-PLGA-NPs was 98 ± 1.9 nm when stabilized with polyvinyl alcohol and 103 ± 4.9 nm when stabilized with Tween-80. Fourier transform-infrared spectroscopy with diamond attenuated total reflectance was used to identify the presence or absence of E2 molecules in PLGA nanocapsules. Cell proliferation was assessed after treating SH-SY5Y neuroblastoma cells with 1 nM-1 µM of E2 and E2-PLGA-NPs. The neuroprotective efficacy against glutamate-induced excitotoxicity was also investigated in SH-SY5Y neuroblastoma cells. Neuroprotection was greater in E2-PLGA-NP-treated cells than in cells treated with the same concentration of E2. Furthermore, E2- and E2-PLGA-NP-treated cells expressed more p-ERK1/2 and p-CREB than cells treated with glutamate only. Moreover, the expression of p-ERK1/2 was higher than that of p-CREB. In this study, p-ERK1/2 had a greater influence on the neuroprotective effect of E2 and E2-PLGA-NPs than p-CREB.

Grape powder supplementation prevents oxidative stress-induced anxiety-like behavior, memory impairment, and high blood pressure in rats.

We examined whether or not grape powder treatment ameliorates oxidative stress-induced anxiety-like behavior, memory impairment, and hypertension in rats. Oxidative stress in Sprague-Dawley rats was produced by using L-buthionine-(S,R)-sulfoximine (BSO). Four groups of rats were used: 1) control (C; injected with vehicle and provided with tap water), 2) grape powder-treated (GP; injected with vehicle and provided for 3 wk with 15 g/L grape powder dissolved in tap water), 3) BSO-treated [injected with BSO (300 mg/kg body weight), i.p. for 7 d and provided with tap water], and 4) BSO plus grape powder-treated (GP+BSO; injected with BSO and provided with grape powder-treated tap water). Anxiety-like behavior was significantly greater in BSO rats compared with C or GP rats (P < 0.05). Grape powder attenuated BSO-induced anxiety-like behavior in GP+BSO rats. BSO rats made significantly more errors in both short- and long-term memory tests compared with C or GP rats (P < 0.05), which was prevented in GP+BSO rats. Systolic and diastolic blood pressure was significantly greater in BSO rats compared with C or GP rats (P < 0.05), whereas grape powder prevented high blood pressure in GP+BSO rats. Furthermore, brain extracellular signal-regulated kinase-1/2 (ERK-1/2) was activated (P < 0.05), whereas levels of glyoxalase-1 (GLO-1), glutathione reductase-1 (GSR-1), calcium/calmodulin-dependent protein kinase type IV (CAMK-IV), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) were significantly less (P < 0.05) in BSO but not in GP+BSO rats compared with C or GP rats. We suggest that by regulating brain ERK-1/2, GLO-1, GSR-1, CAMK-IV, CREB, and BDNF levels, grape powder prevents oxidative stress-induced anxiety, memory impairment, and hypertension in rats.

Luman recruiting factor regulates endoplasmic reticulum stress in mouse ovarian granulosa cell apoptosis.

Follicular atresia is primarily induced by granulosa cell apoptosis; however, the molecular mechanisms that control apoptotic cell death in granulosa cells remain poorly understood. The present studies were undertaken to investigate the role of a novel endoplasmic reticulum stress-regulated gene Luman recruiting factor (LRF) in granulosa cell apoptosis during mouse follicular atresia. Based on immunohistochemistry and confocal laser scanning microscope analysis, LRF protein was localized in the cytoplasm of apoptotic granulosa cells, similar to localization of the LRF, Luman, CCAAT/enhancer-binding protein homologous protein and caspase-12 proteins were localized in apoptotic granulosa cells. However, glucose-regulated protein 78 protein was only present in healthy cells of the mural granulosa cell layers. A spontaneous onset of apoptotic cell death of granulosa cells was induced by thapsigargin or tunicamycin treatment in vitro, which was closely related to the increase of LRF, Luman, CCAAT/enhancer-binding protein homologous protein, and caspase-12 mRNA. Taken together, LRF might be involved in inducing apoptosis of granulosa cells through the endoplasmic reticulum stress pathway and might have a key role in mouse follicular selection.

Neuroprotection provided by dietary restriction in rats is further enhanced by reducing glucocortocoids.

Glucocorticoids (GC)--corticosterone (CORT) in rodents and cortisol in primates--are stress-induced hormones secreted by adrenal glands that interact with the hypothalamic pituitary axis. High levels of cortisol in humans are observed in neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), as well as in diabetes, post-traumatic stress syndrome, and major depression. Experimental models of diabetes in rats and mice have demonstrated that reduction of CORT reduces learning and memory deficits and attenuates loss of neuronal viability and plasticity. In contrast to the negative associations of elevated GC levels, CORT is moderately elevated in dietary restriction (DR) paradigms which are associated with many healthy anti-aging effects including neuroprotection. We demonstrate here in rats that ablating CORT by adrenalectomy (ADX) with replenishment to relatively low levels (30% below that of controls) prior to the onset of a DR regimen (ADX-DR) followed by central administration of the neurotoxin, kainic acid (KA), significantly attenuates learning deficits in a 14-unit T-maze task. The performance of the ADX-DR KA group did not differ from a control group (CON) that did not receive KA and was fed ad libitum (AL). By contrast, the sham-operated DR (SHAM-DR KA) group, SHAM-AL KA group, and ADX-AL KA group demonstrated poorer learning behavior in this task compared to the CON group. Stereological analysis revealed equivalent DR-induced neuroprotection in the SH-DR KA and ADX-DR KA groups, as measured by cell loss in the CA2/CA3 region of the hippocampus, while substantial cell loss was observed in SH-AL and ADX-AL rats. A separate set of experiments was conducted with similar dietary and surgical treatment conditions but without KA administration to examine markers of neurotrophic activity, brain-derived neurotrophic factor (BDNF), transcriptions factors (pCREB), and chaperone proteins (HSP-70). Under these conditions, we noted elevations in both BDNF and pCREB in ADX DR rats compared to the other groups; whereas, HSP-70, was equivalently elevated in ADX-DR and SH-DR groups and was higher than observed in both SH-AL and ADX-AL groups. These results support findings that DR protects hippocampal neurons against KA-induced cellular insult. However, this neuroprotective effect was further enhanced in rats with a lower-than control level of CORT resulting from ADX and maintained by exogenous CORT supplementation. Our results then suggest that DR-induced physiological elevation of GC may have negative functional consequences to DR-induced beneficial effects. These negative effects, however, can be compensated by other DR-produced cellular and molecular protective mechanisms.

Methodology and technology for stabilization of specific states of signal transduction proteins.

The ability to adequately measure the phosphorylation state of a protein has major biological as well as clinical relevance. Due to its variable nature, reversible protein phosphorylations are sensitive to changes in the tissue environment. Stabilizor T1 is a system for rapid inactivation of enzymatic activity in biological samples. Enzyme inactivation is accomplished using thermal denaturation in a rapid, homogeneous, and reproducible fashion without the need for added inhibitors. Using pCREB(Ser133) as a model system, the applicability of the Stabilizor system to preserve a rapidly lost phosphorylation is shown.