GSK-3β Is Required for Memory Reconsolidation in Adult Brain

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimer''s disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/−) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/− mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/− mice, suggesting that GSK+/− mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/− mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/− mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain.     

The biological effects of CRP are not attributable to endotoxin contamination: evidence from TLR4 knockdown human aortic endothelial cells

C-reactive protein (CRP) is the prototypic marker of inflammation and a strong predictor of cardiovascular events in humans. There are questions regarding the validity of the biological effects reported for CRP, in spite of adherence to rigorous control measures minimizing endotoxin [lipopolysaccharide (LPS)] contamination in these in vitro studies. In this study, we addressed the key question of endotoxin contamination in CRP preparations using Toll-like receptor 4 (TLR4) knockdown endothelial cells. Human aortic endothelial cells (HAECs) transfected with prevalidated TLR4 small interfering RNA (siRNA) and scrambled siRNA controls were challenged with pleural fluid-derived CRP or LPS for 12-16 h. Secreted interleukin-6 (IL-6), IL-1beta, IL-8, and plasminogen activator inhibitor-1 (PAI-1) levels and endothelial Nitric oxide synthase (eNOS) activity were determined. TLR4 knockdown in HAECs significantly decreased LPS-induced IL-1beta, IL-6, and IL-8, whereas the stimulatory effects of CRP were similar in both scrambled control and TLR4 knockdown cells. Furthermore, CRP significantly stimulated PAI-1 levels in both control and TLR4-transfected cells and inhibited eNOS activity, whereas LPS effects were negated in TLR4-transfected cells. The data presented cogently demonstrate and further confirm that the biological effects of CRP on HAECs are independent of LPS and thus are attributable to native protein per se. This is the first study to positively authenticate the significance of earlier in vitro reports on CRP biological effects.     

Design,synthesis, and in vitro evaluation of N-(3-(3-alkyl-1H-pyrazol-5-yl) phenyl)-aryl amide for selective RAF inhibition

Notorious oncogenic BRAF V600E plays a significant role in the signal transduction of the MAPK pathway, which is involved in tumor growth, especially in melanoma. Much effort has been made to suppress BRAF V600E through small molecules like vemurafenib and dabrafenib, but the MAPK pathway remains active through paradoxical activation, where CRAF transmits the signal of the MAPK pathway either alone or along with BRAF V600E. Therefore, we designed and synthesized a new series of N-(3-(3-alkyl-1H-pyrazol-5-yl) phenyl)-aryl amide/urea analogues that showed potent inhibitory activities against BRAF V600E and CRAF. Compound 7c exhibited particularly superior selectivity toward BRAF V600E and CRAF over 30 other protein kinases, implying that this chemotype could be investigated as a BRAF paradox breaker. © 2019 Elsevier Ltd. All rights reserved.     

High glucose induces IL-1beta expression in human monocytes: mechanistic insights

Previously, IL-1beta secretion from Type 2 diabetic patients has been shown to be increased compared with controls. In this study, we aimed to delineate the mechanism of IL-1beta induction under high-glucose (HG) conditions in human monocytes. THP-1 cells cultured in normal glucose were treated with increasing concentrations of d-glucose (10-25 mM) for 6-72 h. IL-1beta and IL-1 receptor antagonist levels were measured by ELISA and Western blots, whereas mRNA was quantitated by RT-PCR. Specific inhibitors and small interfering RNAs of PKC, p38, ERK1/2, NF-kappaB, and NADPH oxidase were used to determine the mediators in parallel experiments under HG conditions. IL-1beta-secreted protein, cellular protein, and mRNA increase under HG conditions is time and dose dependent, with maximum increase at 15 mM (48 h; P < 0.05). IL-1 receptor antagonist release was time and dose dependent, similar to IL-1beta expression pattern; however, the molar ratio of IL-1beta to IL-1RA was increased. Data from inhibitor and small interfering RNA experiments indicate that IL-1beta release under HG is mediated by PKC-alpha, via phosphorylation of p38 MAPK, and ERK1/2 leading to NF-kappaB activation, resulting in increased mRNA and protein for IL-1beta. At the same time, it appears that NADPH oxidase via p47phox activates NF-kappaB, resulting in increased IL-1beta secretion. Data suggest that, under HG conditions, monocytes release significantly higher amounts of IL-1beta through multiple mechanisms, further compounding the disease progression. Targeting signaling pathways mediating IL-1beta release could result in the amelioration of inflammation and possibly diabetic vasculopathies.     

Novel conformation-sensitive antibodies specific to three- and four-repeat tau

Two types of tau isoform, three- and four-repeat tau, are found in neurofibrillary tangles--a pathological hallmark of tauopathies. Which isoform is deposited in the affected tissues depends on the tauopathy. To study how and which tau isoforms contribute to neuronal degeneration, we have developed and characterized two novel conformation-sensitive antibodies, T3R and T4R. Two closely related synthetic peptides, PGGGKVQIVYK and PGGGSVQIVYK, respectively, were designed as antigens. The isoform-specific residues, (305)K in three-repeat tau or (305)S in four-repeat tau, and the PHF6 motif (VQIVYK) were identified as critical sequences. Despite the high similarity of the antigens, there was no cross-reactivity between T3R and T4R. Furthermore, T3R and T4R showed reduced binding to the thioflavin-positive beta-structural form of their target. These features may enable these antibodies to act as novel indicators that allow us to observe and evaluate conformational changes in each distinct isoform of tau.     

Knockout of toll-like receptor-4 attenuates the pro-inflammatory state of diabetes

Type 1 diabetes (T1DM) is associated with increased vascular complications and is a pro-inflammatory state. Recent findings have shown increased TLR2 and 4 expression, signaling, ligands, and functional activation in T1DM subjects compared to controls and further accentuated in T1DM with microvascular complications. Thus, the aim of this study was to examine if genetic deficiency of TLR4 attenuates the increased inflammation associated with T1DM using the streptozotocin-induced diabetic mouse model. C57BL/6 and TLR4(-/-) mice were obtained and studied in the native state and following induction of diabetes using streptozotocin. Diabetic (WT+STZ) mice had increased expression of both TLR2 and TLR4, while TLR4(-/-) STZ mice had increased expression only of TLR2, but not TLR4 compared to the non-diabetic mice TLR2 expression was significantly increased with STZ-induced diabetes and was unaffected by knockout of TLR4. Also, levels of MyD88, IRAK-1 protein phosphorylation, Trif, IRF3, and NF-κB activity were significantly reduced in TLR4(-/-) +STZ mice compared to the WT+STZ mice. WT+STZ mice exhibited significantly increased levels of serum and macrophage IL-1β, IL-6, KC/IL-8, IP-10, MCP-1, IFN beta and TNF-α compared to WT mice and this was significantly attenuated in TLR4(-/-) +STZ mice (P<0.01). Thus, TLR4 contributes to the pro-inflammatory state and TLR4KO attenuates inflammation in diabetes.     

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.     

3D-QSAR studies of 1,2-diaryl-1H-benzimidazole derivatives as JNK3 inhibitors with protective effects in neuronal cells

JNKs (c-Jun N-terminal kinases) have the potential to serve as a therapeutic target for various inflammatory, vascular, neurodegenerative, metabolic and oncological diseases. In particular, ATP-competitive JNK3 inhibitors act as neuroprotective agents. Here we introduce 1,2-diaryl-1H-benzimidazole derivatives as selective JNK3 inhibitors from among our in-house compounds and describe our elucidation of their SAR using 3D-QSAR models. A predictive CoMFA model (q² = 0.795, r² = 0.931) and a CoMSIA model (q² = 0.700, r² = 0.937) were used to describe the non-linearly combined affinity of each functional group in the inhibitors.     

Heterologous expression of a tannic acid-inducible laccase3 of <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

A tannic acid-inducible and mycoviral-regulated laccase3 (lac 3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae.