Lack of the Histone Deacetylase SIRT1 Leads to Protection against Endoplasmic Reticulum Stress through the Upregulation of Heat Shock Proteins.

Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1α branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins.

Impact of Prolonged Ischemia on the Immunohistochemical Expression of Programmed Death Ligand 1 (PD-L1).

Antibodies targeting programmed death receptor 1 or programmed death ligand 1 (PD-L1) have become a standard of care to treat different cancers; for some of these tumors, there is a correlation between tissue expression of PD-L1 and response rates in patients. Although most of the analytical challenges in the evaluation of PD-L1 expression have been standardized, preanalytical issues have been less explored. The objective of this study was to evaluate the impact of time of ischemia on the performance of 2 commonly used antibodies against PD-L1.

The Immunohistochemical Expression of Programmed Death Ligand 1 (PD-L1) Is Affected by Sample Overfixation.

Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been approved for the treatment of different cancers. Some of these antibodies show a correlation between the tissue expression of PD-L1 and response. Evaluation of PD-L1 expression presents multiple challenges, but some preanalytical issues such as tissue fixation have been scarcely evaluated.

Dual-reporter in vivo imaging of transient and inducible heat-shock promoter activation.

Gene promoter activity can be studied in vivo by molecular imaging methods using reporter gene technology. Transcription of the reporter and the reported genes occurs simultaneously. However, imaging depends on reporter protein translation, stability, and cellular fate that may differ among the various proteins.

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

Background: Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization.

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes.

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type.

Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between MyD88-dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways.

There is increasing evidence that astrocytes play important roles in immune regulation in the brain. Astrocytes express toll-like receptors (TLR) and build up responses to innate immune triggers by releasing proinflammatory molecules. We investigate signaling pathways and released molecules after astrocyte TLR4 activation.

Extended ischemia prevents HIF1alpha degradation at reoxygenation by impairing prolyl-hydroxylation: role of Krebs cycle metabolites.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that activates the cellular response to hypoxia. The HIF1alpha subunit is constantly synthesized and degraded under normoxia, but degradation is rapidly inhibited when oxygen levels drop. Oxygen-dependent hydroxylation by prolyl-4-hydroxylases (PHD) mediates HIF1alpha proteasome degradation.

Depressed glucose consumption at reperfusion following brain ischemia does not correlate with mitochondrial dysfunction and development of infarction: an in vivo positron emission tomography study.

Glucose consumption is severely depressed in the ischemic core, whereas it is maintained or even increased in penumbral regions during ischemia. Conversely, glucose utilization is severely reduced early after reperfusion in spite that glucose and oxygen are available. Experimental studies suggest that glucose hypometabolism might be an early predictor of brain infarction.

Astrocytes are very sensitive to develop innate immune responses to lipid-carried short interfering RNA.

Short interfering RNA (siRNA) inhibits the synthesis of specific proteins through RNA interference (RNAi). However, siRNA can induce innate immune responses that are mediated by toll-like receptors (TLRs) in cells of the immune system. Here, we sought to evaluate whether siRNA can induce such responses in glial cells.

Glucose promotes caspase-dependent delayed cell death after a transient episode of oxygen and glucose deprivation in SH-SY5Y cells.

Brain ischemia causes neuronal cell death by several mechanisms involving necrotic and apoptotic processes. The contributions of each process depend on conditions such as the severity and duration of ischemia, and the availability of ATP. We examined whether glucose affected the development of apoptosis after transient ischemia, and whether this was sensitive to caspase inhibition.

The RNA binding protein Zfp36l1 is required for normal vascularisation and post-transcriptionally regulates VEGF expression.

The Zfp36l1 gene encodes a zinc finger-containing mRNA binding protein implicated in the posttranscriptional control of gene expression. Mouse embryos homozygous for a targeted mutation in the Zfp36l1 locus died mid-gestation and exhibited extraembryonic and intraembryonic vascular abnormalities and heart defects. In the developing placenta, there was a failure of the extraembryonic mesoderm to invaginate the trophoblast layer.

T3 strongly regulates GLUT1 and GLUT3 mRNA in cerebral cortex of hypothyroid rat neonates.

Experimental hypothyroidism alters the expression of the GLUT1 and GLUT4 glucose transporters in brown adipose tissue, skeletal muscle and heart. Congenital hypothyroidism disrupts the development and function of the CNS, and the importance of GLUT1 for proper brain function has been dramatically evidenced in the cases of GLUT1 deficiency syndrome. Because of this, we hypothesised that the expression of GLUT1 and GLUT3, glucose transporters expressed in brain cortex, may be altered in congenital hypothyroidism.

Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178.

Phenylephrine requires the TATA box to activate transcription of GLUT1 in neonatal rat cardiac myocytes.

Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and metabolism. Phenylephrine, among other hypertrophic agonists, has been reported to increase expression of GLUT1 in neonatal rat cardiac myocytes by activating transcription. However, the specific cis- or trans-acting factors in the GLUT1 gene that are targeted by this agonist remain elusive.

Structural and functional dissection of a conserved destabilizing element of cyclo-oxygenase-2 mRNA: evidence against the involvement of AUF-1 [AU-rich element/poly(U)-binding/degradation factor-1], AUF-2, tristetraprolin, HuR (Hu antigen R) or FBP1 (far-upstream-sequence-element-binding protein 1).

COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability.

Hypertrophic agonists induce the binding of c-Fos to an AP-1 site in cardiac myocytes: implications for the expression of GLUT1.

Objectives: Serum is among the agents known to induce hypertrophy of cardiac myocytes, which occurs concomitant with an increase in AP-1-mediated transcription. We have examined if this effect correlates with changes in the relative abundance of particular AP-1 heterodimers, as their exact composition under these conditions is unknown. Furthermore, we obtained insight on the specific role of c-Fos from studying the induction of the glucose transporter GLUT1 by serum in fibroblasts.

Differential regulation of the muscle-specific GLUT4 enhancer in regenerating and adult skeletal muscle.

We have reported a novel functional co-operation among MyoD, myocyte enhancer factor-2 (MEF2), and the thyroid hormone receptor in a muscle-specific enhancer of the rat GLUT4 gene in muscle cells. Here, we demonstrate that the muscle-specific enhancer of the GLUT4 gene operates in skeletal muscle and is muscle fiber-dependent and innervation-independent. Under normal conditions, both in soleus and in extensor digitorum longus muscles, the activity of the enhancer required the integrity of the MEF2-binding site.