Targeted Mutation of the Mouse Grp94 Gene Disrupts Development and Perturbs Endoplasmic Reticulum Stress Signaling

Glucose-regulated protein 94 (GRP94) is one of the most abundant endoplasmic reticulum (ER) resident proteins and is the ER counterpart of the cytoplasmic heat shock protein 90 (HSP90). GRP94, a component of the GRP78 chaperone system in protein processing, has pro-survival properties with implicated function in cancer progression and autoimmune disease. Previous studies on the loss of GRP94 function showed that it is required for embryonic development, regulation of toll-like receptors and innate immunity of macrophages. Here we report the creation of mouse models targeting exon 2 of the Grp94 allele that allows both traditional and conditional knockout (KO) of Grp94. In this study, we utilized the viable Grp94+/+ and +/− mice, as well as primary mouse embryonic fibroblasts generated from them as experimental tools to study its role in ER chaperone balance and ER stress signaling. Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response. To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94−/− embryonic stem cells. In contrast to Grp94 heterozygosity, complete knockout of GRP94 leads to compensatory upregulation of the ER chaperones GRP78, calnexin and calreticulin but not protein disulphide isomerase. Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway. Furthermore, from analysis of microarray database and immunohistochemical staining, we present predictions where GRP94 may play an important role in specific adult organ homeostasis and function

Occlusion of Regulatory Sequences by Promoter Nucleosomes In Vivo

Nucleosomes are believed to inhibit DNA binding by transcription factors. Theoretical attempts to understand the significance of nucleosomes in gene expression and regulation are based upon this assumption. However, nucleosomal inhibition of transcription factor binding to DNA is not complete. Rather, access to nucleosomal DNA depends on a number of factors, including the stereochemistry of transcription factor-DNA interaction, the in vivo kinetics of thermal fluctuations in nucleosome structure, and the intracellular concentration of the transcription factor. In vitro binding studies must therefore be complemented with in vivo measurements. The inducible PHO5 promoter of yeast has played a prominent role in this discussion. It bears two binding sites for the transcriptional activator Pho4, which at the repressed promoter are positioned within a nucleosome and in the linker region between two nucleosomes, respectively. Earlier studies suggested that the nucleosomal binding site is inaccessible to Pho4 binding in the absence of chromatin remodeling. However, this notion has been challenged by several recent reports. We therefore have reanalyzed transcription factor binding to the PHO5 promoter in vivo, using ‘chromatin endogenous cleavage’ (ChEC). Our results unambiguously demonstrate that nucleosomes effectively interfere with the binding of Pho4 and other critical transcription factors to regulatory sequences of the PHO5 promoter. Our data furthermore suggest that Pho4 recruits the TATA box binding protein to the PHO5 promoter

GRP78/BiP Is Required for Cell Proliferation and Protecting the Inner Cell Mass from Apoptosis during Early Mouse Embryonic Development

GRP78, also known as BiP, is a central regulator of endoplasmic reticulum (ER) homeostasis due to its multiple functional roles in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. ER stress induction of GRP78/BiP represents a major prosurvival arm of the unfolded protein response (UPR). However, the physiological role of GRP78 in development is not known. Using a transgenic approach, we discovered that the Grp78 promoter is activated in both the trophectoderm and inner cell mass (ICM) of embryos at embryonic day 3.5 via a mechanism requiring the ER stress elements. To reveal the function of the GRP78 in vivo, we created a tri-loxP Grp78 mutant allele, which was further crossed with EIIA-cre to create a knockout allele. The Grp78(+/)(−) mice, which express 50% of the wild-type level of the GRP78 protein, are viable. Interestingly, the heterozygous Grp78 cells up-regulate the ER proteins GRP94 and protein disulfide isomerase at both the transcript and protein levels, while other UPR targets such as CHOP and XBP-1 are not affected. Further studies revealed that mouse embryonic fibroblasts from Grp78(+/)(−) mice are capable of responding to ER stress. However, Grp78(−)(/)(−) embryos that are completely devoid of GRP78 lead to peri-implantation lethality. These embryos do not hatch from the zona pellucida in vitro, fail to grow in culture, and exhibit proliferation defects and a massive increase in apoptosis in the ICM, which is the precursor of embryonic stem cells. These findings provide the first evidence that GRP78 is essential for embryonic cell growth and pluripotent cell survival

Effect of Sintering Temperature on Adhesion Property and Electrochemical Activity of Pt/YSZ Electrode

The (Pt/YSZ)/YSZ sensor unit is the basic component of the NOx sensor, which can detect the emission of nitrogen oxides in exhaust fumes and optimize the fuel combustion process. In this work, the effect of sintering temperature on adhesion property and electrochemical activity of Pt/YSZ electrode was investigated. Pt/YSZ electrodes were prepared at different sintering temperatures. The microstructure of the Pt/YSZ electrodes, as well as the interface between Pt/YSZ electrode and YSZ electrolyte, were observed by SEM. Chronoamperometry, linear scan voltammetry, and AC impedance were tested by the electrochemical workstation. The results show that increasing the sintering temperature (≤1500 °C) helped to improve adhesion property and electrochemical activity of the Pt/YSZ electrode, which benefited from the formation of the porous structure of the Pt/YSZ electrode. For the (Pt/YSZ) electrode/YSZ electrolyte system, O2− in YSZ is converted into chemisorbed O2 on Pt/YSZ, which is desorbed into the gas phase in the form of molecular oxygen; this process could be the rate-controlling step of the anodic reaction. Increasing the sintering temperature (≤1500 °C) could reduce the reaction activation energy of the Pt/YSZ electrode. The activation energy reaches the minimum value (1.02 eV) when the sintering temperature is 1500 °C

Hydrophobic Wafer-Scale High-Reproducibility SERS Sensor Based on Silicon Nanorods Arrays Decorated with Au Nanoparticles for Pesticide Residue Detection

High sensitivity and reproducibility are highly desirable to a SERS sensor in diverse detection applications. Moreover, it is a great challenge to determine how to promote the target molecules to be more concentrated on the hotspots of the SERS substrate by engineering a surface with switching interfacial wettability. Along these lines, wafer-scale uniformly hydrophobic silicon nanorods arrays (SiNRs) decorated with Au nanoparticles were designed as the SERS substrate. Typically, the SERS substrate was fabricated by enforcing the polystyrene (PS) sphere self-assembly, as well as the plasma etching and the magnetron sputtering techniques. Consequently, the SERS substrate was treated by soaking within a n-dodecyl mercaptan (NDM) solution at different times in order to obtain adjustable wettabilities. By leveraging the electromagnetic enhancement resulted from the Au nanostructures and enrichment effect induced by the hydrophobicity, the SERS substrate is endowed with efficient SERS capabilities. During the detection of malachite green (MG), an ultralow relative standard deviation (RSD) 4.04–6.14% is achieved and the characteristic signal of 1172 cm−1 can be detected as low as 1 ng/mL. The proposed SiNRs’ structure presents outstanding SERS activity with sensitivity and reproducibility rendering thus an ideal candidate for potential application in analytical detection fields

Yeast strains list.

<p>Yeast strains list.</p