4,6-Dibromo-3-Hydroxycarbazole (an Analogue of Caffeine-like Ca2+ Releaser), a Novel Type of Inhibitor of Ca2+-Induced Ca2+ Release in Skeletal Muscle Sarcoplasmic Reticulum

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A Phos-Tag-Based Approach Reveals the Extent of Physiological Endoplasmic Reticulum Stress

Cellular response to endoplasmic reticulum (ER) stress or unfolded protein response (UPR) is a key defense mechanism associated with many human diseases. Despite its basic and clinical importance, the extent of ER stress inflicted by physiological and pathophysiological conditions remains difficult to quantitate, posing a huge obstacle that has hindered our further understanding of physiological UPR and its future therapeutic potential. Here we have optimized a Phos-tag-based system to detect the activation status of two proximal UPR sensors at the ER membrane. This method allowed for a quantitative assessment of the level of stress in the ER. Our data revealed quantitatively the extent of tissue-specific basal ER stress as well as ER stress caused by the accumulation of misfolded proteins and the fasting-refeeding cycle. Our study may pave the foundation for future studies on physiological UPR, aid in the diagnosis of ER-associated diseases and improve and facilitate therapeutic strategies targeting UPR in vivo

Milk Lacking α-Casein Leads to Permanent Reduction in Body Size in Mice

The major physiological function of milk is the transport of amino acids, carbohydrates, lipids and minerals to mammalian offspring. Caseins, the major milk proteins, are secreted in the form of a micelle consisting of protein and calcium-phosphate

Yokukansan Inhibits Neuronal Death during ER Stress by Regulating the Unfolded Protein Response

Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death.We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein.Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs.Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD

Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

BACKGROUND: Glycan biosynthesis occurs though a multi-step process that requires a variety of enzymes ranging from glycosyltransferases to those involved in cytosolic sugar metabolism. In many cases, glycan biosynthesis follows a glycan-specific, linear pathway. As glycosyltransferases are generally regulated at the level of transcription, assessing the overall transcriptional profile for glycan biosynthesis genes seems warranted. However, a systematic approach for assessing the correlation between glycan expression and glycan-related gene expression has not been reported previously. METHODOLOGY: To facilitate genetic analysis of glycan biosynthesis, we sought to correlate the expression of genes involved in cell-surface glycan formation with the expression of the glycans, as detected by glycan-recognizing probes. We performed cross-sample comparisons of gene expression profiles using a newly developed, glycan-focused cDNA microarray. Cell-surface glycan expression profiles were obtained using flow cytometry of cells stained with plant lectins. Pearson's correlation coefficients were calculated for these profiles and were used to identify enzyme genes correlated with glycan biosynthesis. CONCLUSIONS: This method, designated correlation index-based responsible-enzyme gene screening (CIRES), successfully identified genes already known to be involved in the biosynthesis of certain glycans. Our evaluation of CIRES indicates that it is useful for identifying genes involved in the biosynthesis of glycan chains that can be probed with lectins using flow cytometry

Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses

<p>Abstract</p> <p>Background</p> <p>Hereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y <it>HFE </it>associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH.</p> <p>Results</p> <p>Vector constructs of Wild type HFE and Mutant C282Y HFE were made and transfected into HEK293 cell lines. We have shown that expression of C282Y HFE protein triggers both an unfolded protein response (UPR), as revealed by the increased GRP78, ATF6 and CHOP expression, and an ER overload response (EOR), as indicated by NF-κB activation. Furthermore, C282Y HFE protein induced apoptotic responses associated with activation of ER stress. Inhibition studies demonstrated that tauroursodeoxycholic acid, an endogenous bile acid, downregulates these events. Finally, we found that the co-existence of both C282Y HFE and Z alpha 1-antitrypsin protein (the protein associated with the liver disease of Z alpha 1-antitrypsin deficiency) expression on ER stress responses acted as potential disease modifiers with respect to each other.</p> <p>Conclusion</p> <p>Our novel observations suggest that both the ER overload response (EOR) and the unfolded protein response (UPR) are activated by mutant C282Y HFE protein.</p

Structure-activity Relationship of Bromoeudistomin D, a Powerful Ca2+ Releaser in Skeletal Muscle Sarcoplasmic Reticulum

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