An integrated PKD1-dependent signaling network amplifies IRE1 prosurvival signaling.

Following the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), a condition known as ER stress in this compartment triggers an adaptive signaling pathway referred to as the unfolded protein response (UPR). The UPR aims at restoring ER homeostasis; if the ER stress cannot be resolved, apoptosis is triggered. However, the mechanisms responsible for regulating the balance between cell life and death decisions that occur after exposure to ER stress remain unclear. Protein kinase D1 (PKD1) has been reported to initiate protective signaling against oxidative stress or ischemia, two conditions that impinge on the induction of ER stress. In addition, the high levels of expression of PKD1, observed in highly proliferative cancers and tumors with poor prognosis, contribute to enhanced resistance to chemotherapy. In this study, we show that the ER stress inducers tunicamycin and thapsigargin lead to the activation of PKD1 in human prostate cancer PC-3 cells and in hepatoma HepG2 cells through a PKCδ-dependent mechanism. Moreover, our data indicate that PKD1 is required for the stabilization of inositol-requiring enzyme 1 (IRE1) and the subsequent regulation of its activity. PKD1 activation contributes to the phosphorylation of mitogen-activated protein kinase phosphatase 1, resulting in decreased IRE1-mediated c-Jun N-terminal kinase activation. This study unveils the existence of a novel PKD1-dependent prosurvival mechanism that is activated upon ER stress and selectively enhances IRE1 prosurvival signaling.

Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges.

G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.

Inhibition efficiency of 304-Cu stainless steel against oral bacterial biofilm.

PURPOSE: This study aims to evaluate the antibacterial properties of 304 Cu-bearing stainless steel (SS) with different Cu contents (0, 2.5, 4.5 wt.%) against oral biofilms of Streptococcus mutans (S. mutans), Streptococcus sanguinis (S. sanguinis), and their mixture. METHODS: Bacterial biofilms on the surface of 304-Cu SS were characterized by plate counting, 4', 6-diamidino-2-phenylindole (DAPI) staining with aid of sanning electron microscopy (SEM) and 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT). In addition, the inhibition zone method was also employed to evaluate the antibacterial properties of 304-Cu SS. Cell Counting Kit-8 (CCK-8) and flow cytometry were used to assess the cytotoxicity and apoptosis rate of 304-Cu SS, respectively. RESULTS: 304-4.5Cu SS could effectively inhibit the attachment, formation, activity, and metabolism of bacterial biofilm, possessing the best antibacterial properties exceeding 99.9% of antibacterial rate against S. mutans, S. sanguinis, and their mixture. The diameters of inhibition zones to S. mutans and S. sanguinis on the surface of 304-4.5Cu SS were 21.7 and 14.7 mm, respectively. The results of cell experiments in vitro showed that both 304-2.5Cu SS and 304-4.5Cu SS had no evident cytotoxicity with an identical grade 1. The apoptosis rate exhibited a gradually increased tendency with increase of the Cu content in 304 SS. CONCLUSIONS: 304-4.5Cu SS without cytotoxic effect on NIH3T3 cells has obvious antibacterial activity against S. mutans, S. sanguinis and their mixture. CLINICAL SIGNIFICANCE: The Cu-bearing stainless steel provides a new solution to be used as oral orthodontic devices for inhibiting oral microflora imbalance and enamel demineralization.