Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88

AbstractMyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88

Adverse effects of advanced glycation end products on embryonal development

We studied the effects of advanced glycation end products (AGEs), which are known to accumulate in patients with diabetes, autoimmune diseases, or those who smoke, on embryonal development. Pronuclear (PN) embryos were obtained by flushing the fallopian tubes of rats after superovulation and mating. The cleavage rate and blastocyst yield were evaluated at 24, 72, 96, and 120 h of culture. Glyoxal, an AGE-forming aldehyde, suppressed embryonal development at every stage from PN to blastocyst in a concentration-dependent manner. The cleavage rate of the embryo was also signifi cantly decreased by treatment with glyoxal at concentrations of 1 mM or higher. The blastocyst yield was significantly decreased by treatment with glyoxal at concentrations of 0.5 mM or higher. N-acetyl-L-cysteine (L-NAC) at 1 mM significantly suppressed the glyoxal-induced embryonal toxicity. BSA-AGEs at 5 microg/ml or higher concentration signifi cantly reduced the cleavage rate and blastocyst yield compared to those for BSA-treated embryos. L-NAC at 1 mM significantly suppressed BSAAGE-induced embryonal toxicity. Because AGEs are embryo-toxic, AGE contamination may influence the pregnancy rate of in vitro fertilization and embryo transfer. AGEs, which are increased in women under pathological conditions, may also be involved in their infertility.</p

Increased Anti-HSP60 and Anti-HSP70 Antibodies in Women with Unexplained Recurrent Pregnancy Loss

　Vascular dysfunction has been reported in women with recurrent pregnancy loss (RPL). We investigated the severity of vascular dysfunction in non-pregnant women with RPL and its correlation with anti-heat shock protein (HSP) antibodies that are known to induce arteriosclerosis. We measured the serum anti-HSP60 antibodies, anti-HSP70 antibodies, and anti-phospholipid antibodies (APA) in 68 women with RPL and 29 healthy controls. Among the women with RPL, 14 had a diagnosis of antiphospholipid syndrome (APS), and in the remaining 54, the causes for RPL were unexplained. Compared to the controls, the brachial-ankle pulse wave velocity (baPWV), carotid augmentation index (cAI), and uterine artery pulsatility index (PI) were all significantly higher in the women with both APS and unexplained RPL. Compared to the controls, the anti-HSP60 antibody levels were significantly higher in the APA-positive group of women with unexplained RPL, and the anti-HSP70 antibody levels were significantly higher in APS and APA-positive group of women with unexplained RPL. However, the anti-HSP60 and anti-HSP70 antibody levels did not correlate with the values of baPWV or cAI. Our results demonstrated anti-HSP60 and anti-HSP70 antibodies are increased in women with unexplained RPL. Further studies are needed to elucidate the roles of anti-HSP antibodies and their pathophysiology in unexplained RPL

Carbonyl Compounds in the Gas Phase of Cigarette Mainstream Smoke and Their Pharmacological Properties

Cigarette mainstream smoke is composed of gas and tar phases and contains >4000 chemical constituents, including nicotine and tar. The substances in the gas phase but not in the tar phase can pass through the airway epithelial barrier, enter the systemic circulation via the pulmonary circulation, and increase systemic oxidative damage, leading to the development of cigarette smoking-related diseases such as atherosclerosis. Recently, we identified some stable carbonyl compounds, including acrolein (ACR) and methyl vinyl ketone (MVK), as major cytotoxic factors in nicotine- and tar-free cigarette smoke extract (CSE) of the gas phase. CSE, ACR, and MVK induce protein kinase C (PKC)-dependent activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) via NOX, causing plasma membrane damage and cell apoptosis. CSE, ACR, and MVK also trigger carbonylation of PKC, which is an irreversible oxidative modification. Cell damage and PKC carbonylation in response to treatment with CSE, ACR, or MVK are abolished by thiol-containing antioxidants such as N-acetyl-L-cysteine and reduced glutathione. Thus pharmacological modulation of PKC and NOX activities and the trapping of ROS are potential strategies for the prevention of diseases related to cigarette smoking

Comparison of cytotoxicity of cigarette smoke extract derived from heat-not-burn and combustion cigarettes in human vascular endothelial cells

The present study compared the properties of mainstream smoke generated from heat-not-burn (HNB) cigarettes and a combustion cigarette (hi-lite (TM) brand). Three types of cigarette heating devices were used to generate cigarette smoke at different heating temperatures [Ploom S (TM) (200 degrees C), glo (TM) (240 degrees C), and IQOS (TM) (300-350 degrees C)]. Mainstream smoke was generated using the following puffing regimen: volume, 55 mL; duration, 3 s; and interval, 30 s. The rank order of particulate phase (nicotine and tar) amounts trapped on a Cambridge filter was Ploom S < glo < IQOS < hi-lite. Heated cigarette-derived smoke extract (hCSE) from the devices except for Ploom S, and burned CSE (bCSE) decreased mitochondria) metabolic activity (glo < IQOS < hi-lite) in human vascular endothelial cells. Furthermore, the cytotoxicity was reduced by removing the particulate phase from the mainstream smoke. Endothelial nitric oxide synthase activity was reduced by nicotine- and tar-free CSE of IQOS and hi-lite (IQOS < hi-lite), but not Ploom S and glo. These inhibitory effects were diminished by removing the carbonyl compounds from the mainstream smoke. These results indicated that the cytotoxicity of hCSE was lower than that of bCSE in vascular endothelial cells. (C) 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society

Title page. Adenylate cyclase-cAMP-protein kinase A signaling pathway inhibits endothelin type A receptor-operated Ca 2+ entry mediated via TRPC6 channels. Running title page. Running title: PKA-mediated inhibition of ET A R-induced Ca 2+ entry via TRPC6

The number of text pages: 30 The number of tables: 1 The number of figures: 6 The number of supplemental figures: 4 The number of references: 34 The number of words in the Abstract: 248 The number of words in the Introduction: 718 The number of words in the Discussion: 1,39

Endothelin-1 decreases [Ca2+]i via Na+/Ca2+ exchanger in CHO cells stably expressing endothelin ETA receptor.

Endothelin ETA receptor couples to Gq/11 protein that transduces a variety of receptor signals to modulate diverse cellular responses including Ca2+ mobilization. Stimulation of endothelin ETA receptor with endothelin-1 is generally believed to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) via Gq/11 protein. Here we provide the first convincing evidence that endothelin-1 elicited Gq/11 protein-dependent and -independent ‘decrease’ in [Ca2+]i via Na+/Ca2+ exchanger (NCX) in Chinese hamster ovary (CHO) cells stably expressing human endothelin ETA receptor. In the cells treated with 1 μM thapsigargin, an inhibitor of endoplasmic Ca2+ pump, that induces an increase in [Ca2+]i via capacitative Ca2+ entry, endothelin-1 induced a decrease in [Ca2+]i which was partially inhibited by YM-254890, a specific inhibitor of Gq/11, indicating that Gq/11-dependent and independent pathways are involved in the decrease. The endothelin-1-induced decrease in [Ca2+]i was markedly suppressed by 3′,4′-dichlorobenzamil hydrochloride, a potent NCX inhibitor, and also by a replacement of extracellular Na+ with Li+, which was not transported by NCX, indicating a major role of NCX operating in the forward mode in the endothelin-1-induced decrease in [Ca2+]i. Molecular approach with RT-PCR demonstrated the expression of mRNA for NCX1, NCX2 and NCX3. These results suggest that stimulation of endothelin ETA receptor with endothelin-1 activates the forward mode NCX through Gq/11-dependent and -independent mechanisms: the NCX exports Ca2+ out of the cell depending on Na+ gradient across the cell membrane, resulting in the decrease in [Ca2+]i

Jab1 regulates levels of endothelin type A and B receptors by promoting ubiquitination and degradation

Endothelin type A receptor (ETAR) plays an important role in some cardiovascular disorders where ETAR levels are increased. However, regulatory mechanisms for ETAR levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ETAR-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ETAR as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ETAR-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ETAR and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ETAR, whereas Jab1 overexpression decreased them. jab1 overexpression accelerated disappearance rate of ETAR after protein synthesis inhibition as an index of a degradation rate. ETAR was constitutively ubiquitinated, and the level of ubiquitination was enhanced by jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ETAR degradation and increased the amount of Jab1 bound to ETAR with a maximal level of 500% at 3 h. In the absence of ET-1 stimulation, the level of ETBR was lower than that of ETAR and the degradation rate of ETBR was markedly faster than that of ETAR. Notably, the amount of Jab1 bound to ETBR and ubiquitination level of ETBR were markedly higher than those for ETAR. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ETAR and ETBR by modulating ubiquitination of these receptors, leading to changes in ETAR and ETBR levels