Covalent adduction of serotonin-derived quinones to the SARS-CoV-2 main protease expressed in a cultured cell.

The SARS-CoV-2 main protease is an essential molecule for viral replication and is often targeted by medications to treat the infection. In this study, we investigated the possible inhibitory action of endogenous quinones on the enzyme. Recombinant SARS-CoV-2 main protease was exposed to tryptamine-4,5-dione (TD) or quinone from 5-hydroxyindoleacetic acid (Q5HIAA). As a result, the protease activity was considerably decreased in a dose-dependent manner. The IC50 values of the quinones toward the enzyme were approximately 0.28 µM (TD) and 0.49 µM (Q5HIAA). Blot analyses using specific antibodies to quinone-modified proteins revealed that quinones were adducted to the enzyme at concentrations as low as 0.12 µM. Intact mass analyses showed that one or two quinone molecules were covalently adducted onto the main protease. Chymotrypsin-digested main protease analyses revealed that the quinones bind to thiol residues at the enzyme's active site. When TD or Q5HIAA were exposed to cultured cells expressing the viral enzyme, quinone-modified enzyme was identified in the cell lysate, suggesting that even extracellularly generated quinones could react with the viral enzyme expressed in an infected cell. Thus, these endogenous quinones could act as inhibitors of the viral enzyme.

Long-Term Exposure to Morphine Induces Cross-Tolerance to Acute Antinociceptive Effect of Remifentanil on Somatic and Visceral Stimuli in Rats.

Purpose: Remifentanil is one of the most commonly used opioids intraoperatively. Previous reports indicate that long-term use of opioids may lead to cross-tolerance to remifentanil, which poses a challenge in the control of acute pain intraoperatively. However, there is limited information regarding cross-tolerance to remifentanil, especially in visceral pain. Therefore, this study aimed to examine cross-tolerance to remifentanil in somatic and visceral tolerance using morphine-tolerant rats. Methods: Six male Sprague-Dawley rats were allocated to the morphine and saline groups each. Tolerance to the antinociceptive effect of morphine was induced in rats in the morphine group. Remifentanil was continuously infused intravenously at 10 mcg/kg/min for 120 min to assess cross-tolerance from morphine to remifentanil. The antinociceptive effects on somatic and visceral nociceptive stimuli were measured using the tail-flick (TF) and colorectal distension (CD) tests, respectively. The antinociceptive efficacy was evaluated by converting the response threshold to the percentage maximal possible effect (%MPE). Results: Remifentanil increased the %MPE in the morphine and saline groups in both the tests; however, the increase in %MPE was attenuated significantly in the morphine group compared with that in the saline group at 60, 90, and 120 min (all P < 0.01) in the TF test and at 90 and 120 min in the CD test (all P <0.05). Conclusion: Our results indicate that morphine-tolerant rats exhibit cross-tolerance to remifentanil's acute antinociceptive effects on somatic and visceral stimuli. Cross-tolerance to remifentanil should be considered in the perioperative management of patients using morphine.

Association of Antihypertensive Effects of Esaxerenone with the Internal Sodium Balance in Dahl Salt-Sensitive Hypertensive Rats.

BACKGROUND: The nonsteroidal mineralocorticoid receptor blocker esaxerenone is effective in reducing blood pressure (BP). OBJECTIVE: In this study, we investigated esaxerenone-driven sodium homeostasis and its association with changes in BP in Dahl salt-sensitive (DSS) hypertensive rats. METHODS: In the different experimental setups, we evaluated BP by a radiotelemetry system, and sodium homeostasis was determined by an approach of sodium intake (food intake) and excretion (urinary excretion) in DSS rats with a low-salt diet (0.3% NaCl), high-salt diet (HSD, 8% NaCl), HSD plus 0.001% esaxerenone (w/w), and HSD plus 0.05% furosemide. RESULTS: HSD-fed DSS rats showed a dramatic increase in BP with a non-dipper pattern, while esaxerenone treatment, but not furosemide, significantly reduced BP with a dipper pattern. The cumulative sodium excretion in the active period was significantly elevated in esaxerenone- and furosemide-treated rats compared with their HSD-fed counterparts. Sodium content in the skin, skinned carcass, and total body tended to be lower in esaxerenone-treated rats than in their HSD-fed counterparts, while these values were unchanged in furosemide-treated rats. Consistently, sodium balance tended to be reduced in esaxerenone-treated rats during the active period. Histological evaluation showed that esaxerenone, but not furosemide, treatment attenuated glomerulosclerosis, tubulointerstitial fibrosis, and urinary protein excretion induced by high salt loading. CONCLUSIONS: Collectively, these findings suggest that an esaxerenone treatment-induced reduction in BP and renoprotection are associated with body sodium homeostasis in salt-loaded DSS rats.

Effects of an intrathecal TRPV1 antagonist, SB366791, on morphine-induced itch, body temperature, and antinociception in mice.

PURPOSE: Transient receptor potential vanilloid 1 (TRPV1) not only is activated by multiple stimuli but also is involved with histamine-induced itch. The effects of TRPV1 on morphine-induced itch are unknown. We examined the effects of intrathecal administration of TRPV1 antagonist on morphine-induced itch, body temperature, and antinociception for mice. METHODS: Each C57/BL6j mouse was intrathecally administered with one of the following solutions: morphine, SB366791 (as the TRPV1 antagonist), morphine + SB366791, saline, or vehicle. For each mouse, each instance of observed scratching behavior was counted, the body temperature was measured, and the nociceptive threshold was determined using the tail-immersion test. RESULTS: SB366791 dose-dependently reduced the scratching behavior induced by the administration of morphine. SB366791 and the morphine + SB366791 groups did not manifest an increase in body temperature. Antinociceptive effects were observed to occur dose-dependently for morphine but not for SB366791. Compared with morphine alone, the administration of morphine + SB366791 did not reduce significant antinociceptive effects. CONCLUSION: We propose that an intrathecal TRPV1 antagonist, SB366791, reduced morphine-induced itch without causing hyperthermia and did not suppress morphine-induced antinociception for mice.

In-frame Val216-Ser217 deletion of KIT in mild piebaldism causes aberrant secretion and SCF response.

BACKGROUND: Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. OBJECTIVE: The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. METHODS: We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. RESULTS: A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val216 and Ser217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. CONCLUSION: These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism.

Global DNA hypomethylation and hypoxia-induced expression of the ten eleven translocation (TET) family, TET1, in scleroderma fibroblasts.

The precise mechanisms of tissue fibrosis have not yet been elucidated in systemic sclerosis (SSc). However, studies of the regulation of DNA methylation, the most widely studied epigenetic mechanism, have confirmed the involvement of the TET family proteins, recently identified DNA demethylases, in the pathogenesis of SSc. The mRNA levels of TET family members were compared in normal and SSc fibroblasts. The effects of hypoxia and siRNA specific to HIF-1α on TET expression were also examined. Global methylation status was analysed by LUMA. The presence of 5-hydroxymethylcytosine (5hmC) in SSc was examined by immunohistochemistry. The level of TET1 mRNA in SSc fibroblasts was elevated by 1.68 fold compared with that of normal fibroblasts, but the expression levels of TET2 and TET3 were comparable between both cell types. The expression levels of DNMT1 and DNMT3B mRNA have a tendency to elevate in SSc fibroblasts. Among TET family members, the expression of TET1 was exclusively induced by hypoxia via HIF-1α-independent pathways in SSc fibroblasts, but not in normal fibroblasts. The methylation level was decreased in SSc fibroblasts relative to normal fibroblasts, and 5hmC was present in dermal fibroblasts of skin sections from patients with SSc. TET1 expression in SSc fibroblasts was abnormally regulated in the hypoxic environment and accompanied by global DNA hypomethylation, suggesting the involvement of aberrant DNA methylation in the pathogenesis of SSc.