A recent mutation analysis suggested that Non-Structural Protein 6 (NSP6) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a key determinant of the viral pathogenicity. Here, by transcriptome analysis, we demonstrated that the inflammasome-related NOD-like receptor signaling was activated in SARS-CoV-2-infected lung epithelial cells and Coronavirus Disease 2019 (COVID-19) patients’ lung tissues. The induction of inflammasomes/pyroptosis in patients with severe COVID-19 was confirmed by serological markers. Overexpression of NSP6 triggered NLRP3/ASC-dependent caspase-1 activation, interleukin-1β/18 maturation, and pyroptosis of lung epithelial cells. Upstream, NSP6 impaired lysosome acidification to inhibit autophagic flux, whose restoration by 1α,25-dihydroxyvitamin D3, metformin or polydatin abrogated NSP6-induced pyroptosis. NSP6 directly interacted with ATP6AP1, a vacuolar ATPase proton pump component, and inhibited its cleavage-mediated activation. L37F NSP6 variant, which was associated with asymptomatic COVID-19, exhibited reduced binding to ATP6AP1 and weakened ability to impair lysosome acidification to induce pyroptosis. Consistently, infection of cultured lung epithelial cells with live SARS-CoV-2 resulted in autophagic flux stagnation, inflammasome activation, and pyroptosis. Overall, this work supports that NSP6 of SARS-CoV-2 could induce inflammatory cell death in lung epithelial cells, through which pharmacological rectification of autophagic flux might be therapeutically exploited.

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In this study we have investigated the effects of administration of procyanidins from grape seeds on serum uric acid levels in a model of hyperuricaemia in mice pretreated with oxonate, as well as the xanthine dehydrogenase and xanthine oxidase activities in mouse liver in vivo. The procyanidins, when orally administered to the oxonate-pretreated hyperuricaemic mice, were able to elicit a dose-dependent hypouricaemic effect. At a dose of 400 mg/kg for 3 days, the serum urate levels of the oxonate-pretreated mice were not different from the normal mice. In addition, the hepatic activities of xanthine dehydrogenase and xanthine oxidase in the procyanidins-treated mice were found to decrease significantly. However, the hypouricaemic effects observed in the experimental animals did not seem to parallel the changes in xanthine dehydrogenase and xanthine oxidase activities, implying that the procyanidins might be acting via other mechanisms apart from simple inhibition of enzyme activities. Furthermore, the procyanidin-treated animals exhibited normal growth while the allopurinol-treated animals exhibited some retarded growth. These results demonstrated for the first time that the procyanidins from grape seeds possess in vivo urate-lowering activities. The potential application of these natural compounds in the treatment of hyperuricaemia is discussed.Gout is a common metabolic disorder in human, reportedly afflicting more than 2 million men and women in the United States alone (Kramer & Curhan 2002). The disease is also rising rapidly in China (Li et al. 1997) due probably to recent changes in dietary habits. Gout is often associated with elevated serum levels of uric acids, resulting in the deposition of urate crystals in the joints and kidneys, causing inflammation as well as gouty arthritis and uric acid nephrolithiasis (Kramer & Curhan 2002). Thus, the treatment of gout entails the use of non-steroidal antiinflammatory drugs (NSAIDs) for symptomatic relief as well as xanthine oxidase inhibitor (Liote 2003) to curtail the endogenous production of uric acid. However, NSAIDs produce characteristic side effects, especially in patients with preexisting renal insufficiency, peptic ulceration, and gastric problems (Taylor et al. 2001;Wardi & Shirin 2003). Allopurinol is the most common clinically used xanthine oxidase inhibitor prescribed for the treatment of gout. However its use is sometimes limited by hypersensitivity problems , and even fatal liver necrosis (Pereira et al. 1998).To circumvent such problems of allopurinol and to look for possible alternatives, attempts have been made to identify compounds with xanthine oxidase inhibitory activities Author for correspondence: Ling Dong Kong, School of Life Sciences, Nanjing University, Nanjing 210093, China (fax π86 25 83594691, e-mail kongld/nju.edu.cn). from natural sources (Kong et al. 2000;Zhou et al. 2001). In the present study we report that administration of procyanidins from grape seeds to oxonate-pretreated hyperuricaemic mice results in the reducti...

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Secretin is a 27-amino acid long peptide hormone that regulates pancreatic water, bicarbonate, enzymes, and potassium ion secretion. The human secretin receptor (hSR) is a glycoprotein consisting of 440 amino acids, of which there are 5 putative N-linked glycosylation sites at positions Asn72, Asn100, Asn106, Asn128 (N-terminal ectodomain), and Asn291 (second exoloop). Through functional analysis of the hSR-transfected cells cultured in the presence of various glycosylation inhibitors, it was found that tunicamycin and castanospermine were able to significantly reduce the secretin-stimulated cAMP response. On the other hand, the effects of other inhibitors, swainsonine and deoxymannojirimycin, were much lower, suggesting that the high mannose-type carbohydrate side-chain is essential to the expression of a fully functional hSR. The role of individual N-linked glycosylation sites was studied by mutation analysis (Asn to Leu or Ser to Ala) coupled to measurements of cAMP accumulation and extracellular acidification rate. The ED50 values of the wild-type receptor in these two assay systems were 0.25 and 0.11 nM, respectively, and mutation at position 100, 106, or 291 did not affect either the ED50 values or the maximal responses in the two assays. However, the Asn72Leu and Ser74Ala mutations reduced the maximal responses and increased the ED50 values in both assays, suggesting that this site is a true glycosylation signal. This hypothesis was further supported by competitive binding studies, the same mutants were found to be defective in binding with [125I]secretin. To evaluate whether the change in receptor function of the mutants is caused by the change in the process of presenting the receptor to the cell surface, the mutants and the wild-type receptor were tagged with a c-Myc epitope at the C-termini. Using an anti-c-Myc monoclonal antibody and confocal microscopy, all of the mutant receptors were found to be expressed and delivered to the plasma membrane.