Acid-Catalyzed Photosubstitution of 5-Fluoro-1,3-Dimethyluracil with Substituted Benzenes

Abstract

The substantial photosubstitution of 5-fluoro-1,3-dimethyluracil with substituted benzenes is first achieved by the addition of trifluoroacetic acid to the reaction mixture. The presence of protons is essential for the formation of 5-aryl-1,3-dimethyluracils.     

TGF-β1 Downregulates COX-2 Expression Leading to Decrease of PGE2 Production in Human Lung Cancer A549 Cells,Which Is Involved in Fibrotic Response to TGF-β1

Transforming growth factor-ß1 (TGF-β1) is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL) induced downregulation of cyclooxygenase-2 (COX-2), leading to reduced synthesis of prostaglandin E2 (PGE2), in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT), a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components). Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells.     

Unfolding of CBP21, a lytic polysaccharide monooxygenase,without dissociation of its copper ion cofactor

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu²⁺ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, t_p, for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of t_p in the presence of excess Cu²⁺ was around 75°C, indicating that Cu²⁺ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu²⁺ ⇌ U∙Cu²⁺, where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol⁻¹ in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu²⁺ complex itself.     

Anti-barnacle activity of novel simple alkyl isocyanides derived from citronellol

Twenty novel simple alkyl isocyanides derived from citronellol were synthesized and evaluated for their antifouling activity and toxicity against cypris larvae of the barnacle, Balanus amphitrite. The anti-barnacle activity of the synthesized isocyanides was in the EC₅₀ range of 0.08–1.49 μg ml^?1. Simple isocyanides containing a benzoate and chloro group showed the most potent anti-barnacle activity. In addition, none of the synthesized compounds showed significant toxicity and LC₅₀ values were <10 μg ml^?1. The LC₅₀/EC₅₀ ratios of almost all of the synthesized compounds were >10². The results indicate that these simple isocyanides are promising low-toxicity antifouling agents.     

Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.     

JosD1, a Membrane-targeted Deubiquitinating Enzyme,Is Activated by Ubiquitination and Regulates Membrane Dynamics,Cell Motility,and Endocytosis

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.     

Synthesis and biological evaluation of pyrrolidine derivatives as novel and potent sodium channel blockers for the treatment of ischemic stroke

A novel series of pyrrolidine derivatives as Na⁺ channel blockers was synthesized and evaluated for their inhibitory effects on neuronal Na⁺ channels. Structure–activity relationship (SAR) studies of a pyrrolidine analogue 2 led to the discovery of 5e as a potent Na⁺ channel blocker with a low inhibitory action against human ether-a-go-go-related gene (hERG) channels. Compound 5e showed remarkably neuroprotective activity in a rat transient middle cerebral artery occlusion (MCAO) model, suggesting that 5e would act as a neuroprotectant for ischemic stroke.