COLONY-FORMING ABILITY OF BONE MARROW CELLS OF W ANAEMIC MICE ON MACROPHAGE LAYER FORMED IN PERITONEAL CAVITY OF MICE

The colony-forming ability of haematopoietic cells of W anaemic mice was examined on the macrophage layer formed in the peritoneal cavity of mice. Bone marrow cells of W anaemic mice formed a considerable number of colonies on the macrophage layer, notwithstanding they did not form any colonies in the spleen of the same recipients. As the colony-forming ability of the bone marrow cells was not reduced by the incubation with ³H-thymidine, most of the cells which formed colonies on the macrophage layer seemed to stay in G₀ state. The interrelationship between the spleen colony-forming cells, the macrophage-layer colony-forming cells, and in vitro colony-forming cells was discussed.     

A simple method for observation of phagocytosis on bacterial thin-layer

A simple method was developed to visually present the phagocytic activity of leukocytes by using adherent Staphylococcus aureus cells and blood applied on a plastic dish. When heparinized blood was applied on thin-layer of heat-killed S. aureus cells on the plastic dish, plaques due to the phagocytic activity of leukocytes were observed with a microscope under a low magnification. Fewer and smaller plaques were observed when plasma-deprived rather than whole blood was used. Some analyses were made in respect to the fundamental conditions required for optimal results. This method was considered to be useful for conveniently evaluating the serum opsonin activities and phagocytic function of leukocytes in various kinds of diseases.     

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.     

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.     

Synthesis of Isoprenyl Chalcone “Sophoradin”

Nicotine has been found an effective photosensitizer for DDT. At DDT: nicotine (1:5), DDT along with its formed degradation products DDD, DDE and DBP disappeared within 18 and 60 days under UV and sunlight respectively. Because of persistence, nicotine proved a superior photosensitizer to N,N′-diethylaniline. In DDT emulsifiable concentrates it led to high alkalinity but no DDT degradation up to 60 days at 20~25°C. 0.1 and 0.5% DDT emulsions from these formulations showed no adverse effect on Daucus carrota, Vicia faba, Brassica oleracea var. botrytis, and Dahlia sp., but showed mild to severe phytotoxicity against Pisum sativum and Cicer arietinum; caused by high concentration of nicotine. On Clerodendrum sp. in sunlight, these formulations showed over 20% faster DDT loss between 3~15 days of application. DDT-nicotine mixtures showed no synergism against Tribolium castaneum Herbst.