Regulation of inducible nitric oxide synthase

One of the major functions of macrophages is to provide the body with an immediate innate defence against pathogenic micro-organisms. This defence is largely dependent on the generation of nitric oxide (NO) and superoxide by macrophages which leads to the killing of these pathogens. NO is also important in many other biological functions. It is derived from L-arginine and molecular oxygen by the enzyme NO synthase (NOS). There are a number of classes of NOS including neuronal NOS (nNOS), endothelial NOS (eNOS) and cytokine-inducible NOS (iNOS). iNOS is upregulated and activated by several immunological stimuli including interferon gamma (IFN-gamma; lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha), such activation leads to the production of large quantities of NO which can be cytotoxic. The potential toxicity of NO makes it important to understand the regulation of its production. In the study presented here, J774 cells, a murine macrophage cell line, were used as a model system for studying the induction and regulation of iNOS activation. These cells and murine peritoneal macrophages produce large amounts of NO in response to the T cell-derived lymphokine, IFN- and/or the potent macrophage activator, LPS in a dose-dependent manner. Northern and Western blotting revealed the process of induction of NO synthesis in J774 cells: The maximal induction of NO synthase mRNA was at 4 h while the maximum levels of NOS protein was observed at 8 to 12 h after treatment with IFN-gamma and LPS. IFN-? LPS-induced NO2- accumulation was abolished in the culture supernatants of samples that were pre-treated with cycloheximide. These suggest that iNOS is regulated transcriptionally in a manner that requires de novo protein synthesis. Protein phosphorylation plays a crucial role in regulating the signal transduction cascades leading to many biological responses in eukaryotes. Signals that are reversibly controlled by protein phosphorylation are modulated not only by a protein kinase but also by a protein phosphatase. In this project, I have shown that the induction of iNOS activity in J774 cells by IFN-gamma and LPS was reduced by more than 50% if the cells were pretreated with protein tyrosine kinase (PTK) inhibitors such as Tyrphostin 25, Tyrphostin AG126, and Herbimycin A. In contrast, iNOS was unaffected by pre-incubation with Tyrphostin 1, an inert analogue of these PTK inhibitors. Consistent with these findings, IFN-gamma and LPS-induced iNOS activity was enhanced by 30% in the presence of vanadate, a protein tyrosine phosphatase inhibitor. These results suggested that the activation of tyrosine kinase(s) plays a role in induction of NO synthesis. (Abstract shortened by ProQuest.)

Identification of metabolites of gardenin A in rat liver microsomes using ultra-high performance liquid chromatography coupled with linear ion-trap Orbitrap mass spectrometry

Purpose: To identify the metabolites of gardenin A (GA) in rat liver microsomes (RLMs) using ultra-high performance liquid chromatography coupled with linear ion-trap Orbitrap mass spectrometry (UHPLCLTQ- Orbitrap).Methods: The sample was prepared by incubating GA (100 μg/mL) with RLMs (0.5 mg/mL) for 8 h. Then 5 μL of the sample was injected into UHPLC-LTQ- orbitrap mass spectrometer. The metabolites of GA were tentatively identified based on accurate mass measurements, fragmentation patterns, chromatographic retention times, and bibliography data.Results: A total of 12 metabolites were detected and identified. Based on their  structures, the main reactions in the metabolism of GA are de-methoxylation and de-methylation.Conclusion: This is the first report on in vitro metabolites of GA. These results are considered very helpful for better comprehension of the metabolism of GA and its pharmacological effects.Keywords: Gardenin A, Metabolites, UHPLC-LTQ-Orbitrap, Rat Liver microsome

Role of Bacterial Lipopolysaccharide in Enhancing Host Immune Response to Candida albicans

Human infections involving yeast of the genus Candida often occur in the presence of bacteria, and, as such, it is important to understand how these bacteria influence innate host immunity towards Candida. Dectin-1 is a cell receptor of macrophages for Candida albicans recognition. The aim of this study was to examine dectin-1 expression by monocytes after stimulation with bacterial lipopolysaccharide (LPS), followed by heat-killed C. albicans (HKC). Freshly isolated human peripheral blood monocytes (PBMCs) and human monocytes cell line (THP-1) cells expressed low levels of dectin-1. Stimulation with LPS and GM-CSF/IL-4 was found to increase dectin-1 expression in both CD14(+) human PBMC and THP-1 cells. Enhanced dectin-1 expression resulted in increased phagocytosis of Candida. When THP-1 cells were challenged only with HKC, detectable levels of IL-23 were not evident. However, challenge by LPS followed by varying concentrations of HKC resulted in increased IL-23 expression by THP-1 cells in HKC dose-dependent manner. Increased expression of IL-17 by PBMC also occurred after stimulation with Candida and LPS. In conclusion, bacterial LPS induces an enhanced immune response to Candida by immune cells, and this occurs through increasing dectin-1 expression

Pseudospin symmetry and its approximation in real nuclei

The origin of pseudospin symmetry and its broken in real nuclei are discussed in the relativistic mean field theory. In the exact pseudospin symmetry, even the usual intruder orbits have degenerate partners. In real nuclei, pseudospin symmetry is approximate, and the partners of the usual intruder orbits will disappear. The difference is mainly due to the pseudo spin-orbit potential and the transition between them is discussed in details. The contribution of pseudospin-orbit potential for intruder orbits is quite large, compared with that for pseudospin doublets. The disappearance of the pseudospin partner for the intruder orbit can be understood from the properties of its wave function.Comment: 10 pages, 3 figure

Self-controlled growth of highly uniform Ge/Si hut wires for scalable qubit devices

Semiconductor nanowires have been playing a crucial role in the development of nanoscale devices for the realization of spin qubits, Majorana fermions, single photon emitters, nanoprocessors, etc. The monolithic growth of site-controlled nanowires is a prerequisite towards the next generation of devices that will require addressability and scalability. Here, combining top-down nanofabrication and bottom-up self-assembly, we report on the growth of Ge wires on pre-patterned Si (001) substrates with controllable position, distance, length and structure. This is achieved by a novel growth process which uses a SiGe strain-relaxation template and can be generalized to other material combinations. Transport measurements show an electrically tunable spin-orbit coupling, with a spin-orbit length similar to that of III-V materials. Also, capacitive coupling between closely spaced wires is observed, which underlines their potential as a host for implementing two qubit gates. The reported results open a path towards scalable qubit devices with Si compatibility

WNT-3A regulates an Axin1/NRF2 complex that regulates antioxidant metabolism in hepatocytes

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. Results: WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. Innovation: NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. Conclusion: These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism