In vivo role of Arabidopsis arginase in arginine metabolism and abiotic stress response

Nitric oxide (NO) and polyamines play essential roles in many developmental processes and abiotic stress responses in plants. NO and polyamines are metabolized from arginine through NO synthase (NOS) and arginine decarboxylase (ADC), respectively. Function of arginase, another important enzyme involved in arginine metabolism, in abiotic stress remains largely unknown. In the recent study, we have dissected the impact of arginase on arginine metabolism and abiotic stress responses through manipulating AtARGAHs expression. The results suggested that manipulation of arginase expression modulated accumulation of arginine and direct downstream products of arginine catabolism. AtARGAHs knockout lines exhibited increased accumulation of polyamines and NO and enhanced abiotic stress tolerance, while AtARGAHs overexpressing lines displayed the opposite results. Notably, we highlighted that Arabidopsis arginase plays distinctive and dual roles in the crosstalk between polyamines and NO signaling during abiotic stress responses, mediating both arginine metabolism and reactive oxygen species (ROS) accumulation. It is likely that accumulation of both NO and polyamines might activate abiotic stress responses in the plant.     

一氧化氮在炎性疼痛中的作用

一氧化氮（nitric oxide,NO）是细胞内重要的信使分子和神经递质,它参与多种生命活动,包括炎性疼痛.NO对炎性疼痛的发展和维持起到了重要的作用.研究NO在疼痛中所起到的作用及其机制有利于阐明痛觉生理和发现疼痛治疗的新手段.目前研究表明,脊髓水平NO参与炎性疼痛调制的可能机制主要有NO/cGMP途径、参与调控即刻早期基因、与其他神经递质的协同作用.另外研究表明,3种类型的一氧化氮合酶（nitric oxide synthases,NOS）在炎性疼痛过程中被激活或者有不同程度的增强表达.     

Studies on the inactivation of superoxide dismutase activity by nitric oxide from rat peritoneal macrophages

Rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) and Phorbol myristate acetate (PMA) generated increased levels of superoxide anions (O2ú-) by 122% as compared to those stimulated with PMA alone. However, Nitric oxide (NO) synthase inhibitors-n-monomethyl arginine (nMMA) or spermine-HCI lowered the enhanced levels of O2ú- released by LPS treated macrophages. The Superoxide dismutase (SOD) activity in LPS treated macrophages was 51% lower than that observed in resident cells. NO synthase inhibitors prevented the loss of SOD activity in LPS treated cells. Exogenously added SOD during sensitization of cells with LPS also inactivated the enzyme. This inactivation of SOD is inhibited by Nitric oxide synthase inhibitors. PMA alone did not affect SOD activity. NO synthase inhibitors also did not affect PMA activated superoxide anion generation in macrophages. These studies indicate that nitric oxide generated by LPS treated macrophages can inactivate SOD activity.     

Pattern of macrophage activation in yersinia-resistant and yersinia-susceptible strains of mice

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFbeta-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-gamma and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-beta1 and IL-4 production by BALB/c mice and to an increase in the IFN-gamma levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.     

Molecular and in silico evidences explain the anti-inflammatory effect of Trachyspermum ammi essential oil in lipopolysaccharide induced macrophages

This study presents the anti-inflammatory potential of Trachyspermum ammi essential oil (TAEO) against Escherichia coli lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. TAEO displayed the anti-inflammatory activity by reducing nitric oxide production and impact on the expression of nitric oxide synthases (iNOS), cyclooxygenase-2 (COX-2), and heme oxygenase-1 (HO-1). Besides, TAEO effectively inhibited the COX-2 enzyme activity with IC₅₀ value at 4.49 μg/mL. Furthermore, the molecular docking and simulation studies suggest a strong interaction between COX-2 and the important TAEO components thymol, (-5.88 kcal/mol) and carvacrol (-6.30 kcal/mol). The thymol and carvacrol docked complexes are stabilized by hydrogen bonds (at alanine 188 and tyrosine 371) and several hydrophobic interactions at phenylalanine 196, tyrosine 371, tryptophan 373, and leucine 376 and alanine 185, alanine 188, phenylalanine 196, tryptophan 373, leucine 376, and leucine 377, respectively. These results collectively suggest the anti-inflammatory role of TAEO.     

No effect of exposure to static and sinusoidal magnetic fields on nitric oxide production by macrophages

The effects of exposure to static (1–100 mT) or sinusoidal (1 Hz, 1.6 mT) magnetic fields on the production of nitric oxide (NO) by murine BCG-activated macrophages were investigated. In these cells, the inducible isoform of NO synthase is present. No significant differences were observed in nitrite levels among exposed, sham-exposed, or control macrophages after exposure for 14 h to static fields of 1, 10, 50, and 100 mT and to sinusoidal 1.6 mT, 1 Hz magnetic fields. © 1996 Wiley-Liss, Inc.     

Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei.

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not. These results suggested that O2- played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2- does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.     

Cytokine-induced production of NO by macrophages induces apoptosis and immunological rejection of AK-5 histiocytic tumor

The immunological rejection of the AK-5 histiocytoma in syngeneic hosts involves the participation of NK cells and the upregulation of Th1 type cytokine response. The tumor cells are killed by necrosis and apoptosis. We have studied the role of host peritoneal macrophages in tumor regression. Activated macrophages from tumor- bearing animals produce cytokines like IL-1, TNF-, IL-12 and free radicals like nitric oxide during tumor regression. IL-12 and IFN- played a crucial role in the induction of NO production by the host macrophages, since administration of anti IL-12 and anti IFN- antibodies in AK-5 tumor-bearing animals suppressed NO production by the macrophages. Similarly the cytotoxic activity of the host macrophages which is dependent on NO production was also affected in antibody injected animals. These studies indicate an important role for cytokines in the activation of host macrophages which in turn produce nitric oxide that is involved in the induction of apoptosis in AK-5 cells, leading to the regression of the tumor.     

Effects of cyclic AMP-elevating hormones and autacoids on LPS-activated rat peritoneal, bronchoalveolar and hepatic (Kupffer) macrophages

Peritoneal, bronchoalveolar and hepatic (Kupffer) macrophages activated in vitro by endotoxin, exhibit alterations in nitric oxide production when certain hormones or other biologically active agents (autacoids) are present in the culture medium. They also show changes in acid beta-glucuronidase activities and morphological changes concerning cell size and general appearance. Agents known to elevate the intracellular levels of cyclic AMP, e.g. adrenalin, prostaglandin E2 and dopamine, increase the nitric oxide production in all three types of macrophage. The addition of H-89, an inhibitor of protein kinase A, abolishes the increase in nitric oxide production. Adrenalin also increases the extracellular activity of beta-glucuronidase. The results of this work suggest that cyclic AMP-elevating hormones and autacoids affect the functions of endotoxin-activated macrophages, such as the production of nitric oxide and the activity of acid beta-glucuronidase.     

一氧化氮对心肌的抑制性保护作用

内皮型与神经型一氧化氮合酶(eNOS,nNOS)在心肌细胞内持续表达,而细胞应激可引起诱导型NOS(iNOS)表达.心肌细胞结构型eNOS与nNOS源性NO,在生理条件下对心肌主要发挥4方面的抑制作用:减缓心肌细胞搏动频率,轻度抑制心肌细胞收缩功能,加速心肌细胞舒张并增加顺应性,以及轻度抑制线粒体电子传递而增强氧利用效...     

Cryptococcus neoformans inhibits nitric oxide synthesis caused by CpG-oligodeoxynucleotide-stimulated macrophages in a fashion independent of capsular polysaccharides

Cryptococcus neoformans is eradicated by macrophages via production of NO. Unmethylated CpG-ODN protect mice from infection with this fungal pathogen by inducing IFN-gamma. The present study was designed to elucidate the effect of C. neoformans on the synthesis of NO by alveolar macrophages. For this purpose, MH-S, an alveolar macrophage cell line, was stimulated with CpG-ODN in the presence of IFN-gamma. A highly virulent strain of C. neoformans with thick capsule suppressed the production of NO. Capsular polysaccharides were not essential for this suppression, because there was no difference between acapsular mutant (Cap67) and its parent strain. Physical or close interaction of Cap67 with MH-S was necessary, as shown by the loss of such effect when direct contact was interfered by nitrocellulose membrane. Similar effects were observed by disrupted as well as intact Cap67. Whereas the inhibitory effect of intact Cap67 was completely abrogated by heat treatment, disrupted Cap67 did not receive such influence. Finally, disrupted Cap67 did not show any inhibitory effect on the TLR9-mediated activation of NF-kappaB in a luciferase reporter assay with HEK293T cells, although the TLR4-mediated activation was suppressed. These results revealed that C. neoformans suppressed the synthesis of NO by CpG-ODN and IFN-gamma-stimulated macrophages in a fashion independent of capsular polysaccharides, although the precise mechanism remains to be elucidated.     

Nitric Oxide Cycle in Mammals and the Cyclicity Principle

This paper continues a series of reports considering nitric oxide (NO) and its cyclic conversions in mammals. Numerous facts are summarized with the goal of developing a general concept that would allow the statement of the multiple effects of NO on various systems of living organisms in the form of a short and comprehensive law. The current state of biological aspects of NO research is analyzed in term of elucidation of possible role of these studies in the system of biological sciences. The general concept is based on a notion on cyclic conversions of NO and its metabolites. NO cycles in living organisms and nitrogen turnover in the biosphere and also the Bethe nitrogen–carbon cycle in star matter are considered. A hypothesis that the cyclic organization of processes in living organisms and the biosphere reflects the evolution of life is proposed: the development of physiological functions and metabolism are suggested to be closely related to space and evolution of the Earth as a planet of the Solar System.     

The inhibitory effect of various indolyl amino acid derivatives on arginase activity in macrophages

Summary. Numerous indolyl amino acids and their derivatives inhibited arginase activity. The inhibition was found to be non-competitive, – at least partly – allosteric, and independent on manganese ions in the active site, and it cannot be explained by the dissociation of arginase homotrimers. Indole alone is weakly inhibitory; however, the presence of three-carbon side chains and their net charges is favorable for the inhibition. The binding of the inhibitory compounds caused only minor changes in the steric structure of arginase: a slight increase in α-helix content was detected by circular dichroism together with a decrease in parallel pleated sheet and β-turn sections. A slight alteration in the tertiary structure was also found using tryptophane fluorescence studies, but buried apolar side chains were not transposed to the protein surface. Computer studies that were performed did not provide additional structural information. Authors’ address: András Hrabák, Department of Medical Chemistry, Molecular Biology and Patho-biochemistry, Semmelweis University Medical School, Budapest, VIII. Puskin u. 9., H-1444 POB 260, Hungary     

Itaconate and itaconate derivatives target JAK1 to suppress alternative activation of macrophages

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The production of nitric oxide and tumor necrosis factor by murine macrophages infected with mycobacterial strains differing by hemolytic activity.

In this study, we compared the secretion of nitric oxide (NO) and tumor necrosis factor (TNF-alpha) by murine macrophages infected in vitro with hemolytic or unhemolytic mycobacteria isolates. We observed that unhemolytic mycobacteria induced more intensive NO production by macrophages and were more susceptible to bactericidal effect of mononuclear phagocytes than hemolytic mycobacterial strains. In contrast, the high-virulence hemolytic isolates induced significantly stronger TNF-alpha production by infected macrophages than the low-virulence unhemolytic bacilli.     

Induction of inducible nitric oxide (NO) synthase mRNA and NO production in macrophages infected with influenza A/PR/8 virus and stimulated with its ether-split product

We investigated the inductive activity of infective influenza A/PR/8/34 (PR8) virus and its ether-split product (ESP) on the expression of inducible nitric oxide (NO) synthase (iNOS) and NO production in RAW264.7 (RAW) cells, a murine macrophage (M psi) cell line, and thioglycolate-elicited peritoneal M psi (TPM). In both cells, PR8 virus infection induced iNOS mRNA between 4 hr and 24 hr, attaining a peak value at 12 hr. In correlation with induction of iNOS mRNA, NO amounts increased significantly from 12 to 24 hr. Moreover, this study demonstrated that ESP with the same hemagglutination titer as PR8 virus could induce iNOS mRNA and NO production, although the inductive activity of ESP was weaker than that of PR8 virus. Considering the dual role (beneficial and detrimental roles) of NO on certain inflammatory disorders and virus infections, the inductive activity of influenza virus on the iNOS-mediated NO production independent of its infectivity might contribute to a modification of influenza virus infection.     

Oxidized phospatidylcholine but not native phosphatidylcholine inhibits inducible nitric oxide synthase in RAW 264.7 macrophages

This study was designed to compare the effects of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and native PAPC on the inducible nitric oxide synthase (iNOS) in the macrophage cell line RAW 264.7. Macrophages stimulated by bacterial lipopolysaccharide (1 microg/ml) were incubated with increasing amounts of native or oxidized PAPC (oxPAPC, 10-20 microg/ml). Cells incubated with oxPAPC showed a dose-dependent inhibition of inducible nitric oxide synthesis, as well as reduced iNOS protein expression and mRNA levels. Additionally, chromatin immunoprecipitation assay revealed that oxPAPC reduced the interaction of the active NF-kappaB subunit p65 with the iNOS promoter region when compared to native PAPC.     

Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages

Immunosenescence is an age-associated dysregulation of the immune function, which contributes to increased susceptibility to disease in the elderly. Alveolar macrophages (AM) are known phagocytes that generate reactive oxygen species (ROS) and nitric oxide (NO), essential mediators for host defence. We studied phagocytosis, ROS and NO production in AM obtained from young, adult and senescent rats (1-2, 9-12 and 18-24 months old, respectively) after exposure to lipopolysaccharide (LPS, 0.1-10 microg mL(-1)), 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 microg mL(-1)) or LPS + TPA in culture. Phagocytosis was significantly lower in control AM from adult rats than in AM from young animals. Nevertheless, AM from adult animals pretreated with LPS exhibited higher phagocytic capacity than AM from younger animals. ROS was identified by the NBT test at single cell level and quantified by automated image analysis. When TPA was added to all three populations, AM from adult and senescent animals responded more than AM from young animals. All LPS-stimulated AM produce more NO than controls. However, NO production increased three-, four- and two-fold in young, adult and senescent animals, respectively. Our results demonstrate that AM from young, adult and senescent animals display differential responsiveness to inflammatory mediators. Therefore, aging processes markedly affect AM metabolic functions and may further compromise the lung immune defence response, increasing adverse long-term health effects.     

Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells

Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells.