Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages

Nitric oxide (NO) reacts with superoxide to produce peroxynitrite, a potent oxidant and reportedly exerts cytotoxic action. Herein we validated the hypothesis that interaction of NO with superoxide exerts protection against superoxide toxicity using macrophages from mice with a knockout (KO) of inducible NO synthase (NOS2) and superoxide dismutase 1 (SOD1), either individually or both. While no difference was observed in viability between wild-type (WT) and NOS2KO macrophages, SOD1KO and SOD1-and NOS2-double knockout (DKO) macrophages were clearly vulnerable and cell death was observed within four days. A lipopolysaccharide (LPS) treatment induced the formation of NOS2, which resulted in NO production in WT and these levels were even higher in SOD1KO macrophages. The viability of the DKO macrophages but not SOD1KO macrophages were decreased by the LPS treatment. Supplementation of NOC18, a NO donor, improved the viability of SOD1KO and DKO macrophages both with and without the LPS treatment. The NOS2 inhibitor nitro-l-arginine methyl ester consistently decreased the viability of LPS-treated SOD1KO macrophages but not WT macrophages. Thus, in spite of the consequent production of peroxynitrite in LPS-stimulated macrophages, the coordinated elevation of NO appears to exert anti-oxidative affects by coping with superoxide cytotoxicity upon conditions of inflammatory stimuli.     

Stimulation of the brain NO/cyclic GMP pathway by peripheral administration of tetrahydrobiopterin in the hph-1 mouse

Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH4), a cofactor necessary for the synthesis of catecholamines and serotonin. Current therapy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neurological deficits. The reason might be that BH4 is also a cofactor for nitric oxide synthase. Using a strain of mutant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH4 deficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH4 (100 micromol/kg s.c.) in hph-1 mice significantly elevated the brain BH4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.     

Bcl-2/Beclin-1复合体在自噬中的调节作用

自噬(autophagy)是一种进化保守的溶酶体依赖性分解代谢途径,是细胞维持自稳态的重要机制之一,并参与多种疾病的发生. Beclin-1作为自噬体成核的关键分子之一,是1个调节自噬的关键靶点. Beclin-1有1个BH3结构域,Bcl-2、Bcl-XL等可以通过这个BH3结构域与Beclin-1结合而影响其活性. 抗凋亡Bcl-2家族蛋白和Beclin-1的表达水平、磷酸化、分子的亚细胞定位以及BH3-only蛋白等,均可调节Beclin-1蛋白和Bcl-2家族蛋白结合水平,进而调控自噬的发生,并可能对细胞最终走向自噬还是凋亡起着关键作用.     

BH(4) (tetrahydrobiopterin)-dependent activation, but not the expression, of inducible NOS (nitric oxide synthase)-2 in proinflammatory cytokine-stimulated, cultured normal human astrocytes is mediated by MEK-ERK kinases

Nitric oxide (NO) from astrocytes is one of the signalers used by the brain's extensive glial-neuronal-vascular network, but its excessive production by pro-inflammatory cytokine-stimulated glial cells can be cytodestructive. Here, we show how three pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma) together stimulated the activation, but not the prior expression, of NOS-2 protein via a mechanism involving MEK-ERKs protein kinases in astrocytes from adult human cerebral temporal cortex. The cytokines triggered a transient burst of p38 MAPK activity and the production of NOS-2 mRNA which were followed by bursts of MEK-ERK activities, synthesis of the NOS-2 co-factor tetrahydrobiopterin (BH(4)), a build-up of NOS-2 protein and from it active NOS-2 enzyme. Selectively inhibiting MEK1/MEK2, but not the earlier burst of p38 MAPK activity, with a brief exposure to U0126 between 24 and 24.5 h after adding the cytokine triad affected neither NOS-2 expression nor NOS-2 protein accumulation but stopped BH(4) synthesis and the assembly of the NOS-2 protein into active NOS-2 enzyme. The complete blockage of active NOS-2 production by the brief exposure to U0126 was bypassed by simply adding BH(4) to the culture medium. Therefore, this cytokine triad triggered two completely separable, tandem operating mechanisms in normal human astrocytes, the first being NOS-2 gene expression and accumulation of NOS-2 protein and the second being the synthesis of the BH(4) factor needed to dimerize the NOS-2 protein into active, NO-making NOS-2 enzyme.     

PAF is involved in the Mycoplasma arthritidis superantigen-triggering pathway for iNOS and COX-2 expression in murine peritoneal cells

We investigated the capacity of Mycoplasma arthritidis mitogen (MAM) to induce (a) expression of the inducible enzymes cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS), (b) production of prostaglandin E2 (PGE2) and nitric oxide (NO), and (c) involvement of platelet-activating factor (PAF) in the MAM-induced activation pathway. Resident peritoneal cells from C3H/HePas mice were incubated with MAM in the presence or absence of a PAF-antagonist (WEB2170) or COX-2 inhibitors (nimesulide or NS398). Enzyme expression was evaluated by immunoblotting, PGE2 by EIA, and NO by Griess reaction. Following MAM-stimulation of peritoneal cells, expression of COX-2 was detected at 3 h (peak levels at 12 h) and of iNOS at 6 h (peak levels at 20 h). PGE2 increased till 20 h, decreasing thereafter, whereas NO increased with time. WEB2170 (5 x 10(-5) M) treatment caused 44% inhibition of NO output and reduced iNOS expression (48% at the peak of expression). Concomitant treatment with WEB2170 and nimesulide (10(-5) M) reversed these inhibitory effects. WEB2170 reduced COX-2 expression (43% at the peak of expression) and prevented the decline in PGE2 levels after 20 h. These results suggest the involvement of PAF in the signaling pathway triggered by MAM that leads to expression of iNOS and COX-2, and show that PAF regulates the production of NO, possibly by controlling levels of PGE2.     

Regulation of SOBIR1 accumulation and activation of defense responses in bir1–1 by specific components of ER quality control

Receptor‐like kinases play diverse roles in plant biology. Arabidopsis BAK1‐INTERACTING RECEPTOR‐LIKE KINASE 1 (BIR1) functions as a negative regulator of plant immunity. bir1‐1 mutant plants display spontaneous cell death and constitutive defense responses that are dependent on SUPPRESSOR OF BIR1,1 (SOBIR1) and PHYTOALEXIN DEFICIENT4 (PAD4). Here we report that mutations in three components of ER quality control, CALRETICULIN3 (CRT3), ER‐LOCALIZED DnaJ‐LIKE PROTEIN 3b (ERdj3b) and STROMAL‐DERIVED FACTOR‐2 (SDF2), also suppress the spontaneous cell death and constitutive defense responses in bir1‐1. Further analysis revealed that accumulation of the SOBIR1 protein is reduced in crt3‐1 and erdj3b‐1 mutant plants. These data suggest that ER quality control plays important roles in the biogenesis of SOBIR1, and is required for cell death and defense responses in bir1‐1.     

ESR detection of 1O2 reveals enhanced redox activity in illuminated cell cultures

Low-energy visible light (LEVL) has previously been found to modulate various processes in different biological systems. One explanation for the stimulatory effect of LEVL is light-induced reactive oxygen species formation. In the present study, both sperm and skin cells were illuminated with LEVL and were found to generate singlet oxygen (¹O₂). The detection of ¹O₂ was performed using a trapping probe, 2,2,6,6-tetramethyl-4-piperidone, coupled with electron paramagnetic resonance spectroscopy. In addition, we have shown that, together with ¹O₂ generation, LEVL illumination increases the reductive capacity of the cells, which explains the difficulties encountered in ¹O₂ detection. The potential of visible light to change the cellular redox state may explain the recently observed biostimulative effects exerted by LEVL.     

Disruption of vascular endothelial homeostasis in systemic juvenile idiopathic arthritis-associated macrophage activation syndrome: The dynamic roles of angiopoietin-1 and -2

To assess the role of angiopoietin (Ang)-1 and Ang-2 and to investigate the clinical significance of serum levels of them in systemic juvenile idiopathic arthritis (s-JIA)-associated macrophage activation syndrome (MAS), we determined these levels in 51 patients with s-JIA, 11 patients with polyarticular JIA (poly-JIA), 12 patients with virus associated hemophagocytic syndrome (VAHS), 12 patients with Kawasaki disease (KD), and 15 age-matched healthy controls (HC). The results were compared with clinical features of MAS. During the MAS phase, serum Ang-1 levels were significantly decreased compared with those during the active and inactive phases. Serum Ang-2/1 ratio were significantly elevated during the MAS phase, compared with those during the active and inactive phases. There was a rapid increase in the Ang-2/1 ratio at the onset of MAS. Serum Ang-1 and the Ang-2/1 ratio significantly correlated with measures of disease activity, including AST and LDH. Ang-2/1 dysregulation was also observed in patients with VAHS, whereas not observed in most cases of KD. The homeostasis of vascular endothelial function by Ang-1 and Ang-2 is disrupted in MAS. Serum Ang-1 levels and the Ang-2/1 ratio might represent promising indicators of disease activity for MAS.     

Regulation of EGF-induced phospholipase C-gamma1 translocation and activation by its SH2 and PH domains

Translocation of phospholipase C-γ1 is essential for its function in response to growth factors. However, in spite of recent progress, the phospholipase C-γ1 translocation pattern and the molecular mechanism of the translocation are far from fully understood. Contradictory results were reported as to which domain, PH or SH2, controls the epidermal growth factor-induced translocation of phospholipase C-γ1. In this communication, we studied epidermal growth factor-induced translocation of phospholipase C-γ1 by using comprehensive approaches including biochemistry, indirect fluorescence and live fluorescence imaging. We provided original evidence demonstrating that: (i) endogenous phospholipase C-γ1, similar to YFP-tagged phospholipase C-γ1, translocated to endosomes following its initial translocation from cytosol to the plasma membrane in response to epidermal growth factor; (ii) phospholipase C-γ1 remained phosphorylated in endosomes, but phospholipase C-γ1 activity is not required for its translocation, which suggests a signaling role for phospholipase C-γ1 in endosomes; (iii) the PH domain was not required for the initial translocation of phospholipase C-γ1 from cytosol to the plasma membrane, but it stabilizes phospholipase C-γ1 in the membrane at a later time; (iv) the function of the phospholipase C-γ1 PH domain in stabilizing phospholipase C-γ1 membrane association is very important in maintaining the activity of phospholipase C-γ1; and (v) the role of the PH domain in phospholipase C-γ1 membrane association and activation is dependent on PI3K activity. We conclude that the phospholipase C-γ1 SH2 and PH domains coordinate to determine epidermal growth factor-induced translocation and activation of phospholipase C-γ1.     

Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affect cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins.     

Cardioprotection of H2S by downregulating iNOS and upregulating HO-1 expression in mice with CVB3-induced myocarditis

Aims

To explore the effects and potential mechanisms of hydrogen sulfide (H₂S) in CVB3-induced mice with myocarditis.

Main methods

A total of 75 six-week-old inbred male Balb/c mice were divided randomly into four groups (N, C, P and S). Group N was the negative control. The others were inoculated intraperitoneally (i.p.) with CVB3. Subsequently, groups P and S were injected i.p. once a day with DL-Proparglygylcine (PAG) and NaHS respectively. Group C was the positive control. Inducible nitric oxide synthase (iNOS) and heme oxygenase-1(HO-1) expression on cardiac tissues were evaluated by histopathological examination, immunohistochemistry, RT-PCR and Western blot.

Key findings

The heart-weight to body-weight (HW/BW) ratio, the histologic scores and the iNOS mRNA and protein expression levels were higher, and the HO-1 mRNA and protein expression levels were lower in mice treated with PAG than those mice solely inoculated with CVB3. Mice in group S had a significant decreased in the HW/BW ratio, the histologic scores and the iNOS mRNA and protein expression levels, and had a significant increased in the HO-1 mRNA and protein expression levels compared to the mice in group C. H₂S can attenuate inflammatory cell infiltration, alleviate cardiac edema, and limit myocardial lesions.

Significance

Our data support that H₂S can inhibit iNOS overexpression and induce HO-1 expression, both of which contribute to the cardioprotection of H₂S in CVB3-induced mice myocarditis.