Enhanced NO Production Research Articles

The arginine/ornithine binding protein ArgT plays an essential role in Brucella neotomae/Brucella melitensis to prevent intracellular killing and contribute to chronic persistence in the host

ABSTRACT Brucella species are facultative intracellular bacterial pathogens that cause the contagious zoonotic disease, brucellosis. Brucella spp. infect a wide range of animals, including livestock, wild animals, and marine mammals. Compared with other invasive bacterial pathogens, partial information is available on the virulence factors of Brucella that enable them to survive in the host. Here, we performed transposon-based random mutagenesis of B. neotomae and identified the arginine/ornithine binding protein, ArgT, as one of the crucial virulence determinants of Brucella. Deleting ArgT from B. neotomae or B. melitensis resulted in its attenuation in macrophages, which was restored upon complementation with an ArgT expression plasmid. We observed that macrophages infected with ΔArgT-B. neotomae produced elevated levels of NO due to the inability of these mutants to deplete the host intracellular arginine through their importer. Furthermore, defective survival of ΔArgT B. neotomae and B. melitensis was observed in the infected mice, which correlated with enhanced NO production in the mice. Our studies revealed that ArgT plays a vital role in preventing intracellular killing and contributes to the chronic persistence of B. neotomae/B. melitensis in the host. This study highlights the essential role of arginine in clearing intracellular infections and the subversion of this host defense mechanism by intracellular pathogens for their chronic persistence.

RABV antigenic peptide loaded polymeric nanoparticle production, characterization, and preliminary investigation of its biological activity

Nanoparticle-based antigen carrier systems have become a significant area of research with the advancement of nanotechnology. Biodegradable polymers have emerged as particularly promising carrier vehicles due to their ability to address the limitations of existing vaccine systems. In this study, we successfully encapsulated the G5-24 linear peptide, located between amino acids 253 and 275 in the primary sequence of the rabies virus G protein, into biodegradable and biocompatible PLGA copolymer using the double emulsion solvent evaporation method. The resulting nanoparticles had a size of approximately 230.9 ± 0.9074 nm, with a PDI value of 0.168 ± 0.017 and a zeta potential value of -9.86 ± 0.132 mV. SEM images confirmed that the synthesized nanoparticles were uniform in size and distribution. Additionally, FTIR spectra indicated successful peptide loading into the nanoparticles. The encapsulation efficiency of the peptide-loaded nanoparticles was 73.3%, with a peptide loading capacity of 48.2% and a reaction yield of 30.4%. Peptide release studies demonstrated that 65.55% of the peptide was released in a controlled manner over 28 d, following a 'biphasic burst release' profile consistent with the degradation profile of PLGA. This controlled release is particularly beneficial for vaccine studies. Cytotoxicity tests revealed that the R-NP formulation did not induce cytotoxicity in fibroblast cells and enhanced NO production in macrophages, indicating its potential for vaccine development.

Study of NO and CO Formation Pathways in Jet Flames with CH4/H2 Fuel Blends

The existing natural gas transportation pipelines can withstand a hydrogen content of 0 to 50%, but further research is still needed on the pathways of NO and CO production under moderate or intense low oxygen dilution (MILD) combustion within this range of hydrogen blending. In this paper, we present a computational fluid dynamics (CFD) simulation of hydrogen-doped jet flame combustion in a jet in a hot coflow (JHC) burner. We conducted an in-depth study of the mechanisms by which NO and CO are produced at different locations within hydrogen-doped flames. Additionally, we established a chemical reaction network (CRN) model specifically for the JHC burner and calculated the detailed influence of hydrogen content on the mechanisms of NO and CO formation. The findings indicate that an increase in hydrogen content leads to an expansion of the main NO production region and a contraction of the main NO consumption region within the jet flame. This phenomenon is accompanied by a decline in the sub-reaction rates associated with both the prompt route and NO-reburning pathway via CHi=0–3 radicals, alongside an increase in N2O and thermal NO production rates. Consequently, this results in an overall enhancement of NO production and a reduction in NO consumption. In the context of MILD combustion, CO production primarily arises from the reduction of CO2 through the reaction CH2(S) + CO2 ⇔ CO + CH2O, the introduction of hydrogen into the system exerts an inhibitory effect on this reduction reaction while simultaneously enhancing the CO oxidation reaction, OH + CO ⇔ H + CO2, this dual influence ultimately results in a reduction of CO production.

Azelnidipine protects HL-1 cardiomyocytes from hypoxia/reoxygenation injury by enhancement of NO production independently of effects on gene expression.

It remains to be elucidated whether Ca2+ antagonists induce pharmacological preconditioning to protect the heart against ischemia/reperfusion injury. The aim of this study was to determine whether and how pretreatment with a Ca2+ antagonist, azelnidipine, could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury in vitro. Using HL-1 cardiomyocytes, we studied effects of azelnidipine on NO synthase (NOS) expression, NO production, cell death and apoptosis during H/R. Action potential durations (APDs) were determined by the whole-cell patch-clamp technique. Azelnidipine enhanced endothelial NOS phosphorylation and NO production in HL-1 cells under normoxia, which was abolished by a heat shock protein 90 inhibitor, geldanamycin, and an antioxidant, N-acetylcysteine. Pretreatment with azelnidipine reduced cell death and shortened APDs during H/R. These effects of azelnidipine were diminished by a NOS inhibitor, L-NAME, but were influenced by neither a T-type Ca2+ channel inhibitor, NiCl2, nor a N-type Ca2+ channel inhibitor, ω-conotoxin. The azelnidipine-induced reduction in cell death was not significantly enhanced by either additional azelnidipine treatment during H/R or increasing extracellular Ca2+ concentrations. RNA sequence (RNA-seq) data indicated that azelnidipine-induced attenuation of cell death, which depended on enhanced NO production, did not involve any significant modifications of gene expression responsible for the NO/cGMP/PKG pathway. We conclude that pretreatment with azelnidipine protects HL-1 cardiomyocytes against H/R injury via NO-dependent APD shortening and L-type Ca2+ channel blockade independently of effects on gene expression.

Preparation, characterization and cytotoxicity assessment of a novel selenized polysaccharide from Morchella sextelata

Selenylation modification has been widely developed to improve the biological effects of natural polysaccharides. In this study, a purified new polysaccharide (MSP-4) was isolated from Morchella Sextelata, and selenized into SeMSP-4 using the HNO3-Na2SeO3 method. The selenium (Se) content of SeMSP-4 was 101.81 ± 9.90 mg/kg, and the molecular weight of SeMSP-4 was 1.23 × 105 Da. The FT-IR, XRD and AFM results showed that MSP-4 was successfully combined with the Se element. The structure characters of SeMSP-4 were analyzed by methylation analysis combined with 1D and 2D NMR spectroscopy. And, the radical scavenging test revealed that SeMSP-4 exhibited higher antioxidant capacities in vitro than MSP-4. The cytotoxicity analysis indicated that SeMSP-4 could dose-dependently inhibit the proliferation of HepG2 and HeLa cells, but did not show a cytotoxic effect on normal cells (HEK293). Furthermore, SeMSP-4 stimulation significantly increased the macrophage viability and enhanced NO production in macrophage cells. This study suggested that SeMSP-4 could be utilized as a potential selenium source with antioxidant, antitumor, and immunostimulatory activities.

Roles of radiation reabsorption on flame speed and NO emission during ammonia combustion with syngas blending at elevated pressures

The radiative properties of NH3 and CO have considerable effects on ammonia-syngas flames, which has not been investigated at present. Radiation reabsorption effects on flame speed and NO production of one-dimensional premixed ammonia/syngas/air laminar flames were studied, encompassing a wide syngas content range and elevated pressures up to 25 atm. The improvement of flame speeds by radiation reabsorption reached 25.9% and decreased with rising syngas content, controlled by the direct-radiation effect and the reaction NH2 + NO = N2H + OH (R287). The relative enhancement of NO production decreased from 19.7% to 1.1% with increasing syngas content, affected by N + OH = NO + H (R273). The radiation reabsorption increased the flame speed by 25% at 18 atm. The coupling impact of high pressure and radiation reabsorption on flame speed was dominated by NH + NO = N2O + H (R279) and R287 via NH and OH radicals, respectively.

Research on NO generation characteristics of ammonia-premixed flame

Ammonia is a promising fuel with high energy density, accessible storage, and no CO2 production by combustion, but its combustion produces the pollutant NO. In this study, a Bunsen burner experimental bench was selected to investigate the concentration of NO generated by ammonia combustion at different initial oxygen concentrations. Further, the reaction pathways of NO were analyzed in depth, and sensitivity analysis was performed. The results show that the Konnov mechanism has an excellent predictive effect on NO generated by ammonia combustion. In the ammonia-premixed laminar flame at atmospheric pressure, the NO concentration peaked at an equivalence ratio of 0.9. The high initial oxygen concentration enhanced the combustion of ammonia-premixed flame and increased the conversion of NH3 to NO. NO was not only a product but a contribution to the combustion of NH3. As the equivalence ratio increases, NH2 consumes a large amount of NO and reduces NO production. The high initial oxygen concentration enhanced NO production, and the effect was more pronounced at low equivalents. The study results provide theoretical guidance for the utilization of ammonia combustion and pollutant reduction and help to drive the process of ammonia combustion toward practicality.

Dilution effects analysis on NOx emissions of opposed-jet H2/CO syngas diffusion flames

Syngas is a promising alternative fuel for stationary power generation due to cleaner combustion than convectional fossil fuels. During the gasification processes, the by-products of CO2, H2O, or N2 may be present in the syngas mixture to control the flame temperature and emissions. Several studies indicated that syngas with dilutions is capable of reducing pollutant emissions such as NOx emissions. This work applied a numerical model of opposed-jet diffusion fames to explore the dilution effects on NOx formation and differentiate the inert effect, thermal/diffusion effect, chemical effect, and radiation effect from CO2, H2O, or N2 dilutions. The numerical study was performed by a revised OPPDIF program coupling with narrowband radiation model and detail chemical mechanism. The dilution effects on NOx formation were analyzed by comparing the realistic and hypothetical cases. Regardless the diluent types, the inert effect is the main cause to reduce NO production, followed by chemical effect and radiation effect. The thermal/diffusion effect may promote NO formation because the preferential diffusion due to different diffusivities between diluents and syngas magnifies the reaction rate locally. CO2 dilution reduces NO by radiation effect at low strain rate, and contributes NO reduction by chemical effect at high strain rate. At the same dilution percentage, CO2 dilution reduces NO production the most, followed by H2O and N2. Besides the thermal/diffusion effect, the chemical effect of H2O enhances NO production through thermal route and reburn route.

Enhanced nitric oxide production by macrophages treated with a cell-penetrating peptide conjugate

The SPRY domain-containing SOCS box protein-2 (SPSB2) plays a critical role in the degradation of inducible nitric oxide synthase (iNOS) in macrophages. In this study, we have conjugated a peptide inhibitor of the iNOS-SPSB2 interaction with a cell-penetrating peptide (CPP) for delivery into macrophages, and confirmed its binding to SPSB2. We have assessed the uptake of a fluorophore-tagged analogue by RAW 264.7 and immortalised bone marrow derived macrophage (iBMDM) cell lines, and shown that the CPP-peptide conjugate enhanced NO production. The findings of this study will be useful in further refinement of CPP-peptide conjugates as leads in the development of new antibiotics that target the host innate immune response.

The probiotic properties of different preparations using Lactococcus lactis Z-2 on intestinal tract, blood and hepatopancreas in Cyprinus carpio

Basing on the beneficial effects of live Lactococcus lactis Z-2 and its extracellular polysaccharides on the common carp (Cyprinus carpio L.) in the previous studies, the present study aims may be appropriate to investigate the functions of other forms of L. lactis Z-2 (namely, heat-killed or cellular components) on the intestinal tract, blood and hepatopancreas of common carp. 480 fish [(42.35 ± 0.76) g] were treated with different preparations of L. lactis Z-2 for 7 days and infected with Aeromonas hydrophila for 24 h, following which to detect the levels of NO and cytokines, the activities of lysozyme (LZM) and alkaline phosphatase (AKP) in the serum, the antioxidants (T-AOC, SOD, CAT, GSH, GSH-Px and MDA) in hepatopancreas, the digestive enzymes and the gene expression of nutrient transporters in intestinal. Prior to infection, different treatment groups significantly enhanced NO production, the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), AKP activity, antioxidant content (T-AOC, SOD, CAT, and GSH), and reduced MDA level compared to those in the negative control group (P < .05). In addition, the activities of LZM in the serum and GSH-Px in the hepatopancreas were significantly promoted in most groups (P < .05). Furthermore, different treatment groups significantly increased the activities of digestive enzymes (amylase, lipase and protease), and the genes expression of nutrient transporters (Sglt1, Glut2, Pept1, rBAT, except HK treatment group). While, the levels of NO and pro-inflammatory cytokines in the treatment groups were significantly lower than those in the positive control group after infection (P < .05), but remained relatively stable. Although the LZM and AKP activities decreased significantly upon A. hydrophila infection, the treatment groups still significantly enhanced the LZM and AKP activities (P < .05). In addition, the antioxidant capacity significantly increased in the treatment groups after infection. However, regardless of the infection status, the expression levels of anti-inflammatory cytokines (IL-10, TGF-β) increased significantly in the treatment groups (P < .05). Therefore, these results suggested that the different preparations of L. lactis Z-2 exerted immunomodulatory and antioxidant effects, disease resistance, and promoted nutrient metabolism in common carp; this can form the theoretical basis for the rational use of potential probiotics and provide new perspective in the use of lactic acid bacteria in microecological preparations.

Peculiarities of the Expression of Inducible NO Synthase in Rat Dentate Gyrus in Depression Modeling.

Mild stress exposure contributes to the development of cognitive and emotional deficits, is considered as a model of depressive state, and is characterized by enhanced NO production. In albino mature (12-month-old) male rats, the depressive state was simulated by daily 30-min exposure to stressful stimuli (vibration, loud sound, and strobe light) over 7 days in a special chamber. On paraffin frontal sections of the brain stained with antibodies against inducible NO synthase (iNOS), the expression and distribution pattern of immunoreactive material were evaluated in various layers of the dentate gyrus under normal conditions and after depression modeling. The relative area of iNOS expression in the dentate gyrus of control rats was 8.2 (7.1-9.9)%, while in rats with experimental depression, this parameter was 16.7 (10.5-22.1)%, i.e. increased by 8.5% (p<0.05). In mature rats with modeled depressive state, the expression and relative area of iNOS expression in neuronal perikarya in the granular and subgranular layers of the dentate gyrus increased, which can underlie the mechanisms of damage and determine reduced neuroplasticity and suppressed neurogenesis in the dentate gyrus in rats during adulthood.

Endothelial cell prostaglandin E2 receptor EP4 is essential for blood pressure homeostasis.

Prostaglandin E2 and its cognate EP1-4 receptors play important roles in blood pressure (BP) regulation. Herein, we show that endothelial cell-specific (EC-specific) EP4 gene-knockout mice (EC-EP4-/-) exhibited elevated, while EC-specific EP4-overexpression mice (EC-hEP4OE) displayed reduced, BP levels compared with the control mice under both basal and high-salt diet-fed conditions. The altered BP was completely abolished by treatment with l-NG-nitro-l-arginine methyl ester (l-NAME), a competitive inhibitor of endothelial nitric oxide synthase (eNOS). The mesenteric arteries of the EC-EP4-/- mice showed increased vasoconstrictive response to angiotensin II and reduced vasorelaxant response to acetylcholine, both of which were eliminated by l-NAME. Furthermore, EP4 activation significantly reduced BP levels in hypertensive rats. Mechanistically, EP4 deletion markedly decreased NO contents in blood vessels via reducing eNOS phosphorylation at Ser1177. EP4 enhanced NO production mainly through the AMPK pathway in cultured ECs. Collectively, our findings demonstrate that endothelial EP4 is essential for BP homeostasis.

Structural characterization and immunostimulatory activity of a glucan from Cyclina sinensis

Cyclina sinensis is an edible clam widely distributed along the coastal waters of Asia. In the present study, a polysaccharide (CSP-1) isolated from C. sinensis was purified by a DEAE-Sepharose Fast Flow column, and it had an average molecular weight of 3.8 × 105 Da and a prevalent component monosaccharide of Glc. The results of methylation analysis and 1D/2D NMR indicated that CSP-1 was a glycogen constructed with α-1,4-Glc and branched at C-6 every 9 Glc residues. In addition, Cong red test suggests CSP-1 was not a helical conformation, and irregular and spherical lumps were observed by AFM. Moreover, CSP-1 was found to possess potent immunostimulatory activity on the basis of its significant abilities to enhance NO production and cytokines (TNF-α, IL-1β and IL-6) secretion in RAW 264.7 macrophages.

Immunomodulatory and antioxidant effects of Astragalus polysaccharide liposome in large yellow croaker (Larimichthys crocea)

Astragalus polysaccharides (APS) have been widely used as immunopotentiators in aquaculture, however, the best way of their administration remains to be explored. In the present study, APS liposome (APSL) was prepared by film dispersion-ultrasonic method. The optimal conditions of APSL preparation were determined by response surface methodology, with a ratio of 10:1 (w/w) for soybean lecithin to APS and 8:1 (w/w) for soybean lecithin to cholesterol, and an ultrasound time of 15 min, which produced an encapsulation efficiency of 73.88 ± 0.88% of APSL. In vivo feeding experiments in large yellow croaker showed that both APS and APSL could enhance the contents of serum total protein (TP) and albumin (ALB), activities of serum non-specific immune enzymes such as acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM), and phagocytic activity of head kidney macrophages. Meanwhile, they both increased the activities of serum antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and reduced the content of final lipid peroxidation product malondialdehyde (MDA) in serum, thus exhibiting the antioxidant effects. In vitro experiments on primary head kidney macrophages (PKM) showed that both APS and APSL inhibited ROS production, but obviously enhanced NO production and phagocytic activity of PKM. Furthermore, expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α), IFN-γ, and iNOS in PKM were significantly up-regulated after APS and APSL treatments, but no expression change of IFN-h was observed. Taken together, our results showed that both APS and APSL could improve several immune parameters and antioxidant ability of large yellow croaker either in vivo or in vitro, and the efficacy of APSL was markedly better than APS. These findings therefore indicated that the immunomodulatory and antioxidant activities of APS could be enhanced after encapsulated with liposome, and APSL may represent a potential drug delivery system of APS for development of immunoenhancers in aquaculture.

Sigma‐1 receptor agonists elicit arteriolar dilation via Akt/eNOS activation, and also have endothelial barrier protective properties

Sigma‐1 receptors (σ1) have previously gained considerable attention pertaining to neuroprotection through its actions on calcium homeostasis, and the mitochondria‐associated ER membrane domain. Our laboratory recently showed that the σ1 agonist afobazole causes an endothelial NO‐dependent decrease in contraction amplitude. However, little is known about the mechanisms and functions of σ1 receptor in endothelial cells. In the current study, we investigated how σ1 agonists impact brain arteriolar tone and the Akt/eNOS pathway. Parenchymal arterioles were isolated from brains of Sprague‐Dawley rats and mounted onto glass micropipettes in a 37 °C bath containing albumin‐physiological salt solution. The nonselective σ agonist afobazole was applied to the bath at concentrations of 50, 100, 150, 200, and 250 μM in the presence or absence of the σ1 antagonist BD1047. The selective σ1 agonist, PRE‐084 was used to stimulate human umbilical vein endothelial cells (HUVEC), in which phosphorylation of endothelial NO synthase (eNOS) or Akt phosphorylation on their activation sites (S1177 and S473, respectively) were evaluated by western blotting. NO production was measured by fluorescence microscopy in cells loaded with the indicator dye DAF‐FM. Trans‐endothelial electrical resistance (TER) was used to determine the ability of PRE‐084 to attenuate disruption of endothelial barrier function caused by the the mitochondrial function inhibitors CCCP and antimycin‐A, or the inflammatory cytokine IL‐1β. The results show that afobazole significantly dilated rat brain arterioles, which was inhibited by the σ1 antagonist BD‐1047. Selective activation of σ1 with PRE‐084 significantly increased eNOS phosphorylation on S1177 and this was diminished in the presence of BD‐1047. Akt phosphorylation on S473 following PRE‐084 treatment was also enhanced. In addition, afobazole and PRE‐084 enhanced NO production in endothelial cells. PRE‐084 significantly attenuated decreases in TER caused by IL‐1β or CCCP, but not antimycin‐A, suggesting a potential barrier‐protective role for σ1 receptor, with some limitations that may be related to the source of mitochondrial dysfunction during inflammation. Collectively, our results suggest that σ1 receptor activation can cause vasodilation via the Akt/eNOS pathway in endothelial cells, and that σ1 may enhance endothelial barrier function under inflammatory conditions.Support or Funding InformationSupported by NIH grant R01GM120774.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of bradykinin B2 receptor and dopamine D2 receptor on the oxidative stress, inflammatory response, and apoptotic process in human endothelial cells

Endothelial dysfunction is a hallmark of a wide range of cardiovascular diseases and is often linked to oxidative stress and inflammation. Our earlier study reported the formation of a functional heterodimer between bradykinin receptor 2 (B2R) and dopamine receptor 2 (D2R) that may modulate cell responses, dependent on intracellular signaling. Here, for the first time, we showed a cooperative effect of these receptors on the modulation of processes involved in oxidative stress, inflammation, and apoptosis in endothelial cells. Sumanirole, a specific D2R agonist, was shown to diminish the excessive production of reactive oxygen species induced by bradykinin, a proinflammatory B2R-activating peptide. This effect was accompanied by modified activities of antioxidant enzymes and increased phosphorylation of endothelial nitric oxide synthase, leading to enhance NO production. In turn, endothelial cell co-stimulation with B2R and D2R agonists inhibited the release of interleukin-6 and endothelin-1 and modulated the expression of apoptosis markers, such as Bcl-2, Bcl-xL, Bax, and caspase 3/7 activity. All these observations argue that the D2R agonist counteracts the pro-oxidative, pro-inflammatory, and pro-apoptotic effects induced through B2R, finally markedly improving endothelial functions.

Influence of Apoptotic Bodies and Apoptotic Microvesicles on NO Production in Macrophages.

We studied the effect of extracellular vesicular particles generated during apoptosis by macrophages of M0, M1 and M2 phenotypes on spontaneous and LPS-stimulated production of NO. The fractions of apoptotic bodies and apoptotic microvesicles were obtained in the primary cultures of peritoneal macrophages undergoing apoptosis. The effect of these microparticles on LPS-induced proinflammatory response of recipient macrophages critically depends on the initial phenotype of "donor" macrophages. Microvesicles and especially apoptotic bodies from M1 macrophages stimulate basal NO production. LPS stimulation of these macrophages preincubated with apoptotic bodies was not followed by further growth of NO production; in macrophages preincubated with microvesicles, LPS even suppressed NO production. Apoptotic microparticles obtained from M2 macrophages produced little effect on the basal production of NO. LPS stimulation of macrophages-recipients preincubated with microparticles from M2 macrophages did not enhance NO production. Incubation of macrophages with apoptotic microparticles induces the formation of endotoxic tolerance.

Abstract 588: Corticotropin-releasing Hormone via Its Type2 Receptor Inhibits Insulin-stimulated NO Formation in HUVECs

Background: Stress may provoke endothelial dysfunction thereby inducing atherosclerotic cardiovascular diseases (ASCVD). Corticotropin-releasing hormone (CRH) is recognized as a key regulator of hypothalamo-pituitary-adrenal axis under stress, however, its role in endothelial function and ASCVD is not known well. Insulin can produce NO formation in endothelial cells through eNOS activation. We studied effect of CRH and urocortin 2 (UCN2) on insulin-stimulated NO formation in HUVECs. Methods and Results: HUVECs were incubated in serum-free media for 8 h prior to the experiment. Treatment of HUVECs with insulin (100 nM) enhanced NO production detected by 5 μM 4,5-diaminofluorescein-diacetate. Fluorescent and phase contrast images were obtained using an fluorescence microscope (Em max : 515 nm), using an exposure time based on control conditions, and mean fluorescence intensity was calculated with ImageJ. Insulin-stimulated NO production was markedly inhibited by either CRH or UCN2 (100 nM each), which was reversed mostly by 100 nM astressin 2B (CRH receptor type 2 (CRHR2) inhibitor) and partly by 100 nM NBI35965 (CRHR1 inhibitor). CRH induced Rho-associated kinase (ROCK) and p-IRS-1(Ser). Insulin (2nM) stimulated p-eNOS (Ser1177), which was inhibited by CRH. Conclusions: Taken together, CRH/UCN2 via mainly its type 2 receptor signaling can inhibit insulin-stimulated eNOS/NO pathway. This CRH’s inhibitory effect on insulin signaling is mediated at least partly by activation of ROCK/ p-IRS-1 (Ser).

24S-hydroxycholesterol suppresses neuromuscular transmission in SOD1(G93A) mice: A possible role of NO and lipid rafts

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the initial denervation of skeletal muscle and subsequent death of motor neurons. A dying-back pattern of ALS suggests a crucial role for neuromuscular junction dysfunction. In the present study, microelectrode recording of postsynaptic currents and optical detection of synaptic vesicle traffic (FM1-43 dye) and intracellular NO levels (DAF-FM DA) were used to examine the effect of the major brain-derived cholesterol metabolite 24S-hydroxycholesterol (24S-HC, 0.4 μM) on neuromuscular transmission in the diaphragm of transgenic mice carrying a mutant superoxide dismutase 1 (SODG93A). We found that 24S-HC suppressed spontaneous neurotransmitter release and neurotransmitter exocytosis during high-frequency stimulation. The latter was accompanied by a decrease in both the rate of synaptic vesicle recycling and activity-dependent enhancement of NO production. Inhibition of NO synthase with L-NAME also attenuated synaptic vesicle exocytosis during high-frequency stimulation and completely abolished the effect of 24S-HC itself. Of note, 24S-HC enhanced the labeling of synaptic membranes with B-subunit of cholera toxin, suggesting an increase in lipid ordering. Lipid raft-disrupting agents (methyl-β-cyclodextrin, sphingomyelinase) prevented the action of 24S-HC on both lipid raft marker labeling and NO synthesis. Together, these experiments indicate that 24S-HC is able to suppress the exocytotic release of neurotransmitter in response to intense activity via a NO/lipid raft-dependent pathway in the neuromuscular junctions of SODG93A mice.

Truncated hemoglobin 1 is a new player in Chlamydomonas reinhardtii acclimation to sulfur deprivation.

Truncated hemoglobins constitute a large family, present in bacteria, in archaea and in eukaryotes. However, a majority of physiological functions of these proteins remains to be elucidated. Identification and characterization of a novel role of truncated hemoglobins in the model alga provides a framework for a more complete understanding of their biological functions. Here, we use quantitative RT-PCR to show that three truncated hemoglobins of Chlamydomonas reinhardtii, THB1, THB2 and THB12, are induced under conditions of depleted sulfur (S) supply. THB1 underexpression results in the decrease in cell size, as well in levels of proteins, chlorophylls and mRNA of several S-responsive genes under S starvation. We provide evidence that knock-down of THB1 enhances NO production under S deprivation. In S-deprived cells, a subset of S limitation-responsive genes is controlled by NO in THB1-dependent pathway. Moreover, we demonstrate that deficiency for S represses the nitrate reduction and that THB1 is involved in this control. Thus, our data support the idea that in S-deprived cells THB1 plays a dual role in NO detoxification and in coordinating sulfate limitation with nitrate assimilation. This study uncovers a new function for the Chlamydomonas reinhardtii THB1 in the control of proper response to S deprivation.