NO和钙信使系统在草酸诱导黄瓜叶片抗霜霉病中的作用

通过草酸及其与不同抑制剂亚甲基蓝、EGTA、氯丙嗪和Li+组合处理黄瓜叶片,研究了草酸与抑制剂不同处理组合方式对黄瓜叶片POD活性和叶片病情指数的影响,探讨NO、钙信使系统在草酸诱导叶片抗霜霉病中的作用.结果显示,10～70mmol/L草酸均能不同程度诱导黄瓜叶片POD活性的升高,提高叶片对黄瓜霜霉病的抗病性,降低叶片病情指数,并以30mmol/L效果最好.4种抑制剂分别与30mmol/L草酸同时或先于草酸处理,或草酸处理后一定时间再用抑制剂处理,均明显抑制黄瓜叶片POD活性的升高及病情指数的降低.研究表明,NO、Ca2+、钙调素(CaM)和磷酸肌醇均可能参与了草酸诱导黄瓜霜霉病抗性的信号转导过程.     

Denitrosylation of S-nitrosylated OGT is triggered in LPS-stimulated innate immune response

O-linked N-acetylglucosaminyltransferase (OGT)-mediated protein O-GlcNAcylation has been revealing various aspects of functional significance in biological processes, such as cellular signaling and activation of immune system. We found that OGT is maintained as S-nitrosylated form in resting cells, and its denitrosylation is triggered in innate immune response of lipopolysaccharide (LPS)-treated macrophage cells. S-nitrosylation of OGT strongly inhibits its catalytic activity up to more than 80% of native OGT, and denitrosylation of OGT leads to protein hyper-O-GlcNAcylation. Furthermore, blockage of increased protein O-GlcNAcylation results in significant loss of nitric oxide and cytokine production. We propose that denitrosylation of S-nitrosylated OGT is a direct mechanism for upregulation of OGT activity by which immune defense is critically controlled in LPS-stimulated innate immune response.     

Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/cGMP (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250, and 1000 μM) using a label‐free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron‐mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.     

Spectrofluorimetric analysis of the interaction of amyloid peptides with neuronal nitric oxide synthase: Implications in Alzheimer's disease

Background

The deposition of aggregated β-amyloid peptide senile plaques and the accumulation of arginine within the astrocytes in the brain of an Alzheimer's patient are classic observations in the neuropathology of the disease. It would be logical, in the aetiology and pathogenesis, to investigate arginine-metabolising enzymes and their intimate association with amyloid peptides.

Methods

Neuronal nitric oxide synthase (nNOS) was isolated, purified and shown, through fluorescence quenching spectroscopy and fluorescence resonance energy transfer (FRET), to interact with structural fragments of Aβ_1–40 and be catalytic towards amyloid fibril formation.

Results

Only one binding site on the enzyme was available for binding. Two amyloid peptide fragments of Aβ_1–40 (Aβ_17–28 and Aβ_25–35) had Stern–Volmer values (K_SV) of 0.111 μM⁻¹ and 0.135 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. The polarity of this active site precludes binding of the predominantly hydrophobic amyloid peptide fragments contained within Aβ_17–28 and within two glycine zipper motifs [G-X-X-X-G-X-X-X-G] [Aβ_29–37] and bind to the enzyme at a site remote to the active region.

Conclusions

The interaction and binding of Aβ_17–28 and Aβ_25–35 to nNOS causes the movement of two critical tryptophan residues of 0.77 nm and 0.57 nm respectively towards the surface of the enzyme.

General significance

The binding of Aβ-peptide fragments with nNOS has been studied by spectrofluorimetry. The information and data presented should contribute towards understanding the mechanism for deposition of aggregated Aβ-peptides and fibrillogenesis in senile plaques in an AD brain.     

Changes in nitrogen cycling and retention processes in soils under spruce forests along a nitrogen enrichment gradient in Germany

A network of long-term monitoring sites on nitrogen (N) input and output of forests across Germany showed that a number of Germany's forests are subject to or are experiencing N saturation and that spruce (Picea abies) stands have high risk. Our study was aimed at (1) quantifying the changes in gross rates of microbial N cycling and retention processes in forest soils along an N enrichment gradient and (2) relating the changes in soil N dynamics to N losses. We selected spruce sites representing an N enrichment gradient (indicated by leaching : throughfall N ratios) ranging from 0.04–0.13 (low N),≤0.26 (intermediate N enrichment) to≥0.42 (highly N enriched). To our knowledge, our study is the first to report on mechanistic changes in gross rates of soil N cycling and abiotic NO₃⁻ retention under ambient N enrichment gradient. Gross N mineralization, NH₄⁺ immobilization, gross nitrification, and NO₃⁻ immobilization rates increased up to intermediate N enrichment level and somewhat decreased at highly N-enriched condition. The turnover rates of NH₄⁺ and microbial N pools increased while the turnover rates of the NO₃⁻ pool decreased across the N enrichment gradient. Abiotic immobilization of NH₄⁺ did not differ across sites and was lower than that of NO₃⁻. Abiotic NO₃⁻ immobilization decreased across the N enrichment gradient. Microbial assimilation and turnover appeared to contribute largely to the retention of NH₄⁺. The increasing NO₃⁻ deposition and decreasing turnover rates of the NO₃⁻ pool, combined with decreasing abiotic NO₃⁻ retention, possibly contributed to increasing NO₃⁻ leaching and gaseous emissions across the N enrichment gradient. The empirical relationships of changes in microbial N cycling across the N enrichment gradient may be integrated in models used to predict responses of forest ecosystems (e.g. spruce) to increasing N deposition.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail, Helix lucorum

Abstract. The snail Helix lucorum has been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substrate l ‐arginine (l ‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca²⁺, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis in H. lucorum has a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.