TRPA1 Has a Key Role in the Somatic Pro-Nociceptive Actions of Hydrogen Sulfide

Hydrogen sulfide (H₂S), which is produced endogenously from L-cysteine, is an irritant with pro-nociceptive actions. We have used measurements of intracellular calcium concentration, electrophysiology and behavioral measurements to show that the somatic pronociceptive actions of H₂S require TRPA1. A H₂S donor, NaHS, activated TRPA1 expressed in CHO cells and stimulated DRG neurons isolated from Trpa1^+/+ but not Trpa1^−/− mice. TRPA1 activation by NaHS was pH dependent with increased activity at acidic pH. The midpoint of the relationship between NaHS EC₅₀ values and external pH was pH 7.21, close to the expected dissociation constant for H₂S (pK_a 7.04). NaHS evoked single channel currents in inside-out and cell-attached membrane patches consistent with an intracellular site of action. In behavioral experiments, intraplantar administration of NaHS and L-cysteine evoked mechanical and cold hypersensitivities in Trpa1^+/+ but not in Trpa1^−/− mice. The sensitizing effects of L-cysteine in wild-type mice were inhibited by a cystathionine β-synthase inhibitor, D,L-propargylglycine (PAG), which inhibits H₂S formation. Mechanical hypersensitivity evoked by intraplantar injections of LPS was prevented by PAG and the TRPA1 antagonist AP-18 and was absent in Trpa1^−/− mice, indicating that H₂S mediated stimulation of TRPA1 is necessary for the local pronociceptive effects of LPS. The pro-nociceptive effects of intraplantar NaHS were retained in Trpv1^−/− mice ruling out TRPV1 as a molecular target. In behavioral studies, NaHS mediated sensitization was also inhibited by a T-type calcium channel inhibitor, mibefradil. In contrast to the effects of NaHS on somatic sensitivity, intracolonic NaHS administration evoked similar nociceptive effects in Trpa1^+/+ and Trpa1^−/− mice, suggesting that the visceral pro-nociceptive effects of H₂S are independent of TRPA1. In electrophysiological studies, the depolarizing actions of H₂S on isolated DRG neurons were inhibited by AP-18, but not by mibefradil indicating that the primary excitatory effect of H₂S on DRG neurons is TRPA1 mediated depolarization.     

高迁移率族蛋白1在急性酒精中毒中的作用

急性酒精中毒是一种有害的临床病理状态,短时间内摄入大量的乙醇,会造成多脏器功能损害,通常包括中枢神经抑制、呼吸循环功能障碍、代谢紊乱及免疫系统异常,严重者导致死亡.为了探索急性酒精中毒导致死亡的原因,采用腹腔注射的方法构建了重度急性酒精中毒小鼠模型,发现在模型早期(至迟0.5 h)高迁移率族蛋白1(high mobility group box-1 protein,HMGB1)已显著升高,体外实验也证实酒精导致细胞HMGB1释放,应用单克隆抗体阻断HMGB1有显著的保护作用,这种保护作用是通过降低损伤性炎症反应实现的.在此模型中发现,HMGB1在急性酒精中毒中有着重要的中介作用,调控急性系统性炎症反应,并决定了急性酒精中毒疾病的进程与结局.     

GTPase Activity Plays a Key Role in the Pathobiology of LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with late-onset, autosomal-dominant, familial Parkinson''s disease (PD) and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s) underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker''s yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human α-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.     

Effect of Hydrogen Sulfide on Sympathetic Neurotransmission and Catecholamine Levels in Isolated Porcine Iris-Ciliary Body

In the present study, we investigated the pharmacological action of hydrogen sulfide (H₂S, using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na₂S as donors) on sympathetic neurotransmission from isolated, superfused porcine iris-ciliary bodies. We also examined the effect of H₂S on norepinephrine (NE), dopamine and epinephrine concentrations in isolated porcine anterior uvea. Release of [³H]NE was triggered by electrical field stimulation and basal catecholamine concentrations was measured by high performance liquid chromatography (HPLC). Both NaHS and Na₂S caused a concentration-dependent inhibition of electrically evoked [³H]NE release from porcine iris-ciliary body without affecting basal [³H]NE efflux. The inhibitory action of H₂S donors on NE release was attenuated by aminooxyacetic acid (AOA) and propargyglycine (PAG), inhibitors of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), respectively. With the exception of dopamine, NaHS caused a concentration-dependent reduction in endogenous NE and epinephrine concentrations in isolated iris-ciliary bodies. We conclude that H₂S can inhibit sympathetic neurotransmission from isolated porcine anterior uvea, an effect that is dependent, at least in part, on intramural biosynthesis of this gas. Furthermore, the observed action of H₂S donors on sympathetic transmission may be due to a direct action of this gas on neurotransmitter pools.     

Pharmacological Actions of Hydrogen Sulfide Donors on Sympathetic Neurotransmission in the Bovine Anterior Uvea,In Vitro

In the present study, we investigated the effect of three different sources of hydrogen sulfide (H₂S) on sympathetic neurotransmission from isolated superfused bovine iris-ciliary bodies. The three agents under consideration were: ACS67, a hybrid of latanoprost and a H₂S-donating moiety; l-cysteine, a substrate for endogenous production of H₂S and GYY 4137, a slow donor of H₂S. We also examined the contribution of prostaglandins to the pharmacological actions of the H₂S donors on release of [³H]-norepinephrine ([³H]NE) triggered by electrical field stimulation. ACS67, l-cysteine and GYY 4137 caused a concentration-dependent inhibition of electrically-evoked [³H]NE release from isolated bovine iris-ciliary bodies without affecting basal [³H]NE efflux. The cyclooxygenase inhibitor, flurbiprofen enhanced the inhibitory action of ACS67 and l-cysteine on stimulated [³H]NE release. Both aminooxyacetic acid, an inhibitor of cystathionine-β-synthase and glibenclamide, a K_ATP channel blocker reversed the inhibition of evoked NE release induced by the H₂S donors. We conclude that H₂S donors can inhibit sympathetic neurotransmission from isolated bovine iris-ciliary bodies, an effect partially dependent on the in situ production of H₂S and prostanoids, and is mediated by an action on K_ATP channels.     

Olfactory Plays a Key Role in Spatiotemporal Pathogenesis of Cerebral Malaria

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Cytoglobin Up-regulated by Hydrogen Peroxide Plays a Protective Role in Oxidative Stress

Cytoglobin (Cygb) is a recently discovered intracellular respiratory globin, which exists in all types of cells. It has been suggested that Cygb has a role in protecting cells against oxidative stress. In the present study we have tested this hypothesis. The N2a neuroblastoma cells were exposed to various kinds of insults, including hydrogen peroxide (H₂O₂), hypoxia, kainic acid, high extracellular CaCl₂, high osmolarity, UV irradiation and heat shock. Among them, only H₂O₂-treatment induced a significant up-regulation of cytoglobin mRNA level. We stably transfected N2a cells with Cygb-siRNA vectors and successfully knocked down Cygb. The Cygb-siRNA could exacerbate cell death upon H₂O₂-treatment, as demonstrated by MTT cell viability assay. Thus, Cygb in neuronal cells might be specifically induced under oxidative stress to protect them from death.     

Proteasome 19S RP Binding to the Sec61 Channel Plays a Key Role in ERAD

Import of secretory proteins into the Endoplasmic Reticulum (ER) is an established function of the Sec61 channel. The contribution of the Sec61 channel to export of misfolded proteins from the ER for degradation by proteasomes is still controversial, but the proteasome 19S regulatory particle (RP) is necessary and sufficient for extraction of specific misfolded proteins from the ER, and binds directly to the Sec61 channel. In this work we have identified an import-competent sec61 mutant, S353C, carrying a point mutation in ER-lumenal loop 7 which reduces affinity of the cytoplasmic face of the Sec61 channel for the 19S RP. This indicates that the interaction between the 19S RP and the Sec61 channel is dependent on conformational changes in Sec61p hinging on loop 7. The sec61-S353C mutant had no measurable ER import defects and did not cause ER stress in intact cells, but reduced ER-export of a 19S RP-dependent misfolded protein when proteasomes were limiting in a cell-free assay. Our data suggest that the interaction between the 19S RP and the Sec61 channel is essential for the export of specific substrates from the ER to the cytosol for proteasomal degradation.     

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9–10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA₂s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA₂s are key players.     

The PICALM Protein Plays a Key Role in Iron Homeostasis and Cell Proliferation

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.     

气体信号分子CO和H_2S与抑郁症之间的相关研究

为了探讨气体信号分子一氧化碳(carbon monoxide,CO)及硫化氢(hydrogen sulfide,H2S)与抑郁症之间的关系。选取抑郁症患者40例作为实验组,同时选取健康人40例作为对照组,两组在年龄、性别、肝功能、肾功能、血糖浓度等基本资料方面均无统计学差异(P>0.05),同时检测两组血浆CO、H2S的浓度和过氧化氢酶(catalase,CAT)的活性。研究发现实验组血浆CO含量和CAT活性均高于对照组,而H2S的含量低于对照组,差异均具有统计学意义(P<0.05);实验组血浆中CO和H2S的含量呈直线负相关关系,但和CAT没有直接关系。结果提示内源性CO、H2S与抑郁症之间存在着一定关系,氧化应激参与了反应。     

Depletion of Glycolytic Intermediates Plays a Key Role in Glucose-Phosphate Stress in Escherichia coli

In bacteria like Escherichia coli, the accumulation of glucose-6-phosphate (G6P) or its analogs such as α-methyl glucoside-6-phosphate (αMG6P) results in stress that appears in the form of growth inhibition. The small RNA SgrS is an essential part of the response that helps E. coli combat glucose-phosphate stress; the growth of sgrS mutants during stress caused by αMG is significantly impaired. The cause of this stress is not currently known but may be due to either toxicity of accumulated sugar-phosphates or to depletion of metabolic intermediates. Here, we present evidence that glucose-phosphate stress results from depletion of glycolytic intermediates. Addition of glycolytic compounds like G6P and fructose-6-phosphate rescues the αMG growth defect of an sgrS mutant. These intermediates also markedly decrease induction of the stress response in both wild-type and sgrS strains grown with αMG, implying that cells grown with these intermediates experience less stress. Moreover, αMG transport assays confirm that G6P relieves stress even when αMG is taken up by the cell, strongly suggesting that accumulated αMG6P per se does not cause stress. We also report that addition of pyruvate during stress has a novel lethal effect on the sgrS mutant, resulting in cell lysis. The phosphoenolpyruvate (PEP) synthetase PpsA, which converts pyruvate to PEP, can confer resistance to pyruvate-induced lysis when ppsA is ectopically expressed in the sgrS mutant. Taken as a whole, these results provide the strongest evidence thus far that depletion of glycolytic intermediates is at the metabolic root of glucose-phosphate stress.