Sulforaphane inhibits mitochondrial permeability transition and oxidative stress

Exposure of mitochondria to oxidative stress and elevated Ca²⁺ promotes opening of the mitochondrial permeability transition pore (PTP), resulting in membrane depolarization, uncoupling of oxidative phosphorylation, and potentially cell death. This study tested the hypothesis that treatment of rats with sulforaphane (SFP), an activator of the Nrf2 pathway of antioxidant gene expression, increases the resistance of liver mitochondria to redox-regulated PTP opening and elevates mitochondrial levels of antioxidants. Rats were injected with SFP or drug vehicle and liver mitochondria were isolated 40 h later. Respiring mitochondria actively accumulated added Ca²⁺, which was then released through PTP opening induced by agents that either cause an oxidized shift in the mitochondrial redox state or directly oxidize protein thiol groups. SFP treatment of rats inhibited the rate of pro-oxidant-induced mitochondrial Ca²⁺ release and increased expression of the glutathione peroxidase/reductase system, thioredoxin, and malic enzyme. These results are the first to demonstrate that SFP treatment of animals increases liver mitochondrial antioxidant defenses and inhibits redox-sensitive PTP opening. This novel form of preconditioning could protect against a variety of pathologies that include oxidative stress and mitochondrial dysfunction in their etiologies.     

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O₂^•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O₂^•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.     

A reverse method for diversity introduction of benzimidazole to synthesize H(+)/K(+)-ATP enzyme inhibitors

A series of 2-[(2-pyridylmethyl)sulfinyl]benzimidazole derivatives were synthesized via a solution phase synthetic route using a reversal method of diversity introduction. Using this synthetic strategy, we obtained two key intermediates (4-A and 4-B) simultaneously, which allows us to introduce diversity points onto the benzimidazole part of the final product under reliable reaction conditions to identify potent H⁺/K⁺-ATP enzyme inhibitors. Compound 14l (IC₅₀ = 1.6 × 10⁻⁵ M) was comparable with H⁺/K⁺-ATP enzyme inhibitor in vitro.     

The structure of human ubiquitin in 2-methyl-2,4-pentanediol: a new conformational switch

A new crystal structure of human ubiquitin is reported at 1.8 Å resolution. Compared with the other known crystal structure or the solution NMR structure of monomeric human ubiquitin, this new structure is similar in its overall fold but differs with respect to the conformation of the backbone in a surface‐exposed region. The conformation reported here resembles conformations previously seen in complex with deubiquinating enzymes, wherein the Asp52/Gly53 main chain and Glu24 side chain move. This movement exposes the backbone carbonyl of Asp52 to the exterior of the molecule, making it possible to engage in hydrogen‐bond contacts with neighboring molecules, rather than in an internal hydrogen bond with the backbone of Glu24. This particular crystal form of ubiquitin has been used in a large number of solid state NMR studies. The structure described here elucidates the origin of many of the chemical shift differences comparing solution and solid state studies.     

Ribonuclease P

The gene coding for the RNA subunit of ribonuclease P (RNase P) is essential in all free-living organisms. The RNA subunit, itself, is an enzyme and, from its evolutionary tree, we can infer that it is a very ancient molecule. The specificity of this enzyme is that it cleaves other RNA molecules at the junction of single-stranded and the 5' end of double-stranded regions of RNA. One can speculate that this molecule was very useful in an ancient world in cleaving long pieces of RNA, which must have contained hairpin regions in it, into shorter molecules with the capability of different functions from the longer parent. Today, the specificity of the enzyme can be used in designing drug therapies.     

Inhibition studies with anions and small molecules of two novel β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium

Two new β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were characterized kinetically. The two enzymes possess appreciable activity as catalysts for the hydration of CO₂ to bicarbonate, with k_cat of 0.79 × 10⁶ s⁻¹ and 1.0 × 10⁶ s⁻¹, and k_cat/K_m of 5.2 × 10⁷ M⁻¹ s⁻¹ and of 8.3 × 10⁷ M⁻¹ s⁻¹, respectively. A large number of simple/complex inorganic anions as well as other small molecules (sulfamide, sulfamic acid, phenylboronic acid, phenylarsonic acid, dialkyldithiocarbamates) showed interesting inhibitory properties towards the two new enzymes, with several low micromolar inhibitors discovered. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing lead compounds for novel types of antibacterials.     

酶启动和调控自组装超分子水凝胶

超分子间的弱相互作用使自组装超分子水凝胶的结构比较容易改变。用酶启动和调控超分子水凝胶的自组装不仅能在原位上对超分子水凝胶结构进行调整和控制,而且具有很好的生物选择性,有望制造出生物医学上所需要的材料,并能够控制生物体系中一些重要的生物过程。本文对酶启动和调控自组装超分子水凝胶的两类过程进行了总结,并以磷酸酯酶、β-内酰胺酶、嗜热菌蛋白酶、脂肪酶、基质金属蛋白酶和磷酸酯酶/激酶等酶为例,综述了如何设计和使用酶来启动和调控小分子的自组装超分子水凝胶。     

荧光假单胞菌短链脱氢酶的克隆、表达及酶学性质分析

为了研究荧光假单胞菌中短链脱氢酶的生理角色和催化特性,从荧光假单胞菌Pseudomonas fluorescens GIM1.49基因组DNA克隆表达了一个短链脱氢酶的编码基因pfd,并分析了该基因产物的酶学性质。基因pfd全长684 bp,编码227个氨基酸,推算分子量为24.2 kDa。将携带短链脱氢酶基因的重组质粒pET28b-pfd转入大肠杆菌BL21(DE3) 进行表达,得到了28 kDa的表达产物。重组荧光假单胞菌短链脱氢酶 (PFD) 能氧化4-氯-3-羟基丁酸乙酯、1-苯乙醇、苯甲醇、仲丁醇和还原4-氯-乙酰乙酸乙酯、2-溴-苯乙酮、4-溴-苯乙酮等底物。以4-氯-3-羟基丁酸乙酯为底物时活力最高,Km值为186.90 mmol/L,Vmax为89.56 U/mg。氧化4-氯-3-羟基丁酸乙酯时,最适反应温度和pH分别为12 ℃和10.5,倾向于利用NAD+作辅酶;而还原4-氯-乙酰乙酸乙酯时,最适温度和pH为24 ℃和8.8,倾向于利用NADPH作辅酶。重组PFD能耐受50％ (V/V) 的甲醇等有机助溶剂,Ca2+ (1 mmol/L) 和EDTA (5 mmol/L) 对其酶活有一定的促进作用。上述结果表明,重组PFD是一个新型的短链脱氢酶,其代谢角色推测与卤代次级醇的氧化降解有关。     

四季竹对不同浓度NaCl胁迫的生理响应

以夏秋季优良笋用竹种四季竹盆栽苗为材料,研究了土壤中1‰~6‰浓度NaCl(干重)处理对四季竹叶片脱落率、离子渗漏率、丙二醛(MDA)含量、抗氧化酶活性以及渗透调节物质含量的变化特征,以探讨四季竹的抗盐机理。结果表明:(1)随NaCl浓度的增大和处理时间的延长,四季竹叶片脱落率逐渐增加;四季竹在1‰~2‰NaCl浓度下的土壤中生长良好,在3‰~5‰浓度下随处理浓度的增大伤害逐渐加重,但这种伤害是可逆的,6‰是四季竹的半致死盐浓度。(2)叶片离子渗漏率随NaCl浓度的增大而显著增大,在6‰浓度处理下随处理时间的延长近直线升高,在3‰~5‰浓度下先升高后降低,而在1‰~2‰浓度处理下变化平缓且与同期对照无显著差异;叶片MDA含量随NaCl浓度的增大而增加,随处理时间的延长先增加后降低。(3)四季竹叶片SOD和POD活性在胁迫3~15 d均出现先下降后上升的过程,其有一定的锻炼适应性;叶片脯氨酸含量随NaCl处理浓度的增大而增加,其随处理时间的延长在1‰~2‰浓度处理下变化平缓,在3‰~6‰浓度处理下显著增加后减小;各浓度处理的可溶性蛋白含量随处理时间的延长先增加后减小,但均高于对照。研究发现,四季竹是较耐盐竹种,其可以通过调控自身保护酶活性和渗透调节物质含量来有效缓解盐胁迫伤害,从而表现出一定的耐盐能力。