瞬时受体电位香草酸亚型1激活释放的P物质在小鼠急性心肌梗死后凋亡中的保护作用

瞬时受体电位香草酸亚型1 (transient receptor potential vanilloid 1, TRPV1)在心肌缺血激活后可传导心绞痛信号和释放P物质(substance P, SP).SP是速激肽家族成员之一,主要通过结合并激活神经激肽1 (neurokinin 1,NK1)受体发挥作用. TRPV1和SP在缺血性心脏病中对心功能的恢复和重塑有一定保护作用,但对心肌梗死后凋亡的作用及具体机制尚不明确.本研究用TRPV1基因敲除(TRPV1-/- )小鼠和野生型(wide type, WT)小鼠建立心肌梗死模型,并外源性给予SP和NK1受体拮抗剂RP67580,用TTC染色法观察梗死的面积,TUNEL法检测心肌细胞凋亡指数,Western印迹方法检测caspase-3、Bcl-2、Bax、p53的蛋白表达.结果发现,心肌梗死24 h后,TRPV1-/-小鼠比WT小鼠梗死面积更大,凋亡指数和caspase-3活性更高,Bcl-2/Bax和p53蛋白表达更低. SP预处理可以明显缩小TRPV1-/-小鼠梗死面积,降低凋亡指数、caspase-3活性和升高Bcl-2/Bax比值,而在WT小鼠中改善不明显.外源性给予RP67580,阻断SP与NK1受体结合后,与相应对照组相比,WT小鼠梗死面积和凋亡指数更大,caspase-3蛋白表达更高,Bcl-2/Bax比值更低;TRPV1-/-小鼠与相应对照组比较,凋亡指数和caspase-3表达升高,Bcl-2/Bax比值降低.研究结果表明,SP可能介导了TRPV1在急性心肌梗死后凋亡中的保护作用.     

Mechanism and rate constants of the Cdc42 GTPase binding with intrinsically disordered effectors

Intrinsically disordered proteins (IDPs) are often involved in signaling and regulatory functions, through binding to cellular targets. Many IDPs undergo disorder‐to‐order transitions upon binding. Both the binding mechanisms and the magnitudes of the binding rate constants can have functional importance. Previously we have found that the coupled binding and folding of any IDP generally follows a sequential mechanism that we term dock‐and‐coalesce, whereby one segment of the IDP first docks to its subsite on the target surface and the remaining segments subsequently coalesce around their respective subsites. Here we applied our TransComp method within the framework of the dock‐and‐coalesce mechanism to dissect the binding kinetics of two Rho‐family GTPases, Cdc42 and TC10, with two intrinsically disordered effectors, WASP and Pak1. TransComp calculations identified the basic regions preceding the GTPase binding domains (GBDs) of the effectors as the docking segment. For Cdc42 binding with both WASP and Pak1, the calculated docking rate constants are close to the observed overall binding rate constants, suggesting that basic‐region docking is the rate‐limiting step and subsequent conformational coalescence of the GBDs on the Cdc42 surface is fast. The possibility that conformational coalescence of the WASP GBD on the TC10 surface is slow warrants further experimental investigation. The account for the differences in binding rate constants among the three GTPase‐effector systems and mutational effects therein yields deep physical and mechanistic insight into the binding processes. Our approach may guide the selection of mutations that lead to redesigned binding pathways. Proteins 2016; 84:674–685. © 2016 Wiley Periodicals, Inc.     

Non-Newtonian Blood Flow in Left Coronary Arteries with Varying Stenosis: A Comparative Study

This paper presents Computational fluid dynamic (CFD) analysis of blood flow in three different 3-D models of left coronary artery (LCA). A comparative study of flow parameters (pressure distribution, velocity distribution and wall shear stress) in each of the models is done for a non-Newtonian (Carreau) as well as the Newtonian nature of blood viscosity over a complete cardiac cycle. The difference between these two types of behavior of blood is studied for both transient and steady states of flow. Additionally, flow parameters are compared for steady and transient boundary conditions considering blood as non-Newtonian fluid. The study shows that the highest wall shear stress (WSS), velocity and pressure are found in artery having stenosis in all the three branches of LCA. The use of Newtonian blood model is a good approximation for steady as well as transient blood flow boundary conditions if shear rate is above 100 s-1. However, the assumption of steady blood flow results in underestimating the values of flow parameters such as wall shear stress, pressure and velocity.     

Precipitation of porcine insulin with carbon dioxide

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO₂) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO₂ as an acid‐precipitating agent. The solubility of porcine insulin in NaHCO₃ solutions in pressurized CO₂ was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoelectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self‐association reactions were also taken into account. The biological activity of insulin was maintained after precipitation with CO₂, although some activity can be lost if foam is formed in the depressurization step. Biotechnol. Bioeng. 2009;103: 909–919. © 2009 Wiley Periodicals, Inc.     

Crystal structure and aqueous solubility of ammonium D-glucarate

Ammonium D-glucarate, NH(4)(C(6)H(9)O(8)) [ammonium D-saccharate, NH(4)-SAC], has been synthesized, and its crystal structure solved by single-crystal X-ray diffraction methods. NH(4)-SAC crystallizes in the monoclinic space group P2(1) (#4) with cell parameters a = 4.8350(4) Angstroms, b = 11.0477(8) Angstroms, c = 16.7268(12) Angstroms, beta = 90.973(1) degrees, V = 894.34(12) Angstroms(3), Z = 3. The structure was refined by full-matrix least-squares on F(2) yielding final R-values (all data) R1 = 0.0353 and R(w)2 = 0.0870. The structure consists of alternating (NH(4))(+) and (C(6)H(11)O(6))(-) layers parallel to the bc plane. An extended network of N-H...O(SAC) and O(SAC)-H...O(SAC) hydrogen bonds provide the 3-D connectivity. The aqueous solubility (S(w)) has been shown to be pH independent at ambient conditions within the range 4.5 < pH < 10 with S(w) = 2.19 M/L, whose value is about a factor of two lower than that of the ammonium isosaccharate analogue.     

p38MAPK介导的胶质细胞iNOS的转录激活机制

丝裂原激活蛋白激酶(MAPK)酶级联反应系统参与胶质细胞中iNOS的合成.通过瞬时转染p38MAPK途径中上游激酶,MAPK激酶3(MKK3)和MAPK激酶6 (MKK6 )表达质粒,进一步了解p38MAPK级联传导信号系统调节iNOS基因在胶质细胞中的转录激活机制.MKK3或MKK6表达质粒与接有荧光素酶(luciferase ,Luc)的大鼠iNOS启动基因质粒(iNOS Luc)联合转染C6星形胶质细胞株引起iNOS Luc的激活,并且使细胞因子诱导的iNOSmRNA的表达增强.这两种效应都能够被p38MAPK抑制剂SB2 0 35 80所抑制.MKK3 6也可以诱导核因子κB(NFκB Luc)依赖的转录活性.这些分子水平的研究结果为p38MAPK信号级联传导途径在调节大鼠胶质细胞中iNOS基因转录激活中的重要作用,包括转录因子NFκB的作用提供了证据.通过阻断iNOS表达或NO的生成,抑制细胞炎症发生,为防治神经细胞炎症反应性疾病提供实验依据.     

Salting-in characteristics of globular proteins

Protein solubility, and the formation of various solid phases, is of interest in both bioprocessing and the study of protein condensation diseases. Here we examine the the phase behavior of three proteins (chymosin B, β-lactoglobulin B, and pumpkin seed globulin) previously known to display salting-in behavior, and measure their solubility as a function of pH, ionic strength, and salt type. Although the phase behavior of the three proteins is quantitatively different, general trends emerge. Stable crystal nucleation does not occur within the salting-in region for the proteins examined, despite the crystal being observed as the most stable solid phase. Instead, two types of amorphous phases were found within the salting-in region; additionally, an analog to the instantaneous clouding curve was observed within the salting-in region for chymosin B. Also, protein solutions containing sulfate salts resulted in different crystal morphologies depending on whether Li₂SO₄ or (NH₄)₂SO₄ was used.     

Preparation and physicochemical characterization of 5 niclosamide solvates and 1 hemisolvate

The purpose of the study was to characterize the physicochemical, structural, and spectral properties of the 1∶1 niclosamide and methanol, diethyl ether, dimethyl sulfoxide, N,N' dimethylformamide, and tetrahydrofuran solvates and the 2∶1 niclosamide and tetraethylene glycol hemisolvate prepared by recrystallization from these organic solvents. Structural, spectral, and thermal analysis results confirmed the presence of the solvents and differences in the structural properties of these solvates. In addition, differences in the activation energy of desolvation, batch solution calorimetry, and the aqueous solubility at 25°C, 24 hours, showed the stability of the solvates to be in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > methanol solvate > dimethyl sulfoxide solvate > N,N' dimethylformamide solvate. The intrinsic and powder dissolution rates of the solvates were in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > N,N' dimethylformamide solvate > methanol solvate > dimethyl sulfoxide solvate. Although these nonaqueous solvates had higher solubility and dissolution rates than the monohydrous forms, they were unstable in aqueous media and rapidly transformed to one of the monohydrous forms.     

TRPV1 activation impedes foam cell formation by inducing autophagy in oxLDL-treated vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are an important origin of foam cells besides macrophages. The mechanisms underlying VSMC foam cell formation are relatively little known. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1) and autophagy have a potential role in regulating foam cell formation. Our study demonstrated that autophagy protected against foam cell formation in oxidized low-density lipoprotein (oxLDL)-treated VSMCs; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL and activated autophagy–lysosome pathway in VSMCs; activation of TRPV1 by capsaicin impeded foam cell formation of VSMCs through autophagy induction; activation of TRPV1 by capsaicin induced autophagy through AMP-activated protein kinase (AMPK) signaling pathway. This study provides evidence that autophagy plays an important role in VSMC foam cell formation and highlights TRPV1 as a promising therapeutic target in atherosclerosis.     

Mechanisms underlying modulation of podocyte TRPC6 channels by suPAR: Role of NADPH oxidases and Src family tyrosine kinases

The soluble urokinase receptor (suPAR) has been implicated in the pathogenesis of chronic kidney diseases (CKD) and may function as a circulating “permeability factor” driving primary focal and segmental glomerulosclerosis (FSGS). Here we examined the mechanisms whereby suPAR causes mobilization and increased activation of Ca²⁺-permeable TRPC6 channels, which are also implicated in FSGS. Treatment of immortalized mouse podocytes with recombinant suPAR for 24?h caused a marked increase in cytosolic reactive oxygen species (ROS) that required signaling through integrins. This effect was associated with increased assembly of active cell surface NADPH oxidase 2 (Nox2) complexes and was blocked by the Nox2 inhibitor apoycynin. Treatment with suPAR also evoked a functionally measurable increase in TRPC6 channels that was blocked by concurrent treatment with the ROS-quencher TEMPOL as well as by inhibition of Rac1, an essential component of active Nox2 complexes. Elevated ROS evoked by exposing cells to suPAR or H₂O₂ caused a marked increase in the abundance of tyrosine-phosphorylated proteins including Src, and suPAR-evoked Src activation was blocked by TEMPOL. Moreover, mobilization and increased activation of TRPC6 by suPAR or H₂O₂ was blocked by concurrent exposure to PP2, an inhibitor of Src family tyrosine kinases. These data suggest that suPAR induces oxidative stress in podocytes that in turn drives signaling through Src family kinases to upregulate TRPC6 channels. The combination of oxidative stress and altered Ca²⁺ signaling may contribute to loss of podocytes and progression of various forms of CKD.