Antiparallel Conformation of Knob and Hole Aglycosylated Half-Antibody Homodimers Is Mediated by a CH2–CH3 Hydrophobic Interaction

Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating “knob” and “hole” heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of heterodimeric reaction products, we sought crystal structures of aglycosylated heterodimeric and homodimeric “knob” and “hole” Fc fragments derived from bacterial expression. The structure of the knob-into-hole Fc was determined at 2.64 Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a heterodimer interaction kinetic K_D of < 1 nM. Homodimers of the “knob” and “hole” mutants were also obtained, and their X-ray structures were determined at resolutions 2.5 Å and 2.1 Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob/knob or hole/hole CH3 interactions. The head-to-tail arrangement was disfavored by adding site-directed mutations at F241 and F243 in the CH2 domains, leading to increases in both rate and efficiency of bispecific (heterodimer) assembly.     

Diel variation in lacunal CH4 and CO2 concentration and δ13C in Phragmites australis

We tested the hypothesis that the diurnal patterns of variationin lacunal gas concentrations and isotopic fractionationpreviously reported in a single plant genera (Typha)typified the patterns of all through-flow convective plantsby extending our observations to Phragmites australisCav. In daylight, Phragmites CH₄ transport isdriven by internal pressurization which results in gas flowdown young green culms and its exit from one year old deadbrown culms. Flow rates of 10.4 ± 4.0 mL min^–1 weremeasured in this study. At night, CH₄ is transportedfrom the sediments to the atmosphere via the lacunal plantspaces by molecular diffusion. Within green culms, lacunalCH₄ concentrations varied by a factor of 1000, from 3%(parts by volume) pre-dawn to lows of 25 ppmv during midday.Methane in brown culms varied by a factor of 10 diurnally,from 5% pre-dawn to 0.3% at midday. Lacunal CO₂concentrations varied similarly.Concentrations of both gases varied inversely with lacunalpressure. In green culms, large isotopic fractionations wereobserved in CH₄ and CO₂ in the morning and eveningduring transitions in gas transport mode and were associatedwith slight downward flows counter to the upward diffusionof these gases. Methane ¹³C as depletedas –100 was observed. In daylight, lacunal CH₄ wassimilar to or ¹³C depleted relative to sedimentary andemitted CH₄ isotopic values, but at night lacunalCH₄ was ¹³C enriched relative to sedimentarymethane. Overall, the diurnal variations of CH₄concentration and ¹³C value inPhragmiteswere similar to those observed in Typha andindicate that these patterns should be consistent in otherconvective-flow plants. Furthermore, our results demonstratethat the large isotopic fractionations found in aquaticplants can result solely from isotopic fractionationassociated with gas transport.     

Molecular Mechanics Simulations in Structure Analysis of Intercalate VOPO4·2CH3CH2OH

Molecular mechanics simulations using Cerius² combined with X-ray diffraction and supported with vibrational spectroscopy have been used to investigate the layered structure of vanadyl phosphate VOPO₄ intercalated with ethanol. This intercalated structure exhibits certain degree of disorder, which affects the diffraction diagram and obstructs the conventional structure analysis based on diffraction methods only. Present structure analysis is focused to the crystal packing in the interlayer space and layer stacking in the intercalate. The bilayer arrangement of ethanol molecules in the interlayer has been found, giving the basal spacing d = 13.21 Å, experimental d-value obtained from X-ray diffraction is 13.17 Å. One half from the total number of CH₃CH₂OH molecules is anchored with their oxygens to VOPO₄ layers to complete vanadium octahedra and their orientation is not very strictly defined. The second half of ethanoles is linked with hydrogen bridges to the anchored etahanoles and sometimes also to the layer oxygens. Positions and orientations of these unachored ethanoles with respect to VOPO₄ layers exhibit certain degree of disorder, resulting in the disorder in layer stacking. Molecular mechanics simulations revealed the character of this displacement disorder in layer stacking and enabled to determine the components of the displacement vector.     

Roles of electrostatic interaction and dispersion in CH···CH,CH···π, and π···π ethylene dimers

The structural, mechanical, electronic, and optical properties of orthorhombic Bi₂S₃ and Bi₂Se₃ compounds have been investigated by means of first principles calculations. The calculated lattice parameters and internal coordinates are in very good agreement with the experimental findings. The elastic constants are obtained, then the secondary results such as bulk modulus, shear modulus, Young’s modulus, Poisson’s ratio, anisotropy factor, and Debye temperature of polycrystalline aggregates are derived, and the relevant mechanical properties are also discussed. Furthermore, the band structures and optical properties such as real and imaginary parts of dielectric functions, energy-loss function, the effective number of valance electrons, and the effective optical dielectric constant have been computed. We also calculated some nonlinearities for Bi₂S₃ and Bi₂Se₃ (tensors of elasto-optical coefficients) under pressure.

Does richness of emergent plants affect CO2 and CH4 emissions in experimental wetlands?

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Biotic controls on CO2 and CH4 exchange in wetlands – a closed environment study

Wetlands are significant sources of the important greenhouse gas CH₄. Here we explore the use of an experimental system developed for the determination of continuous fluxes of CO₂ and CH₄ in closed ecosystem monoliths including the capture of ¹⁴CO₂ and ¹⁴CH₄ following pulse labelling with ¹⁴CO₂. We show that, in the ecosystem studied, ebullition (bubble emission) may account for 18 to 50% of the total CH₄ emission, representing fluxes that have been difficult to estimate accurately in the past. Furthermore, using plant removal and ¹⁴C labelling techniques, we use the system to detail the direct influence of vascular plants on CH₄ emission. This influence is observed to be dependent on the amount of vascular plants present. The results that may be produced using the presented experimental set-up have implications for an improved understanding of wetland ecosystem/atmosphere interactions, including possible feedback effects on climate change. In recent years much attention has been devoted to ascertaining and subsequently using the relationship between net ecosystem productivity and CH₄ emission as a basis for extrapolation of fluxes across large areas. The experimental system presented may be used to study the complex relationship between vascular plants and CH₄ emission and here we show examples of how this may vary considerably in nature between and even within ecosystems.     

P2X2和P2X4受体的调制位点

P2X受体是非选择性阳离子通道,广泛分布于中枢及外周神经系统。近年来,本实验室侧重研究重金属、质子、酒精等对P2X2和P2X4受体的调制。研究发现不同的因素对同一种受体介导的ATP-激活电流有不同的调制作用,如增强效应、抑制效应或者两种效应兼有,而且同一种因素对这两种受体的调制效应也不相同。因此,我们推测P2X受体上存在不同的结合位点,本文主要阐述P2X2和P2X4受体不同的调制位点。     

Structures and aromaticity of X2Y 2 − (X = C, Si, Ge and Y = N, P, As) anions

The equilibrium geometries, total energies, and vibrational frequencies of anions X₂Y₂ ⁻ (X = C, Si, Ge and Y = N, P, As) are theoretically investigated with density functional theory (DFT) method. Our calculation shows that for C₂N₂ ⁻ species, the D _2h isomer is the most stable four-membered structure, and for other species the C _2v isomer in which two X atoms are contrapuntal is the most stable structure at the B3LYP/6-311 +G_* level. Wiberg bond index (WBI) and negative nucleus-independent chemical shift (NICS) value indicate the existence of delocalization in stable X₂Y₂ ⁻ structures. A detailed molecular orbital (MO) analysis further reveals that stable isomers of these species have strongly aromatic character, which strengthens the structural stability and makes them closely connected with the concept of aromaticity.     

Discovery, synthesis and SAR of azinyl- and azolylbenzamides antagonists of the P2X₇ receptor

The discovery, of a series of 2-Cl-5-heteroaryl-benzamide antagonists of the P2X(7) receptor via parallel medicinal chemistry is described. Initial analogs suffered from poor metabolic stability and low Vd(ss). Multi parametric optimization led to identification of pyrazole 39 as a viable lead with excellent potency and oral bioavailability. Further attempts to improve the low Vd(ss) of 39 via introduction of amines led to analogs 40 and 41 which maintained the favorable pharmacology profile of 39 and improved Vd(ss) after iv dosing. But these analogs suffered from poor oral absorption, probably driven by poor permeability.     

The origin of the generalized anomeric effect: possibility of CH/n and CH/π hydrogen bonds

Ab initio MO calculations were carried out at the MP4/6-311++G(3df,3pd)//MP2/6-311++G(3df,3pd) level to investigate the conformational Gibbs energy of a series of methyl ethers CH₃O-CH₂-X (X = OH, OCH₃, F, Cl, Br, CN, CCH, C₆H₅, CHO). It was found that the Gibbs energy of the gauche conformers is lower in every case than that of the corresponding anti conformers. In the more stable gauche conformers, the interatomic distance between X and the hydrogen atom was shorter than the sum of the van der Waals radii. The natural bonding orbital (NBO) charges of group X were more negative in the gauche conformers than in the anti conformers. We suggest that the CH/n and CH/π hydrogen bonds play an important role in stabilizing the gauche conformation of these compounds.     

植物排放N2O和CH4的研究

N2O和CH4是2种重要的温室气体,但其排放源尚未得到充分鉴别.1990年和2006年先后报道植物能排放N2O和CH4,并日益受到广泛的关注.然而,迄今为止对植物排放这2种气体的研究均是分开单独进行的.该文以8种陆生草本植物为研究对象,首次同步考察了新鲜离体植物地上部排放N2O和CH4的通量.研究结果表明:8种植物均能排放这2种气体.其中,黑麦草(Lolium perenne)、抱茎苦荬菜(Ixendium sonchifolium)和菠菜(Spinacia oleracea)的CH4通量较高,分别为165.38、52.28和21.64 ngCH4.g-1dw·h-1;抱茎苦荬菜、蒙古蒿(Artemisia mongolica)、大豆(Glycine max)和菠菜的N2O通量较高,分别为7.19、6.92、5.44和4.05 ngN2O·g-1dw.h-1.研究结果不仅为植物本身既能排放N2O又能排放CH4在植物中可能具有普遍性提供了进一步的实验依据,而且为深入研究其机理找到了几种适宜的植物种(如抱茎苦荬菜、菠菜).     

植物排放N2O和CH4的研究

N₂O和CH₄是2种重要的温室气体, 但其排放源尚未得到充分鉴别。1990年和2006年先后报道植物能排放N₂O和CH₄, 并日益受到广泛的关注。然而, 迄今为止对植物排放这2种气体的研究均是分开单独进行的。该文以8种陆生草本植物为研究对象, 首次同步考察了新鲜离体植物地上部排放N₂O和CH₄的通量。研究结果表明: 8种植物均能排放这2种气体。其中, 黑麦草(Lolium perenne)、抱茎苦荬菜(Ixeridium sonchifolium)和菠菜(Spinacia oleracea)的CH₄通量较高, 分别为165.38、 52.28和21.64 ngCH₄·g^–1dw·h^–1; 抱茎苦荬菜、蒙古蒿(Artemisia mongolica)、大豆(Glycine max)和菠菜的N₂O通量较高, 分别为7.19、6.92、5.44和4.05 ngN₂O·g^–1dw·h^–1。研究结果不仅为植物本身既能排放N₂O又能排放CH₄在植物中可能具有普遍性提供了进一步的实验依据, 而且为深入研究其机理找到了几种适宜的植物种(如抱茎苦荬菜、菠菜)。     

CO2倍增对稻田CH4排放的影响

 利用开顶室熏气装置,研究了CO2倍增对稻田CH4排放的影响。发现CO2倍增在分蘖初期、孕穗期、抽穗期、灌浆期和成熟期对稻田CH4排放具有明显的促进作用,分别比对照增加了253％、288％、273％、273％和157％。在分蘖末期至灌浆期有一个高峰,排放通量约为1.91mgCH4·d-1·p-1。这种促进作用可能主要是由于CO2倍增使水稻根系生物量以及根系代谢活性发生变化造成的。     

P2X7 receptor agonists pre- and postcondition the heart against ischemia-reperfusion injury by opening pannexin-1/P2X₇ channels

Protection of the heart from ischemia-reperfusion injury can be achieved by ischemic preconditioning and ischemic postconditioning. Previous studies revealed that a complex of pannexin-1 with the P2X(7) receptor forms a channel during ischemic preconditioning and ischemic postconditioning that results in the release of endogenous cardioprotectants. ATP binds to P2X(7) receptors, inducing the formation of a channel in association with pannexin-1. We hypothesized that this channel would provide a pathway for the release of these same cardioprotectants. Preconditioning-isolated perfused rat hearts with 0.4 μM ATP preceding 40 min of ischemia minimized infarct size upon subsequent reperfusion (5% of risk area) and resulted in >80% recovery of left ventricular developed pressure. Postconditioning with ATP after ischemia during reperfusion was also protective (6% infarct and 72% recovery of left ventricular developed pressure). Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X(7) receptors (brilliant blue G and A438079) blocked ATP pre- and postconditioning, indicating that ATP protection was elicited via the opening of a pannexin-1/P2X(7) channel. An antagonist of binding of the endogenous cardioprotectant sphingosine 1-phosphate to its G protein-coupled receptor diminished protection by ATP, which is also consistent with an ATP-dependent release of cardioprotectants. Suramin, an antagonist of binding of ATP (and ADP) to P2Y receptors, was without effect on ATP protection. Benzoyl benzoyl-ATP, a more specific P2X(7) agonist, was also a potent pre- and postconditioning agent and sensitive to blockade by pannexin-1/P2X(7) channel antagonists. The data point out for the first time the potential of P2X(7) agonists as cardioprotectants.     

P2X受体研究进展

P2X受体为配体门控离子通道,属于P2受体家族。P2X受体的配体是ATP,胞外ATP结合时P2X受体通道打开,允许阳离子(Na 、Ca2 等)通过。目前已克隆了7个哺乳动物的P2X(P2X1-7)受体,并阐明了它们的药理学特性。天然P2X受体可以组装成同型或异型聚合体,形成功能性离子通道。对有关P2X受体的结构、分布、功能、生物物理学特性等研究的最新进展进行了综述。     

Temporal Interleukin-1�� Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

The processing and regulated secretion of IL-1β are critical points of control of the biological activity of this important pro-inflammatory cytokine. IL-1β is produced by both monocytes and macrophages, but the rate and mechanism of release differ according to the differentiation status and the origin of these cells. We aimed to study the control of processing and release in human blood monocytes and human monocyte-derived macrophages. Toll-like receptor (TLR)-induced IL-1β production and release were investigated for dependence upon caspase-1, P2X7 receptor activation, and loss of membrane asymmetry associated with microvesicle shedding. TLR agonists induced P2X7 receptor-dependent IL-1β release in both monocytes and macrophages; however, only monocytes also showed P2X7 receptor-independent release of mature IL-1β. Furthermore, in monocytes ATP-mediated PS exposure could be activated independently of IL-1β production. Release of IL-1β from monocytes showed selectivity for specific TLR agonists and was accelerated by P2X7 receptor activation. Human monocytes released more IL-1β/cell than macrophages. These data have important implications for inflammatory diseases that involve monocyte activation and IL-1 release.