An Eight Amino Acid Segment Controls Oligomerization and Preferred Conformation of the two Non-visual Arrestins

G protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP₆). IP₆ interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP₆ on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP₆, arrestin-2 forms “infinite” chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP₆; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP₆, the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP₆ effect. Because IP₆ is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins.     

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

零下低温对杂交杨树皮层膜脂组成的影响

以不耐寒的美洲黑杨（Ｐｏｐｕｌｕｓｄｅｌｔｏｉｄｅｓｃｖ．“Ｌｕｘ”Ｉ－６９／５５,父本）和耐寒性较强的欧美杨（Ｐ．ｅｕｒａｍｅｒｉｃａｎａｃｌｃｖ．Ｉ－４５／５１,母本）的４个杂交Ｆ＿１代无性系（９５杨、５５９杨、６００杨和１３８１杨）为材料,分析了零下低温寒潮前后枝条皮层的脂质组成。结果表明,寒潮影响下,皮层中磷脂含量增加而组成基本不变,膜脂脂肪酸组成的变化规律是：寒潮前脂肪酸不饱和指数（ＩＵＦＡ）值大的无性系,寒潮前后的ＩＵＦＡ值变化量小;寒潮前ＩＵＦＡ值较小的无性系,寒潮前后ＩＵＦＡ值变化量较大。本文借用力学概念,提出相对抗性概念,给出杨树无性系的相对抗性序列。序列表明Ｆ＿１代无性系的耐寒性已较不耐寒的父本提高,这与田间观察基本一致。     

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.     

The rate assay of alpha-toxin assembly in membrane

Abstract A rapid and easy method to determine the 'rate' of the assembly of α-toxin from Staphylococus aureus in erythrocyte membrane was described. Upon addition of a small amount of α-toxins into erythrocyte suspension, absorbance at 700 nm decreased linearly after a short period of lag time. From the linear portion of the record the rate of the assembly of α-toxin was calculated. An optimum temperature and an optimum pH for the assembly of the toxin on erythrocyte membranes were found to be 25–30°C and pH 5.     

The Effect of Temperature on the Fatty Acid Composition of Tetrahymena pyriformis WH-14

SYNOPSIS. A reduction in the growth temperature of Tetrahymena pyriformis strain WH-14 from 35 C to 15 C resulted in distinct alterations in the fatty acid composition of the glycerophospholipids. The proportion of normal saturated acids declined from 26 to 19%; palmitoleic acid increased by 6%, and the composition of the polyunsaturated fatty acids increased in 18:2 Δ^6,11(n) and decreased in 18:2 Δ^9,12(n) and 18:3 Δ^6,9,12(n). The unsaturation index (the average number of double bonds/100 molecules) did not change with a shift in temperature.
Two biosynthetic pathways exist in Tetrahymena for the formation of unsaturated fatty acids. The observed changes in fatty acid composition that accompany a lowering of the environmental temperature can be accounted for by a reduction in the accumulation of products of the fatty acid pathway leading to the formation of γ-linolenic acid [16:0(n) → 18:0(n) → 18:1 Δ⁹(n) → 18:2 Δ^9,12(n) → 18:3 Δ^6,9,12(n)] and an increase in the components of the pathway leading to the formation of 18:2 Δ^6,11(n) [16:0(n) → 16:1 Δ⁹(n) → 18:1 Δ¹¹(n) → 18:2 Δ^6,11(n)]. The data suggest that the regulatory mechanism in Tetrahymena differs from that found in some bacteria where a simple substitution of unsaturated fatty acids for saturated fatty acids occurs at low culture temperatures.     

饲料中n-3 HUFA含量对花尾胡椒鲷亲鱼的生殖性能及血浆性类固醇激素水平季节变化的影响

以n-3 HUFA含量为0．16％、1．27％、2．36％和3．47％的4种人工配合饲料(D1～D4)及天然饲料(冰鲜杂鱼,D5)饲养花尾胡椒鲷(Plectorhynchus cinctus)雌亲鱼一周年,通过比较各饲料组亲鱼的产卵量、卵和仔鱼质量以及各月份的血浆性类固醇激素水平,研究n-3 HUFA对生殖性能及性类固醇激素水平季节性变化的影响。平均每kg雌鱼的产卵量、卵受精率、仔鱼存活率、开口仔鱼体长等,D2和D3组与D5组相近,但D1和D4组显著低于D5组;饲料中n-3 HUFA含量对血浆17β-雌二醇(E2)和睾酮(T)水平的季节性变化规律无明显影响,但亲鱼性腺发育与成熟时期的E2和T水平．D1和D4组较D5组显著降低。n-3 HUFA含量对亲鱼离体卵泡E2和T的分泌也有一定影响：D4组卵泡E2的基础分泌很少;绒毛膜促性腺激素(HCG,100IU／mL)可刺激D2-D5组卵泡分泌E2和T,但D1组卵泡对HCG无反应。结果提示,花尾胡椒鲷亲鱼饲料中n-3 HUFA的适宜含量为1．27％-2．36％,不足或过高对亲鱼的生殖性能均有不利影响;通过影响性类固醇激素的产生可能是饲料中n-3 HUFA含量影响鱼类生殖性能的机制之一。     

饲料中添加裂殖壶菌粉对凡纳滨对虾脂质沉积和血清生化成分的影响

以健康的凡纳滨对虾(Litopenaeus Vannamei)[初重(5.810.24) g]为研究对象,研究裂殖壶菌粉(Extracted-and-dried microfungi Schizochytrium sp.,EDMS)对凡纳滨对虾脂质转运、沉积以及血清生化成分的影响。在饲料中分别添加0、0.5%、1.0%、1.5%和2.0%的裂殖壶菌粉,制成5组(基础组、EMDS05、EMDS10、EMDS15、EMDS20)等氮等能饲料。将750尾健康的凡纳滨对虾随机分成5组(为每组3个平行,每个平行45尾虾),养殖时间45d。结果表明,随着饲料中DHA水平的提高,肌肉中水分、粗脂肪和粗蛋白质含量均无显著改变(P0.05),但灰分随着添加量的增加呈现上升趋势,且EMDS20组显著高于基础组和EMDS05组(P0.05);肌肉和肝胰脏中高不饱和脂肪酸(HUFA)含量显著升高(P0.05),血清总甘油三酯(TG)含量显著升高(P0.05),总胆固醇(TC)含量保持稳定(P0.05),EDMS15组高/低密度脂蛋白(HDL/LDL)显著高于其他各组(P0.05);超氧化物歧化酶(SOD)呈现先下降后升高的趋势,其中EMDS10组(1.0%添加组)显著低于其他各组(P0.05);丙二醛(MDA)含量显著升高(P0.05)。综上所述,饲料中添加EDMS促进了凡纳滨对虾肌肉中n-3 HUFA的沉积,同时也提高了虾体脂质过氧化压力。     

不饱和土壤CH4的吸收与氧化

不饱和土壤是已知唯一的 CH4 生物壑。综述了不饱和土壤 CH4 的吸收、氧化过程及其影响因素。不饱和土壤中 CH4 氧化的临界浓度低 ,因而甲烷氧化菌可氧化大气 CH4 并将其当作唯一的碳源和能源。土壤 CH4 吸收率与土壤湿度通常呈负相关关系。土壤湿度过高 ,大气 CH4 和 O2 向土壤中扩散受阻 ;或土壤湿度过低引起水分胁迫均导致甲烷氧化菌活性下降。NH 4对土壤中 CH4 氧化的抑制作用可归结为 NH3和 CH4 在甲烷单氧酶水平上的竞争、由氧化作用向硝化作用的转移以及 NH 4氧化生成的 NO- 2 的毒性。NH 4对 CH4 氧化的抑制作用与土壤有效氮含量成正比。各类氮肥对 CH4 氧化抑制作用 :化肥 >有机肥 ;铵态氮肥 >尿素。 NO- 3对 CH4 氧化没有抑制效应。阳离子代换量 (CEC)高的土壤 NH 4对 CH4 氧化的抑制作用轻。 CH4 氧化菌对大气 CH4 的高亲和力及 CH4 氧化所需较低的活化能导致其温度系数 Q1 0 较小。地温较低时 ,土壤氧化 CH4 的能力随温度升高而升高。当地温高于 CH4 氧化的最佳温度时 ,CH4 氧化菌难以与硝化细菌及其它微生物竞争利用土壤空气中的 O2 ,导致其活性降低。甲烷氧化菌对 p H值变化不敏感。团粒结构较好的壤土可保护 CH4 氧化菌免受干扰。未受干扰的森林土壤 CH4 氧化率的峰值一般出现在亚表     

Chaperone-Like Activity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Hsp16.3 Does Not Require Its Intact (Native) Structures

Small heat shock proteins (sHsps) were found to exhibit efficient chaperone-like activities under stress conditions although their native structures are severely disturbed. Here, using an alternative approach (site-directed mutagenesis), we obtained two structurally and functionally distinct Mycobacterium tuberculosis Hsp16.3 single-site mutant proteins. The G59W mutant protein (with Gly59 substituted by Trp) is capable of exhibiting efficient chaperone-like activity even under non-stress conditions although its secondary, tertiary, and quaternary structures are very different from that of the wild type protein. By contrast, the G59A mutant protein (with Gly59 substituted by Ala) resembles with the wild type protein in structure and function. These observations suggest that the Gly59 of the Hsp16.3 protein is critical for its folding and assembly. In particular, we propose that the exhibition of chaperone-like activity for Hsp16.3 does not require its intact (native) structures but requires the disturbance of its native structures (i.e., the native structure-disturbed Hsp16.3 retains its chaperone-like activity or even becomes more active). In addition, the behavior of such an active mutant protein (G59W) also strongly supports our previous suggestion that Hsp16.3 exhibits chaperone-like activity via oligomeric dissociation.