核糖体S6蛋白激酶p90rsk与卵母细胞减数分裂

丝裂原活化蛋白激酶(MAPK)信号途径对减数分裂有重要调节作用,p90rsk是迄今研究最清楚的MAPK下游靶分子,介导MAPK途径在卵母细胞减数分裂中的多种功能,包括卵母细胞减数分裂的启动、MⅠ/MⅡ期转化和MⅡ期阻滞的维持等.p90rsk的磷酸化是MAPK激活的结果,而细胞退出减数分裂时,p90rsk的去磷酸化也发生在MAPK失活以后.介绍了在卵母细胞中p90rsk的研究进展.     

p16INK4a基因的功能及其调控

p16^INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16^INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16^INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16^INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

磷酸化和脱磷酸化对牛脑63kD环核苷酸磷酸二酯酶同工酶活性的调节

本文报道了CaM依赖性磷酸化和脱磷酸化对牛脑63kD PDE同工酶活性的调节作用。实验结果如下:(1)在存在Ca²⁺和CaM时,提纯的牛脑Ca²⁺/CaM-PK Ⅱ能催化牛脑63kD PDE同工酶磷酸化。最大磷酸参入量是1mol/mo1亚基;(2)在Ni²⁺和CaM存在时,提纯的牛脑钙调神经磷酸酶能催化磷酸化型63kDPDE同工酶脱磷酸化;(3)CaH²⁺对磷酸化型63kD PDE同工酶的半激活浓度(AC₅₀)高于非磷酸化型。     

p14ARF对人黑色素瘤细胞增殖的影响及其作用机理的初探

ARF(alternative reading frame)作为INK4a/ARF的β转录产物,能够稳定p53, 诱导细胞周期阻断或凋亡.利用高表达p14^ARF的人黑色素瘤细胞模型,探讨了ARF抑制细胞增殖的分子作用机理.研究发现p14^ARF高表达能将细胞周期阻断在G1和G2期, p53, p21^cip1和p27^kip1蛋白水平明显增强, 而p-ERK1/2,CyclinD1和CyclinE蛋白水平下降, 明显抑制细胞生长. 提示p14^ARF能通过ERK(extracellular signal-regulated kinase)信号通路相互协调作用抑制A375细胞增殖.     

p21WAF1在丁酸钠诱导的人成纤维细胞凋亡中的表现

用丁酸钠（NaBu）诱导了人胚肺二倍体成纤维细胞凋亡（2BS）,检测其诱导过程中凋亡相关基因的表达变化,结果表明,p21^WAF1的表达在凋亡发生前即有明显下降,并持续至凋亡发生时, bcl-2的表达仅在凋亡发生时有所下降,c-myc和c-fos的表达有所上升,而p53和HER-2的表达无明显变化.用稳定转染了不同长度p21^WAF1启动子片段和下游绿色荧光蛋白（GFP）报告基因的2BS-WP系列细胞进一步研究发现,其GFP的表达水平在NaBu诱导过程中下降,主要调控区域为p21^WAF1启动子的TATA box上游0～－800 bp.说明NaBu诱导的人胚肺二倍体成纤维细胞凋亡与p21^WAF1启动子的转录活性下降与密切相关,并且可能不依赖于p53.     

细胞内信号分子传导的研究进展

近年来有关细胞内信号传导的研究,着重体现在Ca²⁺信号传导途径及相应的蛋白质分子如蛋白激酶C(PKC)、钙调素(CaM)、钙调素激酶Ⅱ(CaMKⅡ),同时也对Ras途径中出现的Vav、Rap、Crk、C3G等蛋白质分子以及cAMP和NF-κB途径作了有益的补充与修改.细胞外信号分子通过以上4种途径及其相互通讯(cross-talk),激活了某些蛋白激酶,调控了基因转录及其他相关功能,其中磷酸化对蛋白激酶及转录因子活性的调节起到了非常重要的作用.     

ARF——一种新的抑癌因子的研究进展

INK4a/ARF基因位于人染色体9p21,是人类肿瘤中最常见的基因失活位点之一.INK4a/ARF基因有两套各自独立的启动子,通过可变阅读框,能够编码两种蛋白质：p16^INK4a和p14^ARF（ARF在鼠细胞中为p19^ARF）.p16作为CDK4/6的抑制因子,能够阻断pRb磷酸化,将细胞周期阻断在G1期;而ARF可结合原癌蛋白MDM2,稳定p53,将细胞周期阻断在G1期和G2/M转换期,或诱导细胞凋亡.因此ARF蛋白和p16一样也是一种肿瘤抑制因子.     

蛋白激酶在卵母细胞减数分裂和受精中的作用

脊椎动物卵母细胞的减数分裂和受精过程受到多种蛋白激酶的调节。近年来对于卵母细胞成熟、活化和受精的分子机制研究取得了长足进步 ,发现促成熟因子 (MPF)和促分裂原活化蛋白激酶 (MAPK)是调节卵母细胞细胞周期的关键分子 ,二者的激活和失活导致了减数分裂的恢复、阻滞和完成。许多蛋白激酶通过调节MPF和MAPK活性来影响减数分裂。Polo like激酶活化MPF ,Mos激活MAPK而启动成熟分裂并维持中期阻滞。CaMKII通过泛素途径灭活MPF使卵突破MII期阻滞。另外 ,p90 rsk作为MAPK的下游分子参与减数分裂调节 ,蛋白激酶C(PKC)诱导皮质颗粒排放并抑制MAPK激活 ,酪氨酸蛋白激酶家族成员介导受精诱发的Ca2 释放。这些蛋白激酶的协同作用推动了卵母细胞正常的成熟与受精     

G1阻滞中p21-E2F间的相互作用

前列腺素A2(PGA2)具有强的体内、外抗增殖活性,引起细胞周期阻滞,同时,可诱导cdk抑制物p21蛋白的表达,后者亦可介导多种细胞的G1阻滞.提示p21^waf1/cip1在PGA2诱导的细胞周期阻滞中具有重要作用.主要介绍了近两年来有关p21^waf1/cip1与转录因子E2F间的相互作用的研究,阐述p21^waf1/cip1在PGA2介导的细胞周期阻滞中的作用机制.     

基于Ti4+-IMAC富集的分枝菌酸小杆菌深度覆盖磷酸化蛋白质组研究

【目的】本研究以分枝菌酸小杆菌(Mycolicibacterium smegmatis)为研究对象,探索适于原核微生物理想的磷酸化富集方法。【方法】我们比较了二氧化钛(TiO₂)、Fe³⁺-NTA和Ti⁴⁺螯合在磷酸酯修饰的固相微球(Ti⁴⁺-IMAC) 3种不同富集方法磷酸化肽段的富集效率,并用不同分辨率的质谱仪评估富集稳定性。【结果】Ti⁴⁺-IMAC富集效率最高,磷酸化位点数是TiO₂或Fe³⁺-NTA方法的7倍以上;TiO₂和Fe³⁺-NTA方法富集到的磷酸化位点数相差不大,与已报道的用TiO₂方法富集的磷酸化位点数目接近。Ti⁴⁺-IMAC富集结果稳定性很好,高分辨率Lumos质谱仪鉴定到的磷酸化位点数是Velos的2.6倍。【结论】本研究较高效地实现了分枝菌酸小杆菌磷酸化事件的鉴定,共鉴定到2 280个磷酸化蛋白、10 880个磷酸化肽段及4 433个可信磷酸化位点,有望用于其他微生物的磷酸化蛋白质组学研究。     

Na+/K+-ATPase resistance and cardenolide sequestration: basal adaptations to host plant toxins in the milkweed bugs (Hemiptera: Lygaeidae: Lygaeinae)

Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na⁺/K⁺-ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na⁺/K⁺-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na⁺/K⁺-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.     

Na(+) interaction with the pore of Shaker B K(+) channels: zero and low K(+) conditions.

The Shaker B K(+) conductance (G(K)) collapses (in a reversible manner) if the membrane is depolarized and then repolarized in, 0 K(+), Na(+)-containing solutions (Gómez-Lagunas, F. 1997. J. Physiol. 499:3-15; Gómez-Lagunas, F. 1999. Biophys. J. 77:2988-2998). In this work, the role of Na(+) ions in the collapse of G(K) in 0-K(+) solutions, and in the behavior of the channels in low K(+) was studied. The main findings are as follows. First, in 0-K(+) solutions, the presence of Na(+) ions is an important factor that speeds the collapse of G(K). Second, external Na(+) fosters the drop of G(K) by binding to a site with a K(d) = 3.3 mM. External K(+) competes, in a mutually exclusive manner, with Na(o)(+) for binding to this site, with an estimated K(d) = 80 microM. Third, NMG and choline are relatively inert regarding the stability of G(K); fourth, with [K(o)(+)] = 0, the energy required to relieve Na(i)(+) block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707-724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539-550) decreases with the molar fraction of Na(i)(+) (X(Na,i)), in an extent not accounted for by the change in Delta(mu)(Na). Finally, when X(Na,i) = 1, G(K) collapses by the binding of Na(i)(+) to two sites, with apparent K(d)s of 2 and 14.3 mM.     

Influence of exogenous application of glycinebetaine on antioxidative system and growth of salt-stressed soybean seedlings (Glycine max L.)

Glycinebetaine is one of the most competitive compounds which play an important role in salt stress in plants. In this study, the enhanced salt tolerance in soybean (Glycine max L.) by exogenous application of glycinebetaine was evaluated. To improve salt tolerance at the seedling stage, GB was applied in four different concentrations (0, 5, 25 and 50 mM) as a pre-sowing seed treatment. Salinity stress in the form of a final concentration of 150 mM sodium chloride (NaCl) over a 15 day period drastically affected the plants as indicated by increased proline, MDA and Na⁺ content of soybean plants. In contrast, supplementation with 50 mM GB improved growth of soybean plants under NaCl as evidenced by a decrease in proline, MDA and Na⁺ content of soybean plants. Further analysis showed that treatments with GB, resulted in increasing of CAT and SOD activity of soybean seedlings in salt stress. We propose that the role of GB in increasing tolerance to salinity stress in soybean may result from either its antioxidant capacity by direct scavenging of H₂O₂ or its role in activating CAT activity which is mandatory in scavenging H₂O₂.     

Chloroplast-generated ROS dominate NaCl- induced K+ efflux in wheat leaf mesophyll

Mesophyll K⁺ retention ability has been recently reported as an important component of salinity stress tolerance in wheat. In order to investigate the role of ROS in regulating NaCl^-induced K⁺ efflux in wheat leaf mesophyll, a series of pharmacological experiments was conducted using MV (methyl viologen, superoxide radical inducer), DPI (an inhibitor of NADPH oxidase), H₂O₂ (to mimic apoplastic ROS), and EGCG ((−)-Epigallocatechin gallate, ROS scavenger). Mesophyll pre-treatment with 10 μM MV resulted in a significantly higher NaCl^-induced K⁺ efflux in leaf mesophyll, while 50 μM EGCG pre-treatment alleviated K⁺ leakage under salt stress. No significant change in NaCl^-induced K⁺ efflux in leaf mesophyll was found in specimens pre-treated by H₂O₂ and DPI, compared with the control. The highest NaCl^-induced H⁺ efflux in leaf mesophyll was also found in samples pre-treated with MV, suggesting a futile cycle between increased H⁺-ATPase activity and ROS-induced K⁺ leak. Overall, it is suggested that, under saline stress, K⁺ efflux from wheat mesophyll is mediated predominantly by non-selective cation channels (NSCC) regulated by ROS produced in chloroplasts, at least in bread wheat.     

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca²⁺-activated K⁺ channel (BK_Ca, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BK_Ca channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BK_Ca channel. Calculation of an electrostatic surface map of the BK_Ca gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BK_Ca channel activation gating.     

Spatial,Cellular, and Intracellular Localization of Na+/K+-ATPase in the Sterically Disposed Renal Tubules of Japanese Eel

The kidney plays a crucial role in the regulation of water and ion balances in both freshwater and seawater fishes. However, the complicated structures of the kidney hamper comprehensive understanding of renal functions. In this study, to investigate the structure of sterically disposed renal tubules, we examined spatial, cellular, and intracellular localization of Na⁺/K⁺-ATPase in the kidney of the Japanese eel. The renal tubule was composed of the first (PT-I) and second (PT-II) segments of the proximal tubule and the distal tubule (DT), followed by the collecting ducts (CDs). Light microscopic immunocytochemistry detected Na⁺/K⁺-ATPase along the renal tubules and CD; however, the subcellular distribution of the Na⁺/K⁺-ATPase immunoreaction varied among different segments. Electron microscopic immunocytochemistry further revealed that Na⁺/K⁺-ATPase was distributed on the basal infoldings of PT-I, PT-II, and DT cells. Three-dimensional analyses showed that the renal tubules meandered in a random pattern through lymphoid tissues, and then merged into the CD, which was aligned linearly. Among the different segments, the DT and CD cells showed more-intense Na⁺/K⁺-ATPase immunoreaction in freshwater eel than in seawater-acclimated eel, confirming that the DT and CD segments are important in freshwater adaptation, or hyperosmoregulation. (J Histochem Cytochem 58:707–719, 2010)     

Loss of caveolin-1 impairs retinal function due to disturbance of subretinal microenvironment

Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K(+) affinity of the RPE-expressed α1-Na(+)/K(+)-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis.     

Overexpression of SeNHX1 improves both salt tolerance and disease resistance in tobacco

Recently, we found NHX1, the gene encoding a Na⁺/H⁺ exchanger, participated in plant disease defense. Although NHX1 has been confirmed to be involved in plant salt tolerance, whether the NHX1 transgenic plants exhibit both salt tolerance and disease resistance has not been investigated. The T1 progenies of Nicotiana tabacum L. lines expressing SeNHX1 (from Salicornia europaea) were generated for the present study. Compared with PBI-type control plants, SeNHX1 transgenic tobaccos exhibited more biomass, longer root length, and higher K⁺/Na⁺ ratio at post germination or seedling stage under NaCl treatment, indicating enhanced salt tolerance. The vacuolar H⁺ efflux in SeNHX1 transgenic tobacco was increased after treatment of NaCl with different concentration. Meanwhile, the SeNHX1 transgenic tobaccos showed smaller wilted spot area, less H₂O₂ accumulation in leaves after infection of Phytophthora parasitica var. nicotianae. Further investigation demonstrated a larger NAD(P)(H) pool in SeNHX1 transgenic tobacco. These evidences revealed that overexpression of SeNHX1 intensified the compartmentation of Na⁺ into vacuole under salt stress and improved the ability of eliminating ROS after pathogen attack, which then enhanced salt tolerance and disease resistance simultaneously in tobacco. Our findings indicate NHX1 has potential value in creating crops with both improved salt tolerance and disease resistance.     

The Detailed Localization Pattern of Na+/K+/2Cl? Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na⁺/K⁺/2Cl⁻ cotransporter type 2 (NKCC-2) and Na⁺/K⁺-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na⁺/K⁺-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na⁺/K⁺-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)