A protein whose binding to Na,K-ATPase is regulated by ouabain

Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against alpha1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase alpha1-subunit were detected in the precipitate, and the amount of alpha1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.     

Na/K-ATPase as an oligomeric ensemble

Differences in the kinetic behavior and properties of monomeric and oligomeric forms of membrane-bound Na/K-ATPase are analyzed. It is concluded that enzyme molecules within oligomeric complexes are affected by extrinsic signals that result in change of enzyme activity, whereas the individual (protomeric) state is insensitive to these signals. Some of the major factors of such regulation are microviscosity of the lipid environment, reactive oxygen species, and intracellular protein kinases.     

shASPP2 H22稳转肝癌细胞系的构建及ASPP2敲低对H22移植瘤小鼠血管生成的影响

摘要 目的：构建小鼠shASPP2 H22稳转肝癌细胞系,观察ASPP2敲低对血管生成的影响。方法：针对小鼠ASPP2基因设计了3个不同的shRNA干扰序列（Y18421,Y18422,Y18423）及1个对照序列（GL427NC2）,采用双酶切（Age Ⅰ和EcoR Ⅰ）及质粒连接构建重组质粒,使用菌落PCR和测序比对进行鉴定;使用293T细胞将各重组质粒包装慢病毒并测定滴度;将 shASPP2和对照慢病毒质粒转染H22细胞,采用流式细胞术测定转染效率;采用qRT-PCR、Western Blot法观察shASPP2慢病毒对H22细胞ASPP2的干扰效果;采用CCK8法观察ASPP2敲低对H22细胞增殖的影响;采用Western Blot法观察ASPP2敲低对H22细胞及上清VEGF表达和分泌的影响;采用细胞注射法建立小鼠ASPP2敲低H22细胞皮下移植瘤模型,游标卡尺法观察肿瘤体积大小,采用活体激光共聚焦观察肿瘤血管生成情况,采用Western Blot法观察肿瘤组织VEGF的表达。结果：双酶切、菌落PCR和测序鉴定结果表示各重组质粒构建成功;各重组质粒经慢病毒包装后,测定显示Y18421、Y18422、Y18423和GL427NC2慢病毒质粒的滴度分别为3.40×10⁸ TU/mL、4.08×10⁸ TU/mL、5.49×10⁸ TU/mL和1.7×10⁹ TU/mL;Y18421、Y18422、Y18423及GL427NC2慢病毒质粒转染效率分别为：86.2 %、69.6 %、60.8 %和76.9 %。与GL427NC2 H22细胞相比,Y18421 H22细胞的ASPP2 mRNA及蛋白的表达明显降低（P<0.01,P<0.05）;Y18421细胞在培养24,48,72 h后增殖速率显著增加（P<0.0001,P<0.001,P<0.01）;Y18421细胞及上清的VEGF表达显著升高（P<0.001,P<0.01,P<0.05）。与GL427NC2 细胞移植瘤相比,Y18421细胞移植瘤体积明显增大（P<0.05）,总血管长度显著增加（P<0.05）,VEGF蛋白的表达明显上调（P<0.05）。结论：小鼠shASPP2 H22稳转肝癌细胞系构建成功,ASPP2敲低可能通过上调VEGF的表达促进小鼠H22细胞移植瘤血管生成。     

An Ultralong Lifespan and Low‐Temperature Workable Sodium‐Ion Full Battery for Stationary Energy Storage

Presently, commercialization of sodium‐ion batteries (SIBs) is still hindered by the relatively poor energy‐storage performance. In addition, low‐temperature (low‐T) Na storage is another principal concern for the wide application of SIBs. Unfortunately, the Na‐transfer kinetics is extremely sluggish at low‐T, as a result, there are few reports on low‐T SIBs. Here, an advanced low‐T sodium‐ion full battery (SIFB) assembled by an anode of 3D Se/graphene composite and a high‐voltage cathode (Na₃V₂(PO₄)₂O₂F) is developed, exhibiting ultralong lifespan (over even 15 000 cycles, the capacity retention is still up to 86.3% at 1 A g^?1), outstanding low‐T energy storage performance (e.g., all values of capacity retention are >75% after 1000 cycles at temperatures from 25 to ?25 °C at 0.4 A g^?1), and high‐energy/power properties. Such ultralong lifespan signifies that the developed sodium‐ion full battery can be used for longer than 60 years, if batteries charge/discharge once a day and 80% capacity retention is the standard of battery life. As a result, the present study not only promotes the practicability and commercialization of SIBs but also points out the new developing directions of next‐generation energy storage for wider range applications.     

Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion

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盐碱胁迫下西伯利亚蓼体内无机离子的变化

研究了碳酸氢钠溶液处理后西伯利亚蓼营养器官内部无机离子含量的变化。实验结果表明碳酸氢钠溶液处理前西伯利亚蓼茎部认为是“K+库”;碳酸氢钠溶液处理过程中“K+库”转变成为“Na+库”,以维持叶部的正常生理功能,并且“Na+库”具有一定的限度;碳酸氢钠溶液处理后,西伯利亚蓼不同部位对外界盐碱胁迫的反应存在多样性。植株在“Na+库”达到限度之前进行旺盛的光合作用,在有限的时间内向地下组织输送更多的有机养分,用以繁殖后代。     

Transformation and growth related changes in levels of nuclear and cytoplasmic proteins antigenically related to mammalian beta-galactoside-binding lectin

Immunofluorescence and immunoblotting experiments, using a monoclonal antibody to the 13 kDa mammalian beta-galactoside-binding lectin have shown that human lymphocytes contain nuclear and cytoplasmic proteins of apparent molecular masses of 130, 80, 65 and 13 kDa that are antigenically related to the lectin and whose levels and patterns of expression change in association with transformation, or after stimulation with mitogens. These observations, together with the finding that the myeloid cell line K562 is also rich in the 130 kDa component, whereas the mature granulocytes of normal donors and of patients with chronic myeloid leukaemia are lacking in all of the immunoreactive forms, raise the possibility that this family of lectin-related proteins may be components of growth regulatory systems that are variously elicited in the transformed and stimulated cells.