Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Cytokinin biochemistry in relation to leaf senescence I. The metabolism of 6-benzylaminopurine and zeatin in oat leaf segments

Journal of Plant Growth Regulation - The metabolism of zeatin and that of 6-benzylaminopurine (BAP) have been compared in oat leaf segments in relation to the markedly differing ability of these...     

壳聚糖固定化木瓜蛋白酶的研究

以壳聚糖为载体,成二醛为交联剂将木瓜蛋白酶固定化。5％戊二醛在4-6℃下处理载体5h,加酶液(3.5mg/mL蛋白,pH7.2)固定12h,活力回收达32％,作用于酪蛋白的半衰期为36天,其表观K_m(酪蛋白)值为0.075％(W/V),溶液酶的K_m值为0.086％;最适pH7.0～7.5,溶液酶为7.0～8.5。固定化酶在pH8.5以下,溶液酶在9.0以下活力稳定。固定化酶在45℃以下,溶液酶在75℃以下稳定。用6mol/L脲洗脱固定化酶4次(5.5h)活力仍有54.5％。用固定化酶处理啤酒浊度比对照下降了1.5-3.7倍,蛋白质含量下降了44％,冷藏(4℃)120天无冷混浊现象发生并保持了啤酒原有风味和理化性状。     

斑点酶免疫实验检测流行性出血热抗体方法的研究

本文应用斑点酶免疫试验(DEIA),检测流行性出血热病人血清42份,其结果与间接免疫荧光(IFA)进行了比较,符合率100％,其DEIA几何干均滴度为1:880,IFA为1:580,同类风湿因子阳性血清[RF(+)]无交叉反应,证明本方法具有较高的敏感性和特异性,本文还对不同方法处理组织内源酶的效果和不同阻断剂的阻断效果进行了比较。试验结果表明本法不但敏感、特异,而且简单、快速、安全、经济、易于推广。     

Differences in the In Vitro and In Vivo 5-Hydroxytryptamine Extraction Performance Among Three Common Microdialysis Membranes

The present study summarizes the results of an in vitro and in vivo comparison of the apparent 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid, and 3,4-dihydroxyphenylacetic acid dialysis performance of three types of membrane frequently used in intracerebral microdialysis experiments. The dialysis fiber types examined were a regenerated cellulose Cuprophan (GF), a proprietary polycarbonate ether (CMA), and a polyacrylonitrile/sodium methallylsulfonate copolymer (HOSPAL). The experiments unexpectedly revealed that the HOSPAL membrane-equipped probes displayed clearly aberrant 5-HT diffusion dynamics compared with GF and CMA probes, demonstrable not only in vitro, but also in in vivo experiments. In vitro, the GF and CMA membrane-equipped probes exhibited maximum relative recovery for 5-HT already in the first 20-min sample, whereas the 5-HT recovery of HOSPAL probes increased in a very slow and protracted manner over a period of a little less than 2 h. The GF and CMA probes further displayed an immediate washout of 5-HT when the probes were subsequently transferred to artificial CSF only-containing medium (no 5-HT), whereas approximately 2 h was required to yield near-total extinction of dialysate 5-HT with the standard HOSPAL probes. In vivo, the rat ventral hippocampal dialysate 5-HT output responses to K+ (100 mM) infusion, to Ca2+ omission, and to systemic 8-hydroxy-2-(di-n-propylamino)tetralin injection were all markedly retarded and blunted when HOSPAL instead of GF membrane-equipped probes were used. However, the 5-hydroxyindoleacetic acid and 3,4-dihydroxyphenylacetic acid extraction in vitro and in vivo were comparable using either of the membrane types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measuring the microelastic properties of biological material.

We have used the atomic force microscope (AFM) to measure the local rigidity modulus at points on the surface of a section of hydrated cow tibia. These data are obtained either from contrast changes that occur as the contact force is altered, or from force versus distance curves obtained at fixed points. These two methods yield the same values for rigidity modulus (at a given point). At low resolution, the elastic morphology and topography mirror the features seen in optical and electron micrographs. At high resolution we see dramatic variations in elastic properties across distances as small as 50 nm.     

Effects of 2,3-diphosphoglyceric acid on the human erythrocyte membrane phosphorylation system

The effect of 2,3-diphosphoglycerate (2,3-P2-glycerate) on the phosphorylation of spectrin in solution by purified membrane cyclic AMP-independent protein kinase and in membrane preparations by the endogenous kinase was investigated. 2,3-P2-Glycerate inhibited spectrin phosphorylation both in solution and in the intact membrane. Kinetic analyses showed that 2,3-P2-glycerate had no effect on the Km for ATP but appeared to lower the Vmax of the reaction. When the effect of 2,3-P2-glycerate was examined in the presence of varying concentrations of spectrin, competitive inhibition kinetics were obtained. Interestingly, low concentrations of 2,3-P2-glycerate were found to effect the release of the membrane kinase from erythrocyte membranes. This release reaction may be related to the ability of 2,3-P2-glycerate to interfere with the interaction between the kinase and spectrin. The data suggest the possibility that the kinase may be bound to spectrin in the erythrocyte membrane. 2,3-P2-glycerate also caused the solubilization of 3-phosphoglyceraldehyde dehydrogenase, but not of cyclic AMP-dependent protein kinase. Taken together, our data indicate that 2,3-P2-glycerate may have a regulatory role in membrane protein phosphorylation and also may regulate the extent of association of the kinase with the membrane.     

Inhibition of protein phosphorylation and induction of protein cross-linking in erythrocyte membranes by diamide

This report presents studies on the effect of diamide on protein phosphorylation in erythrocyte membranes. Diamide, a thiol-oxidizing reagent, nonspecifically inhibits cyclic Amp-dependent and -independent autophosphorylation of red cell memvranes, but not the activity of the solubilized membrane cycle AMP-independent protein kinases. Analysis of diamide-treated membranes by gel electrophoresis indicates that diamide is capable of inducing cross-linking of membrane proteins. The action of diamide, both in the inhibition of membrane autophosphorylation and in the cross-linking of membrane proteins, is very similar to that of Cu2+. o-phenanthroline complex. Our data indicate that diamide inhibits erythrocyte membrane autophosphorylation by perturbing the protein substrates.     

气候、植被及土壤因素交互作用对宁夏土壤有机碳的影响机制

基于宁夏全区121个土壤剖面样点,研究不同深度(0—30、30—80、80—120cm)土壤有机碳的空间分布特征,通过相关性、方差分解和构建结构方程模型,分析气候、植被和土壤因素对宁夏不同深度土壤有机碳的影响及其作用途径。结果表明：(1)宁夏不同深度土壤有机碳含量均呈现中间低南北高的空间分布趋势,0—120cm剖面土壤有机碳含量随着土壤深度的增加而降低,土壤有机碳含量均值为5.49g/kg,变异系数达90.71%,含量偏低且空间异质性强。(2)各土层土壤有机碳与年均气温、干燥度、碳酸钙、pH均呈现极显著的负相关性(P<0.01),与年均降水量、相对湿度、植被净初级生产力、全氮、全磷、全钾、钙离子、阳离子交换量、粘粒含量均呈现极显著的正相关性(P<0.01)。(3)各因素对不同土层土壤有机碳的作用方式和影响程度有差异。土壤全氮、全磷、碳酸钙、阳离子交换量均能直接影响土壤有机碳,年均气温和植被净初级生产力对土壤有机碳的直接影响效应不显著,主要通过土壤属性间接影响土壤有机碳含量。随着土层深度的增加,气候和植被因素作用明显减弱,土壤因素作用增强并成为主要影响因素。该结果有助于宁夏土...     

抗菌药对鲍曼不动杆菌外膜囊泡产量及主要生物学特性的影响

【背景】细菌耐药性已成为全球健康卫生和经济发展的巨大威胁。替加环素是治疗多重耐药肠杆菌所致严重感染的主要药物之一,但在2019年发现了可介导其高水平耐药的可转移替加环素耐药基因tet（X3）。外膜囊泡作为介导水平基因转移的新型方式,在介导tet（X3）水平转移中的作用目前尚无报道。【目的】以tet（X3）阳性替加环素耐药鲍曼不动杆菌34AB为对象,探究不同抗菌药物对其外膜囊泡产量及主要生物学特性的影响。【方法】采用微量肉汤稀释法测定细菌药物敏感性,超速离心法提取细菌外膜囊泡,BCA法测定外膜囊泡产量,使用马尔文纳米粒度电位仪测定外膜囊泡的粒径与电位,PCR法（定性）及RT-qPCR法（定量）检测外膜囊泡中携带的tet（X3）基因。【结果】相较于无抗生素对照组[（0.64±0.04） mg/mL],在不同抗菌药物亚抑菌浓度（1/2 MIC和1/4 MIC）处理后,34AB外膜囊泡的产量均有所增加,以头孢他啶[1/2 MIC,（2.83±0.57） mg/mL;1/4 MIC,（2.38±0.29） mg/mL]和美罗培南[1/2 MIC,（2.19±0.11） mg/mL;1/4 MIC,（1.96±0.37） mg/mL]作用最为显著（p<0.01）。同时抗菌药物作用后,各组外膜囊泡粒径和电位均有所降低,而携带的tet（X3）基因拷贝数均有所上升（2.80×10⁴-2.63×10⁷copies/μL）。【结论】抗菌药物的临床应用可能会导致耐药细菌外膜囊泡产量及携带的耐药基因丰度增加,进而增强其作为水平基因转移载体传播耐药基因的风险。