Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

The Influence of Low Protein Diet on the Composition of Rat Liver Cell Fractions

Rats were divided into high protein diet and low protein diet fed groups. Their livers were removed, homogenized, and fractionated into nuclei, mitochondria, microsome and supernatant fraction.

Amount of total- and phospho-lipids in each fraction was measured, and simultaneously, total nitrogen was measured. Then, the ratio of total- and phospho-lipids to nitrogen was calculated. The influence of low protein diet on this calculated value was seen in the mitochondrial and supernatant fraction but was not recognized in other fractions.

The relationship between this phenomenon and energy efficiency of diets was discussed.     

Effects of Taurine on Calcium Ion Uptake and Protein Phosphorylation in Rat Retinal Membrane Preparations

The effects of taurine on ATP-dependent calcium ion uptake and protein phosphorylation of rat retinal membrane preparations were investigated. Taurine (20 mM) stimulates ATP-dependent calcium ion uptake by twofold in crude retinal homogenates. In contrast, it inhibits the phosphorylation of specific membrane proteins as shown by acrylamide gel electrophoresis and autoradiography. The close structural analogue of taurine, 2-aminoethylhydrogen sulfate, demonstrates similar effects in both systems, i.e., stimulation of ATP-dependent calcium ion uptake and inhibition of protein phosphorylation, whereas isethionic acid and guanidinoethanesulfonate have no effect on either system. A P1 subcellular fraction of the retinal membrane preparation that contains photoreceptor cell synaptosomes has a higher specific activity for the uptake of calcium ions. Phosphorylation of specific proteins in the P1 fraction is also inhibited by the addition of 20 mM taurine. Taurine has no effect on retinal ATPase activities or on phosphatase activity, thus suggesting that it directly affects a kinase system.     

Effects of Melatonin on Carbonic Anhydrase from Human Erythrocytes In Vitro and from Rat Erythrocytes In Vivo

The in vitro effects of melatonin (N-acetyl-5-methoxytryptamine) on human carbonic anhydrase isozymes (HCA-I and HCA-II) from human erythrocytes and in vivo effects on rat erythrocytes carbonic anhydrase (CA) were determined. Human erythrocyte carbonic anhydrase isozymes were purified by haemolysate preparation and Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The HCA-I enzyme, having a specific activity of 7337.5?EU/mg protein, was purified 843-fold with a yield of 60% and the HCA-II enzyme, having a specific activity of 17067?EU/mg protein, was purified 1962-fold with a yield of 22.7%. For in vitro experiments, the enzyme activity was minimal at 2×10-4?M melatonin concentration and increased above this concentration. Ten mg?kg-1 melatonin was administered intraperitoneally and showed a stimulatory effect on the enzyme. Time-dependent in vivo studies were conducted for melatonin in Sprague–Dawley type rats. It was found that CA activity in the rat erythrocytes was decreased by the melatonin after 1 and 3 hours to 2500±500.0 and 1875±239.4 respectively which were statistically significant (p<0.05) differences to the control (2660±235.8). However, CA activity was restored to its normal level after 6?h (2666±235.7) (p>0.05) probably due to metabolism of the melatonin. The findings indicate that melatonin may be pharmacologically useful in some diseases.     

Protective Effects of Ferulic Acid on High Glucose-Induced Protein Glycation,Lipid Peroxidation,and Membrane Ion Pump Activity in Human Erythrocytes

Ferulic acid (FA) is the ubiquitous phytochemical phenolic derivative of cinnamic acid. Experimental studies in diabetic models demonstrate that FA possesses multiple mechanisms of action associated with anti-hyperglycemic activity. The mechanism by which FA prevents diabetes-associated vascular damages remains unknown. The aim of study was to investigate the protective effects of FA on protein glycation, lipid peroxidation, membrane ion pump activity, and phosphatidylserine exposure in high glucose-exposed human erythrocytes. Our results demonstrated that FA (10-100 μM) significantly reduced the levels of glycated hemoglobin (HbA1c) whereas 0.1-100 μM concentrations inhibited lipid peroxidation in erythrocytes exposed to 45 mM glucose. This was associated with increased glucose consumption. High glucose treatment also caused a significant reduction in Na⁺/K⁺-ATPase activity in the erythrocyte plasma membrane which could be reversed by FA. Furthermore, we found that FA (0.1-100 μM) prevented high glucose-induced phosphatidylserine exposure. These findings provide insights into a novel mechanism of FA for the prevention of vascular dysfunction associated with diabetes.     

低剂量氯胺酮对工作记忆的影响

氯胺酮是一种N-甲基-D-天冬氨酸受体（NMDAR）阻滞剂，低剂量氯胺酮具有良好的镇痛、抗炎和抗抑郁作用，近年来受到了广泛的关注。但是低剂量氯胺酮对于高级认知功能的影响尚未全面阐明。工作记忆是涉及众多复杂认知活动的关键功能，有研究显示低剂量氯胺酮急性或慢性使用均会损伤工作记忆，其神经机制也开始受到关注。深入分析低剂量氯胺酮对于工作记忆的影响及其机制对于指导氯胺酮的临床使用具有重要意义。本文首先介绍了低剂量氯胺酮作用于神经系统的药理作用途径，以及工作记忆本身的神经环路机制，进而回顾了近年来关于低剂量氯胺酮对工作记忆的急性和慢性作用的相关研究，并重点分析了低剂量氯胺酮损伤工作记忆的可能的神经机制，希望对低剂量氯胺酮在临床中的合理使用提供科学依据。     

大鼠脊髓细胞膜胰岛素受体的结合特性

大鼠脊髓细胞膜胰岛素受体的结合特性朱尚权,徐明华,张新堂,叶莺（中国科学院上海生物化学研究所,２０００３１）姜新建,林淑琼（上海市第一人民医院康复科,２０００８５）关键词胰岛素受体,脊髓细胞膜免疫组织化学、放射免疫自显影和放射受体测定技术已证明脑各区...     

奥利亚罗非鱼DMO和DMT蛋白二级结构和B细胞抗原表位的预测

以DMO和DMT氨基酸序列为基础,采用Garnier-Robson法、Chou-Fasman法和Karplus-Schulz法预测蛋白质的二级结构;按Kyte-Doolittle法、Emini法和Jameson-Wolf法预测DMO和DMT蛋白的B细胞抗原表位。预测结果表明：在DMO蛋白N-端第80～112,144～147,193～194,251～255,260～269区段和279～283区段,DMT蛋白N－端61～86,98～105,140～146,239～241区段和第269～273区段,可能是α-螺旋中心;DMO蛋白N-端第59～61,69～70,148～150区段和383～390区段,DMT蛋白的N-端第125～129,207～213,255～264区段和第281～284区段,可能是β-折叠中心;在DMO蛋白分子N-端40～41, 44～45,50～51,128～129,189～192,204～207,216～222,226～233,244～246,298～299区段和第323～326区段和DMT蛋白分子N-端第12～13,26～27,43～44,58～60,93～95,115～120,136～139区段和第149～151区段具有较柔软的结构,这些区段有可能进行一定幅度的摆动或折叠而形成较复杂的三级结构。DMO蛋白N-端第1～5,41～51,65～67,86～89,98～110,154～170,183～203,205～248,258～264,284～291,293～298,270～375,389～392,402～410区域和DMT蛋白N-端第1～9,17～28,77～84,114～123,131～139,157～184,196～207区域可能是B细胞表位优势区域。以蛋白质的二级结构预测作为辅助手段,用抗原指数,亲水性参数和可及性参数预测DMO和DMT蛋白的B细胞表位,为DMO和DMT蛋白单克隆抗体的制备提供了线索,为系统研究奥利亚罗非鱼DMO和DMT基因的性别调控机理研究提供参考。     

SARS病毒M蛋白的二级结构和B细胞表位预测

以SARS病毒基因组序列为基础,采用GarnierRobson方法、ChouFasman方法和KarplusSchulz方法预测蛋白质的二级结构;按KyteDoolittle方案、Emini方案和JamesonWolf方案预测SARS病毒M蛋白的B细胞表位。预测结果表明,在SARS病毒M蛋白N端第11～20、27～36区段和第133～141区段可能是α螺旋中心;M蛋白分子N端第20～27、34～37,44～56,61～64,70～76,79～97,117～132,142～147,165～176区段和第216～221区段可能是β折叠中心。在M蛋白N端第5～6、40～44、105～107、112～116、189～190、202～203区段和第210～215区段具有较柔软的结构,有可能进行一定幅度的摆动或折叠而形成较复杂的三级结构。SARS病毒M蛋白N端第1～15、37～47、99～120、181～192区段和第196～215区段内或附近很可能是B细胞表位优势区域。以蛋白质的二级结构预测作为辅助手段,用抗原指数,亲水性参数和可及性参数预测SARS冠状病毒M蛋白的B细胞表位,为实验确定SARS病毒M蛋白的B细胞表位和免疫识别研究奠定了基础 。     

基于灰狼优化算法的蛋白质二级结构分类

针对传统方法在蛋白质二级结构分类中精度低的问题,介绍了一种基于灰狼优化算法的卷积神经网络图像分类算法.首先,选取卷积神经网络模型中所需优化的参数,并且初始化灰狼优化算法的迭代次数、灰狼数量、搜索边界和空间维数;其次,计算优化参数的个体适应度函数,对个体适应度进行排序,确定历史最优解、优解和次优解,更新灰狼的位置;最后,...     

耗竭性游泳对大鼠心肌线粒体膜功能的影响

耗竭性游泳对大鼠心肌线粒体膜功能的影响王文信,丁树哲,许豪文（华东师范大学体育系运动生化实验室,上海２０００６２）关键词心肌线粒体,内膜表面电位,游离钙,总钙,合成活力长时间耗竭性游泳或跑步引起心肌、骨骼肌、肝脏等线粒体结构和功能变化,这些变化可能与...     

Early Effects of Neonatal Rat Desympathization on Secondary Heart Rhythms

Neonatal Wistar rats for the first 3 weeks of life were injected intraperitoneally with isobarine every other day. The single doze was 40 mg/kg. Control animals were injected with saline. Degenerative changes in sympathetic ganglia were evident as early as in the 10-day old animals and increased by 18–19 days. The heart rate in the desympathized animals was lower than in control from 10–11 to 18–19 days, but by the end of the 3rd week the differences were eliminated. The same occurred with respiration rate. At same terms there was an essential decrease of amplitude of the heart rate high-frequency fluctuations synchronous with respiration and of the periodogram slow waves with the period about 1 min. Using the method of fast Fourier transform, the power spectra of heart rate fluctuations (secondary heart rhythms) in 5 frequency ranges (0–0.01, 0.01–0.03, 0.03–0.1, 0.1–1.0, and 1.0–2.5 Hz) were calculated. Desympathization leads to a decrease of the fluctuation power in all ranges, but in the ultralow-frequency range this decrease is the least pronounced, which suggests the presence of non- sympathetic mechanisms in their genesis. The greatest changes occur in the middle-frequency area. In all cases, differences from control values increase from the 10–11th to the 18–19th days, after which a tendency for restoration is observed, in spite of an enhancement of processes of degeneration of sympathetic neurons. This indicates an activation of the compensatory mechanisms, due to which consequences of desympathization are partially smoothed at distant terms of studies.     

Fast Electromechanical Amplification in the Lateral Membrane of the Outer Hair Cell

Outer hair cells provide amplification within the mammalian cochlea to enhance audition. The mechanism is believed to reside within the lateral membrane of the cell that houses an expansive array of molecular motors, identified as prestin, which drives somatic electromotility. By measuring nonlinear capacitance, the electrical signature of electromotility, at kilohertz rates we have uncovered new details of the early molecular events that arise from voltage perturbations of prestin. We show that dynamic changes in motor state probability occur within the kilohertz range, and signify an amplificatory event. Additionally, we show a lack of effect of Cl driving force, an absence of cell length effect (indicating that the kinetics does not vary across auditory frequency), and the first demonstration of the time dependence of tension induced amplificatory shifts. The process we have identified, where the stimulus-response function shifts in time along the stimulus axis in a multi-exponential manner, bears similarities to those components of adaptation found in the OHC stereociliar transducer identified recently. As with the forward transducer, the speed of the reverse transducer amplificatory event consequently impacts on high frequency peripheral auditory processing.     

重组蛋白转导域-神经肽Y融合蛋白对体外培养大鼠脂肪细胞的影响

目的探讨重组蛋白转导域-神经肽Y融合蛋白对体外培养大鼠脂肪细胞的影响。方法采用剪切消化法分离大鼠前脂肪细胞,培养液中添加重组PTD-NPY融合蛋白,检测前脂肪细胞和成熟脂肪细胞的形态学变化、细胞中甘油三酯含量和甘油磷酸脱氢酶(GPDH)活性。结果重组PTD-NPY融合蛋白处理大鼠前脂肪细胞72 h,细胞体积增大,细胞数量增加,细胞甘油三酯含量和GPDH活性升高;重组PTD-NPY融合蛋白处理成熟脂肪细胞48h后,细胞体积明显增大,细胞内脂肪滴数量增加并融合成较大的脂滴,甘油三酯含量和GPDH活性均显著升高。结论重组PTD-NPY融合蛋白明显促进前脂肪细胞的分化,促进脂肪细胞中甘油三酯的合成与沉积。为重组PTD-NPY融合蛋白在动物生产及人类疾病治疗中的实际应用奠定理论基础。     

Low Copy Numbers of DC‐SIGN in Cell Membrane Microdomains: Implications for Structure and Function

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC‐SIGN, a C‐type lectin, in membrane microdomains. DC‐SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC‐SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1 µm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3 T3 cells contains only 4–8 molecules of DC‐SIGN, consistent with our preliminary super‐resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (～50 nm) pathogen, dengue virus, leading to infection of host cells.      

Effects of Photodynamic Action on the Cell Membrane of Euglena

The effects of photodynamic action on the cell membrane of Euglena gracilis were investigated by means of studies on dye binding and electrophoretic mobility. Molecular species of alkaline or acid dyes can penetrate the membrane to about the same extent. Once the cell has been injured by pbotodynamic action, ils ahility to exclude large ions is partially lost; it becomes greatly more permeable to dye anions. Binding of rose bengal induces an increased negative charge on Euglena cells which is reversed by subsequent photosensitized damage to the cells.     

Effects of Parenteral Lipid Infusion on DNA Damage in Very Low Birth Weight Infants

Very low birth weight (VLBW) infants are known to have poorly developed antioxidant system and may be at increased risk for radical damage. Previous studies have reported higher levels of lipid peroxide products in lipid emulsion used for parenteral nutrition. To examine the direct effects of parenteral lipid infusion on DNA damage in VLBW infants, we measured urinary 8-hydroxydeoxyguanosine (8-OHdG) levels in VLBW infants before, during, and after the parenteral lipid infusion. In both the lipid-infused and lipid-free groups, urinary 8-OHdG excretion levels at 14 days old were significantly ( p <0.01) lower than those at 2 and 7 days old. However, there were no significant differences in urinary 8-OHdG excretion levels between the lipid-infused and lipid-free groups at 2, 7, and 14 days old. Our results suggest that parenteral lipid infusion does not cause oxidative DNA damage, but irrespective of the infusion DNA damage during the first week of life is enhanced when compared with 14 days after birth in VLBW infants.     

Reconstruction and Stability of Secondary Structure Elements in the Context of Protein Structure Prediction

Efficient and accurate reconstruction of secondary structure elements in the context of protein structure prediction is the major focus of this work. We present a novel approach capable of reconstructing α-helices and β-sheets in atomic detail. The method is based on Metropolis Monte Carlo simulations in a force field of empirical potentials that are designed to stabilize secondary structure elements in room-temperature simulations. Particular attention is paid to lateral side-chain interactions in β-sheets and between the turns of α-helices, as well as backbone hydrogen bonding. The force constants are optimized using contrastive divergence, a novel machine learning technique, from a data set of known structures. Using this approach, we demonstrate the applicability of the framework to the problem of reconstructing the overall protein fold for a number of commonly studied small proteins, based on only predicted secondary structure and contact map. For protein G and chymotrypsin inhibitor 2, we are able to reconstruct the secondary structure elements in atomic detail and the overall protein folds with a root mean-square deviation of <10 Å. For cold-shock protein and the SH3 domain, we accurately reproduce the secondary structure elements and the topology of the 5-stranded β-sheets, but not the barrel structure. The importance of high-quality secondary structure and contact map prediction is discussed.     

Expression and Effects of Hyaluronan and of the Hyaluronan-Binding Protein Hyaluronectin in Newborn Rat Brain Glial Cell Cultures

Abstract: Hyaluronan (HA) is a polymerized nonsulfated extracellular matrix glycosaminoglycan that may be involved in brain development. We have tested the expression of HA and the HA-binding protein hyaluronectin (HN) in glial cell cultures from newborn rat brain. HA was secreted into the culture medium by type 1 astrocytes in the first stages of the primary cultures. The secretion was high during cell proliferation, reached a maximum when they were confluent, and then decreased. HA was not secreted at a detectable level by total O-2A lineage cell- enriched cultures. HA labeled small O-2A progenitor cells (GFA-, A2B5⁺, HA⁺), small O-2A progenitorlike (GFA^?, A2B5^?, HA⁺) cells, and type 2 astrocytes (GFA⁺, A2B5⁺, HA⁺), but not mature oligodendrocytes (Galc⁺, HA^?). In contrast to HA, hyaluronectin labeled oligodendrocyte membranes (i.e., more mature cells) from day 8. A2B5⁺ GFA^? cells were found to be either HA⁺ or HN⁺ at days 7–9, suggesting intermediary stages. The addition of HA to primary cultures and to O-2A progenitor-enriched cultures decreased significantly the increase in the number of O-2A progenitors, of mature (Galc⁺) oligodendrocytes proportionally to the decrease of the O-2A progenitor number, and of BrdU⁺ cells, suggesting that HA acts (directly or indirectly) on O-2A cell proliferation. This effect, which was seen for concentrations as low as 0.1 μg/ml, was HA specific and was not observed with other glycosaminogly- cans. When primary cultures were performed in the presence of hyaluronidase-digested or HA-depleted (by passage on a HN column) fetal calf serum, the total number of O-2A lineage cells was dramatically increased (100%, p<10–⁴) in comparison with control cultures in standard fetal calf serum. Platelet-derived growth factor increased the total number of O-2A lineage cells and of (Galc⁺) oligodendrocytes. This effect was opposed by HA dose dependently. The effect of HA was significantly inhibited by HN (30%, p<10–⁴). HN had, however, no effect when it was added to culture in the presence of hyaluronidase in fetal calf serum, suggesting its effect was only due to its binding to HA. During cell maturation, HA disappears as HN appears. This and the fact that HA and PDGF have opposite effects suggest an effect of these factors, or of their balance, on myelination.