神经节苷脂GM3对J6－2细胞蛋白质磷酸化的影响

通过研究神经节苷脂GM₃对国人单核样白血病细胞系J6-2细胞蛋白质磷酸化的影响,在[γ- ³²P]ATP,GM₃,ATP,Mg²⁺与J6-2细胞液及颗粒两部分共同反应,10min(30℃)体系中,观察到GM₃对两部分蛋白质磷酸化的调节作用.GM₃(100μmol/L)促进颗粒部分分子量为180 000,87 000,78 000,67 000,43 000及31 000的蛋白质磷酸化,促进胞液部分分子量为87 000及56 000的蛋白质磷酸化,而且能抑制70 000及43 000蛋白质磷酸化.由于GM₃已被前人证实能对J6-2细胞起分化作用,其作用时间长达4-6d,很可能GM₃对蛋白质磷酸化作用的调节是GM₃促分化作用的早期信号.     

有机锗对巨噬细胞激活与磷脂代谢转换的影响

有机锗(CGS、DGS、Ge-132)于活体内能激活小鼠腹腔巨噬细胞(Mφ),后者于体外对肿瘤细胞Hca-16H₃和J6-2表达Mφ介导的肿瘤细胞毒(MTC),CGS与DGS的激活效应高于Ge-132,CGS为最强.它们激活的Mφ磷脂PC代谢转换较常驻Mφ(R-Mφ)明显增高,表现在［³H］胆碱掺入PC增加,CGS的增加效应最强.Ge-132激活的Mφ(Ge-12-Mφ)与R-Mφ比较,增加［³²P］Pi掺入PC,降低［³²P］Pi或［³H］肌醇掺入PI,但［³²P］Pi或［³H］肌醇掺入PIP、PIP₂未有显著差异.PC代谢转换的增加很可能是有机锗激活Mφ表达MTC的信息传递所需要的.     

GM3在几种哺乳动物肝脏和狗红细胞中的含量及其分离纯化

从正常兔、猪和狗的肝脏及狗红细胞中分离纯化了总神经节苷脂,测定了脂结合唾液酸,进行了高效薄层层析,比较了上述四种组织中GM₃的含量。结果表明狗红细胞中的GM₃的含量较另三种的高,狗肝和兔肝次之,猪肝含量甚微。从狗红细胞中提取和纯化了GM₃,其得量为每毫升压积红细胞351.0μg,纯度为92.2％。     

神经节苷脂GM_3对人单核样白血病J6-2细胞中磷脂代谢的影响

用50μmol/L GM_3处理J6-2细胞6天,经组化与细胞学检查,证实细胞沿单核巨噬细胞途径分化。以[~(32)P]Pi或[CH_3-~(14)C]胆碱冲激(Pulse)后,将洗净的细胞进行Folch分配。取下相进行薄层层析,再做放射自显影。结果表明:GM_3的处理能促进[~(32)P]Pi或[CH_3-~(14)C]胆碱向磷脂酰胆碱的参入,而抑制向其他磷脂组分的参入。用[CH_3-~(14)C]胆碱冲激的Folch分配上相含水溶性胆碱代谢物,经TLC,结果表明CM_3的处理使[CH_3-~(14)C]胆碱向CDP-胆碱的参入减少。本研究证实,GM_3能调节J6-2细胞的磷脂代谢,其调节机制值得研究。     

蛋白激酶C研究的最新进展

作为能使蛋白激酶C(PKC)活化的第二信使甘油二酯(DAG)不仅可由磷脂酰肌醇(PtdIns)水解产生,大量实验表明还可从磷脂酰胆碱(PC)水解而来,其中磷脂酶C(PLC)及磷脂酶D(PLD)参与了这一过程,磷脂酶A₂(PLA₂)的作用产物脂肪酸(FA)也能激活PKC.PKC至少有10种亚型,依据其活化方式可分三大类:典型PKC,新PKC和非典型PKC.PKC参与了基因表达的调控.     

Mg2＋对阿霉素引起心肌线粒体F1F0变化的保护

抗肿瘤药物阿霉素(ADM)对心肌线粒体F₁F₀-复合体呈现抑制而对F₁-ATPase无抑制,这表明ADM可能是通过膜脂起作用的,适当浓度Mg²⁺能降低ADM对复合体的抑制.经 ³¹P-NMR和标记荧光探针NBD-PE,DPH,MC-540以及内源荧光等的测定,结果表明ADM可能首先通过诱导F₁F₀膜脂形成非双层脂结构,继而影响了膜脂的堆积程度和流动性,进而引起F₁F₀-复合体酶蛋白构象的改变,最终导致酶活力的降低.Mg^2＋则可能由于与ADM竞争与心磷脂的结合,而对ADM引起F₁F₀的变化产生保护作用.     

胞浆型磷脂酶A2介导弱氧化修饰低密度脂蛋白诱导的人脐静脉内皮细胞凋亡

探讨弱氧化修饰低密度脂蛋白(MM-LDL)能否诱导人脐静脉内皮细胞(HUVECs)凋亡以及胞浆型磷脂酶A₂(cPLA₂)在此过程中的作用.MTT法测定细胞存活率;相差显微镜、荧光显微镜和流式细胞仪检测细胞凋亡;³H-花生四烯酸(³H-AA)预标法测定PLA₂活性;蛋白质印迹检测cPLA₂磷酸化;激光共聚焦显微镜检测单个细胞内钙离子浓度的变化.结果表明,MM-LDL(100～300 mg/L)作用后的HUVECs呈现凋亡典型的形态特征,凋亡率随MM-LDL浓度的增加而上升.MM-LDL能引起胞内钙离子浓度增加,cPLA₂的活化及磷酸化.15 μmol/L AACOCF₃和5 mmol/L EGTA在抑制cPLA₂活性的同时,部分抑制MM-LDL诱导的HUVECs凋亡.加入外源性AA(50 μmol/L)能逆转AACOCF₃引起的凋亡抑制.结果提示,cPLA₂参与了MM-LDL诱导HUVECs凋亡的信号传递.     

胞外ATP对AlCl3诱导的细胞死亡过程中胞内H2O2和Ca2+水平的影响及其生理机制分析

该实验以烟草悬浮细胞 BY 2 为材料,在烟草悬浮细胞中分别加入0.05、0.10、0.15、0.20 mmol·L^-1AlCl₃,以等体积去离子水处理的悬浮细胞液为对照,并依据前述实验结果选择0.15 mmol·L^-1 AlCl₃,分别添加5 mmol·L^-1 DMTU(H₂O₂ 抑制剂)、20 μmol·L^-1CaCl₂、15 μmol·L^-1 LaCl₃(Ca²⁺通道抑制剂)和50 μmol·L^-1 ATP设计多项处理,分析胞外ATP(eATP)对铝离子(Al³⁺)胁迫引起的植物细胞死亡及其胞内H₂O₂、Ca²⁺的影响,以揭示Al³⁺胁迫下植物调节细胞死亡的可能机制,进一步扩展对eATP功能的认知。结果显示：(1)随着 AlCl₃ 胁迫浓度的提高,细胞死亡水平和胞内H₂O₂水平上升,而胞内Ca²⁺和eATP水平则逐渐降低。(2)外援施加H₂O₂抑制剂 DMTU(二甲基硫脲)和Ca²⁺能够有效缓解AlCl₃诱导的细胞死亡水平的上升;而Ca²⁺通道抑制剂LaCl₃(三氯化镧)则加剧了AlCl₃胁迫下的细胞死亡。(3)在AlCl₃胁迫下对细胞添加外源ATP,能够缓解AlCl₃胁迫下胞内H₂O₂水平上升和Ca²⁺水平下降的同时,并显著降低AlCl₃胁迫导致的细胞死亡。研究表明, Al³⁺以剂量依赖的模式提升细胞死亡和细胞内H₂O₂的水平并降低胞内Ca²⁺和eATP水平,AlCl₃诱导的细胞死亡受到H₂O₂和Ca²⁺水平变化的调节,eATP可以通过调节H₂O₂与Ca²⁺水平缓解AlCl₃诱导的细胞死亡。推测Al³⁺胁迫可能通过抑制钙离子通道而破坏了细胞内H₂O₂和Ca²⁺之间的协同关系,外源ATP对Al³⁺诱导H₂O₂上升的缓解作用可能是由于其提升了细胞的抗氧化能力。     

信息跨膜传递的分子机制

胞外信息作用于质膜表面的受体,转变为胞内信使分子完成信息的跨膜传递。β受体结合配体后活化G蛋白(通过α亚基与β、γ的解离)影响环化酶的活性。而钙联受体则通过触发肌醇磷脂的代谢,生成胞内信使甘油二酯(DG)和三磷酸肌醇酯(IP₃),胞内游离Ca²⁺浓度瞬间增高(Ca²⁺动员过程),通过不同的蛋白激酶引起特定的生理效应。DG活化蛋白激酶-C,IP₃动员胞内Ca²⁺,它们通过二个相互独立而协同的过程调节细胞的代谢。     

胰岛素和胰岛素突变体促进大鼠脂肪细胞摄取葡萄糖

报道了大鼠脂肪细胞摄取葡萄糖测定胰岛素体外活性的简便方法 .在胰岛素存在下 ,脂肪细胞摄取葡萄糖的量比对照增加 5～ 6倍 .当胰岛素浓度在 0 .2～ 1 0μg/L时 ,脂肪细胞摄取D [3 ³H] 葡萄糖的量与胰岛素浓度对数呈线性关系 .葡萄糖和 3 O 甲基葡萄糖抑制指脂肪细胞摄取D [3 ³H]葡萄糖 .利用这一方法测定了[B₂ Lys] 胰岛素和 [B₃ Lys] 胰岛素的体外活力 ,分别为胰岛素的 6 1.6 %和 154% .     

Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis

ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary. Here, we show that with the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the ε subunit, ΔpH imposed by acid-base transition and Δψ produced by valinomycin-mediated K(+) diffusion potential contribute equally to the rate of ATP synthesis within the experimental range examined (ΔpH -0.3 to 2.2, Δψ -30 to 140 mV, pH around the catalytic domain 8.0). Either ΔpH or Δψ alone can drive synthesis, even when the other slightly opposes. Δψ was estimated from the Nernst equation, which appeared valid down to 1 mm K(+) inside the proteoliposomes, due to careful removal of K(+) from the lipid.     

Antioxidant and anticholinesterase investigations of Rumex hastatus D. Don: potential effectiveness in oxidative stress and neurological disorders

Background

Rumex species are traditionally used for the treatment of neurological disorders including headache, migraine, depression, paralysis etc. Several species have been scientifically validated for antioxidant and anticholinestrase potentials. This study aims to investigate Rumex hastatus D. Don crude methanolic extract, subsequent fractions, saponins and flavonoids for acetylcholinestrase, butyrylcholinestrase inhibition and diverse antioxidant activities to validate its folkloric uses in neurological disorders. Rumex hastatus crude methanolic extract (Rh. Cr), subsequent fractions; n-hexane (Rh. Hex), chloroform (Rh. Chf), ethyl acetate (Rh. EtAc), aqueous fraction (Rh. Aq), crude saponins (Rh. Sp) and flavonoids (Rh. Fl) were investigated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at various concentrations (125, 250, 500, 1000 μg/mL) using Ellman’s spectrophotometric analysis. Antioxidant potentials of Rh. Sp and Rh. Fl were evaluated using DPPH, H₂O₂ and ABTS free radical scavenging assays at 62.5, 125, 250, 500, 1000 μg/mL.

Results

All the test samples showed concentration dependent cholinesterase inhibition and radicals scavenging activity. The AChE inhibition potential of Rh. Sp and Rh. Fl were most prominent i.e., 81.67 ± 0.88 and 91.62 ± 1.67 at highest concentration with IC₅₀ 135 and 20 μg/mL respectively. All the subsequent fractions exhibited moderate to high AChE inhibition i.e., Rh. Cr, Rh. Hex, Rh. Chf, Rh. EtAc and Rh. Aq showed IC₅₀ 218, 1420, 75, 115 and 1210 μg/mL respectively. Similarly, against BChE various plant extracts i.e., Rh. Sp, Rh. Fl, Rh. Cr, Rh. Hex, Rh. Chf, Rh. EtAc and Rh. Aq resulted IC₅₀ 165, 175, 265, 890, 92, 115 and 220 μg/mL respectively. In DPPH free radical scavenging assay, Rh. Sp and Rh. Fl showed comparable results with the positive control i.e., 63.34 ± 0.98 and 76.93 ± 1.13% scavenging at 1 mg/mL concentration (IC₅₀ 312 and 104 μg/mL) respectively. The percent ABTS radical scavenging potential exhibited by Rh. Sp and Rh. Fl (1000 μg/mL) were 82.58 ± 0.52 and 88.25 ± 0.67 with IC₅₀ 18 and 9 μg/mL respectively. Similarly in H₂O₂ scavenging assay, the Rh. Sp and Rh. Fl exhibited IC₅₀ 175 and 275 μg/mL respectively.

Conclusion

The strong anticholinesterase and antioxidant activities of Rh. Sp, Rh. Fl and various fractions of R. hastatus support the purported ethnomedicinal uses and recommend R. hastatus as a possible remedy for the treatment of AD and neurodegenerative disorders.     

Role of Microorganisms in Emission of Nitrous Oxide and Methane in Pulse Cultivated Soil Under Laboratory Incubation Condition

Soil from a pulse cultivated farmers land of Odisha, India, have been subjected to incubation studies for 40 consecutive days, to establish the impact of various nitrogenous fertilizers and water filled pore space (WFPS) on green house gas emission (N₂O & CH₄). C₂H₂ inhibition technique was followed to have a comprehensive understanding about the individual contribution of nitrifiers and denitrifiers towards the emission of N₂O. Nevertheless, low concentration of C₂H₂ (5 ml: flow rate 0.1 kg/cm²) is hypothesized to partially impede the metabolic pathways of denitrifying bacterial population, thus reducing the overall N₂O emission rate. Different soil parameters of the experimental soil such as moisture, total organic carbon, ammonium content and nitrate–nitrogen contents were measured at regular intervals. Application of external N-sources under different WFPS conditions revealed the diverse role played by the indigenous soil microorganism towards green house gas emission. Isolation of heterotrophic microorganisms (Pseudomonas) from the soil samples, further supported the fact that denitrification might be prevailing during specific conditions thus contributing to N₂O emission. Statistical analysis showed that WFPS was the most influential parameter affecting N₂O formation in soil in absence of an inhibitor like C₂H₂.     

Comparative Study of Antibiofilm Activity of Copper Oxide and Iron Oxide Nanoparticles Against Multidrug Resistant Biofilm Forming Uropathogens

The effectiveness of the metal oxide nanoparticles viz. CuO and Fe₂O₃ as antibacterial agents against multidrug resistant biofilm forming bacteria was evaluated. CuO nanoparticles were also experimented for antibiofilm and time kill assay. The CuO displayed maximum antibacterial activity with zone of inhibition of (22 ± 1) mm against methicillin resistant Staphylococcus aureus (MRSA) followed by Escherichia coli (18 ± 1) mm. The Fe₂O₃ showed the zone of inhibition against MRSA of (14 ± 1) mm followed by E. coli (12 ± 1) mm. CuO proved to be more toxic than Fe₂O₃ nanoparticles showing significantly high antibacterial activity and found to possess dose dependent antibiofilm properties.     

Extracellular-derived calcium does not initiate in vivo neurotransmission involving docosahexaenoic acid

In vitro studies show that docosahexaenoic acid (DHA) can be released from membrane phospholipid by Ca²⁺-independent phospholipase A₂ (iPLA₂), Ca²⁺-independent plasmalogen PLA₂ or secretory PLA_{2 (sPLA2)}, but not by Ca²⁺-dependent cytosolic PLA₂ (cPLA2), which selectively releases arachidonic acid (AA). Since glutamatergic NMDA (N-methyl-D-aspartate) receptor activation allows extracellular Ca²⁺ into cells, we hypothesized that brain DHA signaling would not be altered in rats given NMDA, to the extent that in vivo signaling was mediated by Ca²⁺-independent mechanisms. Isotonic saline, a subconvulsive dose of NMDA (25 mg/kg), MK-801, or MK-801 followed by NMDA was administered i.p. to unanesthetized rats. Radiolabeled DHA or AA was infused intravenously and their brain incorporation coefficients k*, measures of signaling, were imaged with quantitative autoradiography. NMDA or MK-801 compared with saline did not alter k* for DHA in any of 81 brain regions examined, whereas NMDA produced widespread and significant increments in k* for AA. In conclusion, in vivo brain DHA but not AA signaling via NMDA receptors is independent of extracellular Ca²⁺ and of cPLA₂. DHA signaling may be mediated by iPLA₂, plasmalogen PLA₂, or other enzymes insensitive to low concentrations of Ca²⁺. Greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage.     

Mechanisms involved in the adenosine-induced vasorelaxation to the pig prostatic small arteries

Benign prostatic hypertrophy has been related with glandular ischemia processes and adenosine is a potent vasodilator agent. This study investigates the mechanisms underlying the adenosine-induced vasorelaxation in pig prostatic small arteries. Adenosine receptors expression was determined by Western blot and immunohistochemistry, and rings were mounted in myographs for isometric force recording. A_2A and A₃ receptor expression was observed in the arterial wall and A_2A-immunoreactivity was identified in the adventitia–media junction and endothelium. A₁ and A_2B receptor expression was not obtained. On noradrenaline-precontracted rings, P1 receptor agonists produced concentration-dependent relaxations with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA) = {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 > 2-Cl-IB-MECA = 2-Cl-cyclopentyladenosine = adenosine. Adenosine reuptake inhibition potentiated both NECA and adenosine relaxations. Endothelium removal and ZM241385, an A_2A antagonist, reduced NECA relaxations that were not modified by A₁, A_2B, and A₃ receptor antagonists. Neuronal voltage-gated Ca²⁺ channels and nitric oxide (NO) synthase blockade, and adenylyl cyclase activation enhanced these responses, which were reduced by protein kinase A inhibition and by blockade of the intermediate (IK_Ca)- and small (SK_Ca)-conductance Ca²⁺-activated K⁺ channels. Inhibition of cyclooxygenase (COX), large-conductance Ca²⁺-activated-, ATP-dependent-, and voltage-gated-K⁺ channel failed to modify these responses. These results suggest that adenosine induces endothelium-dependent relaxations in the pig prostatic arteries via A_2A purinoceptors. The adenosine vasorelaxation, which is prejunctionally modulated, is produced via NO- and COX-independent mechanisms that involve activation of IK_Ca and SK_Ca channels and stimulation of adenylyl cyclase. Endothelium-derived NO playing a regulatory role under conditions in which EDHF is non-functional is also suggested. Adenosine-induced vasodilatation could be useful to prevent prostatic ischemia.     

Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V₁ moiety of Enterococcus hirae V-ATPase (EhV₁) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV₁ using single-molecule analysis employing a load-free probe. EhV₁ rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (V_max) of 107 revolutions/s and a Michaelis constant (K_m) of 154 μm at 26 °C. At all ATP concentrations tested, EhV₁ showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V₁-ATPase (TtV₁). At 10 μm ATP (⪡K_m), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10⁶ m⁻¹ s⁻¹. At 40 mm ATP (⪢K_m), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV₁. Our results identify the common properties of rotary catalysis of V₁-ATPases that are distinct from those of F₁-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.     

竹红菌乙素作猝灭剂研究Fo构象方法的建立

线粒体F₁F_o复合体F_o部分a亚基的色氨酸荧光可被竹红菌乙素(hypocrellin B, HB)猝灭.不同温度下测定Stern-Volmer图的结果显示,猝灭常数(K_sv)随温度的增加而加大,时间衰变荧光的结果显示,荧光寿命随HB浓度的增加而减小,加入不同浓度的HB, F₁F_o复合体的吸收峰没有位移.这些实验结果支持动态猝灭机理.HB还具有有效猝灭浓度低,不影响酶的活力;在脂相和水相的分布比率可高达16 560∶1;实验操作简便等优点.因此HB可作为理想的疏水相荧光猝灭剂,研究与膜结合的F₁F_o复合体中镶嵌于膜脂内F_o的构象变化.     

Evolution of C4 plants: a new hypothesis for an interaction of CO2 and water relations mediated by plant hydraulics

C(4) photosynthesis has evolved more than 60 times as a carbon-concentrating mechanism to augment the ancestral C(3) photosynthetic pathway. The rate and the efficiency of photosynthesis are greater in the C(4) than C(3) type under atmospheric CO(2) depletion, high light and temperature, suggesting these factors as important selective agents. This hypothesis is consistent with comparative analyses of grasses, which indicate repeated evolutionary transitions from shaded forest to open habitats. However, such environmental transitions also impact strongly on plant-water relations. We hypothesize that excessive demand for water transport associated with low CO(2), high light and temperature would have selected for C(4) photosynthesis not only to increase the efficiency and rate of photosynthesis, but also as a water-conserving mechanism. Our proposal is supported by evidence from the literature and physiological models. The C(4) pathway allows high rates of photosynthesis at low stomatal conductance, even given low atmospheric CO(2). The resultant decrease in transpiration protects the hydraulic system, allowing stomata to remain open and photosynthesis to be sustained for longer under drying atmospheric and soil conditions. The evolution of C(4) photosynthesis therefore simultaneously improved plant carbon and water relations, conferring strong benefits as atmospheric CO(2) declined and ecological demand for water rose.     

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe₃O₄) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.