Permeability and the hidden area of lipid bilayers

The passive water permeability of a lipid vesicle membrane was studied, related to the hydrostatic (not osmotic) pressure difference between the inner and the outer side of the vesicle in a water environment without additives. Each pressure difference was created by sucking a vesicle into a micropipette at a given sucking pressure. The part of the membrane sucked into the micropipette (the projection length) was measured as a function of time. The time dependence can be divided into two intervals. We put forward the idea that smoothing of membrane defects, accompanied by an increase of the membrane area, takes place during the initial time interval, which results in a faster increase of the projection length. In the second time interval the volume of the vesicle decreases due to the permeability of its membrane and the increase of the projection length is slower. The hidden area and the water permeability of a typical lipid bilayer were estimated. The measured permeability, conjugated to the hydrostatic pressure difference, is an order of magnitude higher than the known value of the permeability, conjugated to the osmotic pressure difference. A hypothesis, based on pore formation, is proposed as an explanation of this experimental result.     

The effect of amphiphilic compounds on the secretion of levansucrase by Zymomonas mobilis

The effect of some aliphatic (n-butanol to n-hexadecanol) and aromatic (benzyl and phenethyl alcohols), anesthetics (procaine) and surfactants (Tween 20 to Tween 80) on the secretion of levansucrase by the levan-producing strain of Gram-negative ethanologenic bacteria Zymomonas mobilis 113S were examined in this study.

During incubation of Z. mobilis cells with sucrose (10 mM) a decrease of the levansucrase activity was observed in the presence of these amphiphilic compounds concomitantly with an increase of a total amount of protein in the medium. Since none of the compounds under study had any effect on enzyme activity in vitro observed structure- and concentration-dependent relationships most probably reflected differently conditioned processes of membrane-associated secretion of levansucrase and total protein by Z. mobilis. The patterns of fluorescence titrations by ANS⁻ indicated to competitive interactions between an amphiphilic compound of varied structure and the probe for the polar and non-polar binding sites of Z. mobilis membrane structures. The effect of 2,4-DNP (protonophore) and sodium azide (an inhibitor of ATPase) alone as well as in combination with aliphatic alcohols suggested to the participation of energy transduction system in the secretion of levansucrase by Z. mobilis cells. Under conditions of abolished proton motive force (PMF) the level of levansucrase decreased whereas the amount of protein elevated significantly in the medium in accordance with the expected requirement of PMF to perform the secretion of levansucrase and to keep intact the permeability barrier of cells.     

Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.     

一种高效的哺乳动物粪便DNA提取通用方法

以粪便为材料提取动物DNA进行动物保护遗传学和分子生态学研究的关键是能否提取到高质量的粪便DNA.然而提取方法通用性不好和产物质量不高等问题阻碍了粪便DNA分析技术的推广.本文介绍的改进型十六烷基三甲基溴化铵提取法可广泛适用于各食性哺乳动物粪便DNA提取,在11种不同食性动物的粪便DNA提取实验中验证了它的可靠性和通用性.本方法成本低廉(3元/样),用实验室常规试剂即可完成粪便DNA提取,其产物纯度高于专用试剂盒QIAamp DNA Stool Kit,在拥有超过专业试剂盒提取效果的同时尽可能的降低了实验成本,有利于粪便DNA技术的推广.     

一种提取橡胶树叶中总DNA的方法

橡胶树叶中富含多糖和多酚类物质,严重影响高质量基因组DNA的提取,文章针对这一特点提出了改良的CTAB法.这一方法可用于DNA的大量或小量提取,小量提取的产量为50μg·g-1(FW),大量提取可达到400μg·g-1(FW);可从橡胶树古铜期、变色期、稳定期等3个生长时期的叶中提取出高质量DNA,所得DNA可用于PCR分析和酶切分析等分子生物学研究.     

马铃薯块茎中总RNA的快速提取

马铃薯块茎中由于含有大量的多糖和酚类物质,因此难以从中提取RNA。本实验采用CTAB法从马铃薯块茎中提取了总RNA,并进行了马铃薯病毒的检验。结果表明:得到的RNA完整性好,A260/A280在1.7～2.0范围内,纯度也比较高。用RT-PCR方法进行马铃薯Y病毒的检验,得到了一条与预计扩增长度相同的一条带,并检验出枣庄地区马铃薯Y病毒的感染率为75%。     

反胶束萃取技术分离胰激肽原酶

研究了用十六烷基三甲基溴化铵（CTAB）/正己醇/正辛烷反胶束溶液萃取和反萃取商业用胰激肽原酶时,水相pH值、离子强度和种类、CTAB浓度和助表面活性剂浓度等因素对分离效率的影响,并从反胶束微观结构给予解释。结果表明：［CTAB］＝0.02 mol•L-1,正己醇／正辛烷(V/V)＝1：5,萃取pH＝9.0,反萃pH＝7.0,萃取［KBr］＝0.1 mol•L-1,反萃［KBr］＝1.5 mol•L-1,反萃取加15％乙醇(V/V)时,萃取率接近100%,反萃取活性回收得率在80%以上。商业用酶的纯化倍数最高为1.97倍,粗酶为7.15倍,且粗酶纯化后比活在200U／mg以上,电泳分析证实了纯化效果,显示了很好的工业前景。     

珍珠黄杨ITS-PCR体系的建立与优化

本研究利用改良CTAB法从珍珠黄杨叶片中提取基因组DNA作为模板,在TaqDNA聚合酶量不变的基础上,利用正交设计L_9(3~4)对4个因素(模板DNA、Mg~(2+)、dNTP和引物)在3个水平上对珍珠黄杨ITS-PCR反应体系进行优化.实验结果表明,在总体积50μL的反应体系中,建立了最佳ITS-PCR扩增条件:Mg~(2+)浓度2.0 mmol/L、引物浓度0.3 μmol/L、dNTP浓度0.3 mmol/L、DNA模板浓度240 ng/50μL、ToqDNA聚合酶的用量1.75 U/50μL和退火温度56℃,该优化体系保证了珍珠黄杨ITS-PCR产物的纯度和质量要求.珍珠黄杨ITS片段克隆测序后获得的序列长度为642 bp,其系统学信息将为珍珠黄杨的起源进化提供有力的分子水平证据.     

同时提取油茶中DNA和RNA的简便方法

介绍了一种以CTAB提取方法为基础,结合其它DNA和RNA提取方法,经反复实验建立的一种同时提取油茶DNA和RNA的简便方法。该方法能有效地去除植物组织中酚类和多糖等次生物质的影响,所得到的DNA和RNA纯度高,完整性好,可以用于进一步的如DNA分析,mRNA的分离,RT-PCR,构建cDNA文库和基因表达分析等实验。     

CTAB结合DNA凝胶回收试剂盒提取食用菌DNA

目的:为探索从食用菌子实体中快速分离和纯化DNA的方法.方法:以三种栽培的食用菌为材料,采用CTAB结合DNA凝胶回收试剂盒进行基因组DNA的分离和纯化.结果:与对照相比,结合法提取DNA样品的电泳条带更规则、清晰,并且DNA的酶切效率显著高于对照.结论:该结合法是一种快捷、高效提取纯化食用菌子实体DNA的方法.     

Superoxide flashes: early mitochondrial signals for oxidative stress-induced apoptosis

Irreversible mitochondrial permeability transition and the resultant cytochrome c release signify the commitment of a cell to apoptotic death. However, the role of transient MPT (tMPT) because of flickering opening of the mitochondrial permeability transition pore remains elusive. Here we show that tMPT and the associated superoxide flashes (i.e. tMPT/superoxide flashes) constitute early mitochondrial signals during oxidative stress-induced apoptosis. Selenite (a ROS-dependent insult) but not staurosporine (a ROS-independent insult) stimulated an early and persistent increase in tMPT/superoxide flash activity prior to mitochondrial fragmentation and a global ROS rise, independently of Bax translocation and cytochrome c release. Selectively targeting tMPT/superoxide flash activity by manipulating cyclophilin D expression or scavenging mitochondrial ROS markedly impacted the progression of selenite-induced apoptosis while exerting little effect on the global ROS response. Furthermore, the tMPT/superoxide flash served as a convergence point for pro- and anti-apoptotic regulation mediated by cyclophilin D and Bcl-2 proteins. These results indicate that tMPT/superoxide flashes act as early mitochondrial signals mediating the apoptotic response during oxidative stress, and provide the first demonstration of highly efficacious local mitochondrial ROS signaling in deciding cell fate.     

Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane

The biochemical and biophysical properties of the membrane and some general characteristics of the response of Lactobacillus casei ATCC 393 (reclassified Lactobacillus zeae) to hyperosmotic conditions were studied. Under hypertonic conditions, the hydrophobicity and the bile salt sensitivity of the cultures were increased. The glycolipid AcylH3DG is only present in membranes of NaCl containing medium, whereas, H4DG undergoes a significant increment and H2DG a significant decrease. The fluidity of both the purified membranes and the total lipid vesicles, as determined with the fluorescent probe DPH, did not change in conditions of high salinity. This was coincident with changes in the fatty acid (FA) composition where an increase in the saturated/unsaturated FA ratio was compensated by a rise in the fluidifying 11,12-methyleneoctadecanoic FA (cyc 19:0). Under osmotic stress conditions, Laurdan and acridine orange in total lipid vesicles showed increased lateral lipid packing and proton permeability, respectively.     

Soluble gangliosides in cultured neurotumor cells

Abstract: The biosynthesis and degradation of glycosphingolipids were studied in cytosolic and membrane fractions obtained from rat glioma C6 cells. Both pools had a similar composition of neutral glycosphingolipids but the soluble pool contained only a few percent of the total. The major ganglioside in C6 cells was GM3, of which only 2% was soluble. Whereas the bulk of the membrane GM3 was accessible to surface labeling procedures, the soluble GM3 was not. Mouse neuroblastoma N18 cells also contained small amounts of cytoplasmic gangliosides corresponding to GM3, GM2, GM1, and GDla. When C6 cells were incubated with medium containing [³H]galactose at 37°C, the specific activity of soluble GM3 initially increased more rapidly than that of membrane GM3; by 4 h, the specific activities in both pools became equal. Total incorporation into the membrane pool, however, was always several-fold greater even at the shortest incubation times examined. The labeling pattern of neutral glycosphingolipids in both soluble and membrane fractions indicated the existence of a precursor-product relationship between glucosylceramide and other glycosphingolipids. When labeled cells were transferred to nonradioactive medium, glucosylceramide disappeared the most rapidly, with a 50% loss within <6 h. The turnover rates of other glycosphingolipids were much slower. Although cytosolic GM3 was degraded more rapidly (t_1/2= 26 h) than membrane-bound GM3 (t_1/2= 44 h), its turnover rate was much slower than the time required for transport of GM3 to the cell surface (20–30 min). Our results are consistent with the existence of a small intracellular pool of soluble gangliosides and neutral glycosphingolipids that is stable and independent of the main membrane-bound pool. Although the role of these cytosolic glycolipids is unknown, they do not appear to represent a transport pool between the site of synthesis and the plasma membrane.     

Synergistic Triggering of Superoxide Flashes by Mitochondrial Ca2+ Uniport and Basal Reactive Oxygen Species Elevation

Mitochondrial superoxide flashes reflect a quantal, bursting mode of reactive oxygen species (ROS) production that arises from stochastic, transient opening of the mitochondrial permeability transition pore (mPTP) in many types of cells and in living animals. However, the regulatory mechanisms and the exact nature of the flash-coupled mPTP remain poorly understood. Here we demonstrate a profound synergistic effect between mitochondrial Ca²⁺ uniport and elevated basal ROS production in triggering superoxide flashes in intact cells. Hyperosmotic stress potently augmented the flash activity while simultaneously elevating mitochondrial Ca²⁺ and ROS. Blocking mitochondrial Ca²⁺ transport by knockdown of MICU1 or MCU, newly identified components of the mitochondrial Ca²⁺ uniporter, or scavenging mitochondrial basal ROS markedly diminished the flash response. More importantly, whereas elevating Ca²⁺ or ROS production alone was inefficacious in triggering the flashes, concurrent physiological Ca²⁺ and ROS elevation served as the most powerful flash activator, increasing the flash incidence by an order of magnitude. Functionally, superoxide flashes in response to hyperosmotic stress participated in the activation of JNK and p38. Thus, physiological levels of mitochondrial Ca²⁺ and ROS synergistically regulate stochastic mPTP opening and quantal ROS production in intact cells, marking the flash as a coincidence detector of mitochondrial Ca²⁺ and ROS signals.     

甜叶菊RNA分离方法研究

甜叶菊组织中酚类、萜类和多糖等代谢产物含量较高,RNA难分离,易降解,使用现有的方法提取甜叶菊组织RNA产率低、质量差,无法应用于实验操作.本文针对甜叶菊组织次生代谢物多的特点,以传统的CTAB法(cetyl trimethyl ammonium bromide)为基础,分别采用不同的方法进行RNA抽提和纯化.结果发现采用CTAB-LiCl法提取的RNA完整性好,且纯度高;传统的CTAB法提取的RNA完整性好,但有DNA和其它杂质的污染;高盐溶液法提取的RNA降解比较严重,同时还有杂质污染.结论说明CTAB-LiCl法适用于多糖类植物RNA的分离.     

麦冬基因组DNA提取方法研究

为了从富含多糖、多酚的顽拗植物麦冬中分离出高质量基因组DNA,分别建立了改良CTAB法和改良SDS法。通过使用核分离液和CTAB/NaCl溶液、提取液中加入0.35倍体积无水乙醇等最大限度地除去杂质。将两种方法提取的8种麦冬DNA与两种离心柱式试剂盒提取的DNA在纯度、产率等方面进行对比,并进行双酶切和SRAP分析。麦冬类植物SRAP反应体系的建立尚属首次。结果表明,改良CTAB法和改良SDS法均能从8种麦冬叶片中提取到高质量的基因组DNA,改良CTAB法提取纯度更高,提取幼叶DNA纯度可以与离心柱式试剂盒媲美,能够满足多种分子生物学试验要求。在后续试验中,用改良CTAB法从20多种沿阶草族植物中提取到了高纯度DNA。该方法通用性好,提取的DNA纯度高,对其他顽拗类植物基因组DNA的提取具有借鉴意义。     

微量大豆种子基因组DNA的快速制备

用改良的CTAB法,从微量大豆种子样品中快速提取了基因组DNA,并从大豆基因组中扩增到了大豆蛋白酶抑制剂基因.