Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide

To understand the role of sphingomyelinase (SMase) in the function of biological membranes, we have investigated the effect of conversion of sphingomyelin (SM) to ceramide (Cer) on the assembly of domains in giant unilamellar vesicles (GUVs). The GUVs were prepared from mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-palmitoly-d-erythro-sphingosine (C₁₆Cer), N-palmitoyl-d-erythro-sphingosylphosphorylcholine (C₁₆SM) and cholesterol. The amounts of DOPC, sum of C₁₆Cer and C₁₆SM, and cholesterol were kept constant (the ratio of these four lipids is shown as 1:X:1-X:1 (molar ratio), i.e., X is C₁₆Cer/(C₁₆Cer + C₁₆SM)). Shape and distribution of domains formed in the GUVs were monitored by a fluorescent lipid, Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (0.1 mol%). In GUVs containing low C₁₆Cer (X = 0 and 0.25), round-shaped domains labeled by the fluorescent lipid were present, suggesting coexistence of liquid-ordered and disordered domains. In GUVs containing intermediate Cer concentration (X = 0.5), the fluorescent domain covered most of GUV surface, which was surrounded by gel-like domains. Differential scanning calorimetry of multilamellar vesicles prepared in the presence of higher Cer concentration (X ≥ 0.5) suggested existence of a Cer-enriched gel phase. Video microscopy showed that the enzymatic conversion of SM to Cer caused rapid change in the domain structure: several minutes after the SMase addition, the fluorescent region spread over the GUV surface, within which regions with darker contrast existed. Image-based measurement of generalized polarization (GP) of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which is related to the acyl chain ordering of the lipids, was performed. Before the SMase treatment domains with high (0.65) and low (below 0.4) GP values coexisted, presumably reflecting the liquid-ordered and disordered domains; after the SMase treatment regions with intermediate GP values (0.5) and smaller regions with higher GP values (0.65) were present. Generation of Cer thus caused a phase transition from liquid-ordered and disordered phases to a gel and liquid phase.     

雄性大熊猫褪黑素与睾酮季节性变化

褪黑素通过调控下丘脑-垂体-性腺内分泌轴使季节性繁殖动物在适宜的季节进行繁殖活动.大熊猫(Ailuropoda melanoleuca)在春季集中繁殖.为探究雄性大熊猫褪黑素和睾酮的季节性变化规律,本研究选取成都大熊猫繁育研究基地3只成年雄性大熊猫作为实验对象,在自然光照下对这3只大熊猫进行每周1次为期1年(2018年...     

小相岭大熊栖息地干扰调查

强烈的人类干扰是大熊猫濒危的主要原因。本于2001年6～7月对四川省小相岭大熊猫栖息地内竹子开花、火灾、滑坡3种自然干扰类型和采伐、放牧、采药、公路、偷猎、割竹采笋、耕作、开矿和旅游9种人类干扰类型进行了调查。调查样方878个,干扰发生在海拔1800～4000m。结果表明：自然干扰都不严重,干扰比例最高的竹子开花,也仅占1．14％。采伐、放牧、采药和公路是主要人类干扰类型,分别占调查总样方的34．9％,26．3％,5．1％和4．2％。干扰最严重的地段是海拔3000～3499m,采伐样方的49．67％,放牧样方的47．19％,采药样方的31．11％均发生在该段,同时有48．89％的大熊猫活动痕迹样方也在该海拔。近年来,由于国家实施“天然林保护工程”,采伐已不再是主要干扰,而放牧、采药和偷猎等活动在短时间内仍不会减少。     

生态补偿对大熊猫栖息地周边农户生态足迹的影响

大熊猫栖息地正面临破碎化和人类活动干扰的威胁,如何减少这些威胁成为保护大熊猫这一生态旗舰物种的关键.通过实施生态补偿项目,充分考虑当地弱势群体生存和发展的权利,帮助其进行生计替代和能源替代,以减小生态系统所承受的压力.以120个项目农户作为研究对象,利用生态足迹方法,探讨了生态补偿项目对生活在关键生态区域内农村人口的影响.结果表明,在各种生态生产性土地中,农户对林地和草地生态占用最大,能源替代可以大幅降低农户的薪柴消耗水平,从而降低农户对林地的生态占用.通过调整农户的牲畜养殖模式,减小了放牧对草地资源的压力和对草地的生态占用.农户人均生态足迹由2005年的2.7346hm2减小至2007年的1.6325 hm2,对周边环境的压力降低,有利于保护和恢复珍稀野生动植物栖息地.     

大熊猫繁殖生物学初步研究

近年来,在动物园内人工饲养下的大熊猫(Ailuropoda melanoleuc),由于雌雄个体常常不能同时发情,甚至都不发情,因而不能正常产仔。故大熊猫的繁殖问题就成为国内外各动物园所关注的问题之一。 北京动物园大熊猫于1963年自然交配产仔首次成功。1978年以后,国内外动物园又相继进行了上百次的人工授精试验,据报道,真正受孕产仔的仅有中国的北京、上海、成都、杭州以及西班牙的马德里和墨西哥等动物园。可见其受精率很低。     

Molecular evidence for a single genetic clone of invasive Arundo donax in the United States

Arundo donax (giant reed) is an aggressive invasive weed of riparian habitats throughout the southern half of the United States from California to Maryland. Native to Asia, the species is believed to have been initially introduced into North America from the Mediterranean region although subsequent introductions were from multiple regions. To provide insight into the potential for biological control of A. donax, genetic variation in plants sampled from a wide geographical area in the United States was analyzed using Sequence Related Amplification Polymorphism (SRAP) and transposable element (TE)-based molecular markers. Invasive individuals from 15 states as well as four populations in southern France were genetically fingerprinted using 10 SRAP and 12 TE-based primer combinations. With the exception of simple mutations detected in four plants, A. donax exhibited a single multilocus DNA fingerprint indicating a single genetic clone. The genetic uniformity of invasive A. donax suggests that classical biological control of the species could be successful. A lack of genetic diversity in the invaded range simplifies identification of native source populations to search for natural enemies that could be used as biocontrol agents.     

Multisegmental cobalt filling of the dorsal giant fibers in the nervous system of the earthworm,Lumbricus terrestris

Summary The anatomical organization of the two dorsal giant fiber systems of the earthworm Lumbricus terrestris is demonstrated in whole mounts and serial-section reconstructions based on backfillings of the ventral nerve cord with cobalt chloride. Both the medial and lateral fiber systems can be labeled selectively over more than ten body segments. They show a characteristic segmental pattern of collaterals with some modification in tail segments and of dorsal plasma protrusions in the unpaired medial giant fiber presumably representing openings in the myelin sheath. We found no multisegmental cobalt transport in other large neurons of the nerve cord. Cobalt passes through the segmentai septa between consecutive axonal elements of the metameric giant fibers and presumably also through commissural contacts between specific collaterals of the lateral giant fibers. Since these sites of contact are known to represent electrical synapses, cobalt coupling may, in L. terrestris, correlate with functional electrotonic coupling.Abbreviations CL collateral of lateral giant fiber - CM collateral of medial giant fiber - GIN giant interneuron - LGF lateral giant fiber - MGF medial giant fiber - SN segmental nerve     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm²) and conductance (23-44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic β₂ receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.