A Bruch’s membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a similar thickness as Bruch’s membrane (3 μm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell). Cultures established on either material developed a cobblestoned morphology with partial pigmentation within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned medium collected from above and below both membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrate that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model.

Protection against UVB inactivation (in vitro) of rat lens enzymes by natural antioxidants

Oxidative damage, through increased production of free radicals, is believed to be involved in UV-induced cataractogenesis (eye lens opacification). The possibility of UVB radiation causing damage to important lenticular enzymes was assessed by irradiating 3 months old rat lenses (in RPMI-1640 medium) at 300 nm (100 mu Wcm(-2)) for 24 h, in the absence and presence of ascorbic acid, alpha-tocopherol acetate and beta-carotene. UVB irradiation resulted in decreased activities of hexokinase, glucose-6-phosphate dehydrogenase, aldose reductase, and Na, K- ATPase by 42, 40, 44 and 57% respectively. While endopeptidase activity (229%) and lipid peroxidation (156%) were increased, isocitrate dehydrogenase activity was not altered on irradiation. In the presence of externally added ascorbic acid, tocopherol and beta-carotene (separately) to the medium, the changes in enzyme activities (except endopeptidase) and increased lipid peroxidation, due to UVB exposure, were prevented. These results suggest that UVB radiation exerts oxidative damage on lens enzymes and antioxidants were protective against this damage.

Lipotoxicidade na leptospirose humana: aspectos prognósticos e potencial efeito benéfico da administração de albumina

A leptospirose humana é uma doença infecciosa aguda de amplo espectro clínico e que cursa com alterações metabólicas e dislipidêmicas envolvendo colesterol total e frações, triglicerídeos e ácidos graxos não esterificados (AGNEs). Dentre os mecanismos celulares envolvidos na sua fisiopatologia encontram-se a inibição da enzima Na, K ATPase pela endotoxina GLP e a lipotoxicidade, ambos agravados pela redução dos níveis circulantes da albumina, molécula que exerce um papel fundamental na adsorção de moléculas lipídicas. Neste estudo observacional, determinamos as concentrações séricas de bilirrubina, creatinina e albumina e, pela técnica de cromatografia líquida de alta performance, a concentração sérica dos AGNEs de cadeia longa (C16: C18) de 27 pacientes com síndrome de Weil durante o período de internação hospitalar, dos quais cinco vieram a falecer. Verificamos correlações significantes (p<0,05) ao longo da internação hospitalar, nas concentrações séricas de marcadores bioquímicos de gravidade da doença (bilirrubina, creatinina e albumina), AGNEs, ácido oléico e ácido linoléico, e relação molar ácido oléico/ albumina, com r (Pearson) de -0,7981, -0,7699, 0,9014, -0,8795 -0,9816, -0,9694, -0,9821, respectivamente. A relação molar ácido oléico/ albumina e ácido oléico+ linoléico/albumina foi significantemente mais elevada nos pacientes que faleceram (p<0,001), retornando aos valores semelhantes aos do grupo controle nos pacientes que evoluíram para a cura. Na análise por Curva Roc, a relação molar ácido oléico/albumina se mostrou um bom teste preditivo, com valor de corte 0,705 associado com maior especificidade e sensibilidade prognóstica. Nossos resultados sugerem que a utilização parenteral da albumina humana em pacientes com leptospirose pode ser uma potente ferramenta terapêutica nos casos mais graves ao interferir positivamente no resgate do equilíbrio bioquímico das relações molares ácido oléico/ albumina e ácido oléico+linoléico/albumina.

Alcohol-Related Brain Damage in Humans

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin beta II, and alpha- and beta-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in a-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in beta-spectrin protein levels, and a significant increase in transmembranous alpha 3 (catalytic) subunit of the Na+, K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of a-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic alpha-and beta-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics

Mechanisms of Toxicity of Ag Nanoparticles in Comparison to Bulk and Ionic Ag on Mussel Hemocytes and Gill Cells

Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag.

Effects of atrazine herbicide on physiological, biochemical and histological biomarkers of developmental stages of larvae and fry in Caspian kutum, Rutilus frisii kutum

To investigation of the toxic effects of atrazine on newly hatched larvae and releasing age fry of the Caspian Kutum, Rutilus frisii kutum, the 96h LC50 was determined as 18.53 ppm and 24.95 ppm, respectively. Newly hatched larvae were exposed to three sublethal concentrations of atrazine (1/2LC50, 1/4LC50 and 1/8LC50) for 7 days. Different histopathological alterations were observed in fins and integument, gills, Kidney, digestive system, liver and the brain of the exposed larvae. Fry’s were exposed to one sublethal concentration of atrazine (1/2LC50) for four days, and like the larvae’s, many histopathological alterations were observed in fins and integument, gills, Kidney, digestive system, liver and the brain of the exposed fry’s, too. Also, measurements of the body ions: Na+, K+, Ca2+, Mg2+ and Cl- in atrazine exposed larvae and fry’s compare to control groups showed that atrazine is changed the body ions composition. No significant differences were found in length growth rate, weight growth rate and the condition factor of the atrazine exposed larvae and fry. Immunohistochemical localization of the Na+, K+-ATPase in integumentary and gill ionocytes, showed no differences in dispersion pattern of the ionocytes in atrazine exposed larvae and fry, compare to control group. Measuring the dimensions of the ionocytes and counting the ionocytes showed that atrazine is affecting on ionocytes by mild increasing in size and mild decreasing in number. Ultrastructural studies, using SEM and TEM, showed that atrazine have significant effects on cellular and subcellular properties. It caused necrosis in surface of the pavement cells in branchial epithelium, necrosis in endoplasmic reticulum of the ionocytes and changed the shape of the mitochondria in these cells. Results showed that sublethal concentrations of atrazine were very toxic to larvae and fry of the Rutilus frisii kutum, and at these levels can made some serious histopathological alterations in their tissues. Related to the severe histopathological alterations in osmoregulatory organs, like gill, kidney and digestive system, and the alterations in the body ion composition, it could be concluded that atrazine could interfere with the osmoregulation process of the Rutilus frisii kutum at the early stages of the life history.

In vivo studies on the toxic effects of microcystins on mitochondrial electron transport chain and ion regulation in liver and heart of rabbit

This study examined the toxic effects of microcystins on mitochondria of liver and heart of rabbit in vivo. Rabbits were injected i.p. with extracted microcystins (mainly MC-RR and -LR) at two doses, 12.5 and 50 MCLReq. mu g/kg bw, and the changes in mitochondria of liver and heart were studied at 1, 3,12, 24 and 48 h after injection. MCs induced damage of mitochondrial morphology and lipid peroxidation in both liver and heart. MCs influenced respiratory activity through inhibiting NADH dehydrogenase and enhancing succinate dehydrogenase (SDH). MCs altered Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities of mitochondria and consequently disrupted ionic homeostasis, which might be partly responsible for the loss of mitochondrial membrane potential (MMP). MCs were highly toxic to mitochondria with more serious damage in liver than in heart. Damage of mitochondria showed reduction at 48 h in the low dose group, suggesting that the low dose of MCs might have stimulated a compensatory response in the rabbits. (C) 2008 Elsevier Inc. All rights reserved.

Effects of La3+ on lipid fluidity land structural transitions in human erythrocyte membranes

The effects of La3+ on the structure and function of human erythrocyte membranes were investigated by fluorescence polarization, spin-labeled electron spin resonance (ESR) and differential scanning calorimetry (DSC). The results showed that increasing concentrations of La3+ inhibited (Na++K+)-ATPase and Mg2+-ATPase activities. La3+ lowered the lipid fluidity of erythrocyte membranes and induced structural transitions in erythrocyte membranes.

INTERACTION OF LANTHANUM AND CALCIUM WITH HUMAN ERYTHROCYTE-MEMBRANES

The effect of lanthanum and calcium on the structure and function of human erythrocyte membranes was investigated by fluorescence polarization, spin- labeled electron spin resonance (ESR) and laser Raman spectroscopy. The results showed that low concentration of La3+ (0.5 mu mol/L) activated a Little (Na++K+)-ATPase and Mg2+-ATPase activities, and it inhibited obvi ously the ATPase activities with increasing its concentrations. La3+ lowered the lipid fluidity of human erythrocyte membranes and decreased the vibration intensity of alpha-helix of the protein in the Amide I '. The effect of Ca2+ on the lipid fluidity and alpha-helix of the protein in the Amide I ' was smaller than that of La3+.

塔玛亚历山大藻(Alexandrium tamarense)对鲈鱼(Lateolabrax japonicus) 的危害机制研究

本论文选择在我国分离得到的一株有毒赤潮甲藻-塔玛亚历山大藻（Alexandrium tamarense，ATHK株），研究了其对一种我国沿海常见和典型养殖鱼类鲈鱼（Lateolabrax japonicus）的危害机制。首先研究了塔玛亚历山大藻（ATHK）对鲈鱼鳃结构的影响及其溶血毒性； 然后采用腹腔注射的方法，研究了高剂量塔玛亚历山大藻毒素（ATHK毒素：约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）和低剂量塔玛亚历山大藻毒素（ATHK毒素： 约为0.16×105 细胞，相应PSP为0.0886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.00118µg STX Equal）在鲈鱼体内代谢过程中对鲈鱼肝脏、肾脏和鳃组织的超微结构、Na+K+-ATPase活性、肝脏功能和肾脏功能的影响、以及对抗氧化系统酶活性和异生物质代谢酶的影响，以期从不同方面了解塔玛亚历山大藻及其所产水溶性毒素（ATHK毒素）对鲈鱼的毒害效应及机制，为有毒赤潮的有效管理提供一定的科学依据。 塔玛亚历山大藻（ATHK）对鲈鱼鳃组织影响的实验结果表明，该藻使鲈鱼鳃组织出现水肿现象，细胞间隙变大；粘液细胞颗粒不规则，颜色加深，颗粒发生凝集，有板结状；氯细胞线粒体内部基质凝集。不产PSP的一种亚历山大藻（AT-6）也使鲈鱼鳃出现水肿，且使细胞出现一定的固缩现象。显微镜观察发现鲈鱼鳃丝间存在有这两种亚历山大藻细胞。由此推测塔玛亚历山大藻ATHK和AT-6的表面结构可能具有能导致鲈鱼鳃组织水肿的机械作用。对人血细胞溶血实验结果表明塔玛亚历山大藻（ATHK）具有较强的溶血毒性，大小与藻的生长阶段和细胞密度都有一定的关系：指数期的溶血毒性最大，随细胞数目的增多，活性逐渐加大；藻细胞、细胞碎片、细胞内容物都有一定的溶血毒性，其中细胞碎片的活性最大。通过11种（株）产PSP的亚历山大藻、不产PSP的亚历山大藻以及标准PSP的实验结果表明这种溶血毒性是由藻细胞的其它非PSP物质造成的，且这种溶血毒性在产PSP的亚历山大藻中具有一定的普遍性。 塔玛亚历山大藻（ATHK）对鲈鱼组织超微结构实验结果表明：ATHK毒素（约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）能导致鲈鱼组织细胞超微结构发生剧烈的变化，主要表现在：肝细胞细胞膜有肿胀现象，部分膜边缘溶解；细胞质糖原颗粒化；核糖体脱落，仅见滑面内质网；细胞质和线粒体内都出现空泡，且线粒体的嵴状结构也发生变化；核膜溶解比较严重，核质外溢，且异染色质边际化。前肾细胞超微结构的变化主要是淋巴细胞核质出现空泡，核膜有溶解迹象；Ⅰ型粒细胞颗粒膨大，伪足增多且变长；Ⅱ型颗粒细胞颗粒增多，内部出现空腔，细胞膜和核膜溶解，胞质、细胞器和核质外溢。鳃组织中氯细胞的核膜局部溶解，核仁弥散，线粒体膜溶解，微细小管膨大；粘液颗粒膜溶解，内部结构遭受破坏；扁平细胞核膜及线粒体膜几乎全部溶解。因此，我们的结果表明，ATHK毒素能作用于鲈鱼细胞的内膜和外膜系统，使膜发生溶解、脱落等变化；比较注射同样剂量大小ATHK毒素120h和240h时鲈鱼组织超微结构发现，细胞超微结构在一定程度上能够恢复。 ATHK毒素对Na+K+-ATPase活性、肝脏功能以及肾脏功能的影响结果表明，0.16×105―1.6×105细胞范围内的ATHK毒素可以显著影响肝脏和鳃组织中的Na+K+-ATPase，使这两种组织中的Na+K+-ATPase活性出现不同程度的下降； 而且还能够显著抑制肝脏中谷丙转氨酶的活性，最大抑制率为95%。但此范围内的ATHK毒素不能显著影响肾脏中的Na+K+-ATPase活性以及尿素氮含量。因此，ATHK毒素对Na+K+-ATPase活性的抑制则会导致细胞能量的缺失，使细胞进一步发生其它变化，而ATHK毒素对肝脏功能完整性的影响则可能会抑制对蛋白质的分解代谢。 ATHK毒素对鲈鱼肝脏、肾脏和鳃组织中的、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱肝肽过氧化物酶（GSH-Px）以及谷胱肝肽转硫酶（GST）活性变化影响的结果表明：高剂量（约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）ATHK毒素能显著诱导鲈鱼肝脏和鳃组织中SOD、GSH-Px以及GST酶活性，最大变化范围为正常状态下的3-4倍，对肝脏中CAT酶活性具有一定的抑制作用，对鳃中的CAT抑制效应则不显著；但此剂量的ATHK毒素仅对肾脏鳃中的GSH-Px活性有一定的诱导作用，对SOD、CAT以及GST的活性没有显著影响。低剂量（约为0.16×105 细胞，相应PSP为0.0886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.00118µg STX Equal）ATHK毒素也能诱导鲈鱼肝脏和鳃组织中SOD、GSH-Px以及GST酶活性，其在第一个24h内的诱导效果与临界致死毒素剂量相似； 且连续注射低剂量ATHK毒素则对肝脏和鳃中这三种酶活性具有累加的诱导作用，使这三种酶活性的变化范围为正常的5倍；低剂量ATHK毒素对肝脏中CAT酶活性也具有抑制作用，但对鳃中CAT酶活性的抑制作用并不显著。 同样，低剂量ATHK毒素除对肾脏中GSH-Px活性具有一定诱导效应外，对SOD、CAT以及GST都没有显著影响。 SOD、GSH-Px以及GST酶活性的显著升高表明ATHK毒素在鲈鱼体内代谢过程中能诱导鲈鱼产生活性氧自由基，且GST活性的升高则说明作为细胞色素P450依赖的异生物质代谢酶，GST在ATHK毒素代谢过程中可能可以加速ATHK毒素的代谢。推测鲈鱼可以通过这三种酶降低ATHK毒素以及次生毒物活性氧自由基对鲈鱼的危害。

两种海藻提取物的化学成分和生物活性的研究及其应用

本论文的研究工作由两部分组成，第一部分研究了海带（Laminaria japonica）水提取物中的活性物质，并研究了提取物对蔬菜促生长的影响及其作用机制。第二部分对三列凹顶藻(Laurencia tristicha)乙醇提取物的乙酸乙酯相进行了活性筛选和化学成分研究，并对其中分离得到的单体化合物进行了生物活性筛选。 第一部分主要以中国人工养殖的海带为原料，使用与海藻多糖生产相结合的提取技术并浓缩其中的有效成分。对浓缩提取物进行了蔬菜的农田效果实验，并对作物抗旱性能的增加、作物硝酸盐积累的减少、作物品质的改善、以及作物抵抗病毒病的能力等影响进行了作用机制方面的研究。海藻浓缩提取物进行的农田效果实验表明：作物抗旱型相对含水量RWC值在92%~94%之间；病毒病的防治效果最高可达到91%；作物的品质有明显的改善，最重要的是首次发现海藻提取物有降低蔬菜中硝酸盐的含量（硝酸盐的含量是与有机蔬菜区别的重要指标之一）的作用。该部分研究工作的创新性主要体现在：（1）首次在国内外提出和采用与海藻多糖生产相结合的提取技术。该技术的应用不但减少了提取成本，使工业化生产成为可能,更重要的是使我国的海藻工业生产可能实现高值化和开辟综合利用的新途径。（2）首次发现海藻中的小分子海藻多糖具有和细胞激动素、甜菜碱、植物生长素等活性物质同样的生物活性。 第二部分的研究是在查阅了大量的近20年来国内外有关红藻凹顶藻中化学成分研究的相关文献的基础上，对凹顶藻中的次生代谢产物进行了综述。该论文主要是通过对红藻三列凹顶藻的95%乙醇提取物的乙酸乙酯相进行化学成分分析和生物活性筛选以期能够发现具有药用前景的活性先导化合物。 为了寻找具有生物活性的化合物，我们对采自我国南海硇洲岛海域的红藻三列凹顶藻的95%乙醇提取物的乙酸乙酯相进行了活性筛选。采用MTT法对其在KB细胞株、Bel-7402细胞株、PC-3M细胞株、MCF-7细胞株、Ketr-3细胞株模型上进行了细胞毒活性测试；采用酶模型对其进行了Na+,K+-ATPase的抑制活性测试；采用MTT法对其在犬主动脉血管模型上进行了血管平滑肌细胞增殖抑制活性测试；结果表明，三列凹顶藻的95%乙醇提取物的乙酸乙酯相对Na+,K+-ATPase和犬血管平滑肌细胞增殖具有一定的抑制活性。 利用正相和反相色谱、Sephadex LH-20色谱以及反相HPLC等手段进行分离纯化，从我国南海海域的红藻三列凹顶藻中分离得到33种化学成分，通过波谱学方法（IR、MS、NMR）以及X-ray单晶衍射试验对其化学结构进行了确证，其中化合物L1~L8为新结构化合物，化合物L5为具有新骨架的全新结构化合物，化合物L9~L13为新天然产物，化合物L18和L22系首次从海洋生物中获得，所有化合物均为首次从该属海藻中得到。新化合物L1~L8均为倍半萜类化合物，命名分别为：(1R,3R)-(-)-3-(3-hydroxy-4-methylphenyl)- 1,3-dimethyl–2-methylidene cyclopentanol (L1), (1R,3R)-(-)-3-(4-methylphenyl)-1,3-dimethyl-2-methylidenecyclopentanol (L2)， (1R, 3R)-(-)-3-(2-hydroxy-4-methylphenyl)-1,3-dimethyl–2–methylidenecyclopentanol (L3)，(+)-(1S,2R)–2-(3–hydroxy–4–methylphenyl)-1,2-[3.1.0]bicy-clohexane (L4)，()-(1S,2R) -5-hydroxy–6–methyl-spiro-dihydrobenzofuran-2(3H),2-{1-methyl-[3.1.0]bicyclohexane} (L5), (+)-6-methyl-2-(p-tolyl)hept-4-en-2,6-diol (L6)，(3R,3aS,8bS)-(-)-2,3,3a,8b–tetrahydro–7-bromo – 3 a– hydroxymethyl - 3, 6, 8b - trimethyl-1H- cyclopenta[b] benzofuran (L7 )，(3R, 3aS, 8bS) - (-) - 2,3,3a,8b–tetrahydro–3 a–hydroxymethyl-3,6,8b -trimethyl -1H – cyclopenta [b] benzofuran (L8)。25个已知结构化合物确定为：(+)-(1R,2R)-4-bromo-1,5, 9–trimethyl–12– methylidene–8–oxa-tricyclo[7.2.1.02]dodeca-2,4,6-triene (L9)，(3S,3aR,8bS)-(-)-2,3,3a, 8b– tetra -hydro–7-bromo–3–hydroxy-3,3a,6,8b-tetramethyl-1H-cyclopenta[b]benzofu- ran (L10 )，(3R, 3aR, 8bS) - (-) - 2, 3, 3a, 8b – tetrahydro – 7 - bromo – 3 – hydroxy - 3,3a,6,8b - tetramethyl - 1H - cyclopenta [b] benzofuran (L11 )，(3S,3aR,8bS) - (-) - 2, 3, 3a, 8b – tetrahydro –3–hydroxy -3, 3a, 6, 8b - tetramethyl-1H-cyclopenta[b]benzofuran (L12 )， ( 3aR, 8bS) - (-) - 3a,8b –dihydro–7 - bromo – 3, 3a, 6, 8b - tetramethyl - 1H - cyclopenta[b]benzofuran (L13 )，aplysinol (L14 ) ，aplysin (L15)，laurebiphenyl (L16)，johnstonol (L17)，gossonorol (L18)，7,10-epoxy-ar- bisabol-11-ol (L19)，10-epi-7,10-epoxyarbisabol-11-ol (L20) 3β-hydroxy- 5α, 6α-epoxy- β- ionone (L21 )，3β-hydroxy-5β,6β-epoxy-β-ionone (L22 )，胆甾醇 (L23 )，胆甾-5-烯-3β,7α二醇胆甾-5-烯-3β,7α二醇 (L24)，β-谷甾醇 (L25)，叶绿醇 (L26 )，玉米黄素 (L27 )，对羟基苯甲醛 (L28 )，3-吲哚甲醛 (L29 )，1-O-十六烷酰基-3-O-β-D-吡喃半乳糖基-丙三醇(L30 )，1-O-十八烷酰基-3-O-β-D-吡喃半乳糖基-丙三醇 (L31 )，丙三醇-1-软脂酸单酯 (L32 )，正十六碳酸 (L33 )。 采用MTT法对其中23个单体化合物在Bel-7402细胞株、BGC-823细胞株、A549细胞株、A2780细胞株、HCT-8细胞株和HELL细胞株模型上进行了细胞毒活性测试；采用MTT法对其中13个单体化合物在犬主动脉血管模型上进行了血管平滑肌细胞增殖抑制活性测试；结果表明，部分单体化合物显示出一定的生物活性。

微生物——铊相互作用的生物地球化学研究——以真菌（Fungus）为例

铊是一种有毒有害的重金属元素，已经引起了广泛的关注。本论文通过对黔西南铊矿区土壤和沉积物样品的菌株分离、铊高耐受性菌株的筛选、胞外吸附、富集、亚细胞水平区系分布、絮凝实验及ITS序列等实验研究分析，并结合铊的地球化学相关研究，较系统地阐述了真菌--铊的生物地球化学过程机理，得出以下结论： 1、与环境背景区相比，黔西南滥木厂铊矿区内的河流、土壤中铊的已有不同程度的积累，直接导致了当地微生物生物量在很大程度上的降低，微生物生物量与铊含量间有显著的负相关关系。研究区内的沉积物、土壤中的微生物区系结构和数量发生了明显变化，细菌、真菌及放线菌数量均出现显著降低，而且三大微生物对重金属污染的敏感性大小也不一样，即放线菌>细菌>真菌。从土壤样品中分离到的主要菌群仍为常见种属，如青霉属（Penicillium）、木霉属（Trichoderma）、拟青霉（Paecilomyces）等。 2、经过初筛菌株的铊耐受性实验，在1000 mg/L水平筛选得到九株高耐受性菌株。吸附实验表明：微生物菌株对铊的吸附效率在4.63～16.89%，且随着环境中铊浓度的上升而降低，这可能是因为铊浓度的升高加大了对微生物生长的抑制作用，所形成的菌丝体（或菌丝球）减少，表面积也相应减少，从而导致了吸附效率的下降。各种常量元素和铊的关系呈显著相关性，钙、钾和钠等常量元素也是微生物赖以维持生存的因子，可能由于微生物细胞对钙、钾的吸附方式与对铊的吸附方式类似。因此，随着铊处理浓度的上升，钙和钾的吸附量也随之减少，而钠则呈现相反的趋势。 3、富集实验表明，九株菌株对铊的富集量随着铊处理浓度上升而降低，其影响趋势与对生物量的影响趋势基本一致，最高可达到7189 mg/kg，最大富集系数为7.2。九株菌株对常量元素的富集与对铊的富集并无明显的相关性，但在考察铊处理浓度对常量元素的富集影响时发现，铊处理浓度的上升与对钙的富集量表现出较强的正相关；而对钾、钠、镁的富集影响并不明显。 4、亚细胞水平上的铊分布研究表明，铊的富集优先顺序为：细胞质>细胞壁>细胞器。亚细胞水平的区隔化作用是微生物对铊的主要耐受机制，细胞质是赋存铊的主要场所（53.83～79.45 %）。结合各亚细胞组分中常量元素与铊之间的相关性，并联系前人的研究，Tl+主要是通过细胞壁的Na+ -K+ ATPase和K+ -电位门通道进入细胞内的从而影响细胞的正常代谢的，而Ca2+的活化更有助于这一过程。 5、絮凝实验表明，培养三天后的发酵液对矿区废水中铊的去除率最高可达到70.49 %，最佳影响因子组合为：pH=8，温度为16℃，搅拌时间为4分钟。菌株的絮凝活性最高可达到57.32%，最佳影响因子组合为：pH=8，温度为14℃，搅拌时间为4分钟。 6、通过对九株铊高耐受性菌株的ITS序列分析及其在Gene Bank中的BLAST比对结果表明，五株菌株同属于木霉属（Trichoderma），两株菌株同属于青霉属（Penicillium）。这表明这两类真菌对铊的适应性较强，为以后寻找铊高耐受性菌株及其资源化利用提供了理论基础。

Invivo Changes In The Activity Of Gill ATPases And Hemolymph Ions Of Carcinus Maenas Exposed To p p'-DDT And Reduced Salinities

1. Haemolymph osmoregulation by the shore crab, Carcinus maenas was confirmed over a range of salinities. 2. Na+K+-ATPase and Mg2+-ATPase activities in gill tissue increased with a decrease in salinity. 3. Na+K+-ATPase and Mg2+-ATPase activities were inhibited after exposure to p,p′-DDT. 4. K+ ion levels of the haemolymph remained unchanged, but Na+ ion levels were affected after 7 hr when transferred from 100 to 5% SW after exposure to DDT.

Higher brain neurons succumb to acute stroke-like insult while lower brain neurons strongly resist

Pyramidal neurons (PyNs) in ‘higher’ brain are highly susceptible to acute stroke injury yet ‘lower’ brain regions better survive global ischemia, presumably because of better residual blood flow. Here we show that projection neurons in ‘lower’ brain regions of hypothalamus and brainstem intrinsically resist acute stroke-like injury independent of blood flow in the brain slice. In contrast `higher` projection neurons in neocortex, hippocampus, striatum and thalamus are highly susceptible. In live brain slices from rat deprived of oxygen and glucose (OGD), we imaged anoxic depolarization (AD) as it propagates through these regions. AD, the initial electrophysiological event of stroke, is a depolarizing front that drains residual energy in compromised gray matter. The extent of AD reliably determines ensuing damage in higher brain, but using whole-cell recordings we found that all CNS neurons do not generate a robust AD. Higher neurons generate strong AD and show no functional recovery in contrast to neurons in hypothalamus and brainstem that generate a weak and gradual AD. Most dramatically, lower neurons recover their membrane potential, input resistance and spike amplitude when oxygen and glucose is restored, while higher neurons do not. Following OGD, new recordings could be acquired in all lower (but not higher) brain regions, with some neurons even withstanding multiple OGD exposure. Two-photon laser scanning microscopy confirmed neuroprotection in lower, but not higher gray matter. Specifically pyramidal neurons swell and lose their dendritic spines post-OGD, whereas neurons in hypothalamus and brainstem display no such injury. Exposure to the Na+/K+ ATPase inhibitor ouabain (100 μM), induces depolarization similar to OGD in all cell types tested. Moreover, elevated [K+]o evokes spreading depression (SD), a milder version of AD, in higher brain but not hypothalamus or brainstem so weak AD correlates with the inability to generate SD. In summary, overriding the Na+/K+ pump using OGD, ouabain or elevated [K+]o evokes steep and robust depolarization of higher gray matter. We show that this important regional difference can be largely accounted for by the intrinsic properties of the resident neurons and that Na+/K+ ATPase pump efficiency is a major determining factor generating strong or weak spreading depolarizations.

ENaC activity in collecting ducts modulates NCC in cirrhotic mice.

Cirrhosis is a frequent and severe disease, complicated by renal sodium retention leading to ascites and oedema. A better understanding of the complex mechanisms responsible for renal sodium handling could improve clinical management of sodium retention. Our aim was to determine the importance of the amiloride-sensitive epithelial sodium channel (ENaC) in collecting ducts in compensate and decompensate cirrhosis. Bile duct ligation was performed in control mice (CTL) and collecting duct-specific αENaC knockout (KO) mice, and ascites development, aldosterone plasma concentration, urinary sodium/potassium ratio and sodium transporter expression were compared. Disruption of ENaC in collecting ducts (CDs) did not alter ascites development, urinary sodium/potassium ratio, plasma aldosterone concentrations or Na,K-ATPase abundance in CCDs. Total αENaC abundance in whole kidney increased in cirrhotic mice of both genotypes and cleaved forms of α and γ ENaC increased only in ascitic mice of both genotypes. The sodium chloride cotransporter (NCC) abundance was lower in non-ascitic KO, compared to non-ascitic CTL, and increased when ascites appeared. In ascitic mice, the lack of αENaC in CDs induced an upregulation of total ENaC and NCC and correlated with the cleavage of ENaC subunits. This revealed compensatory mechanisms which could also take place when treating the patients with diuretics. These compensatory mechanisms should be considered for future development of therapeutic strategies.