In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Use of a crude extract or purified antigen from first‐instar cattle grubs,Hypoderma lineatum,for the detection of anti‐Hypoderma antibodies in free‐ranging cervids from southern Spain

During the 2003–2005 hunting seasons, a total of 120 Cervidae, including 39 red deer (Cervus elaphus hispanicus) and 81 fallow deer (Dama dama), were examined for subcutaneous myiasis. Animals were shot from January to June in southern Spain. Specific antibodies against Hypodermatinae (Diptera: Oestridae) were detected by indirect enzyme‐linked immunosorbent assay (iELISA) using a crude larval extract (CLE) and a purified antigen [hypodermin C (HC)] obtained from first instars of Hypoderma lineatum (De Villers) (Diptera: Oestridae). Hypoderma actaeon Brauer was the only species detected in this study, which represents the first confirmation of this species in fallow deer from Spain. The overall prevalence of animals presenting subcutaneous larvae (14.2%) was considerably lower than the prevalences determined by iELISA with CLE (43.3%) and HC (40.0%). Red deer showed a higher prevalence of Hypoderma than fallow deer. The concordance between larval examination during the hunting season and iELISA using both antigens was low, whereas the concordance between the CLE and HC ELISAs was good. Larval antigens obtained from H. lineatum constitute a good tool for the diagnosis of H. actaeon in Cervidae, especially when the hunting season does not coincide with the maximum presence of larvae on the back.     

The Dutch N-cascade in the European perspective

The Netherlands is "well known" for its nitrogen problems; it has one of the highest reactive nitrogen (Nr) emission densities in the world. It is a small country at the delta of several large European rivers. Ever since the industrial revolution, there has been a growing excess of nutrients and related emissions into the atmosphere (ammonia, nitrogen oxides and nitrous oxide) and into groundwater and surface water (nitrate), leading to a large range of cascading environmental impacts. Vehicular traffic, sewage and animal husbandry are the main sources of oxidized and reduced forms of Nr. This paper provides an overview of the origin and fate of nitrogen in the Netherlands, the various reported impacts of nitrogen, the Dutch and European policies to reduce nitrogen emissions and related impacts. In addition, ways are presented to go forward to potentially solve the problems in a European perspective. Solutions include the improvement of nitrogen efficiencies in different systems, technological options and education.     

Effect of the Pro-Gly-Pro peptide on resistance of rats to acute hypobaric hypoxia and posthypoxic behavioral disorders

Outbred white rats were subjected to acute hypobaric hypoxia in an altitude chamber at a reduced pressure of 145 mm Hg. Parameters of behavioral activity were evaluated in the tests of elevated cross maze and hole chamber and by the Porsolt method. Posthypoxic behavioral changes were characterized by an increase in the level of anxiety and depression and by a decrease in the level of orienting and exploratory activity. Preliminary intraperitoneal administration of the peptide 15 min before the hypoxia at a dose of 3.7 mol/kg increased the coefficient of individual resistance of the animals owing to a decrease in restitution time and significantly decreased posthypoxic behavioral disorders. We previously found that the Pro-Gly-Pro peptide had positive influence on homeostasis and maintained an adequate blood supply to organs and tissues. It was proposed that this ability of the Pro-Gly-Pro peptide could be one of the important mechanisms for its protective antihypoxic effect.     

Prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin administration

The possibility of prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin devoid of proteolytic activity and capable of stimulating the reaction of anticoagulation system was studied. The injection of lethal thromboplastin dose was shown to produce a sharp increase in soluble fibrin blood content, total disappearance of fibrinolytic activity and intravascular blood coagulation. The animals died of thrombosis in 90% of cases. It was established that the injection of lethal thromboplastin dose 5 min after DIP-alpha-thrombin injection caused a 13% lethality from thrombosis. No reliable changes in fibrinolytic activity and soluble fibrin content were observed. A significant increase in thrombin and recalcification time was recorded. It is suggested that DIP-alpha-thrombin prevents intravascular blood coagulation induced by lethal thromboplastin dose due to mobilization of the reserve capacities of neuro-humoral anticoagulation system.     

河西走廊天然固沙植被区地表甲虫多样性及其对沙漠化的指示作用

干旱、半干旱区沙漠化强烈影响动植物分布及多样性,地表甲虫是荒漠中主要的动物类群,它们对沙漠化引起的植被和土壤环境变化响应十分敏感。鉴于此,以河西走廊中部张掖绿洲外围的天然固沙植被区作为研究区,依据沙漠化发育程度选择流动沙丘（ASD）、丘间低地（IL）、半固定（SFSD）和固定沙丘（FSD）4种生境,调查了地表甲虫群落组成及影响甲虫分布的植被和土壤环境。研究发现,4种生境地表甲虫群落组成明显不同并存在季节变异,5月ASD与IL、SFSD和FSD生境地表甲虫群落的相异性大于8月。5月和8月SFSD生境地表甲虫活动密度均显著高于其他生境,8月FSD生境地表甲虫多样性指数显著高于其他生境。不同大小甲虫对沙漠化的响应模式不同,大中型甲虫对沙漠化的响应较小型甲虫敏感,这在5月表现尤为明显。地表甲虫与环境因子的RDA分析结果表明,12个植被和土壤环境因子解释了49.8%的地表甲虫群落变异,其中植被环境解释了甲虫群落变异的16.3%,土壤环境解释了甲虫群落变异的4.2%,植被和土壤环境相互作用解释了甲虫群落变异的29.3%。pRDA分析结果表明,草本物种丰富度、灌木盖度、土壤有机碳含量和粗砂含量是影响地表甲虫分布的主要环境因子,它们解释了43.7%的地表甲虫群落变异。Pearson相关分析表明,草本物种丰富度与地表甲虫活动密度呈显著正相关,而与地表甲虫均匀度呈显著负相关;灌木盖度与地表甲虫多样性呈显著正相关;地表甲虫物种丰富度与灌木盖度和草本物种丰富度均呈显著正相关。此外,研究还发现戈壁琵甲、克氏扁漠甲、中华砚甲和甘肃齿足象可以用于指示FSD生境,东鳖甲属昆虫可以用于指数SFSD生境,谢氏宽漠王可以用于指示IL及ASD生境。     

The effect of PGP on β-hexosaminidase and histamine secretion in rat peritoneal mast cells in vitro

The investigation of the effect of peptide prolyl-glycyl-proline (PGP) on β-hexosaminidase and histamine secretion by mast cells in primary culture has shown that incubation of mast cells with PGP (6 × 10⁻⁵ M) before their activation by synacten significantly decreased the amount of secreted histamine and β-hexosaminidase in comparison with the action of synacten only. The peptide in investigated concentration had no influence on the level of spontaneous secretion. Incubation of cells with PGP did not prevent their activation by compound 48/80. Therefore, PGP can have a direct effect on isolated rat mast cells in vitro and diminish their secretory activity under activation by synacten.     

肠内营养对乳腺癌术后化疗患者肠道菌群及生活质量的影响

目的研究肠内营养对乳腺癌术后化疗患者肠道菌群、生活质量、营养指标水平的影响。方法选取2012年7月至2018年1月于我院进行乳腺癌术后化疗的91例女性乳腺癌患者为研究对象,根据是否进行肠内营养将患者分为肠内营养组和对照组,观察化疗前后2组患者肠道菌群、生活质量、营养指标水平的变化及不良反应发生情况。结果治疗前2组患者肠道菌群、生活质量、营养指标水平差异无统计学意义(均P0.05)。治疗后,肠内营养组患者肠道双歧杆菌数量为(6.7±0.5)lg CFU/g、乳杆菌数量为(8.5±0.4)lg CFU/g,均显著高于治疗前,同时显著高于对照组(均P0.05)。治疗后肠内营养组患者出现反酸、腹胀、便秘等胃肠道不良反应的比例与对照组比较差异无统计学意义(均P0.05)。治疗后两组患者总体健康、认知功能、情绪功能、角色功能、社会功能、活力、躯体疼痛、躯体功能评分显著高于治疗前(均P0.05),且肠内营养组患者生活质量各指标的评分显著高于对照组(均P0.05)。治疗后肠内营养组患者血清总蛋白(TB)、血清清蛋白(ALB)、血清前清蛋白(PA)、转铁蛋白(TF)水平均显著高于对照组(均P0.05)。结论肠内营养可预防乳腺癌术后化疗患者肠道菌群的紊乱及营养不良的发生,安全有效,有助于提高乳腺癌患者的生活质量。     

天然和人工固沙灌木林蜘蛛和甲虫分布与环境因子的关系

干旱区人工植被恢复驱动的土地利用变化强烈影响了地表和土栖的节肢动物群落结构及多样性。然而,我们对地表节肢动物群落关键类群-蜘蛛和甲虫对固沙植被恢复的响应及与环境变化关系的认识还很有限。以天然固沙灌木林和2种人工固沙灌木林为研究对象,运用方差分析和多变量分析等方法定量研究了干旱区天然和人工固沙植被区地表蜘蛛和甲虫分布特征及影响要素。结果表明,天然灌木林与人工梭梭、柽柳林地表蜘蛛和甲虫群落组成明显不同,人工梭梭、柽柳林地表蜘蛛活动密度和甲虫多样性均显著高于天然灌木林,而地表甲虫密度和蜘蛛多样性变化与之相反。两种人工固沙灌木林之间蜘蛛和甲虫群落组成也存在一定差异,人工柽柳林地表蜘蛛活动密度、多样性和甲虫物种丰富度均显著高于人工梭梭林。进一步分析发现,蜘蛛群落中狼蛛科、平腹蛛科、皿蛛亚科和球蛛科与甲虫群落中拟步甲科、步甲科和象甲科等一些甲虫种属对3种生境的选择模式不同决定了蜘蛛和甲虫群落聚集结构。植被、土壤环境因子与蜘蛛和甲虫pRDA和pCCA结果表明,草本生物量、凋落物量、土壤含砂量、电导率和灌木盖度是影响蜘蛛分布的主要环境因子,它们解释了82.1%的蜘蛛群落变异;灌木盖度、草本生物量、土壤pH和砂含量是影响甲虫群落分布的主要环境因子,它们解释了60.6%的甲虫群落变异。总之,人工固沙灌木恢复影响了植被和土壤环境,它们相互作用改变了荒漠-绿洲过渡区蜘蛛和甲虫等地表节肢动物的分布格局。     

Hybridization between diploid Centaurea pseudophrygia and tetraploid C. jacea (Asteraceae): the role of mixed pollination,unreduced gametes,and mentor effects

We studied hybridization between the diploid Centaurea pseudophrygia and the tetraploid C. jacea by performing crossing experiments and screening natural populations using flow cytometry. The experiments confirm that the studied species exhibit strong reproductive isolation. Interspecific hybrids were formed at a low frequency, including triploids (originating from reduced gametes) and tetraploids (involving unreduced gametes of the diploids). In contrast, hybrids were almost absent among seeds and adult plants of natural mixed populations and among the offspring from experimental pollinations with a mixture of pollen of both ploidy levels. We found that mixed pollination is an important mechanism for preventing hybridization between plants of different ploidy levels and sustaining the reproduction of the tetraploids. A mentor effect (induced selfing in the presence of pollen of different ploidy levels) was observed in both diploids and tetraploids, reinforcing the reproductive isolation between cytotypes. Higher ploidy levels (pentaploid, hexaploid) involving unreduced gametes of the tetraploid species were identified. Notably, pentaploids were discovered for the first time in Centaurea sect. Jacea. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 93–106.