A laboratory study on risk assessment of microcystin-RR in cropland

The persistence time and risk of microcystin-RR (MC-RR) in cropland via irrigation were investigated under laboratory conditions. In order to evaluate the efficiency of the potential adsorption and biodegradation of MC-RR in cropland and the persistence time of MC-RR for crop irrigation, high performance liquid chromatography (HPLC) was used to quantify the amount of MC-RR in solutions. Our study indicated that MC-RR could be adsorbed and biodegraded in cropland soils. MC-RR at 6.5 mg/L could be completely degraded within 6 days with a lag phase of 1 - 2 days. In the presence of humic acid, the same amount of MC-RR could be degraded within 4 days without a lag phase. Accordingly, the persistence time of MC-RR in cropland soils should be about 6 days. This result also suggested the beneficial effects of the organic fertilizer utilization for the biodegradation of MC-RR in cropland soils. Our studies also demonstrated that MC-RR at low concentration (< 10 mu g/L) could accelerate the growth of plants, while high concentration of MC-RR (> 100 mu g/L) significantly inhibited the growth of plants. High sensitivity of the sprouting stage plants to MC-RR treatments as well as the strong inhibitory effects resulting from prolonged irrigation further indicated that this MC-RR growth-inhibition may vary with the duration of irrigation and life stage of the plants. (c) 2007 Published by Elsevier Ltd.

在盐胁迫下外源维生素浸种对小麦发芽及幼苗生长的影响

维生素(Vitamin)又称维他命，为“万年青”产品，是维持人体生命健康必需的一类低分子有机化合物质。维生素对人体健康的作用人们研究很多, 维生素可以增强人体对感染的抵抗力，降低出生缺陷及降低癌症和心脏病发病率等，一旦缺乏，肌体代谢就会失去平衡，免疫力下降，各种疾病，病毒就会趁虚而入；而维生素对作物影响的研究却很少。目前为止，尚无对用维生素浸种的方法来研究外源维生素是否对小麦种子萌发及幼苗生长起调节作用的报道，且对其在小麦抗逆性方面影响的研究甚少，对盐的胁迫抗性研究尚未有人报道。小麦（Triticum aestivum L.）属于拒盐的淡土性作物。盐害不利于小麦生长，严重影响小麦的产量和品质。本研究采用4 种不同维生素VB1、VC、VB6、VPP，分别对供试小麦品种川育12（红皮）、川育16（白皮）小麦浸种后，在一般自然条件下和逆境（盐胁迫条件）下，进行试验。探讨在正常情况下与在不同盐浓度条件下，各维生素及盐浓度对小麦发芽及幼苗生长的影响，并且比较两种不同皮色的小麦在相同盐胁迫条件下的差异表现，同时研究维生素处理的特异性，且哪种维生素对盐害缓解作用最佳。研究结果表明：在无盐胁迫（自然）条件下，对用4 种不同维生素VB1、VC、VB6、VPP 浸种小麦川育12、川育16 后的种子萌发及幼苗生长（幼苗的根长、根重、苗高、苗鲜重）的研究结果表明：4 种外源维生素浸种均对小麦发芽有调节作用，都能提高其最终发芽率。但是提高幅度有所差异。用VB6 浸种后的小麦提高幅度最多，VC 次之，VPP 提高幅度最小。同时，4 种外源维生素浸种对小麦种子的出芽速度及芽后长势也有一定的影响。VB6、VC 处理的小麦种子出芽速度最快，萌发后长势最好；VB1 出芽速度相对较慢，VPP 最慢,但都大于对照；VB1 处理长势略高于对照，VPP 处理的小麦长势则低于对照。从整体来看，VB6、VC处理促进效应明显， VB1 次之，而VPP 在某些方面无效甚至产生负效应。此外，相同的维生素处理对不同的品种的种子萌发、生长效果也存在差异，各种维生素作用于川育12 的效应均强于对川育16。进一步对幼苗根系TTC 还原力及幼苗叶片中硝酸还原酶活性进行测定、分析。研究发现：并非所有种类的维生素对幼苗根系TTC 还原力及幼苗叶片中硝酸还原酶活性的提高都有帮助。幼苗根系TTC 还原力在不同维生素处理下存在显著差异，而与小麦品种关系甚微。经VB6、VC 处理后，根系TTC 还原力测定值均显著高于对照，VB1 不明显，VPP 则略低于对照。VB6、VC 处理的幼苗叶片中硝酸还原酶的含量大于对照，VB1 与对照相差无几，而VPP 处理的川育12 幼苗叶片中的硝酸还原酶活性比对照CK 略高，而在川育16 中则略比对照CK 有所下降，呈现出抑制效应。综上结果表明：VB6、VC 具有促进种子发芽，幼苗生长及根系生长的作用，是较好的促生长剂；VPP 具有抑制作用，是较好的抑制剂，可进一步研究、开发利用。在盐胁迫条件下，对用4 种不同维生素VB1、VC、VB6、VPP 浸种川育12、川育16 后的种子萌发及幼苗生长（幼苗的根长、根重、苗高、苗鲜重）的研究结果表明：在不同盐浓度胁迫条件下， 各处理的种子萌发及幼苗生长均受到不同程度的抑制。随着盐浓度的增加, 发芽率、发芽指数和活力指数成下降趋势；幼苗的根长、根重、苗高、苗鲜重不断降低。4 种维生素处理间也表现出较大差异。VB6、VC 在每个处理中均保持对盐害的缓解作用，VB6 较VC 更易于促进发芽及幼苗生长。最终发芽率高，根系多、长、重，苗高高、重。而VB1、VPP 则表现出抑制作用。在高盐浓度150mM 时，4 种维生素浸种后的种子，其最终发芽率均不能达到40％，但VB6、VC 处理最终发芽率、苗重、根重均高于对照，VPP 最终发芽率、苗重、根重均低于对照。进一步对幼苗根系TTC 还原力及幼苗叶片中脯氨酸含量进行测定、分析。研究发现：不同盐浓度，不同维生素处理、不同品种间存在差异。随着盐浓度的增加（75mM，100mM，150mM），幼苗根系TTC 还原力活性成下降趋势，幼苗叶片中脯氨酸的积累量成上升趋势。VB6 处理脯氨酸含量增加最为明显，VC 次之，VPP 与对照接近，其变化幅度最小。经VB6、VC 处理后的幼苗根系还原强度，在不同盐浓度下，测定值均显著高于对照，VB1 不明显，VPP 则低于对照，产生负效应。此外，品种间表现不尽相同，相同的维生素处理，相同的盐浓度对不同的品种的种子萌发、生长效果也存在差异， 4 种维生素对川育16 的作用均强于川育12，但其影响趋势是一致的。说明VB6、VC 具有耐（抗）盐性，可以促进种子发芽和幼苗生长，是较好的耐（抗）盐拌种剂。 Vitamin is one kind of necessary low molecular compound for humans tosustain health and life. Lots of Studies have been done on the effectc of the vitaminsfor people. Vitamin can help people improve the body's natural resistance to disease,Drop the rate of birth defects、cacers and the incidence of the heart diseases. Ifpeople have less of them, the metabolism of the organism may throw off balance,immunity may drop off, and catch disease; Though the effects for Vitamin to thecrops are limited. up to now, there’s no one use soking seeds of wheats with vitaminsas a method, to study on how the effects will happen on the wheat seed germinationand seedling growth, and there are only few reserches on antireversion force forwheats ,none for the antireversion force in Sault stress condition.Wheat（Triticum aestivum L.）is sensitive to the salt, so the salt damage will doharm to wheat’s growth, it will have an unfavorable impact on the output and thequality of wheat.On this reaserch, we Soaking CHY12(red)、CHY16 (white) wheat seeds withVitamin C, B1, PP, B6 (50mg/L) as a pretreatment first. Then under two condition: one is in the normal environment the other is in different Salinity, we begin ourexperiments. Then disscuss on if the vitamin and salinity affect the wheat seedgermination and seedling growth, and what is the different between the two of them,the result shows that:Under the normal condition, after soaking seeds with VB1、VC、VB6、Vpp，we study on the their seed germination and the seeding growth(the root length andweights, The seedling heights and weights), it shows that all of those four kinds ofvitamin can adjust the seed germination, but different in The growth rate. VB6 isbest for increase, VC comes second,VPP is the worst. Meanwhile, those four vitaminalso have effect on the speed of the sprouting of the wheat. VB6、Vc can faster theseed germination most, and the seedlings are all doing well; VB1 do little effects onthe budding, Vpp is the worst, but all treatments are better than CK; but in Vi, VB1some what above the CK, while VPP lower than that. On the whole, the acceleratingeffect of VB6、VC are obvious, VB1 takes second place, but VPP in some aspects arenoneffective even have negative effect. Furthermore, different kind of seeds with thesame vitamin may different in seed germination and seedling growth, four vitaminson CHY16 is better than CHY12.More studies on TTC reductive capacity of roots and the activity of nitratereductase in the leaves, the reasult shows not all the vitamin can help the seedlings toimprove the TTC reductive capacity and the activity of nitrate reductase. TTCreductive capacity in different treatments shows significant differences,but notcorrelate to the variety of the wheat. The TTC reductive capacity of VB6、Vctreatments are all higher than CK, VB1 is nearly the same as CK, VPP is a littlelower than CK. Through the study of acivity of nitrate reductase, it shows that,VB6、VC are higher than CK ,VB1 is nearly the same as CK also, VPP is a little higher inthe CK of CHY12 but lower in CHY16. Through all the results above: VB6、Vc helpthe wheat seed germination, seedling growth and the growth of roots, is theperfectable factor of stimulating the growth; Vpp is a inhibition, that’ll be furtherreserch,and well develop and utilize in the future.Under the different Salinity condition, after soaking seeds with VB1、VC、VB6、Vpp，we study on the their seed germination and the seeding growth(the root lengthand weights, The seedling heights and weights), it shows that: under differentsalinity, the seed germination and the seedling growth of any treatment are inhibited.With the increase of the concentration, the germination rate, Vi、Gi all had fallen; theroot length and weight, the seedling heights and weights steadily sank down. There are also have pronounced difference between all treatments with four differentvitamins.VB6、VC in all treatments are alleviative the salt damage, VB6 is easier tocause to put forth buds than VC, and it’s quantitative value is the highest in theultimate germination rate, in root and seedlings’ hight and weight. Though the VPP、VB1 are seems to inhibite its growth. Under the high concentration150mM Nacl, theultimate germination rate in all treatments are below the 40%, but VB6、VC’squantitative values in any experiments are higher than CK,while VPP lower thanCK.Then we study on the TTC reductive capacity of roots and the content of Polinein leaves, the result shows that between the different salinity, different vitamintreatments, different varieties of the wheat have discrepancy.along with theincreasing concentraion of the salinity（75mM，100mM，150mM），TTC reductivecapacity of roots decreases, the accumulation of the content of Poline in leaves havean upward trend. The increase of VB6’s treatment are obviously, VC comessecond,VPP is nearly come up with CK, changes a little. In TTC reductive capacity of roots’s reserch, VB6、VC are higher than CK at any time,VB1 is not palpable,VPP is lower than CK, makes negative affect on wheat. In addition, varieties of thewheats are remain different, no matter it shows promoting or inhibiting, all fourvitamins have moreobvious effects on CHY16 than CHY12, but the tendency of theeffection are the same. It is say that VB6、VC can help wheat to standwith the saultwell, and promot in growth，they are the better reagent to mix with the seed.

中国青藏高原青稞抗穗发芽资源评价与α-淀粉酶基因的克隆及表达

穗发芽（PHS，preharvest sprouting）是影响禾本科作物生产的重要的灾害之一。收获时期如遇潮湿天气容易导致穗发芽发生。发生穗发芽的种子内部水解酶（主要是α-淀粉酶）活性急剧升高，胚乳贮藏物质开始降解，造成作物产量和品质严重降低。因此，选育低穗发芽风险的品种是当前作物育种工作中面临的重要任务。 青稞(Hordeum vulgare ssp. vulgare)主要分布于青藏高原，自古以来就是青藏高原人民的主要粮食。近年来，由于青稞丰富的营养成分和特有的保健品质、在燃料工业中的潜力以及在啤酒酿造工业中的利用前景，在发达国家日趋受到重视，掀起综合研究利用的热潮。我国拥有占全世界2/3 以上的青稞资源，具有发展青稞产业的得天独厚的条件。然而，由于青稞收获期间恰逢青藏高原雨季来临，常有穗发芽灾害发生，使青稞生产损失巨大。目前对青稞穗发芽研究很少，适用于育种的穗发芽抗性材料相对缺乏，不能很好的满足青稞穗发芽抗性育种的需要。本研究以青藏高原青稞为材料，对其穗发芽抗性的评价指标和体系进行构建，同时筛选青稞抗穗发芽品种并对其抗性进行评价，还利用分子生物学手段对青稞穗发芽抗性的分子机理进行了初步探讨。主要研究结果如下： 1. 本试验以来自于我国青藏高原地区的青稞为材料，对休眠性测定的温度范围进行探讨，并对各种穗发芽抗性测定方法的对青稞的适用性进行评测。通过探讨温度对13 个不同基因型的青稞籽粒发芽和休眠性表达的影响，对筛选青稞抗穗发芽资源的温度条件进行探索，并初步分析了其休眠性表达的机理。在10，15，20，25，30℃的黑暗条件下，选用新收获的13 个青稞品种为材料进行籽粒发芽实验，以发芽指数（GI）评价其休眠性。结果发现，不同品种对温度敏感性不同，其中温度不敏感品种，在各温度条件下均表现很低的休眠性；而温度敏感品种，其休眠性表达受低温抑制，受高温诱导。15℃至25℃是进行青稞休眠性鉴定的较适宜的温度范围。通过对供试材料发芽后的α-淀粉酶活性，发现温度对青稞种子的休眠性表达的影响至少在一定程度上表现在对α-淀粉酶活性的调控上。随后，对分别在马尔康和成都进行种植的34 份青稞穗发芽指数（SI），穗发芽率（SR），籽粒发芽指数（GI）和α-淀粉酶活性（AA）进行了测定和分析，发现它们均受基因型×栽培地点的极显著影响，且四个参数之间具有一定相关性。GI 参数由于其变异系数较低，在不同栽培地点稳定性好，且操作简便，是较可靠和理想的穗发芽评价参数。SI 参数可作为辅助，区别籽粒休眠性相似的材料（基因型）或全面评价材料（基因型）的穗发芽抗性特征。AA 参数稳定性较差，并且检测方法复杂，因此不建议在育种及大量材料筛选和评价时使用。此外，青稞穗发芽抗性受环境影响较大，评价时应考虑到尽可能多的抗性影响因素及其在不同栽培条件下的变异。 2. 对来自青藏高原的青稞穗发芽抗性特征及其与其它农艺性状间的关系进行研究。通过测定穗发芽指数（SI）、籽粒发芽指数（GI）和α-淀粉酶活性（AA），表明113 份青稞材料的穗发芽抗性具有显著差异。SI、GI 和AA 参数的变幅分别为1.00~8.86、0.01~0.97 和0.00~2.76，其均值分别为4.72、0.63 和1.22。根据SI 参数，六个基因型，包括‘XQ9-5’，‘XQ33-9’，‘XQ37-5’，‘XQ42-9’，‘XQ45-7’和‘JCL’被鉴定为抗性品种。综合SI、GI 和AA 参数，可以发现青稞的穗发芽抗性机制包含颖壳等穗部结构的抗性和种子自身的抗性（即种子休眠性），且供试材料中未发现较强的胚休眠品种，除‘XQ45-7’外，所有品种在发芽第四天均能检测出α-淀粉酶活性。穗部结构和种子休眠的抗性机制因基因型不同而不同，在穗发芽抗性中可单独作用或共同作用。农家品种和西藏群体分别比栽培品种和四川群体的穗发芽抗性强，而在不同籽粒颜色的青稞中未发现明显差异。相关性检验发现，青稞的穗发芽抗性，主要是种子休眠性，与百粒重、开花期、成熟期、穗长、芒长和剑叶长呈显著负相关关系，与株高相关性不显著。农艺性状可以作为穗发芽抗性材料选育中的辅助指标。本试验为青稞穗发芽抗性育种研究提供了必要的理论基础和可供使用的亲本材料。 3. α-淀粉酶是由多基因家族编码的蛋白质，在植物种子萌发时高度表达，与植物种子的萌发能力密切相关。在大麦种子发芽时，高等电点α-淀粉酶的活性远大于低等电点的α-淀粉酶。为了研究不同穗发芽抗性青稞品种中编码高等电点α-淀粉酶Amy1 基因结构与抗性间的关系，我们以筛选得到的抗性品种‘XQ32-5’（TR1）、‘XQ37-5’（TR2）、‘XQ45-7’（TR3），易感品种‘97-15’（TS1）、‘9657’（TS2）以及强休眠大麦品种‘SAMSON’（SAM）为材料，对其Amy1 基因的编码区序列进行克隆和结构分析，并对它们推导的氨基酸序列进行比较。结果显示，青稞Amy1 基因具有三个外显子、两个内含子，编码区中有13 个核苷酸变异位点，均位于2、3 号外显子，2 个变异位点位于2 号外显子。SAM 和TS1 分别在2 号外显子相应位置有5 个相同的碱基(GAACT)的插入片段。相应α-淀粉酶氨基酸序列推导发现，所有核苷酸变异中有8 个导致相应氨基酸残基的改变，其余位点为同义突变。青稞Amy1 基因编码区序列品种间相似度高达99%以上，部分序列变异可能与其穗发芽抗性有关。随后，我们又通过SYBR Green 荧光定量技术对该基因在不同发芽时间（1d~7d）的相对表达水平进行了差异性检测。结果发现，7 天内不能检测到SAM 的Amy1 基因表达，5 个青稞品种间的Amy1 基因的相对表达量均随着发芽时间延长而上升，但上升方式有所不同。弱抗品种该基因表达更早，转录本增加速率更大，且在4~5 天可达到平台期。发芽7 天中，抗性品种总转录水平明显低于易感品种。本研究结果表明，青稞Amy1 基因的转录水平是与其穗发芽抗性高度相关。 我国青藏高原青稞，尤其是农家品种的穗发芽抗性具有丰富的变异，蕴藏着穗发芽抗性育种的宝贵资源。本研究为青稞穗发芽抗性育种建立了合理抗性评价体系，筛选出可供育种使用的特殊材料，阐明了农艺性状可辅助穗发芽抗性育种，同时还对穗发芽抗性与α-淀粉酶基因的结构和表达关系进行分析，为青稞穗发芽抗性资源筛选奠定了基础。 Preharvest sprouting (PHS) is a serious problem in crop production. It often takes place when encountering damp, cold conditions at harvest time and results in the decrease of grain quality and great loss of yield by triggering the synthesis of endosperm degrading enzymes (mostly the α-amylase). Therefore, PHS is regarded as an important criterion for crop breeding. In order to minimize the risk of PHS, resistant genotypes are highly required. Hulless barley (Hordeum vulgare ssp. vulgare) is the staple food crop in Qinghai-Tibetan Plateau from of old, where is one of the origin and genetic diversity centers of hulless barley. Recently, interest in hulless barley has been sparked throughout the world due to the demonstrations of its great potential in health food industry and fuel alcohol production. Indeed, hulless barley can also be utilized to produce good quality malt if the appropriate malting conditions are used. In China, overcast and rainy conditions often occur at maturity of hulless barley and cause an adverse on its production and application. PHS resistant genotypes, therefore, are highly required for the hulless barley breeding programs. However, few investigations have been made so far on this issue. The objectives of this study were: 1) to assessment of methods used in testing preharvest sprouting resistance in hulless barley; 2) to evaluate the variability and characteristics of PHS resistance of hulless barley from Qinghai-Tibet Plateau in China; 3) to select potential parents for PHS resistance breeding; 4) to primarily study on the molecular mechanism of PHS resistance of hulless barley. Our results are as followed: 1. We investigated the temperature effects on seed germination and seed dormancy expression of hulless barley, discussed appropriate temperature range for screening of PHS resistant varieties, and analyzed the mechanism of seed dormancy expression of hulless barley. The dormancy level of 13 hulless barley were evaluated by GI (germination index) values calculating by seed germination tests at temperature of 10，15，20，25，30℃ in darkness. There were great differences in temperature sensitivity among these accessions. The insensitive accessions showed low dormancy at any temperature while the dormancy expression of sensitive accessions could be restrained by low temperature and induced by high temperature. The temperature range of 15℃ to 25℃ was workable for estimating of dormancy level of hulless barley according to our data. Analysis of α-amylase activity showed that the temperature effects on seed germination and the expression of seed dormancy be achieved probable via regulating of α-amylase activity. Furthermore, we evaluated the differences in sprouting index (SI), sprouting rate (SR), germination index (GI) and α-amylase activity (AA) between Maerkang and Chengdu among 34 accessions of hulless barley from Qinghai-Tibetan Plateau in China. These PHS sprouting parameters were significantly affected by accession×location, and they had correlation between each other. GI was the most reliable parameter because of its low CV value, good repeatability and simple operation. SI could assist in differentiating between accessions of similar dormancy or overall evaluation of the resistance. AA was bad in repeatability and had relatively complex testing method, therefore, not appropriate for breeding and evaluation and screening of PHS resistant materials. Besides, since PHS resistance of hulless barley was greatly influenced by its growth environment, possibly much influencing factors and variations between cultivated conditions should be considered. 2. In this study, large variation was found among 113 genotypes of hulless barley (Hordeum vulgare ssp.vulgare) from Qinghai-Tibetan Plateau in China, based on the sprouting index (SI), germination index (GI) and α-amylase activity (AA) which derived from sprouting test of intact spikes, germination test of threshed seeds and determination of α-amylase activity, respectively. The range of SI, GI and AA was 1.00~8.86, 0.01~0.97 and 0.00~2.76，the mean was 4.72, 0.63 and 1.22 espectively. Six resistant genotypes, including ‘XQ9-5’, ‘XQ33-9’, ‘XQ37-5’, ‘XQ42-9’, ‘XQ45-7’ and ‘JCL’, were identified based on SI. Integrating the three parameters, it was clear that both hulls and seeds involved in PHS resistance in intact spikes of hulless barley and there was no long-existent embryo dormancy found among the test genotypes. All the genotypes, except ‘XQ45-7’, had detectable α-amylase activity on the 4th day after germination. There was PHS resistance imposed by the hull and seed per se and the two factors can act together or independent of each other. Besides, landraces or Tibet hulless barley had a wider variation and relatively more PHS resistance when compared with cultivars or Sichuan hulless barley. No significant difference was found among hulless barley of different seed colors. The correlation analysis showed PHS resistance was negatively related to hundred grain weight, days to flowering, days to maturity, spike length, awn length and flag length but not related to plant height. This study provides essential information and several donor parents for breeding of resistance to PHS. 3. Alpha-amylase isozymes are encoded by a family of multigenes. They highly express in germinating seeds and is closely related to seed germination ability. In barley germinating seeds, the activity of high pI α-amylase is much higher than low pI α-amylase. The aim of this study was to determine the relationship between preharvest sprouting resistance of hulless barley and the gene structure of Amy1 gene which encodes high pI α-amylase. The coding region and cDNA of Amy1 gene of three resistant accessions, including ‘XQ32-5’ (TR1), ‘XQ37-5’ (TR2), ‘XQ45-7’ (TR3), two susceptible accessions ‘97-15’ (TS1), ‘9657’ (TS2) and one highly dormant barley accession ‘SAMSON’ (SAM) was cloned. Analysis of their DNA sequences revealed there were three exons and two introns in Amy1 gene. Thirteen variable sites were in exon2 and exon3, 2 variable sites were in intron2. SAM and TS1 had a GAACT insert segment in the same site in intron2. Only 8 variable sites caused the change of amino acid residues. There were 99% of similarity between the tested hulless barley and some of the variable sites might be related with preharvest sprouting resistance. Then, we investigated the expression level of Amy1 gene in the 7-day germination test. Results of quantitative real-time PCR indicated that the relative expression trends of Amy1 gene were the same but had significant differences in the increase fashion between hulless barleys and no detectable expression was found in SAM. Susceptible accessions had earlier expression and faster increase and reached the maximum on day 4 ~ day 5. Besides, total transcripts level was found lower in resistant accessions than susceptible accessions. This study indicated that α-amylase activity was highly related to the transcription level of Amy1 gene which not correlated to missense mutation sites. In conclusion, hulless barley, especially the landraces from Qinghai-Tibetan Plateau in China possesses high degree of variation in PHS performance, which indicates the potential of Tibetan hulless barley as a good source for breeding of resistance to PHS. This study provides several donor parents for breeding of resistance to PHS. Our results also demonstrate that agronomic traits may be used as assistants for PHS resistance selection in hulless barley. Besides, analysis of high pI α-amylase coding gene Amy1 revealed the relative high expression of was Amy1 one of the mainly reason of different PHS resistance level in hulless barley.

Caracteristicas de brotacoes de tuberculos de clones de batata resistentes a pinta-preta.

1999

Evaluation of the antiangiogenic potential of AQ4N.

Purpose: A number of cytotoxic chemotherapy agents tested at low concentrations show antiangiogenic properties with limited cytotoxicity, e.g., cyclophosphamide, tirapazamine, and mitoxantrone. AQ4N is a bioreductive alkylaminoanthraquinone that is cytotoxic when reduced to AQ4; hence, it can be used to target hypoxic tumor cells. AQ4N is structurally similar to mitoxantrone and was evaluated for antiangiogenic properties without the need for bioreduction.

Experimental Design:The effect of AQ4N and fumagillin on human microvascular endothelial cells (HMEC-1) was measured using a variety ofin vitro assays, i.e., 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide, wound scrape, tubule formation, rat aortic ring, and invasion assays. Low-dose AQ4N (20 mg/kg) was also given in vivo to mice bearing a tumor in a dorsal skin flap.

Results:AQ4N (10-11to10-5mol/L) hadno effect on HMEC-1viability. AQ4N (10-9to10-5mol/L) caused a sigmoidal dose-dependent inhibition of endothelial cell migration in the wound scrape model. Fumagillin showed a similar response over a lower dose range (10-13 to 10-9 mol/L); however, the maximal inhibition was less (25% versus 43% for AQ4N). AQ4N inhibited HMEC-1 cell contacts on Matrigel (10-8 to 10-5 mol/L), HMEC-1 cell invasion, and sprouting in rat aorta explants. Immunofluorescence staining with tubulin, vimentim, dynein, and phalloidin revealed that AQ4N caused disruption to the cell cytoskeleton. When AQ4N (20 mg/kg) was given in vivo for 5 days, microvessels disappeared in LNCaP tumors grown in a dorsal skin flap.

Conclusions:This combination of assays has shown that AQ4N possesses antiangiogenic effects in normoxic conditions, which could potentially contribute to antitumor activity

Antibody-Mediated Inhibition of Cathepsin S Blocks Colorectal Tumor Invasion and Angiogenesis

Purpose: Cathepsin S is a cysteine protease that promotes the invasion of tumor and endothelial cells during cancer progression. Here we investigated the potential to target cathepsin S using an antagonistic antibody, Fsn0503, to block these tumorigenic effects.
Experimental Design: A panel of monoclonal antibodies was raised to human cathepsin S. The effects of a selected antibody were subsequently determined using invasion and proteolysis assays. Endothelial cell tube formation and aorta sprouting assays were done to examine antiangiogenic effects. In vivo effects were also evaluated using HCT116 xenograft studies.
Results: A selected cathepsin S antibody, Fsn0503, significantly blocked invasion of a range of tumor cell lines, most significantly HCT116 colorectal carcinoma cells, through inhibition of extracellular cathepsin S–mediated proteolysis. We subsequently found enhanced expression of cathepsin S in colorectal adenocarcinoma biopsies when compared with normal colon tissue. Moreover, Fsn0503 blocked endothelial cell capillary tube formation and aortic microvascular sprouting. We further showed that administration of Fsn0503 resulted in inhibition of tumor growth and neovascularization of HCT116 xenograft tumors.
Conclusions: These results show that blocking the invasive and proangiogenic effects of cathepsin S with antibody inhibitors may have therapeutic utility upon further preclinical and clinical evaluation.

VEGF-induced retinal angiogenic signalling is critically dependent on Ca2+ signalling via Ca2+/calmodulin-dependent protein kinase II

Purpose. The authors conducted an in vitro investigation of the role of Ca2+-dependent signaling in vascular endothelial growth factor (VEGF)-induced angiogenesis in the retina.

Methods. Bovine retinal endothelial cells (BRECs) were stimulated with VEGF in the presence or absence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; intracellular Ca2+ chelator), U73122 (phospholipase C (PLC) inhibitor), xestospongin C (Xe-C), and 2-aminoethoxydiphenyl borate (2APB) (inhibitors of inositol-1,4,5 triphosphate (IP3) signaling). Intracellular Ca2+ concentration ([Ca2+]i) was estimated using fura-2 Ca2+ microfluorometry, Akt phosphorylation quantified by Western blot analysis, and angiogenic responses assessed using cell migration, proliferation, tubulogenesis, and sprout formation assays. The effects of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 were also evaluated on VEGF-induced Akt signaling and angiogenic activity.

Results. Stimulation of BRECs with 25 ng/mL VEGF induced a biphasic increase in [Ca2+]i, with an initial transient peak followed by a sustained plateau phase. VEGF-induced [Ca2+]i increases were almost completely abolished by pretreating the cells with BAPTA-AM, U73122, Xe-C, or 2APB. These agents also inhibited VEGF-induced phosphorylation of Akt, cell migration, proliferation, tubulogenesis, and sprouting angiogenesis. KN93 was similarly effective at blocking the VEGF-induced activation of Akt and angiogenic responses.

Conclusions. VEGF increases [Ca2+]i in BRECs through activation of the PLC-IP3 signal transduction pathway. VEGF-induced phosphorylation of the proangiogenic protein Akt is critically dependent on this increase in [Ca2+]i and the subsequent activation of CaMKII. Pharmacologic inhibition of Ca2+-mediated signaling in retinal endothelial cells blocks VEGF-induced angiogenic responses. These results suggest that the PLC/IP3/Ca2+/CaMKII signaling pathway may be a rational target for the treatment of angiogenesis-related disorders of the eye.

Docosahexaenoic Acid Improves the Nitroso-Redox Balance and Reduces VEGF-Mediated Angiogenic Signaling in Microvascular Endothelial Cells

Purpose. Disturbances to the cellular production of nitric oxide (NO) and superoxide (O2-) can have deleterious effects on retinal vascular integrity and angiogenic signaling. Dietary agents that could modulate the production of these signaling molecules from their likely enzymatic sources, endothelial nitric oxide synthase (eNOS) and NADPH oxidase, would therefore have a major beneficial effect on retinal vascular disease. The effect of ?-3 polyunsaturated fatty acids (PUFAs) on angiogenic signaling and NO/superoxide production in retinal microvascular endothelial cells (RMECs) was investigated.

Methods. Primary RMECs were treated with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 48 hours. RMEC migration was determined by scratch-wound assay, proliferation by the incorporation of BrdU, and angiogenic sprouting using a three-dimensional model of in vitro angiogenesis. NO production was quantified by Griess assay, and phospho-eNOS accumulation and superoxide were measured using the fluorescent probe dihydroethidine. eNOS localization to caveolin-rich microdomains was determined by Western blot analysis after subfractionation on a linear sucrose gradient.

Results. DHA treatment increased nitrite and decreased superoxide production, which correlated with the displacement of eNOS from caveolar subdomains and colocalization with the negative regulator caveolin-1. In addition, both ?-3 PUFAs demonstrated reduced responsiveness to VEGF-stimulated superoxide and nitrite release and significantly impaired endothelial wound healing, proliferation, and angiogenic sprout formation.

Conclusions. DHA improves NO bioavailability, decreases O2- production, and blunts VEGF-mediated angiogenic signaling. These findings suggest a role for ?-3 PUFAs, particularly DHA, in maintaining vascular integrity while reducing pathologic retinal neovascularization.

The Pleiotropic Effects of Simvastatin on Retinal Microvascular Endothelium Has Important Implications for Ischaemic Retinopathies

Background: Current guidelines encourage the use of statins to reduce the risk of cardiovascular disease in diabetic patients; however the impact of these drugs on diabetic retinopathy is not well defined. Moreover, pleiotropic effects of statins on the highly specialised retinal microvascular endothelium remain largely unknown. The objective of this study was to investigate the effects of clinically relevant concentrations of simvastatin on retinal endothelium in vitro and in vivo.

Methods and Findings: Retinal microvascular endothelial cells (RMECs) were treated with 0.01–10 µM simvastatin and a biphasic dose-related response was observed. Low concentrations enhanced microvascular repair with 0.1 µM simvastatin significantly increasing proliferation (p<0.05), and 0.01 µM simvastatin significantly promoting migration (p<0.05), sprouting (p<0.001), and tubulogenesis (p<0.001). High concentration of simvastatin (10 µM) had the opposite effect, significantly inhibiting proliferation (p<0.01), migration (p<0.01), sprouting (p<0.001), and tubulogenesis (p<0.05). Furthermore, simvastatin concentrations higher than 1 µM induced cell death. The mouse model of oxygen-induced retinopathy was used to investigate the possible effects of simvastatin treatment on ischaemic retinopathy. Low dose simvastatin(0.2 mg/Kg) promoted retinal microvascular repair in response to ischaemia by promoting intra-retinal re-vascularisation (p<0.01). By contrast, high dose simvastatin(20 mg/Kg) significantly prevented re-vascularisation (p<0.01) and concomitantly increased pathological neovascularisation (p<0.01). We also demonstrated that the pro-vascular repair mechanism of simvastatin involves VEGF stimulation, Akt phosphorylation, and nitric oxide production; and the anti-vascular repair mechanism is driven by marked intracellular cholesterol depletion and related disorganisation of key intracellular structures.

Conclusions: A beneficial effect of low-dose simvastatin on ischaemic retinopathy is linked to angiogenic repair reducing ischaemia, thereby preventing pathological neovascularisation. High-dose simvastatin may be harmful by inhibiting reparative processes and inducing premature death of retinal microvascular endothelium which increases ischaemia-induced neovascular pathology. Statin dosage should be judiciously monitored in patients who are diabetic or are at risk of developing other forms of proliferative retinopathy.

Association Study of SNAP25 and Schizophrenia in Irish Family and Case-Control Samples

SNAP25 occurs on chromosome 20p12.2, which has been linked to schizophrenia in some samples, and recently linked to latent classes of psychotic illness in our sample. SNAP25 is crucial to synaptic functioning, may be involved in axonal growth and dendritic sprouting, and its expression may be decreased in schizophrenia. We genotyped 18 haplotype-tagging SNPs in SNAP25 in a sample of 270 Irish high-density families. Single marker and haplotype analyses were performed in FBAT and PDT. We adjusted for multiple testing by computing q values. Association was followed up in an independent sample of 657 cases and 411 controls. We tested for allelic effects on the clinical phenotype by using the method of sequential addition and 5 factor-derived scores of the OPCRIT. Nine of 18 SNPs had Pvalues

Development of the nervous system in Dugesia tigrina during regeneration after fission and decapitation

In the present study of Dugesia tigrina the development of the nervous system is followed and compared during regeneration after fission and after decapitation. Immunocytochemistry was used, with antisera raised against the biogenic amine, 5-hydroxytryptamine (5-HT) and the two neuropeptides, neuropeptide F (NPF), and FMRF amide. The results indicate that two processes are involved in the formation of the new cerebral ganglion. First, new processes sprouting from the original main longitudinal nerve cords bend transversely, indicating the position of the developing horseshoe-shaped anterior cerebral commissure. Then new nerve cells in front of the commissure differentiate from neoblasts and their growth cones fasciculate with the fibres from the old main longitudinal nerve cords. In the cerebral ganglion, 5-HT-IR cells appear before NPF-IR cells, in contrast to the pharynx where NPF-IR cells differentiate before the 5-HT-IR cells. In the peripheral nervous system, NPF-IR fibres and cells appear at a very early stage and dominate the whole regeneration process. A role for the PNS in early pattern formation is suggested.

FKBPL Is a Critical Antiangiogenic Regulator of Developmental and Pathological Angiogenesis

OBJECTIVE: The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models.

APPROACH AND RESULTS: FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL's critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl(+/-) mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish.

CONCLUSIONS: FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes.

The Role of Brain-Derived Neurotrophic Factor (BDNF) in the Development of Neurogenic Detrusor Overactivity (NDO)

Neurogenic detrusor overactivity (NDO) is a well known consequence of spinal cord injury (SCI), recognizable after spinal shock, during which the bladder is areflexic. NDO emergence and maintenance depend on profound plastic changes of the spinal neuronal pathways regulating bladder function. It is well known that neurotrophins (NTs) are major regulators of such changes. NGF is the best-studied NT in the bladder and its role in NDO has already been established. Another very abundant neurotrophin is BDNF. Despite being shown that, acting at the spinal cord level, BDNF is a key mediator of bladder dysfunction and pain during cystitis, it is presently unclear if it is also important for NDO. This study aimed to clarify this issue. Results obtained pinpoint BDNF as an important regulator of NDO appearance and maintenance. Spinal BDNF expression increased in a time-dependent manner together with NDO emergence. In chronic SCI rats, BDNF sequestration improved bladder function, indicating that, at later stages, BDNF contributes NDO maintenance. During spinal shock, BDNF sequestration resulted in early development of bladder hyperactivity, accompanied by increased axonal growth of calcitonin gene-related peptide-labeled fibers in the dorsal horn. Chronic BDNF administration inhibited the emergence of NDO, together with reduction of axonal growth, suggesting that BDNF may have a crucial role in bladder function after SCI via inhibition of neuronal sprouting. These findings highlight the role of BDNF in NDO and may provide a significant contribution to create more efficient therapies to manage SCI patients.

Phenotype-Based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis

Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge Diverset^TM library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4–10 μM in zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT_1–2) at micromolar IC₅₀ values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.

Pkd1 Regulates Lymphatic Vascular Morphogenesis during Development.

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.