Tank cultivation of the red alga Palmaria palmata: Year-round induction of tetrasporangia, tetraspore release in darkness and mass cultivation of vegetative thalli

Field-collected tetrasporophytes of Palmaria palmata were tumbled in 300-L outdoor tanks from January to August at ambient daylength or in a constant short-day (SD) regime (8 h light per day), both at 10 or 15 degrees C. Tetrasporangia were massively induced after 2.5 months under SD conditions at 10 degrees C and completely lacking at 15 degrees C, both under SD or ambient daylength conditions, with a few tetrasporangia present at 10 degrees C and ambient daylength. Elongation rates of tagged tetrasporophytic thalli peaked from March to April in all four conditions, when the biomass densities in the outdoor tanks were close to 2.5 kg fresh weight m(-2). Under all four conditions, juvenile proliferations started to appear in June from the margins of the old fronds, and attained approximately 1 cm in length by the end of July. Approximately 80% of the tetraspores were released during the first three dark phases in a light/dark regime, and the remaining 20% during the light phases. A minimum of 10 min darkness was observed to trigger spore release. White light inhibited tetraspore release, while a similar number of spores were released in continuous red light or in the light/dark regime, although with no significant differences of spore release during subjective days and nights. Sporelings were successfully derived from the released tetraspores for mass propagation of the male gametophyte in 2000-L outdoor tanks in a greenhouse. Mass production of male gametophytic sporelings of P. palmata was completed two times by SD induction of tetrasporangia at 10 degrees C, release of spores in darkness and culturing the sporelings until they were ready to be propagated vegetatively in greenhouse tanks. One experiment lasted from January to October 2001, with spore release in June, and the second from September to April 2003, with spore release in January. These results may support the development of sustainable, year-round Palmaria farming. (c) 2005 Elsevier B.V. All rights reserved.

Growth of Gracilaria lemaneiformis under different cultivation conditions and its effects on nutrient removal in Chinese coastal waters

Gracilaria lemaneiformis (Bory) Daws has been extensively cultivated as a source of commercial agar and the ecomaterials in Shenao Bay, Guangdong Province, Jiaozhou Bay, Shandong Province and other waters in China. This paper examines the in situ suspended farming of G. lemaneiformis using raft cultivation under different conditions and its effects on nutrient removal in the laboratory. The results showed that cultivated Gracilaria grew well in both Shenao Bay and Jiaozhou Bay. The biomass of Gracilaria increased from 50 to 775 g m(-1) (fresh weight) during 28 days, with special growth rate (SPG) 13.9% d(-1) under horizontal cultivation in Jiaozhou Bay. Light, temperature, nutrient supply, as well as cultivation treatments such as initial density, and depth of suspension seaweed were important to the growth of Gracilaria. The highest biomass production was observed in the horizontal culture condition (0.0 m) and 0.5-1.5 m deep layer in Jiaozhou Bay. However, the highest growth rate in Shenao Bay appeared under the lowest initial stocking density treatment. In the laboratory, the aquarium experiments (fish and seaweed culture systems) demonstrated that Gracilaria was able to remove inorganic nutrients effectively. The concentration of NH4+-N decreased by 85.53% and 69.45%, and the concentration of PO4-P decreased 65.97% and 26.74% in aquaria with Gracilaria after 23 days and 40 days, respectively. The results indicate that Gracilaria has the potential to remove excess nutrient from coastal areas, and the large-scale cultivation of G. lemaneiformis could be effective to control eutrophication in Chinese coastal waters. (c) 2005 Elsevier B.V. All rights reserved.

Optimization of conditions for cell cultivation of Porphyra haitanensis conchocelis in a bubble-column bioreactor

Process conditions for cell cultures derived from conchocelis of female red macroalga Porphyra haitanensis were optimized in an illuminated 0.3-l bubble-column photobioreactor, using CO2 in air as the sole carbon source during a 20-day cultivation period. It reached the highest growth rate when the initial cell density was 700 mg l(-1)(dry weight), the optional aeration rate was 1.2 v/v/min, inorganic nitrate concentration was 15 mM and inorganic phosphate concentration was 0.6 mM. This is the first reported bioreactor cultivation study of cell cultures derived from conchocelis of Porphyra haitanensis.

Optimal light regime for the cultivation of transgenic Laminaria japonica gametophytes in a bubble-column bioreactor

Fluctuating light intensity had a more significant impact on growth of gametophytes of transgenic Laminaria japonica in a 2500 ml bubble-column bioreactor than constant light intensity. A fluctuating light intensity between 10 and 110 mu E m(-2) s(-1), with a photoperiod of 14 h:10 h light:dark, was the best regime for growth giving 1430 mg biomass l(-1).

开垦和放牧对内蒙古草原土壤碳库和温室气体通量的影响

本文采用野外观测和室内测定相结合的方法，研究了内蒙古草原两种主要的土地利用方式（开垦和放牧）对土壤碳库和温室气体通量的影响，结果如下： 1．内蒙古草甸草原开垦后30年后，与天然草原相比，在休闲年份0～20 cm土壤有机碳和土壤微生物量碳含量均没有显著下降，但开垦导致土壤易分解碳下降了24%。土壤易氧化碳受季节性水热因子的驱动，表现出明显的季节变化动态。因此，土壤易分解碳的较土壤碳库的其他组分对开垦更敏感，是表征土壤管理措施引起有机质变化的一个重要指标。 草原开垦后，土壤-植物系统氧化大气甲烷的能力明显提高，农田和天然草原CH4平均吸收通量分别是48.9 和 29.0 μg C m-2 h-1，开垦后增加了1.7倍。开垦没有改变CH4吸收通量 “夏季高秋季低” 的季节变化规律（由降水决定）;开垦使N2O的平均释放通量增加了47%，农田和天然草原N2O平均吸收通量分别是56.6 和 38.6 ugN m-2 h-1;开垦同时也增大了通量的变异幅度;但没有改变N2O季节变化规律，只是出现高峰的时间较天然草原推后约10天左右。 2．开垦后的农田土壤在模拟添加厩肥后，刺激了土壤微生物的呼吸代谢，使CO2的释放量增加了5-7倍。试验期间总体排放的CO2中，约60%来源于羊粪，40% 来源于土壤。两种土壤即羊草顶级草原土壤（高碳高氮）和冷蒿－小禾草退化草原土壤（低碳低氮），在CO2的释放总量和释放比例上都没有显著性差异。添加厩肥均造成两种土壤碳库的净碳损失，并且退化草原土壤（7.0%）的土壤净碳损失要大于羊草草原（2.6%）。说明与开垦后的高C土壤相比，在已经退化草原的低C土壤上施厩肥将趋向于土壤更大的净碳损失。 3．自由放牧22年后，羊草草原0～10 cm土壤有机碳、微生物量碳和易分解碳分别下降了14.1%、27.9%和22.0%;大针茅草原0～5 cm土壤有机碳和微生物量碳分别下降了27.6%和38.2%。两类草原土壤碳组分的季节变化受水热因子的驱动，大针茅草原季节波动出现高峰的时间较羊草草原迟。土壤微生物量碳在表征羊草草原和大针茅草原土壤碳素的动态变化时，要敏感于土壤总有机碳。放牧对冷蒿-小禾草草原土壤各碳素组分影响不明显。在表征放牧对冷蒿-小禾草草原土壤的影响指示上，MB-C/ Org-C和Lab-C/ Org-C要比MB-C和Lab-C更加敏感。这说明在研究放牧对草原土壤碳库影响时，不同的草原类型应使用不同的指标来表征其变化。 内蒙古羊草草原是大气CH4的汇，自由放牧增加土壤对CH4的吸收。CH4平均吸收通量增加了27%，但CH4吸收的季节变化形式没有改变;放牧使。自由放牧还增大了N2O的排放通量，将原来N2O源、汇的双重功能改变为单一的源功能;放牧使N2O平均释放通量增加了1倍;放牧显著增加了羊草草原向大气排放CO2的量（p<0.05），并且年度排放量范围也有所增大。 4．草原羊尿斑土壤的pH和NH4+浓度在施后显著升高，但土壤微生物C和N没有明显变化，尿斑N素会发生大量的流失。粪斑和厩肥斑中各有46%和27%的N素分解后转移到植物中。羊草种群斑块上粪尿斑引起CO2和N2O通量的变化，要大于星毛委陵菜种群斑块。与植被类型的影响相比，羊粪尿斑尤其是尿斑对温室气体通量的影响更大。尿斑既降低了土壤对CH4的吸收，又增加了CO2和N2O的释放，使粪尿斑上相当于CO2的净排放量比对照土壤增加了15%。 在内蒙古草原中等放牧条件下，家畜粪尿斑在放牧草地上的覆盖面积约只有2%，与未被家畜排泄物覆盖的草原土壤 相比，粪尿斑对内蒙古草原温室气体总体收支产生的影响可以忽略不计。因此内蒙古草原地区温室气体减排措施的重点，应放在家畜的食性食量对温室气体的影响以及厩肥的科学利用上。但随放牧强度的加大，家畜排泄物覆盖草地的面积将大大增加，加之放牧生态系统中家畜瘤胃代谢产生的的大量温室气体，其对草原温室气体的核算将会产生的影响也是不容忽视的。

开垦和放牧对内蒙古半干旱草原蒸发散的影响

为了研究开垦和放牧对内蒙古半干旱草原生态系统蒸发散的影响，我们以内蒙古锡林郭勒盟多伦县的草地和农田以及锡林浩特围封草地和退化草地为实验样地，利用涡度协方差技术对四个样地2006-2007年的蒸发散和微气象因子进行连续测定，探讨开垦和放牧对内蒙古半干旱草原生态系统蒸发散的影响及其驱动机制。同时利用稳定同位素技术区分多伦半干旱草原生态系统蒸发散的组分，初步探讨生态系统蒸发散各组分的变化特征及其驱动因子。 在观测的两年里，四个生态系统的年蒸发散接近或超过了年降雨量。在较湿润年份（2006年）的生长季（5-9月），开垦使得半干旱草原的蒸发散降低了15%，而在较干旱年份（2007年）降低了7%。放牧在2006年生长季期间使草原生态系统蒸发散降低了13%，而在2007年生长季里，围封草地和退化草地的蒸发散没有显著性差异。开垦和放牧造成的土壤含水量显著下降是生态系统蒸发散降低的最主要的原因。此外，农田开垦改变了植被的种类和物候特征，缩短了植物的生长期（农作物的生长期主要是6-8月，而草地的生长季一般为5-9月），是造成农田整个生长季的蒸发散比草地低的另一个原因。干旱年份降雨量少、土壤含水量低，限制了植被的生长，降低了冠层导度，从而导致了植物蒸腾的下降。另一方面，在干旱年份，开垦和放牧增加了草原生态系统蒸发散对土壤含水量变化的敏感性，表明未来降雨格局的变化不仅直接通过影响土壤含水量来改变蒸发散，而且会影响蒸发散和土壤含水量之间的相关性。 通过研究生态系统对不同强度和不同时间降雨的响应表明，> 3 mm的降雨会增加内蒙古半干旱草原生态系统的土壤含水量和蒸发散，对内蒙古半干旱草原生态系统关键过程有效。雨后1-2天蒸发散达到峰值，之后下降。多数大的降雨事件后，农田和退化草地蒸发散峰值比相对应的草地高，之后下降的也快，这与植被的生长状况、凋落物的量和地面的裸露程度有关。雨后土壤含水量和蒸发散的变化与降雨事件的大小呈正相关的关系，并且蒸发散的变化与土壤含水量的变化有显著的线性相关性，说明了土壤含水量是影响蒸发散变化的主要因子。土壤含水量初始值和干旱期的长短会影响内蒙古半干旱草原生态系统蒸发散对降雨的响应。土壤含水量初始值低会增强蒸发散对降雨的响应，而土壤含水量初始值高时，蒸发散对降雨的响应会下降。干旱期长会增强蒸发散对降雨的瞬时响应。围封草地由于有凋落物的缓冲和保水作用，未来降雨强度的增加会促进围封草原植被的生长;而退化草地地表裸露，对大的降雨事件缓冲作用小，地表径流和土壤蒸发强烈，不利于保水和植被的生长。因此，未来极端降雨事件频率的增加和干旱期的延长势必会对半干旱草原生态系统产生影响，尤其对退化草地产生不利影响。 为了进一步了解蒸发散组分的变化规律及其驱动因子，我们利用稳定同位素与微气象技术相结合的方法，区分半干旱草原生态系统蒸发散的组分。区分结果表明，在2006年和2007年整个生长季期间，植物蒸腾均为蒸发散的主要形式，在2006年和2007年生长季期间植物蒸腾占蒸发散的比例分别为88%和73%。影响植物蒸腾的因子主要是净辐射、大气相对湿度、土壤含水量和叶面积指数;影响蒸发的因子主要是大气相对湿度、土壤含水量和风速;而影响蒸发散的因子主要是净辐射、大气相对湿度、土壤含水量和风速，说明了这些水分流失过程受到不同环境因子的驱动。此外，我们根据一个简单的模型把同位素方法区分的蒸发散组分外推到整个生长季，结果表明，在2006年和2007年整个生长季蒸腾与蒸发散的比例高，并且蒸腾与降雨量的比例高，由此说明，半干旱草原生态系统植被能够有效地利用水分，雨水利用效率高。生态系统总初级生产力（GPP）与植物蒸腾（T）存在明显的正相关关系，从5-8月，生态系统水分利用效率（GPP/T）逐渐升高。 我们的研究结果表明：人类的活动如开垦和放牧通过改变植被类型、土壤持水能力和蒸发散对土壤含水量的响应，降低了草原生态系统的蒸发散，增加了生态系统蒸发散对降雨响应的敏感性，不利于土壤的保水和植被对水分的利用。半干旱草原生态系统蒸发散组分的稳定同位素技术区分，为进一步从新的角度探讨土地利用方式的改变对草原生态系统蒸发散影响的生理机制提供了可能。

玉簪品种的引种，栽培和组织培养

本研究通过对玉簪属植物的组织培养，发现以腋芽、花序、试管苗的叶片为外植体，均可达到迅速繁殖的目的，但对于不同的玉簪品种来说，所能采用的外植体的种类又不尽相同：对于非嵌合体玉簪品种，以腋芽、花序、试管苗的叶片作外植体都可以;而对于嵌合体玉簪品种，如果以花序或试管苗的叶片为外植体，通过诱导不定芽进行快繁，在所得的试管苗中，超过50%的个体失去斑叶特性，这在实际生产中是不可行的，如果以腋芽为外植体，所得试管苗中多于95%的可保持原有嵌合体特征，故为理想的外植体。 在以腋芽为外植体的离体培养中，取嵌合玉簪品种H. ‘Francee’和H. ‘Ground Master’的芽作材料，通过对试验结果的分析、比较，选出了合适的繁殖培养基：MS ＋ BA0.5mg/L + NAA0.5mg/L + KH2PO4150mg/L + 水解乳蛋白500mg/L + 蔗糖30g/L + 琼脂 5.6g/L，其可提高腋芽分化率，达到了快速繁殖的目的，此外，该培养基还可保持原来的生长势，降低斑叶特性分离比;同时选出了生根培养基：MS + IAA1mg/L + 蔗糖30g/L + 琼脂5.6g/L。 以花葶为外植体的试验，证实了此种方式仅适合非嵌合体玉簪的快速繁殖。 在以叶片为外植体的研究中，取了嵌合体品种H. ‘Francee’、H. ‘Ground Master’、H. ‘Gold Standard’、H. ‘Color Glory’和H. ‘Little Ming’的再生叶征作材料，通过对试验结果的分析、比较、观察到在培养基：MS + BA4mg/L + NAA0.1mg/L + 蔗糖 30g/L + 琼脂 5.6g/L上只有芽的形成，培养基：MS + BA0.4mg/L + NAA0.4mg/L + 蔗糖 30g/L + 琼脂 5.6g/L则利于根的发生，而培养基：MS + BA4mg/L + NAA0.4mg/L + 蔗糖 30g/L + 琼脂 5.6g/L促进根或（和）不定芽的产生，则因品种的不同而变化，另外还发现，经诱导不定芽途径所得到的试管苗，仅少于5%的个体保持斑叶特性，所以不可用于嵌合体玉簪的组织培养，但可快速繁殖非嵌合体玉簪。

猕猴胚胎干细胞的分离培养和鉴定

目的　从体外培养成熟囊胚中分离并鉴定猕猴胚胎干细胞(embryonic stem cell , ES cell) 。方法　猕猴卵母细 胞经体外成熟培养、体外受精和早期胚胎体外成熟培养后,获得猕猴囊胚。当囊胚由透明带自然孵出后,用细玻璃针剥离 囊胚中的内细胞团(inner cell mass , ICM) 并与饲养细胞进行共培养。由ICM分离,培养并鉴定胚胎干细胞集落。结果　由 4 只FSH 超排猕猴中共取得92 个处于GV 期的猕猴卵母细胞,选取其中的22 个用HECM210 培养基培养后,获得6 个高质 量的囊胚,由此6 个囊胚中分离得到3 个内细胞团,并由此最终获得1 株猕猴ES 细胞,即RS5 细胞。RS5 细胞具高比例核P 质比,核仁多,其细胞集落边缘平整,其内各单个细胞清晰。经约5 个月的连续传代后,仍保持了正常二倍体的核型,其染 色体数目为42 条。碱性磷酸酶细胞组织化学染色为阳性,说明RS5 细胞为未分化态的胚胎干细胞。经高密度和长时间 培养后,RS5 细胞可进一步分化为多种类型细胞。结论　RS5 细胞株具有自我更新能力和多分化潜能,属于胚胎干细胞。

Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro

Aim: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. Methods: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPELC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-I-127 iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC50) of sifuvirtide on cell fusion was determined by co-cultivation assay. Results: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 mu g/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10626 +/- 2886 mu g/L and 528 +/- 191 mu g/L, and the terminal elimination half-lives (T,12) were 6.3 +/- 0.9 h and 5.5 +/- 1.0 h, respectively. After sc, T-max was 0.25-2 h, and the absolute bioavailability was 49% +/- 13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC50 of 0.33 mu g/L. Conclusion: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T-1/2 of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC50 in vitro.

Duplication and independent selection of cell-wall invertase genes GIF1 and OsCIN1 during rice evolution and domestication

Background: Various evolutionary models have been proposed to interpret the fate of paralogous duplicates, which provides substrates on which evolution selection could act. In particular, domestication, as a special selection, has played important role in crop cultivation with divergence of many genes controlling important agronomic traits. Recent studies have indicated that a pair of duplicate genes was often sub-functionalized from their ancestral functions held by the parental genes. We previously demonstrated that the rice cell-wall invertase (CWI) gene GIF1 that plays an important role in the grain-filling process was most likely subjected to domestication selection in the promoter region. Here, we report that GIF1 and another CWI gene OsCIN1 constitute a pair of duplicate genes with differentiated expression and function through independent selection. Results: Through synteny analysis, we show that GIF1 and another cell-wall invertase gene OsCIN1 were paralogues derived from a segmental duplication originated during genome duplication of grasses. Results based on analyses of population genetics and gene phylogenetic tree of 25 cultivars and 25 wild rice sequences demonstrated that OsCIN1 was also artificially selected during rice domestication with a fixed mutation in the coding region, in contrast to GIF1 that was selected in the promoter region. GIF1 and OsCIN1 have evolved into different expression patterns and probable different kinetics parameters of enzymatic activity with the latter displaying less enzymatic activity. Overexpression of GIF1 and OsCIN1 also resulted in different phenotypes, suggesting that OsCIN1 might regulate other unrecognized biological process. Conclusion: How gene duplication and divergence contribute to genetic novelty and morphological adaptation has been an interesting issue to geneticists and biologists. Our discovery that the duplicated pair of GIF1 and OsCIN1 has experiencedsub-functionalization implies that selection could act independently on each duplicate towards different functional specificity, which provides a vivid example for evolution of genetic novelties in a model crop. Our results also further support the established hypothesis that gene duplication with sub-functionalization could be one solution for genetic adaptive conflict.

抚仙金线鲃人工繁殖与鱼苗培育技术

抚仙金线鲃(Sinocyclocheilus tingi)是抚仙湖的特有种.2009年3-4月,有4次繁殖试验取得了成功.先后试验了40尾雌鱼和20尾雄鱼,成功催产19尾雌鱼和15尾雄鱼,分别占雌雄鱼的47.5%和75.0%.获得鱼卵25 547粒,并实施干法受精.有16810粒受精,平均受精率为65.8%.孵化出鱼苗约6040尾,平均孵化率为23.6%.经过20目的饲养,仔鱼存活3 056尾,存活率为50.6%.抚仙金线鲃较低的催产率,可能是生殖功能紊乱所致:但也不排除是人工营造的条件离其自然环境需求尚有距离.要使抚仙金线鲃成为鱼鱇<鱼良>白鱼(Anabarilius grahami)之后又一当地特色养殖鱼种,突破其人工繁殖瓶颈,培育高品质鱼苗是必由之路.突破其人工繁殖,还可为研究抚仙金线鲃的生活史和保护该物种免于灭绝奠定良好基础.

滇池铜绿微囊藻(M．aeruginosa zosa Kutz)的分离培养与总DNA提取的改进

利用稀释法结台平板分离培养法将采自滇池水华中的铜绿微囊藻进行了成功的 分离纯化对比纯化培养前后，发现纯化后该藻的生长速度明显加快，密度大为提高，约为纯化前 的5倍用乙醇乙醚混合液剐该藻作预处理后，破坏了其胶质被．再经常规方法提取总DNA，其得 率提高3 4倍

人胚胎神经干细胞的分离培养及鉴定研究

目的:研究人胚胎神经干细胞的培养条件及体外分化情况。方法:从12周龄人胚胎脑皮质分离细胞,采用无血清培养技术,协同应用碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和重组人白血病抑制因子(rhLIF)进行培养;5-溴脱氧尿嘧啶核苷(BrdU)标记检测细胞的增殖能力,间接免疫荧光化学法检测细胞的分化情况。结果:培养得到的大量半悬浮生长的神经干细胞球能够传代培养;BrdU标记阳性,可诱导分化为神经元、星形胶质细胞和少突胶质细胞。结论:人胚胎脑皮质分离细胞培养得到的细胞群具有神经干细胞的基本特征,可进一步用于基础及临床研究。

PRELIMINARY INVESTIGATION OF THE HABITATS OF PRESBYTIS-FRANCOISI AND PRESBYTIS-LEUCOCEPHALUS, WITH NOTES ON THE ACTIVITY PATTERN OF PRESBYTIS-LEUCOCEPHALUS

In this paper the habitat structure and ecology of Presbytis francoisi and Presbytis leucocephalus are compared. Observations were made of the two langur species in areas of southwest Guangxi province in which the langurs occur but are not sympatric. The results showed that the habitat of P. leucocephalus differs from that of P. francoisi and that the habitat in western areas was different from that in eastern areas in terms of vegetation and other criteria. P. francoisi was limited in its distribution to localities at higher altitudes, in contrast to P. leucocephalus. This may be due to human activities such as crop cultivation and logging. With respect to its activity pattern, P. leucocephalus spent 51.8% of its day in the trees and 48.2% on the rocky substrate. The results of this study suggest that Presbytis may best be regarded as a semiarboreal form.