Characterization of GDP-mannose dehydrogenase from the brown alga Ectocarpus siliculosus providing the precursor for the alginate polymer

Alginate is a major cell wall polymer of brown algae. The precursor for the polymer is GDP-mannuronic acid, which is believed to be derived from a four-electron oxidation of GDP-mannose through the enzyme GDP-mannose dehydrogenase (GMD). So far no eukaryotic GMD has been biochemically characterized. We have identified a candidate gene in the Ectocarpus siliculosus genome and expressed it as a recombinant protein in Escherichia coli. The GMD from Ectocarpus differs strongly from related enzymes in bacteria and is as distant to the bacterial proteins as it is to the group of UDP-glucose dehydrogenases. It lacks the C-terminal ～120 amino acid domain present in bacterial GMDs, which is believed to be involved in catalysis. The GMD from brown algae is highly active at alkaline pH and contains a catalytic Cys residue, sensitive to heavy metals. The product GDP-mannuronic acid was analyzed by HPLC and mass spectroscopy. The K(m) for GDP-mannose was 95 μM, and 86 μM for NAD(+). No substrate other than GDP-mannose was oxidized by the enzyme. In gel filtration experiments the enzyme behaved as a dimer. The Ectocarpus GMD is stimulated by salts even at low molar concentrations as a possible adaptation to marine life. It is rapidly inactivated at temperatures above 30 °C.     

Effects of fermented rooibos (Aspalathus linearis) on adipocyte differentiation

Rooibos (Aspalathus linearis) contains a rich complement of polyphenols, including flavonoids, considered to be largely responsible for its health promoting effects, including combatting obesity. The purpose of this study was to examine the effect of fermented rooibos hot water soluble solids on in vitro adipocyte differentiation by using differentiating 3T3-L1 adipocytes. Hot water soluble solids were obtained when preparing an infusion of fermented rooibos at “cup-of-tea” strength. The major phenolic compounds (>5 mg/g) were isoorientin, orientin, quercetin-3-O-robinobioside and enolic phenylpyruvic acid-2-O-β-d-glucoside. Treatment of 3T3-L1 adipocytes with 10 μg/ml and 100 μg/ml of the rooibos soluble solids inhibited intracellular lipid accumulation by 22% (p < 0.01) and 15% (p < 0.05), respectively. Inhibition of adipogenesis was accompanied by decreased messenger RNA (mRNA) expression of PPARγ, PPARα, SREBF1 and FASN. Western blot analysis exhibited decreased PPARα, SREBF1 and AMPK protein expression. Impeded glycerol release into the culture medium was observed after rooibos treatment. None of the concentrations of rooibos hot water soluble solids was cytotoxic, in terms of ATP content. Interestingly, the higher concentration of hot water soluble solids increased ATP concentrations which were associated with increased basal glucose uptake. Decreased leptin secretion was observed after rooibos treatment. Our data show that hot water soluble solids from fermented rooibos inhibit adipogenesis and affect adipocyte metabolism, suggesting its potential in preventing obesity.     

Selenium addition alters mercury uptake,bioavailability in the rhizosphere and root anatomy of rice (Oryza sativa)

Background and Aims

Mercury (Hg) is an extremely toxic pollutant, especially in the form of methylmercury (MeHg), whereas selenium (Se) is an essential trace element in the human diet. This study aimed to ascertain whether addition of Se can produce rice with enriched Se and lowered Hg content when growing in Hg-contaminated paddy fields and, if so, to determine the possible mechanisms behind these effects.

Methods

Two cultivars of rice (Oryza sativa, japonica and indica) were grown in either hydroponic solutions or soil rhizobags with different Se and Hg treatments. Concentrations of total Hg, MeHg and Se were determined in the roots, shoots and brown rice, together with Hg uptake kinetics and Hg bioavailability in the soil. Root anatonmy was also studied.

Key Results

The high Se treatment (5 μg g^–1) significantly increased brown rice yield by 48 % and total Se content by 2·8-fold, and decreased total Hg and MeHg by 47 and 55 %, respectively, compared with the control treatments. The high Se treatment also markedly reduced ‘water-soluble’ Hg and MeHg concentrations in the rhizosphere soil, decreased the uptake capacity of Hg by roots and enhanced the development of apoplastic barriers in the root endodermis.

Conclusions

Addition of Se to Hg-contaminated soil can help produce brown rice that is simultaneously enriched in Se and contains less total Hg and MeHg. The lowered accumulation of total Hg and MeHg appears to be the result of reduced bioavailability of Hg and production of MeHg in the rhizosphere, suppression of uptake of Hg into the root cells and an enhancement of the development of apoplastic barriers in the endodermis of the roots.     

中亚热带天然林土壤CH_4吸收速率对模拟N沉降的响应

陆地森林土壤是重要的大气甲烷(CH4)汇,大气氮(N)沉降增加对森林土壤CH4吸收速率影响突出。运用静态箱-气相色谱法对中亚热带天然林土壤CH4吸收速率对模拟N沉降的响应进行连续3a的观测;试验作3种N处理,分别为对照(CK,0 kg N·hm-2·a-1)、低氮(LN,50 kg N·hm-2·a-1)和高氮(HN,100 kg N·hm-2·a-1),每种处理重复3次,每个月采集气体1次,同时测定0—5 cm土壤温度和0—12 cm土壤含水量;分析不同N沉降水平土壤CH4吸收速率的差异、动态变化以及对土壤含水量和土壤温度响应,并探讨N沉降对土壤理化性质的影响。结果显示:天然林土壤(CK)平均CH4吸收速率为(-62.78±14.39)μg·m-2·h-1,LN和HN土壤平均CH4吸收速率分别下降了30.21%、7.24%,CK、LN和HN处理土壤CH4吸收速率季节变化趋势相似;观测期间土壤CH4吸收速率对LN响应达到显著水平(P0.05),对HN响应则不显著(P0.05);LN、HN处理前两年对土壤CH4吸收速率抑制作用均不显著(P0.05),但在第3年LN极显著降低了土壤CH4吸收速率(P0.01),HN处理对土壤CH4吸收速率的影响则在第3年表现为显著抑制作用(P0.05),表明土壤CH4吸收速率对N沉降的响应随着N沉降时间的持续呈抑制效应加剧的趋势。相关分析表明:CK与HN土壤CH4吸收速率与土壤温度和土壤含水量均有显著相关性(P0.05),但LN土壤CH4吸收速率仅与土壤含水量显著相关(P0.05),表明土壤含水量是控制各N沉降处理土壤CH4吸收速率动态的主要环境因子。此外,LN、HN处理下土壤pH均极显著降低(P0.01),但LN土壤pH极显著低于HN(P0.01);LN处理极显著提高了土壤C/N比(P0.01),HN处理则相反;LN和HN处理对土壤NH+4-N、NO-3-N、可溶性总N(TDN)、可溶性有机碳(DOC)、地面凋落物量、地下0—10 cm细根生物量影响均不显著(P0.05),表明一定时期内N沉降首先引起了土壤pH和土壤C/N比的显著变化。     

利用分析Unigene在转录组中表达模式的方法拼接盐角草铵转运基因

RNA-seq技术能够全面快速地获得物种在某一状态下的转录本序列信息,但测序并组装后的大量Unigene往往不包含完整ORF(Open reading frame)。转录组库具有一定的冗余性,存在着属于同一个转录本的Unigene,这些Unigene因为无重叠区不能拼接而存在转录组库中。基于这种情况,为了拼接铵转运蛋白家族Unigene,首先挑选注释为AMT(Ammonium transporter)且ORF不完整的所有Unigene(5条),通过分析Unigene在4个转录组的表达模式,其中2条Unigene(Uni4和Uni5)具有相同的表达模式,推测可能来自同一转录本。然后通过NCBI blastx将这2条Unigene与参考物种的AMT蛋白质比对,确定其在转录本的位置及序列相互间没有交叠(如果两条编码序列相互交叠则不能组成同一个转录本)。结果发现Uni4和Uni5分别位于参考转录本5′端和3′端位置,因此假定它们属于同一个转录本,中间空缺约120 bp未知序列。通过试验验证,分别在Uni4和Uni5上设计单正向引物和单反向引物,PCR扩增得到约800 bp片段,将其测序并与两条Unigene比对,证实Uni4和Uni5属于同一转录本且获得了缺失的未知序列。最终拼接得到1 667 bp序列,包含1 482 bp完整ORF,编码494个氨基酸,通过系统进化分析将其归类为amt1亚家族,命名为Seamt1。生物信息学手段预测Se AMT1蛋白与已知的其他物种AMT性质相似。本研究采用转录组Unigene表达模式聚类的方法挖掘潜在的同一转录本Unigene,并且通过另外两组Unigene检验了该方法的可行性。这一便捷方法有助于转录组中Unigene的延伸和拼接,有助于完整ORF的获得及后期基因功能研究。     

The effects of pulsed magnetic field exposure on the permeability of leukemia cancer cells

Purpose: The newer methods of cancer treatment require new idea of drug delivery in cancer cells. Due to numerous researches electromagnetic field affect on cell function and cell membrane for possible therapeutic and drug delivery. In this article, we determined in vitro uptake of fluorescent dyes into the attached K562 cells due to time-varying magnetic field exposure. Method and material: The K562 cells were exposed to magnetic pulses via Magstim stimulator and double 70?mm coil. The strength and duration of pulses in all experiments were the same and three different frequencies of 0.25, 1 and 10?Hz pulses for 56, 112 and 28 numbers of pulses were applied (nine experimental groups) and uptake of Ly and PI was measured in each group. Result: Our results show that magnetic field can efficiently increase permeability. Among the treatment groups, the system gives the optimal permeabilization when cells are exposed to a train of 28 pulses with 1?Hz frequency.