Biochemical modeling of an autonomously oscillatory circadian clock in Euglena

Eukaryotic microorganisms, as well as higher animals and plants, display many autonomous physiological and biochemical rhythmicities having periods approximating 24 hours. In an attempt to determine the nature of the timing mechanisms that are responsible for these circadian periodicities, two primary operational assumptions were postulated. Both the perturbation of a putative element of a circadian clock within its normal oscillatory range and the direct activation as well as the inhibition of such an element should yield a phase shift of an overt rhythm generated by the underlying oscillator. Results of experiments conducted in the flagellate Euglena suggest that nicotinamide adenine dinucleotide (NAD+), the mitochondrial Ca2+-transport system, Ca2+, calmodulin, NAD+ kinase, and NADP+ phosphatase represent clock "gears" that, in ensemble, might constitute a self-sustained circadian oscillating loop in this and other organisms.     

Changes of Activities in NAD Kinase and NADP Phosphatase During Ripening and Senescence of Tomato and Strawberry Fruits

Activities of NAD kinase(NADK)and NADP phosphatase and relationship between the two enzymes and temperature, respiration, ethylene production and trifluoperazine(TFP) were studied during ripening and senescence of strawberry and tomato frnits after harvest at 4℃and 20℃. The activity of NAD kinase in strawberry decreased slowly during first four days, then increased gradually. The NADP phosphatase activity increased at the second day, decreased the next day,then increased again. In tomato fruit, the activities of NAD kinase and NADP phosphatase increased at the second day, decreased with the ripening and senescence of the fruit. The change trend of NAD kinase and respiration in the two fruits were similar, the same were NADP phosphatase and ethylene production. TFP enhanced the activity of NAD kinase and had little effect on NADP phosphatase. Low temperature(4℃ ) activated the NAD kinase and reduced the activity of NADP phosphatase. These results indicated that the NAD kinase and NADP phosphatase were related to the ripening and senescence of strawberry and tomato fruits. The activation of NAD kinase probably postponed the ripening and senescence of the fruits.     

Lactate dehydrogenase isozymes: further kinetic studies at high enzyme concentration

By competition with lactate dehydrogenase (LDH) for nicotinamide adenine dinucleotide (NAD), commonly occurring intracellular proteins, such as glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and albumin, can protect LDH-1 and LDH-5 from inhibition and ternary complex formation with NAD and pyruvate. The existence of intracellular proteins that compete with LDH for NAD renders unphysiological a model for estimating the extent of intracellular LDH inhibition based on incubations of only LDH, NAD, and pyruvate.     

单坡面塑料日光温室优化结构模拟设计——日光温室采光优化初探

本研究对单坡面塑料日光温室结构进行了计算机模拟优化设计,并与生产上应用面积大的“琴弦式”日光温室进行采光性能比较。结果证明：优化结构日光温室在“冬至”节日,地面日辐射总量比对照日光温室提高１４．０％,光透过率提高８．６９％,光质成分及光分布也有改善。     

苎麻NAD7基因的克隆与分析

线粒体中泛醌氧化还原酶的7亚基基因(NAD7)是与植物呼吸代谢和植物细胞质雄性不育相关的重要基因之一。本研究利用NAD7的保守序列设计引物,用RT-PCR方法克隆了苎麻(Boehmeria nivea)NAD7的cDNA片段,其长度为969 bp,编码322个氨基酸。序列比对表明该cDNA序列及预测的蛋白序列与拟南芥、油菜有98%的同源性。聚类分析表明苎麻NAD7基因的进化处在很古老的地位。为深入研究苎麻雄性不育的分子生物学机理提供依据。     

烟草中依赖于NAD+的山梨醇脱氢酶基因的克隆及序列分析

以珊西烟(Nicotiana tabacum L.cv Xanthi NN)为材料,通过RT-PCR,首次克隆了烟草中山梨醇脱氢酶基因(NAD+-SDH),该基因全长1119bp,编码372个氨基酸残基,其氨基酸序列与番茄和葡萄的山梨醇脱氢酶同源性分别为92%和87%。     

Yeast life-span extension by calorie restriction is independent of NAD fluctuation

Calorie restriction (CR) slows aging in numerous species. In the yeast Saccharomyces cerevisiae, this effect requires Sir2, a conserved NAD+-dependent deacetylase. We report that CR reduces nuclear NAD+ levels in vivo. Moreover, the activity of Sir2 and its human homologue SIRT1 are not affected by physiological alterations in the NAD+:NADH ratio. These data implicate alternate mechanisms of Sir2 regulation by CR.     

不同NAD含量条件下培养的猪胸膜肺炎放线杆菌对小白鼠致病性和免疫保护性的影响

将在含体积分数为0.050%、0.001%的尼克酰胺腺嘌呤二核苷酸(NAD)的LB培养基中培养的血清5型猪胸膜肺炎放线杆菌(APP),分别以3×109CFU.只-1的菌量对30日龄小白鼠进行接种,观察不同NAD含量条件下培养的APP对小白鼠致病性的影响.结果表明:用0.001%NAD培养的血清5型APP对小白鼠的致病性较强,小白鼠接种APP后,多数表现肺脏严重出血;而用0.050%NAD培养的血清5型APP接种小白鼠后,只有少数小白鼠肺脏出血严重,多数表现为轻度出血.将上述条件培养的APP按常规方法制备油乳剂灭活疫苗,分别在第3周龄和第6周龄对小白鼠进行免疫接种,在2免后3周对免疫的小白鼠进行同源攻击,并对免疫前后小白鼠的抗体效价进行检测.结果表明:分别用0.050%、0.001%NAD培养的血清5型APP制备的灭活疫苗,对小白鼠的保护率差异不显著;用0.001%NAD培养的血清5型APP制备的灭活疫苗免疫小白鼠,用血清5型APP同源攻击后,发病的小白鼠肺脏出血较轻,而用0.050%NAD培养的疫苗免疫小白鼠攻毒后,发病的小白鼠肺脏出血严重.可见在不同NAD含量条件下培养的APP对小白鼠的致病性和免疫保护力不同.     

仁用杏新优品种引种栽培试验

以龙王帽为对照,按照随机区组设计方法对引进的仁用杏4个品种的繁育方法、幼树生长量、抗性能力等指标进行了调查研究。结果表明,嫁接方法采用皮下接比芽接成活率提高40．8％。4a生幼树平均高生长量3．27m,丰仁、超仁、油仁、国仁分别比龙王帽高17．1％、15．1％、14．4％、14．0％;胸径生长平均2．69cm,丰仁、超仁、油仁、国仁分别比龙王帽高25．5％、21．6％、18．2％、15．6％。对幼树进行了观察,丰仁生长量高于其它品种,是一个很有引种前景的优良树种。     

红麻线粒体基因NAD3和COB的RNA编辑与表达分析

以红麻细胞质线粒体近等基因系UG93A(不育系)、UG93B(保持系)及UG93A×福红992(恢复系)的F1代为材料,研究花药线粒体基因NAD3和COB的转录与表达。结果发现,在3种供试材料中,基因NAD3的CDS区在DNA水平和RNA编辑水平上均无差异;不育系与保持系的COB基因DNA编码序列无差异,但在RNA水平上发生了编辑,8个位点的编辑均发生于密码子的第一或第二位碱基,且导致氨基酸种类变化,主要为亲水性氨基酸转变为疏水性氨基酸;COB基因在UG93A中的RNA编辑频率低于UG93B;NAD3和COB基因在3种材料中的转录本大小基本相同,但在表达水平上表现为不育系显著低于保持系和F1代。由此推测NAD3和COB基因在红麻花药发育过程中有着重要的作用。     

不同饲料不同龄期小蚕人工饲料育比较试验

用32个蚕品种杂交形式供试,比较了新老2种稚蚕人工饲料配方的疏毛率;结果表明新配方的24 h疏毛率普遍优于老配方,差异达到极显著水平;24 h疏毛率,老配方中只有6个蚕品种超过了98%,新配方中有21个蚕品种超过了98%,增加了15个蚕品种型式,新配方明显扩大了一代杂交种杂交率检验和小蚕共育的适用蚕品种范围.从不同龄期小蚕人工饲料育与全龄桑叶育的养蚕成绩和茧丝成绩看,用人工饲料进行小蚕共育基本可行,综合考虑各方面因素,共育到2龄后再改喂桑叶或分蚕出售给农民较好.     

Direct demonstration of an adaptive constraint

The role of constraint in adaptive evolution is an open question. Directed evolution of an engineered beta-isopropylmalate dehydrogenase (IMDH), with coenzyme specificity switched from nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide phosphate (NADP), always produces mutants with lower affinities for NADP. This result is the correlated response to selection for relief from inhibition by NADPH (the reduced form of NADP) expected of an adaptive landscape subject to three enzymatic constraints: an upper limit to the rate of maximum turnover (kcat), a correlation in NADP and NADPH affinities, and a trade-off between NAD and NADP usage. Two additional constraints, high intracellular NADPH abundance and the cost of compensatory protein synthesis, have ensured the conserved use of NAD by IMDH throughout evolution. Our results show that selective mechanisms and evolutionary constraints are to be understood in terms of underlying adaptive landscapes.     

氮胁迫条件下水稻理想株型基因ipa1对氮代谢的影响

利用水稻(Oryza sativa L.)近等基因系NIL-IPA1和NIL-ipa1,研究在施氮与不施氮条件下,理想株型调控基因ipa1在水稻快速分蘖期对氮代谢的影响。结果发现,在施氮条件下,相对于NIL-IPA1植株,NIL-ipa1水稻植株中谷氨酰胺合成酶(Glutamine synthetase,GS)、NADH谷氨酸合酶(NADH-glutamate synthase,NADH-GOGAT)的活性降低,叶片中游离氨基酸含量减少,但NADH谷氨酸脱氢酶(NADHglutamate dehydrogenase,NADH-GDH)、NAD+谷氨酸脱氢酶(NAD+-glutamate dehydrogenase,NAD+-GDH)活性升高,表明ipa1能够降低水稻植株氮素同化效率,同时提高氮素重复利用率。在不施氮条件下,NIL-IPA1和NIL-ipa1植株GS活性提高,NADH-GOGAT、NADH-GDH、NAD+-GDH活性降低,可溶性蛋白和游离氨基酸含量减少,同时发现在NIL-ipa1植株中GS、NADH-GOGAT活性变化相对较小,NAD+-GDH活性相对较低,游离氨基酸含量相对较高,表明ipa1能够降低氮胁迫对水稻植株氮同化效率的影响,提高水稻植株对低氮胁迫的抗性。     

卵形短须螨为害西番莲研究

首次在福建省南部发现一种为害西番莲的新害螨 ,形态学鉴定为卵形短须螨 (Brevipalpusobovatus Donnadieu) .1 991-1 992年在室内饲养该螨 ,共 8-9代 ,卵期 6.0 -2 5 .0 d,幼螨期 3 .0 -1 4.0 d,若螨 期 2 .0 -1 2 .0 d,若螨 期 5 .0 -2 1 .5 d,产卵前期 2 .0 -3 0 .0 d,以雌成螨越冬 ,其寿命超过 3 0 d,最长达 2个月 .该螨在西番莲上雌雄比大于 2 0 0 0∶ 1 ,主要为孤雌生殖 .调查表明 ,卵形短须螨分布在漳州地区的紫色、黄色种西番莲和新引进的无性系“台农 1号”上 ,主要为害植株成龄叶片 ,田间种群数量高峰期出现在 1 1月份 .以前尚未有该螨为害世界其它地区西番莲的报道     

草莓果实采后NAD激酶活性与NAD（H）、NADP（H）含量及活性氧代谢的关系

【目的】研究草莓采后成熟衰老过程中NADK活性与NAD（H）、NADP（H）及活性氧代谢和膜氧化产物变化的关系，以探讨NADK在非跃变型果实成熟衰老过程中的作用，为调控果实的成熟衰老提供理论依据。【方法】将从果园采回的草莓果实贮藏于不同的温度下并进行每天取样，研究草莓果实在低温（4℃）、常温（20℃）贮藏期间成熟衰老过程中NAD激酶（NADK）活性及其底物NAD（H）、产物NADP（H）以及超氧阴离子O2- •过氧化氢（H2O2）、膜氧化产物丙二醛（MDA）含量的变化并分析NADK 与上述指标的关系。【结果】草莓果实在低温（4℃）贮藏时，其NADK活性比常温（20℃）贮藏的高，NAD（H）含量则相应比常温贮藏的低，NADP（H）含量则高于常温下的；同时，在常温贮藏期间果实O2- •生成速率和H2O2含量、膜氧化产物MDA含量均比低温贮藏的高，暗示NADK可能通过影响NAD（H）、NADP（H）的含量及比例来调控O2- •生成速率和H2O2含量，从而调控果实的成熟衰老。【结论】非跃变型果实草莓采后成熟衰老过程中，保持较高的NADK活性有利于延缓果实的成熟衰老，降低NADK活性可导致NAD和NAD（H）含量的积累，进而加速电子传递，产生大量的活性氧O2- •和H2O2，从而促进膜过氧化作用和积累较多的MDA，最终导致果实衰老变质。     

苏云金芽孢杆菌NAD(P)H:醌氧化还原酶基因的克隆和敲除载体的构建

以苏云金芽孢杆菌库斯塔克亚种(Bacillus thuringiensis subsp.kurstaki)8010为研究材料,通过PCR技术获得Bt 8010菌株NAD(P)H:醌氧化还原酶基因上下游2个片段,以含有卡那霉素抗性基因的pDG780质粒为中间载体,通过酶切、连接和PCR,构建含待敲除NAD(P)H:醌氧化还原酶基因上下游片段、卡那霉素抗性基因的重组质粒pRN5101DKU,为后续Bt8010菌株NAD(P)H:醌氧化还原酶基因的敲除做准备.     

Conformational variability of NAD+ in the free and bound states: a nicotinamide sandwich in NAD+ crystals

X-ray analysis of the free-acid crystal form of the coenzyme nicotinamide adenine dinucleotide (NAD+) revealed a conformational difference between the free NAD+ molecule and one bound in enzymes or complexed to Li+ ions. The pyrophosphate group showed asymmetry in the phosphate-oxygen bonds of the phosphate-oxygen-phosphate link; this bond at the nicotinamide side of the link is longer than that at the adenosine side by 0.04 angstrom. The crystal structure showed a novel intermolecular stacking of adenine and water molecules on opposite sides of nicotinamide that gives rise to a nicotinamide sandwich.