水稻生育期对稻水象甲成虫种群空间格局影响及抽样技术

[目的] 明确四川省西南浅丘稻区稻水象甲成虫种群在水稻不同生育期的空间格局及抽样技术，为获取准确稻水象甲虫情调查资料和制定有效的综合防控措施提供理论依据。[方法] 调整水稻播栽时间，错开2组试验田水稻的生育期，用聚集度指标法、回归模型法和频次卡方检验法分析稻水象甲成虫种群的空间格局及水稻生育期对空间聚集特性的影响，并对田间序贯抽样技术和抽样方法进行研究。[结果] 不同田块稻水象甲成虫平均密度为0.48~5.83头·丛^-1，分蘖期水稻田虫口密度显著高于抽穗期。稻水象甲成虫在不同水稻生育期稻田间呈负二项聚集分布，基本成分为个体群，个体间相互吸引，聚集强度随种群密度的升高而增加。当种群密度较低时，其聚集由环境因素引起；种群密度较高时，其聚集为其自身的聚集习性与环境因素共同引起。双对角线抽样法是稻水象甲成虫田间抽样的最佳方法，当稻水象甲成虫防治指标为1头·丛^-1时，Iwao序贯抽样模型为T_1（n），T_0（n）=n±1.96√2.286n，结合Kuno序贯抽样模型建立了用于田间抽样的复序贯抽样图。[结论] 稻水象甲成虫在不同水稻生育期稻田间呈负二项聚集分布，分蘖期水稻田虫口密度显著高于抽穗期，双对角线抽样法是稻水象甲成虫田间抽样的最佳方法。     

低致死剂量氯虫苯甲酰胺对沟金针虫食物利用和相关生理生化指标的剂量和时间效应

【目的】明确氯虫苯甲酰胺对沟金针虫Pleonomus canaliculatus亚致死效应的生理生化机制,阐明氯虫苯甲酰胺低致死剂量对沟金针虫食物利用、能量物质含量以及体内消化酶、保护酶和解毒酶活力的影响。【方法】室内采用土壤混药法测定氯虫苯甲酰胺对沟金针虫3龄幼虫毒力,并测定了氯虫苯甲酰胺LC₁₀, LC₂₅和LC₄₀低致死剂量对沟金针虫3龄幼虫营养指标和体内能量物质含量的影响;采用酶动力学法检测了氯虫苯甲酰胺低致死剂量处理1, 6, 12, 24, 48和72 h后沟金针虫3龄幼虫体内消化酶(蛋白酶、α-淀粉酶、脂肪酶、海藻糖酶)、保护酶(CAT, POD和SOD)以及解毒酶(CarE, MFO和GST)活力的动态变化。【结果】氯虫苯甲酰胺对沟金针虫3龄幼虫有较高毒力,其LC₅₀值为1.2397 mg/kg。LC₁₀和LC₄₀剂量氯虫苯甲酰胺处理沟金针虫3龄幼虫后,平均相对生长率(MRGR)和近似消化率(AD)显著降低,严重干扰其对食物的利用;LC₁₀, LC₂₅和LC₄₀剂量处理后沟金针虫3龄幼虫体内主要的能量物质(蛋白质、脂质、碳水化合物、海藻糖)含量和消化酶活力均明显降低,而解毒酶和保护酶活力显著增加,最终延缓其生长发育。【结论】氯虫苯甲酰胺对沟金针虫幼虫具有很高的杀虫活性,低致死剂量氯虫苯甲酰胺处理沟金针虫幼虫后,通过抑制消化酶活性,使其对食物的利用能力降低和生长发育延缓,以及诱导解毒酶和保护酶活性来阻止外界毒物侵害。研究结果为阐明氯虫苯甲酰胺对沟金针虫的亚致死效应机制及作用机理提供了一定的理论基础。     

小菜蛾对氯虫苯甲酰胺抗药性与田间防治效果的相关性研究

【目的】明确小菜蛾Plutella xylostella(L.)田间种群对氯虫苯甲酰胺抗药性变化与田间防治效果的相关性。【方法】2011—2015年在田间进行了氯虫苯甲酰胺防治小菜蛾田间药效试验,并测定了该地小菜蛾田间种群对氯虫苯甲酰胺的敏感性。【结果】氯虫苯甲酰胺在相同剂量下对小菜蛾田间防治效果从2011的97.74%下降到2015年的63.53%;而小菜蛾对氯虫苯甲酰胺的相对毒力指数从2011年的1上升到2015的11.93。【结论】小菜蛾对氯虫苯甲酰胺的敏感性与其抗药性总体上呈现负相关。     

高原春油菜田小菜蛾种群动态和抗药性的监测

【目的】为了准确掌握典型春油菜种植区小菜蛾Plutella xylostella(L.)种群变化动态和抗药性现状。【方法】诱捕法调查了青海高原小菜蛾成虫发生动态、室内用浸渍法测定了小菜蛾田间种群的抗性倍数,并进行了田间药效试验。【结果】青海省小菜蛾一般一年发生3代,但2 500 m以上的地区第3代成虫数量较第1代、第2代明显下降。在我省高原春油菜区,每日20:00至次日晨4:00是小菜蛾成虫发生主要的时间段。小菜蛾在青海省不能越冬。湟中点小菜蛾对溴虫腈产生低水平抗性;对多杀菌素、丁醚脲产生中等抗性水平;对Bt、高效氯氰菊酯、茚虫威产生高水平的抗性;对阿维菌素、啶虫隆、氯虫苯甲酰胺产生极高水平抗性。互助点小菜蛾对溴虫腈、丁醚脲产生低水平抗性;对多杀菌素、啶虫隆产生中等抗性水平;对Bt、氯虫苯甲酰胺、茚虫威产生高水平抗性;对阿维菌素产生极高水平抗性。小菜蛾的抗性监测结果与田间药效结果基本一致,溴虫腈的抗性倍数最低,田间防治效果好于其他参试药剂。【结论】青海省小菜蛾年发生代数较少,且不能越冬。春油菜田小菜蛾已对大部分农药产生了抗药性。     

新疆和云南番茄潜叶蛾种群对六种杀虫剂的敏感性及其与解毒酶活性的关系

【目的】番茄潜叶蛾Tuta absoluta 是新入侵我国的对番茄具有毁灭性危害的入侵害虫,目前入侵我国的番茄潜叶蛾种群对杀虫剂的抗性尚无报道。本研究旨在明确新疆和云南番茄潜叶蛾田间种群对6种常用杀虫剂的敏感性及其与解毒酶活性的关系。【方法】采用浸叶法测定6种常用杀虫剂对番茄潜叶蛾新疆和云南种群2龄幼虫的室内毒力。通过对2龄幼虫的生物测定确定3种增效剂［CYP450抑制剂胡椒基丁醚(PBO)、酯酶抑制剂磷酸三苯酯(TPP)和GST抑制剂丁烯二酸二乙酯(DEM)］对氯虫苯甲酰胺的增效作用。采用酶活性分析测定室内敏感种群和田间抗性种群(新疆种群) 2龄幼虫体内解毒酶［细胞色素P450酶(CYP450)、谷胱甘肽S-转移酶(GST)和羧酸酯酶(CarE)］活性,以确定杀虫剂抗性与解毒酶活性的关系。【结果】番茄潜叶蛾云南种群对6种杀虫剂的敏感性由高到低依次为甲维盐、溴虫腈、多杀菌素、茚虫威、氯虫苯甲酰胺和高效氯氰菊酯。新疆种群对6种杀虫剂的敏感性由高到低依次为甲维盐、溴虫腈、氯虫苯甲酰胺、多杀菌素、茚虫威和高效氯氰菊酯。与室内敏感种群相比,云南和新疆种群对氯虫苯甲酰胺的抗性水平最高,抗性倍数分别为212.7和169.3倍。生物测定结果表明,3种增效剂PBO, TPP和DEM均对氯虫苯甲酰胺无明显增效作用。酶活性测定结果表明,番茄潜叶蛾室内敏感种群和田间抗性种群之间2龄幼虫中CYP450, GST和CarE活性无显著差异。【结论】番茄潜叶蛾新疆和云南种群对测试的6种杀虫剂产生不同程度的抗性,对氯虫苯甲酰胺的抗性最高,番茄潜叶蛾对杀虫剂的抗性与解毒酶活性无关。本研究的结果对番茄潜叶蛾的田间防治和杀虫剂抗性治理具有指导意义。     

氯虫苯甲酰胺对非靶标害虫褐飞虱实验种群的亚致死效应

在稻田中,氯虫苯甲酰胺是以鳞翅目幼虫为主要防治对象的新型杀虫剂,而褐飞虱 是该药剂的重要非靶标害虫.本文采用稻茎浸渍法测定氯虫苯甲酰胺对其非靶标害虫褐飞虱3龄若虫和成虫的毒力.结果表明:氯虫苯甲酰胺对褐飞虱3龄若虫和成虫的LC50分别为26.85和35.53 mg·L-1;以氯虫苯甲酰胺亚致死浓度LC10和LC25分别处理褐飞虱3龄若虫后,对当代褐飞虱雌虫寿命无显著影响,但LC25剂量处理后,当代褐飞虱雌虫产卵量显著降低45.6粒.亚致死剂量处理褐飞虱3龄若虫后,显著影响F1代褐飞虱的产卵量和雌虫寿命,雌虫产卵量分别减少43.5和72.9粒,雌虫寿命分别缩短1.35和2.87 d;两个剂量处理后F1代的各虫态发育历期均有所延长;施药后各项种群参数也发生了变化,种群内禀增长率rm分别降低12.8％和23.5％,净增殖率R0分别降低37.4％和68.7％,而世代平均历期T和种群加倍时间t均延长.表明氯虫苯甲酰胺亚致死剂量对褐飞虱种群增长具有一定的抑制作用.     

6种杀虫剂对南美番茄潜叶蛾的毒力及田间防效

[目的]研究6种杀虫剂对南美番茄潜叶蛾卵、幼虫和成虫的毒力及其在温室番茄上的防治效果,为南美番茄潜叶蛾防治提供高效杀虫剂和施药技术。[方法]采用浸渍法和药膜法测定了6种杀虫剂对南美番茄潜叶蛾卵、幼虫和成虫的毒力,田间调查毒力较高杀虫剂对温室番茄上南美番茄潜叶蛾防效。[结果] 6种杀虫剂中的乙基多杀菌素、甲氨基阿维菌素苯甲酸盐和阿维菌素对南美番茄潜叶蛾卵有毒力作用,致死中浓度（LC₅₀）分别为1.415、13.588和23.194 mg·L^-1。6种杀虫剂对南美番茄潜叶蛾幼虫的LC₅₀分别为：阿维菌素0.026 mg·L^-1、四唑虫酰胺0.052 mg·L^-1、甲氨基阿维菌素苯甲酸盐0.057 mg·L^-1、乙基多杀菌素0.072 mg·L^-1、氯虫苯甲酰胺0.484 mg·L^-1和呋虫胺2.039 mg·L^-1。对于南美番茄潜叶蛾成虫,24 h时,仅甲氨基阿维菌素苯甲酸盐和四唑虫酰胺对成虫有较高毒力;72 h时,6种杀虫剂对成虫的LC₅₀分别为：甲氨基阿维菌素苯甲酸盐0.390 mg·L^-1、乙基多杀菌素1.646 mg·L^-1、四唑虫酰胺2.630 mg·L^-1、呋虫胺5.577 mg·L^-1、阿维菌素22.502 mg·L^-1和氯虫苯甲酰胺39.636 mg·L^-1。在成虫盛发期第4天施药,阿维菌素、四唑虫酰胺、甲氨基阿维菌素苯甲酸盐、乙基多杀菌素在南美番茄潜叶蛾危害严重的温室番茄上防效达80%以上。[结论]阿维菌素、四唑虫酰胺、甲氨基阿维菌素苯甲酸盐、乙基多杀菌素、氯虫苯甲酰胺和呋虫胺对南美番茄潜叶蛾卵、幼虫或成虫有较高毒力,其中阿维菌素、四唑虫酰胺、甲氨基阿维菌素苯甲酸盐和乙基多杀菌素田间防效较好。     

滇中稻区稻水象甲的卵巢发育及防治

【目的】开展稻水象甲卵巢发育、扩散能力和防治药剂的筛选研究,为云南稻水象甲的防治提供依据。【方法】对成虫采用卵巢解剖法,确定卵巢的发育进度和产卵期;采用田间系统调查法和染色法明确扩散范围;采用浸叶法对常用杀虫剂进行室内筛选。【结果】2009年4月下旬至5月上旬秧苗期,稻水象甲Ⅱ级卵巢达100%,Ⅲ级卵巢的平均抱卵量为8粒·头~(-1),最大抱卵量为23粒·头~(-1)。秧田返栽田的幼虫、成虫虫口密度明显高于大田,秧田返栽田的幼虫虫量是大田的4.82倍,第一代成虫是大田的6.50倍。烯啶虫胺、毒死蜱、辛硫磷、三唑磷、锐劲特对稻水象甲成虫防效在96.11%以上。【结论】滇中稻水象甲自然传播的范围小,随稻秧移栽是稻水象甲的主要传播方式,秧苗揭膜1周后至移栽前是化学防治的关键期,建议选用烯啶虫胺、辛硫磷、三唑磷、锐劲特在此期间开展田间防控。     

噻虫啉亚致死剂量对斜纹夜蛾解毒酶系活性与生长繁殖的影响

【目的】斜纹夜蛾Spodoptera litura(Fabricius)是一种重要农业害虫,因药物、外界环境等选择压力的不同,造成其生长发育有所差异。噻虫啉是第二代氯代烟碱类杀虫剂,对昆虫烟碱型乙酰胆碱受体有很强的激动作用。本实验旨在探讨噻虫啉对斜纹夜蛾的亚致死效应,为斜纹夜蛾的综合防治和噻虫啉的合理使用提供理论依据。【方法】采用饲料混毒法,测定噻虫啉对斜纹夜蛾3龄幼虫的毒力,确定其亚致死剂量LC25和LC50。通过离体酶活性测定,分析噻虫啉亚致死剂量对斜纹夜蛾体内羧酸酯酶、谷胱甘肽-S转移酶和多功能氧化酶3种代谢解毒酶活性的影响。记录各个年龄阶段的生长发育、繁殖力以及种群增长等数据,应用特征年龄-龄期及两性生命表方法分析LC25和LC50剂量处理和对照的斜纹夜蛾子代(F1)两性生命表参数的差异。【结果】以LC25和LC50剂量处理斜纹夜蛾3龄幼虫48 h,羧酸酯酶活性均受到明显诱导,分别升高了14.2%和45.1%;谷胱甘肽-S转移酶活性受到明显的抑制,分别降低了9.8%和37.1%;而多功能氧化酶活性均低于对照,其抑制作用与浓度成正比,但差异不显著。LC25和LC50剂量处理斜纹夜蛾幼虫后,子代(F1)的成虫前期分别比对照延长了3.05 d和4.80 d,雄成虫寿命分别缩短了2.06 d和3.31 d,雌成虫寿命分别缩短了0.13 d和0.92 d;其单雌产卵量均显著减少,分别降低了17.7%和33.3%;化蛹率分别降低了10.7%和11.4%;两个处理的内禀增长率(r)、周限增长率(λ)和净增殖率(R0)均显著小于对照,平均世代时间(T)均明显延长。【结论】羧酸酯酶可能为斜纹夜蛾对噻虫啉解毒代谢过程中的主要解毒酶,在其后续抗性形成中起主要作用;噻虫啉亚致死剂量对斜纹夜蛾的生长、发育、繁殖有显著的抑制作用。     

云南稻区稻水象甲Lissorhoptrus oryzophilus Kuschel种群发生特征

【背景】2007年6月,云南省首次在嵩明县发现稻水象甲,为掌握其年发生世代、成虫和幼虫种群消长动态及其越冬特点开展此项研究。【方法】2008—2010年,采用田间系统调查法对云南省昆明市嵩明县大桥村稻区稻水象甲的种群发生规律进行了研究。【结果】每年4月初气温回升(4月气温9.0~24.1℃,均温16.1℃),稻水象甲越冬成虫开始出土活动,从稻田周边越冬场所迁移至稻埂或秧田取食杂草和秧苗。5月中旬,随水稻的移栽,迁移至大田为害并产卵,孵化后的幼虫取食水稻根系。6月中旬水稻分蘖期,幼虫发生量达到高峰,虫量为3.17~11.33头·丛-1;7月中下旬水稻孕穗期,也是形成土茧的高峰期,土茧量5.90~9.00头·丛-1;7月下旬—8月中旬水稻抽穗和扬花期,成虫大量出现,数量达0.21~4.85头·网-1;8月中旬水稻乳熟期,成虫逐渐迁移至稻田附近向阳坡面的田埂和沟埂上,主要集中在0~3 cm有杂草覆盖的浅土层越冬,直至翌年的4月底,最大越冬虫量98.33~266.00头·m-2。【结论与意义】稻水象甲在云南省嵩明县水稻区年发生1~1.5代;种群发生动态与水稻的生育期紧密相关,4月初越冬成虫为害秧苗,7月份是防治成虫的关键时期。     

Thermotolerant strain of Bacillus licheniformis producing lipase

A thermotolerant variant of Bacillus licheniformis strain H1 (isolated from Jordan valley soil), was highly active in degrading macromolecules, and possessed a lipase activity with a half life of 30 min at 70°C. This activity was produced during exponential growth. The extracellular crude lipase showed maximal activity at pH 10, and retained 65% of its stability at pH 12.The author is with the Department of Biological Sciences, University of Jordan, P.O. Box 2686, Amman 11181, Jordan     

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to -galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.Issued as NRCC No. 31634     

Identification of a fibrinolytic enzyme by <Emphasis Type="Italic">Bacillus vallismortis</Emphasis> and its potential as a bacteriolytic enzyme against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

A potent fibrinolytic enzyme-producing bacterium was isolated from the traditional Korean condiment Chungkook-jang and identified as Bacillus vallismortis Ace02. The extracellular fibrinolytic enzyme was purified with a 18% recovery of activity from supernatant cultures using CM-Sepharose column chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme was 757 kFU mg⁻¹. Its molecular mass was about 28 kDa and the initial amino acids of the N-terminal sequence were AQSVPYGVSQ. The full amino acid sequence of fibrinolytic enzyme Ace02 corresponded with bacteriolytic enzyme, L27, from Bacillus licheniformis, which has strong lytic activity against Streptococcus mutans, a major causative strain of dental caries. This suggests that the purified enzyme should be used for prevention of dental caries as well as being an effective thrombolytic agent.     

小叶锦鸡儿抗沙埋生长与抗氧化酶及同工酶变化的关系

小叶锦鸡儿(Caraganas tenophylla L.)是广泛应用于流动沙丘治理的优良固沙植物。然而关于其抗沙埋生理机理目前尚不清楚。选择生长在科尔沁沙地的小叶锦儿为试验材料,依据株高对其进行不同程度沙埋(轻度、中度、重度沙埋), 并通过测定沙埋过程中植株高度、不同部位叶片丙二醛(MDA)含量、抗氧化酶活力、抗氧化酶同工酶谱变化, 以揭示其抗沙埋生理适应机理和基因调控机理。结果表明:沙埋6d,植株各部位生长加快,尤其是顶部和基部生长更快。叶片MDA含量降低、整株植物叶片平均过氧化氢酶(CAT)、过氧化物酶(POD)、过氧化物歧化酶(SOD)活力增加,但重度沙埋使抗氧化酶活力下降。沙埋12d,植株各部位生长继续加大, 沙下叶片凋落。与对照相比,沙上叶片MDA含量成倍增加,并与叶片POD、SOD和CAT活力的大幅度提高呈正相关,并与对照差异显著(P < 0.01)。同时,不同厚度沙埋6d,叶片CAT同工酶出现两新带CAT III和CATII;POD同工酶谱带(6条酶带)随沙埋厚度增加,叶片PODII区带加宽、色加深,POD I 和POD III酶带消失。但是,不同厚度沙埋下,沙上和沙下叶片CAT、SOD和POD酶谱带数和活力均相同。这表明在沙埋应激适应反应期(6d),叶片抗氧化酶活力的增强与抗氧化酶基因表达增强和基因启动有关。受到沙埋重力胁迫的成熟叶可能将胁迫信号传递给沙上没有沙埋的叶子及生长点,导致整株叶片产生整体适应性反应,激活抗氧化酶系统,以致加速生长。因此,小叶锦鸡儿萌蘖生物学特性和抗氧化酶对沙埋胁迫快速响应在维护氧自由基代谢平衡和植株快速恢复生长中起重要保护作用。     

Enzyme function and polymorphism: A test in two Anolis lizard species

Electrophoretic surveys of two lizard species were used to test hypotheses that relate levels of enzyme variability to enzyme function (single-substrate versus multiple-substrate enzymes, regulatory versus nonregulatory enzymes). Anolis roquet behaviorally regulates its body temperature, but its congener A. gundlachi is passive to variations in the thermal environment. As a result, populations of A. gundlachi probably experience the thermal environment as temporally coarse-grained, whereas populations of A. roquet do not. We therefore predicted that A. gundlachi would exhibit greater enzyme heterozygosity than A. roquet and that different enzyme classes would contribute disproportionately to this interspecific difference. The data show (1) that A. gundlachi does have a higher heterozygosity and (2) that this difference appears to result from high levels of heterozygosity at loci coding for multiple-substrate enzymes. The dichotomy between regulatory and nonregulatory enzymes offers no explanation for the variability in heterozygosity among enzyme loci in these species.E.Z. was supported by a grant from the Natural Sciences and Engineering Council of Canada. The study was accomplished while P.E.H. held a postdoctoral fellowship from the Killam Trust of Dalhousie University and a grant from the Research Development Fund of Dalhousie University. The collection of material was made possible by grants (to P.E.H.) from National Science Foundation (DEB 75-16334), the Explorers Club of New York, Sigma Xi, and the Richmond and Anderson Funds of Harvard University. We thank Dr. D. W. Foltz for his help with the calculations.     

Characterization of Polyphenol Oxidase from the Latex of Opium Poppy

Polyphenol oxidase from the latex of opium poppy was purified to the electrophoretic homogeneity by affinity chromatography using p-aminobenzoic acid as a ligand coupled to Sepharose CL-4B by divinyl sulphone activation method. The purified enzyme was used to prepare the polyclonal antibodies. The purified latex PPO exhibited high diphenolase activity in comparison with almost unmeassurable monophenolase activity. Both of these activities were sensitive to the activation with sodium dodecyl sulphate. Two isoforms (65 and 40 kDa) of latex PPO were separated by the gel filtration. There were no differences in substrate specifity (weak monophenolase and high diphenolase activity) and sensitivity to inhibitors between these isoforms, but they showed differences in electrophoretic mobility. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

Characterization of DNA restriction-modification systems in Spirulina platensis strain pacifica

Four unique restriction enzymes were identified in the soluble protein fraction of Spirulina platensis strain pacifica, a commercially important strain of marine cyanobacterium that is used as a supplement in a human diets. These are SpaI, SpaII, SpaIII and SpaIV, which are isoschizomers of Tth111I, Pvul, PvuII and HindIII, respectively. The recognition sites of each of these four enzymes were identified by restriction digests of different plasmid DNAs of known sequence and determining the cleavage sites by sequencing. SpaI is the most predominant restriction enzyme present in S. platensis strain pacifica. It shows high activity at 37 °C compared to 65 °C for its isoschizomer Tth111I.Department of Plant Molecular Physiology     

Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.     

Similarities and differences in the properties of multiple isoforms of maize endosperm casein kinase I (CK-I)

Two novel type I casein kinases named CK-1B and CK-1C have been purified from maize endosperm (three weeks after anthesis) by a six step procedure involving ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-75, Heparin-sepharose, and ATP-agarose chromatography. The catalytic subunits of both enzymes were identified as a 35-37 kDa polypeptide doublet by in situ phosphorylation after SDS/PAGE in active casein gel. Both enzymes required 5-10 mmol · L⁻¹ Mg²⁺ for maximal activity, could utilize only ATP as phosphate donor, were insensitive to heparin, were not autophosphorylated, had a pH optimum at pH 7 to 8.5, and exclusively phosphorylated acidic proteins (casein, phosvitin). Regarding the enzyme differences, their properties were as follows: a) CK-1B could bind on ATP-agarose affinity column, while CK-1C could not; b) the activity of CK-1C was strongly stimulated at low concentrations (1 mmol/L) of spermine, while that of CK-1B was inhibited; c) CK-1B and CK-1C Km values for ATP were 11 μmol · L⁻¹ and 26 μmol · L⁻¹, respectively; d) Mg²⁺ could substituted by Mn²⁺ in the CK-1B catalytic activity (by about 80 percnt;); e) CK-1B phosphorylated serine, while CK-1C both serine and threonine on casein. The combination of these results with those from Babatsikos and Yupsanis (2000) brings the number of investigated maize endosperm CK-I isoforms to three (CK-1B, CK-1C, and CK-1E). This is the first biochemical approach demonstrating that multiple isoforms of CK-I casein kinases are present in the same plant tissue.