铜离子胁迫对两种地衣细胞结构的影响

采用光学显微镜徒手切片技术和透射电子显微镜超薄切片制备技术,以两种叶状地衣即中国树花(Ramalina sinensis)和地卷(Peltigera rufescens)为实验材料,研究了不同浓度CuSO4(0、1、2、3、4mmol/L)处理24h后地衣体细胞的存活率以及Cu2+胁迫对细胞超微结构的影响。结果显示:(1)光学显微镜下可初步确定,Cu2+浓度越大中国树花共生藻细胞存活率越小,而同样处理条件下地卷共生藻细胞的存活率则基本保持不变。(2)低浓度Cu2+(1mmol/L)对中国树花细胞结构基本无影响,细胞壁、细胞膜及细胞内的线粒体、叶绿体完整;随着Cu2+浓度增加,当处理Cu2+浓度为2mmol/L时,细胞壁无损,但细胞膜开始破坏形成小空泡,线粒体嵴变凌乱,叶绿体也出现皱缩,类囊体膨胀,基粒排列紊乱;当Cu2+处理浓度大于2mmol/L时,共生藻的细胞膜和细胞器出现不同程度的损伤;当Cu2+浓度为3mmol/L时,细胞壁变薄,细胞膜形成的空泡变大,细胞内部结构变松散,蛋白核消失,叶绿体与细胞质混在一起,基粒片层扭曲,分布混乱,线粒体变形;当Cu2+浓度达4mmol/L时,细胞结构完全受到破坏。(3)不同浓度Cu2+处理对地卷共生藻细胞结构无明显的影响,在所有处理条件下地卷共生藻细胞壁、细胞膜都完整,且大多数共生藻细胞处于分裂状态。研究认为,中国树花对Cu2+胁迫较敏感,Cu2+耐受在1~2mmol/L之间,Cu2+浓度与中国树花细胞结构的损伤程度存在着明显的剂量效应关系,Cu2+浓度越高,其属于共球藻的真核共生藻细胞受损程度越大;地卷对Cu2+胁迫具有一定的耐性,Cu2+胁迫下地卷属于蓝藻的原核共生藻细胞仍能繁殖分裂产生子代细胞。     

两种地衣共生藻对铜和锌的耐受性及吸附特性研究

重金属污染生物修复中,藻类对重金属的吸附潜力引起诸多研究者的关注。为了探讨从地衣体分离培养的地衣共生藻对重金属Cu~(2+)、Zn~(2+)吸附特性及其耐受性的差异,以2种地衣共生藻为研究材料,采用Evan’s blue染色法、BCO和双硫腙分光光度法测定地衣共生藻细胞活率,培养液及藻体内的Cu~(2+)、Zn~(2+)含量。结果显示:不同浓度Cu~(2+)、Zn~(2+)胁迫下,2种地衣共生藻的细胞活力及重金属吸附特性有所差异,对Cu~(2+)胁迫的耐受性及吸附性为:P.EB;Zn~(2+)胁迫下,培养至9d时耐受为:P.E B,随着培养时间的延长P.E的细胞活力急剧下降并低于B,但吸附率还是处于P.E B;并且2种地衣共生藻对Zn~(2+)胁迫的耐性及吸附性明显高于Cu~(2+)。研究发现,来自菌藻共生的特殊生物-地卷属地衣的两种藻类比一些自由生长的藻类对铜和锌胁迫具有较高的耐受性及吸附性。     

地卷属5株共生藻的分离鉴定及抗逆性分析

[目的]了解地卷属的5种地衣共生藻分离、鉴定,并对其响应干旱、重金属、盐和温度等非生物胁迫因子的抗逆特性进行研究。[方法]采用涂布法进行分离培养及其结合形态与分子学方法初步鉴定分类地位;并采用PEG模拟法和重金属检测法研究对共生藻耐受性。[结果](1)在绿皮地卷(Peltigera apthosa)、犬地卷(Peltigera canina)、平盘软地卷(Peltigera elisabethae)、地卷(Peltigera rufescens)和膜地卷(Peltigera membranacea)等5种地卷中分离培养了5株藻(编号为1、2-1、4-2、6和7号),均属于杆裂丝藻属(Stichococcus)。(2)PEG模拟法对共生藻耐干旱特性的研究结果表明,1号、2-1号、6号和7号共生藻的干旱耐受性可达到30%,其中1号共生藻、2-1号和6号共生藻的PEG耐受性达40%。共生藻对不同重金属离子的耐受性存在显著差异:对1.0 mmol/L浓度Cu~(2+)、Cd~(2+)和Fe~(2+)均有一定的耐受性藻株为1号、2-1号、6号和4-2号共生藻;对1.0 mmol/L Zn~(2+)具有耐受性的藻株为6号、7号和4-2号共生藻。其中6号藻株对Cu~(2+)和Fe~(2+)的耐受性可高达1.5 mmol/L,7号藻株也对Cu~(2+)耐受性达到1.5 mmol/L',6号藻株对不同重金属的耐受性显著高于其它藻株。5株共生藻中,4-2号和7号藻对0.3 mol/L氯化钠具有一定的耐受性,1号和6号共生藻对Na Cl的耐受性最为强,可达到0.4 mol/L。其中(6号、7号、2-1号)在35℃条件下也能生长,所有藻株均显示敏感性。[结论]对干旱、重金属、盐和温度等非生物胁迫抗逆特性最高的藻株为地卷中分离的6号藻株。研究结果证实了地卷属地衣中包含了具有较强的生存力和耐干旱、重金属和盐胁迫适应能力的藻类品种。     

三种地卷响应Cu~(2+)胁迫的差异分析

基于谷胱甘肽(Glutathione,GSH)含量变化和铜吸附性分析地卷、平盘软地卷和犬地卷响应Cu~(2+)胁迫的差异,以了解谷胱甘肽在低等生物地衣抗重金属胁迫中的作用。分光光度法和火焰原子吸收方法分别测定地衣体的GSH与细胞内、外的铜含量。结果显示,低浓度Cu~(2+)胁迫下,3种地卷GSH均呈上升趋势,4 mmol/L时达最高;5-8 mmol/L时,地卷和犬地卷的GSH逐步下降较明显,但仍显著高于对照组,而平盘软地卷GSH在3-5 mmol/L Cu~(2+)范围差异不显著,Cu~(2+)5 mmol/L时才出现较明显的下降(P0.05)。4 mmol/L Cu~(2+)处理时间不同时,犬地卷的GSH变化与处理时间正相关,而地卷和平盘软地卷的GSH出现波动式变化(6 h增加,12 h下降,18-24 h上升)。3种地卷胞外铜与Cu~(2+)浓度和处理时间呈正相关并显著高于胞内铜(P0.01),而胞内富集的铜与地衣GSH变化相对应呈高-低-高的变化趋势。菌藻共生的特殊生物-地卷类似于高等植物可诱导合成GSH缓解胁迫产生的伤害,3种地卷的铜耐受性差异与其铜吸附性和GSH合成能力密切相关。     

抗重金属土壤微藻F1对Cu~(2+)胁迫的耐受生理机制研究

以从新疆富蕴县金属矿区土壤筛选出的抗重金属微藻F1为材料,测定在不同浓度(0、0.5、1.0、1.5和2.0mmol/L)Cu~(2+)胁迫下F1微藻叶绿素a、可溶性蛋白、MDA和谷胱甘肽(GSH)含量,以及谷胱甘肽硫转移酶(GST)、谷胱甘肽过氧化酶(GSH-PX)和谷氨酰半胱氨酸合成酶(GCL)的活性,同时用红外光谱仪检测细胞壁上参与重金属螯合的官能团,用扫描电镜分析细胞壁上的离子交换情况,探讨F1土壤微藻对Cu~(2+)胁迫的耐受生理机制,为利用微藻生态修复技术去除污染区土壤重金属奠定基础。结果显示:(1)F1土壤微藻对Cu~(2+)具有较强的耐抗性。(2)F1土壤微藻细胞中参与吸附Cu~(2+)的基团分别为-OH、-CH2、-RCONH2和C-OH等。(3)F1土壤微藻在吸附Cu~(2+)的过程中,Cu~(2+)与细胞壁上的Al~(3+)、Fe~(2+)、Mg~(2+)、Ca~(2+)、K~+、Na~+和Zn~(2+)发生了交换。(4)藻细胞中的GSH和GSH-PX在F1土壤微藻耐受Cu~(2+)胁迫时起主要作用。研究认为,F1微藻种对Cu~(2+)的耐受性首先与其细胞壁外表细微结构及其离子交换特性有关,并且细胞壁上-OH、-CH_2、-RCONH_2和C-OH等化学基团起着重要作用,其次细胞内的谷胱甘肽以及谷胱甘肽相关酶类能够有效清除活性氧的过量积累,最终保护细胞免受重金属胁迫损伤。     

Cu~(2+)胁迫对罗氏沼虾血细胞毒性及caspase-3、α-2M基因表达的影响

本研究以Cu~(2+)(0、100μg·L~(-1))对罗氏沼虾Macrobrachium rosenbergii进行处理,分别在胁迫后的0 h、3 h、6 h、12 h、24 h和48 h收集血淋巴,检测血细胞中的细胞凋亡率、活性氧(ROS)含量、酯酶活性、一氧化氮(NO)含量以及细胞凋亡执行分子caspase-3和免疫因子α-2M基因的表达。结果显示,血细胞凋亡率和ROS含量的变化趋势一致,在胁迫后的6~48 h显著升高(P0.05)。酯酶活性在胁迫后的3~48 h均显著下降(P0.05),最低值出现在48 h。在胁迫后的6~48 h,NO含量持续增加,并与对照组的差异有统计学意义(P0.05)。caspase-3的相对基因表达量在胁迫后的6~48 h均显著升高(P0.05),并在24 h达到最高值。在胁迫后的12 h,α-2M基因的表达量显著升高(P0.05)并达到峰值,在24 h有短暂降低,但与对照组相比仍显著增加(P0.05),在48 hα-2M基因的表达量显著降低(P0.05)。相关性分析显示,血细胞凋亡率和NO以及ROS都呈显著的正相关(P0.001)。实验表明,Cu~(2+)胁迫显著抑制了机体的酯酶活性,诱导NO和ROS的产生以及免疫相关基因α-2M的表达来对抗Cu~(2+)胁迫带来的损伤。随着胁迫时间的延长,过高浓度的NO和ROS对机体自身造成了伤害,并诱导凋亡相关基因caspase-3的表达,最终导致细胞凋亡,说明Cu~(2+)对虾类具有免疫抑制性和毒性。NO和ROS的升高可能是细胞凋亡的主要诱因。     

PUMA对高糖诱导的大鼠H9C2心肌细胞凋亡的作用及机制

目的:观察促凋亡蛋白p53上调凋亡调控因子(PUMA)在高糖所致H9C2心肌细胞凋亡中的作用及机制。方法:H9C2心肌细胞随机分为对照组(使用5.5mmol/L葡萄糖作用于细胞)和高糖组(使用35 mmol/L葡萄糖作用于细胞,HG组)分别刺激6 h, 12 h, 24 h和48 h,每组设复孔5个,TUNEL染色检测细胞凋亡率;RT-PCR及Western blot法分别测定PUMA mRNA及蛋白表达情况;JC-1法检测线粒体膜电位;Western blot测定caspase-3表达和细胞色素c(Cyt C)释放。H9C2细胞随机分为四组,对照组、高糖(35 mmol/L)、HG+si-scramble组(使用si-scramble转染心肌细胞24 h,使用35mmol/L葡萄糖作用于细胞)和Si-PUMA组(使用si-PUMA转染心肌细胞24 h,使用35mmol/L葡萄糖作用于细胞),观察抑制PUMA表达对高糖诱导细胞凋亡率、线粒体膜电位、Cyt C的影响。结果:与对照组相比,高糖刺激心肌细胞组TUNEL染色阳性率、活化caspase-3和PUMA表达明显升高(P<0.0...     

重金属铜、锌、镉复合胁迫对麻疯树幼苗生理生化的影响

该研究以Cu~(2+)、Zn~(2+)、Cd~(2+)单一胁迫为对照,探讨不同浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫对麻疯树幼苗生理生化指标的影响。结果表明:随着Cu~(2+)、Zn~(2+)、Cd~(2+)浓度的增加,麻疯树幼苗叶片中的蛋白质(Pro)、丙二醛(MDA)含量均逐渐增加,其叶片叶绿素含量随着Zn~(2+)胁迫浓度的增加呈现出先降后升的趋势,在中等浓度(100 mg·L-1)的Zn~(2+)胁迫时含量最低、随着Cu~(2+)胁迫浓度的增加叶绿素含量先升高后降低,在Cu~(2+)浓度为200 mg·L-1时含量最高,达到1 200 mg·g-1FW; Cd~(2+)胁迫对叶绿素含量和根系活力无明显影响。根系活力在Zn~(2+)浓度为100 mg·L~(-1)时最强,随着Cu~(2+)浓度的增加而减弱。低浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)对过氧化物酶活性和可溶性糖含量都具有促进作用。Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫时对可溶性蛋白、叶绿素和丙二醛含量均无明显影响,随着复合胁迫时浓度的增加,可溶性糖含量和根系活力先增后减。这表明麻疯树对三种重金属的胁迫具有一定的抗性,过高浓度的胁迫会影响麻疯树幼苗生理生化的一些指标,但是麻疯树可以通过自身的防御系统使伤害降到最小。此外,重金属复合胁迫可以在一定程度上减轻单一胁迫对麻疯树幼苗造成的毒害作用。     

一株虾池来源的螺旋拟柱孢藻藻株的分离鉴定及重金属离子Cu~(2+)、Cd~(2+)和Pb~(2+)对其生长的影响

【目的】探讨凡纳滨对虾养殖水体中入侵蓝藻拟柱孢藻的生长生理特性。【方法】从汕头澄海人工对虾养殖池分离纯化藻株,通过形态及其16SrRNA基因鉴定,之后在CT与BG11两种蓝藻通用培养基的基础上优化最佳培养条件,最后分析了不同浓度的3种重金属离子即Cu~(2+)(0–0.8 mg/L)、Cd~(2+)(0–4 mg/L)和Pb~(2+)(0–80 mg/L)对藻株生长的影响。【结果】澄海虾池来源的分离纯化藻株形态呈卷曲螺旋型,16S rRNA基因序列与多株其他来源的拟柱孢藻相似度均达98%以上。实验室培养,藻株最佳生长状态的培养条件是在BG11培养基的基础上调整氮浓度及氮磷比分别为N 62 mg/L,N︰P=9︰1,在此条件下,藻丝生物量可达(0.632±0.170)×107/L,藻丝比平均生长速率最高为(0.063±0.001)/d。本分离藻株活体对重金属Cu~(2+)、Cd~(2+)和Pb~(2+)具有一定的耐受性,其耐受浓度范围分别为0–0.2、0–0.5和1–40 mg/L,其中,Cu~(2+)和Cd~(2+)对藻的生长具有抑制作用,而且此抑制作用随着金属离子剂量的增加及作用时间的延长更加显著,Cu~(2+)和Cd~(2+)对藻体的半数抑制浓度(96 h EC50)分别为0.125和0.551 mg/L;而浓度范围为0–80 mg/L的Pb~(2+)对藻体的生长则表现为低剂量(≤40 mg/L)呈促进,高剂量(≥80 mg/L)则抑制。【结论】从凡纳滨对虾养殖池中分离鉴定出一株形态呈螺旋型的拟柱孢藻,命名为螺旋拟柱孢藻(Cylindrospermopsis raciborskii helix),本藻株活体能够在一定浓度的Cu~(2+)、Cd~(2+)和Pb~(2+)中生长,为螺旋拟柱孢藻活藻生物吸附重金属离子而改善虾池水体环境提供了可能性。     

钙与渗透胁迫下大豆细胞透性的关系

缺钙(加EGTA 1mmol/L)处理的大豆下胚轴和幼根细胞透性增大,K~+外漏;钙浓度提高时,膜透性降低。在渗透胁迫下,细胞透性是缺钙处理大干低钙(1mmol/L)处理大于高钙(5mmol/L)处理,且高钙下细胞透性明显改变的时间延迟。Mg~(2+)不能代替Ca~(2+);La~(3+)对钙的作用有拮抗性。     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Annular and non-annular binding sites on the (Ca + Mg)-ATPase

Quenching of the fluorescence of the (Ca²⁺ + Mg²⁺)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

The phospholipid requirement of the (Ca + Mg)-ATPase from human platelets

The phospholipid requirement of the (Ca²⁺ + Mg²⁺)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.     

Synthesis of N-monomethylagmatine, a decarboxylation product of N-monomethyl- -arginine

N^G-Monomethylagmatine, a decarboxylation product of N^G-monomethyl- -arginine, has been synthesized by reacting putrescine with N,S-dimethylthiopseudouronium iodide. The structural identity of the product was confirmed by proton NMR and mass spectroscopy, and its properties were determined on thin-layer and electrophoretic chromatography.     

Molecular analysis of two genes between let-653 and let-56 in the unc 22(IV) region of Caenorhabditis elegans

Summary A previous study of genomic organization described the identification of nine potential coding regions in 150 kb of genomic DNA from the unc-22(IV) region of Caenorhabditis elegans. In this study, we focus on the genomic organization of a small interval of 0.1 map unit bordered on the right by unc-22 and on the left by the left-hand breakpoints of the deficiencies sDf9, sDf19 and sDf65. This small interval at present contains a single mutagenically defined locus, the essential gene let-56. The cosmid C11F2 has previously been used to rescue let-56. Therefore, at least some of C11F2 must reside in the interval. In this paper, we report the characterization of two coding elements that reside on C11F2. Analysis of nucleotide sequence data obtained from cDNAs and cosmid subclones revealed that one of the coding elements closely resembles aromatic amino acid decarboxylases from several species. The other of these coding elements was found to closely resemble a human growth factor activatable Na⁺/H⁺ antiporter. Pairs of oligonucleotide primers, predicted from both coding elements, have been used in PCR experiments to position these coding elements between the left breakpoint of sDf19 and the left breakpoint of sDf65, between the essential genes let-653 and let-56.     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

Direct evidence for an ADP-sensitive phosphointermediate of (K + H-ATPase

Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K⁺ + H⁺)-ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 μM [γ-³²P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7–15 μM Mg²⁺. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non-radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of ³²P-labeled enzyme. The relative proportion of the ADP-sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined.