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1.
活性氧参与生物体内复杂的代谢过程。本文对它在家蚕个体发育过程中的生物学功能进行了初步探讨。 取1岁龄家蚕蛹分别注射H2O2、脱皮激素(Ecdn)、保幼激素(JH)和还原型谷胱苷肽(GSH),培养;定期取样测定家蚕体内H2O2含量。 对家蚕整个幼虫期(Fig.1),尤其是大眠期(Fig.2)、化蛹期(Table1)、蛹期(Fig.3-a,b,c)以及成虫期(Fig.4, Table 2)的研究结果表明,家蚕体内H2O2代谢具有如下特点:(1)入眠、化蛹、化蛾和死亡前H2O2含量都显著下降;(2)幼虫期1-3龄H2O2含量逐步下降,4~5龄H2O2含量回升,蛹期和成虫期H2O2含量与4~5龄接近;(3)每个龄期的中期H2O2含量最高;(4)CAT活性与H2O2含量呈负相关变化,前者迟于后者;另外,CAT活性远远大于SOD活性;(5)Ecdn、GSH处理可以降低家蚕蛹期H2O2含量,并使其提前下降,JH、H2O2处理含量下降,并相应地提早推迟化蛾;(6)成虫期雄蛾H2O2含量、SOD和CAT活性都显著高于同时期雌蛾。家蚕体内H2O2含量的变化与其发育密切相关。H2O2含量下降是变态的信号;家蚕成虫期H2O2 在性别上的  相似文献   

2.
The ionophoretic activity of PGBx, an oligomeric mixture synthesized from 15-dehydro PGB1, with different cations was measured using arsenazo III-entrapped liposomes. The order of ionophoretic activity was Zn2+ greater than Co2+ greater than Mn2+ greater than Cu2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. The intrinsic fluorescence of PGBx was quenched by the binding of divalent cations as well as by La3+ and H+. Quenching by K+ and Na+ was minimal. The order of quenching strength of divalent cations was Zn2+ greater than Co2+ greater than Cu2+ = Mn2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. Binding affinities of these cations determined by a murexide indicator method were in good agreement with that determined by the fluorescence quenching reaction. The cation binding affinity of PGBx in aqueous solutions correlates with the ionophoretic activity in liposomes. The binding affinity for K+ was estimated from the inhibition by K+ of Ca2+ binding by PGBx. Although PGBx has a lower selectivity for divalent cation binding than the ionophore A23187, the characteristics of the binding affinity of these two compounds for various ions were similar. The pK of PGBx as determined by fluorescence quenching was 6.7. The molecular weight of the divalent cation binding unit was estimated to be about 680, with each PGBx molecule having three such binding sites. The binding of Ca2+ to such a site is one-to-one.  相似文献   

3.
The nucleotide sequence of bkdB, the structural gene for E2b, the transacylase component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida has been determined and translated into its amino acid sequence. The start of bkdB was identified from the N-terminal sequence of E2b isolated from branched-chain-oxoacid dehydrogenase of the closely related species, P. aeruginosa. The reading frame was composed of 65.5% G + C with 82.3% of the codons ending in G or C. There was no intergenic space between bkdA2 and bkdB. No codons requiring minor tRNAs were utilized and the codon bias index indicated a preferential codon usage. The bkdB gene encoded 423 amino acids although the N-terminal methionine was absent from E2b prepared from P. aeruginosa. The relative molecular mass of the encoded protein was 45,134 (45,003 minus methionine) vs 47,000 obtained by SDS/polyacrylamide gel electrophoresis. There was a single lipoyl domain in E2b compared to three lipoyl domains in E2p, and one domain in E2o, the transacylases of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli respectively. There was significant similarity between the lipoyl domain of E2b and of E2p and E2o as well as between the E1-E2 binding domains of E2b, E2p and E2o. There was no similarity between the E3 binding domain of E2b to E2p and E2o which may reflect the uniqueness of the E3 component of branched-chain-oxoacid dehydrogenase of P. putida. The conclusions drawn from these comparisons are that the transacylases of prokaryotic pyruvate, 2-oxoglutarate and branched-chain-oxoacid dehydrogenases descended from a common ancestral protein probably at about the same time.  相似文献   

4.
重金属对油菜种子萌发和胚根生长的影响   总被引:5,自引:0,他引:5  
分析了Hg2 、Cd2 、Ni2 、Co2 、Zn2 5种重金属离子对油菜种子萌发和胚根伸长的影响,以及金属离子K 、Mg2 和Ca2 与重金属的交互作用。结果表明:(1)重金属对油菜种子萌发的抑制作用依次为Hg2 >Cd2 和Co2 >Ni2 >Zn2 ,而对胚根生长的毒害作用依次为Hg2 >Cd2 >Co2 >Ni2 >Zn2 。(2)萌发率为40%以上时,K 和Ca2 可以提高Ni2 、Zn2 和Co2 胁迫下油菜种子的萌发率,却进一步降低了Hg2 、Cd2 胁迫下种子的萌发;Mg2 可以提高Ni2 、Zn2 、Cd2 和Co2 胁迫下种子的萌发率,但对Hg2 毒害却没有缓解。(3)胚根伸长率达到60%以上时,K 和Mg2 增强了Ni2 、Hg2 、Cd2 和Co2 对胚根生长的抑制,而Ca2 则缓解了Zn2 、Ni2 和Co2 对胚根生长的抑制作用。研究结果对于重金属复合污染土壤中植物种子的萌发和定植具有理论和实践意义。  相似文献   

5.
目的:克隆小鼠紧密连接蛋白ZO-2(zonula occludens protein2)基因构建其真核表达载体,并验证其在293T细胞系中的表达,为进一步研究其功能奠定基础。方法:从小鼠淋巴结来源的cDNA中分三段分别扩增,通过基因克隆方法获得紧密连接蛋白ZO-2基因全长,连接至pMD-18T载体中,酶切测序正确后,插入pEGFP-C2载体中,构建真核表达载体pEGFP-C2-ZO2。酶切正确后,瞬时转染入293T细胞中,48h后荧光显微镜观察其绿色荧光GFP融合蛋白的表达,并用Western blot检测其在转染细胞中的蛋白水平表达。结果:通过酶切鉴定和测序结果证明成功克隆了ZO-2基因,western blot结果表明成功构建了真核表达载体pEGFP-C2-ZO2。结论:小鼠紧密连接蛋白ZO-2基因的获得和真核表达载体pEGFP-C2-ZO2的成功构建为下一步研究其生物学功能奠定了基础。  相似文献   

6.
The ultrasonic disinfection of Escherichia coli was carried out in the presence of anatase-type TiO2 particles, and the effectiveness of the combination of ultrasonic irradiation with TiO2 addition was verified. The rate constant was determined from the plot of the common logarithm of the survival cell ratio versus the ultrasonic irradiation time using first-order kinetics. In the absence of particles, the rate constant was proportional to the ultrasonic power. When ultrasonic disinfection was carried out in the presence of TiO2 particles, which have a radical generation ability, the rate of disinfection was remarkably faster than that in the absence of TiO2. In the presence of silica particles, which have no radical generation ability, the rate of disinfection was the same as that in the absence of TiO2. These results suggest that the radical generation ability of TiO2 appeared as a result of the ultrasonic irradiation. The effect of the amount of TiO2 on the rate of disinfection was also examined. The rate constant for disinfection in the presence of TiO2 was saturated to a certain value and was represented using the Langmuir-type equation. The proposed model well describes the effects of the ultrasonic power and the amount of TiO2 on the rate constant for disinfection.  相似文献   

7.
Calcium ionophores are generally assumed to directly facilitate the transport of Ca2+ across the plasma membrane. The ability of Ca2+ ionophores ionomycin and A23187 to increase Ca2+ concentration in the cytosol ([Ca2+]i) in different cells was analyzed in detail using fluorescent Ca2+ probes. In fura-2-loaded cells, the dependence of the level of [Ca2+]i on ionomycin and A23187 concentrations had a complex character and could not be explained by ionophoric properties only. The Ca2+ signal induced by the Ca2+ ionophores consisted of three components. The first component was due to the activation of Ca2+ influx through native Ca2+ channels and was sensitive to drugs which inhibited the receptor-operated Ca2+ influx. The second component originated from phospholipase C-dependent mobilization of Ca2+ from intracellular stores. An additional influx of Ca2+ into the cells was activated in this case by a store-regulated mechanism. The third ionophoric component was very small at low concentrations of the ionophores. The effect of the ionophores on Ca2+ influx and Ca2+ mobilization was demonstrated on different cells such as Ehrlich ascites tumour cells, murine peritoneal neutrophils, macrophages, and T-lymphocytes. Thymocytes, neutrophils, and Ehrlich ascites tumour cells were more sensitive to the Ca2+ ionophores. Memory T-cells and brown preadipocytes were ionophore-resistant. The insensitivity to Ca2+ ionophores correlated with the absence of Ca2+ in the intracellular Ca2+ stores and the low activity of plasma membrane store-regulated Ca2+ channels.  相似文献   

8.
A new phosphorescence imaging method (Rumsey et al. Science Wash. DC 241: 1649-1651, 1988) has been used to continuously monitor the PO2 in the blood of the cerebral cortex of newborn pigs. A window was prepared in the skull and the brain superfused with artificial cerebrospinal fluid. The phosphorescent probe for PO2, Pd-meso-tetra(4-carboxyphenyl)porphine, was injected directly into the systemic blood. The phosphorescence of the probe was imaged, and the lifetimes were measured using flash illumination and a gated video camera. The PO2 in the blood of the veins and capillary beds of the cortex was calculated from the lifetimes. Systemic blood pressure was continuously monitored while the systemic arterial PCO2, PO2, and blood pH were measured periodically. The PO2 in the blood was quantitated for 60- to 200 microns2 regions within the image (from a total field of approximately 3 mm diam). The PO2 in the microvasculature was not uniform across the viewing field but increased or decreased in each region independently of the other regions. Thus at any point in time the PO2 in a region could be substantially above or below the average value. During hyperventilation, which lowered arterial PCO2 and increased pH of the blood, the average PO2 decreased in proportion to the decrease in arterial PCO2. For example, hyperventilation, which decreased arterial PCO2 from its normal value of 40 Torr to 10 Torr, caused a rapid (within 5 min) decrease in PO2 in the blood of capillaries and veins to approximately one-third of normal.  相似文献   

9.
The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-gamma synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-gamma, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-gamma was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with anti-Tac remained low throughout the first week of culture and interferon-gamma synthesis was not observed. Nonetheless, interleukin 2-induced proliferation was inhibited by anti-Tac on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-gamma synthesis, and for the growth of thymocytes in vitro.  相似文献   

10.
【目的】构建传染性法氏囊病毒VP2蛋白展示禽流感M2e抗原表位的重组蛋白,研发预防H5或H9亚型禽流感和传染性法氏囊的基因工程疫苗。【方法】根据现有禽流感疫苗株M2e的氨基端12个氨基酸多肽序列(nM2e)序列,结合GenBank中H5和H9亚型禽流感病毒nM2e的比对结果,确定nM2e序列。用融合PCR分别将1拷贝H5或H9的nM2e序列插入IBD B87株VP2基因的PBC区,获得VP2BCnM2e重组基因。将重组基因克隆至杆状病毒表达系统,转染Sf9细胞进行表达。经间接免疫荧光和Western blotting检测Sf9细胞表达重组基因后,扩繁重组病毒,制备疫苗,间隔4周对非免鸡作2次重复免疫,用间接ELISA和鸡胚成纤维细胞中的病毒血清中和试验检测血清中VP2和nM2e的抗体效价。【结果】成功构建含H5或H9 nM2e的VP2BCnM2e重组基因,该重组基因在Sf9细胞中得到表达。经免疫鸡,两重组蛋白均能激发针对VP2和nM2e的抗体,VP2BCnM2eH5组抗体效价高于VP2BCnM2eH9组。【结论】两重组蛋白均具有免疫原性,VP2BCnM2eH5免疫原性更佳。  相似文献   

11.
The biosynthetic mechanism of prostaglandin D2 in human platelet-rich plasma has been investigated. Platelet-rich plasma was separated into washed platelets and platelet-poor plasma, and [1-14C]prostaglandin H2 was incubated with each fraction. The enzymatic conversion of the endoperoxide to prostaglandin D2 was found only in platelet-poor plasma and not in washed platelets or platelet lysate. This prostaglandin D synthetase activity was purified to homogeneity and identified as serum albumin by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and immunoelectrophoresis. The optimal pH and Km value for prostaglandin H2 were 9.0 and 6 microM, respectively. Glutathione was not required for the activity. Although prostaglandin H2 ws converted to prostaglandin D2 and E2 in the reaction, only the prostaglandin D2 formation was dependent on the protein amount and abolished by prior boiling. The action of this activity under physiological conditions was examined in a model system constituted of serum albumin and washed platelets. Prostaglandin D2 formation was observed in association with thrombin-evoked platelet aggregation in this system and was proportional to the number of platelets and the concentration of serum albumin, suggesting that thrombin-stimulated platelets released prostaglandin H2, and the latter compound was then converted to prostaglandin D2 by the action of serum albumin. Consistent with this interpretation, prostaglandin H2 added to platelet-rich plasma was converted in part to prostaglandin D2, and the aggregation caused by this endoperoxide was greatly enhanced by neutralizing the action of prostaglandin D2 with anti-prostaglandin D2 antiserum.  相似文献   

12.
CO2对牟氏角毛藻高密度培养的影响   总被引:5,自引:0,他引:5  
以牟氏角毛藻为材料,通过对比实验研究了CO2和CO2通入方式对光合生物反应器中高密度培养的牟氏角毛藻的生长速度和细胞密度的影响;并以水体pH值为指标,控制CO2的通入数量,进而研究了牟氏角毛藻在我们自行研制的卧式光合生物反应器中的生长情况.结果表明细胞悬浮培养达到一定密度之后,CO2的供应,可以有效地提高生长速度和细胞密度.对CO2通入方式的实验结果表明,不同的CO2通入方式,产生的效果不同,其中以空气和纯CO2混合后通入,效果最好.  相似文献   

13.
An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.  相似文献   

14.
本研究通过PCR扩增出猪圆环病毒2型(PCV—2)的全基因组(1768bp),克隆入pcDNA3载体的EcoR I酶切应点,获得含有PCV-2全基因组的重组质粒,命名为pcDNApcv2。将重组质粒大量扩增后,用EcoR I切出1768bp的PCV—2全基因组,在体外用T4DNA连接酶使其连接环化。用脂质体法将体外连接产物转染无PCV污染的PK—15细胞,经4次连续传代,用间接免疫荧光实验(IFA)及电镜观察证实已获得复制能力的PCV—2病毒。由此可见,本试验构建的环化的PCV—2全基因组DNA具有感染性。  相似文献   

15.
The ribonucleotide reductase (RR) 2 gene of the HSV-2 strain G was cloned, sequenced, and expressed in an E. coli cell. The RR2 gene was located on the PstI 2.4 kb fragment, which was cloned and sequenced. The ORF of the gene was 1,011 bp and its termination codon was TAG; also, the CATATAA sequence was present in the promoter of the RR2 gene. A Poly A signal sequence (AATAAA) was found in the 3'-noncoding region. The RR2 proteins that were produced in the E. coli and Vero cells were confirmed using a Western blot analysis. SDS-PAGE revealed that the molecular weights of the fusion-RR2 that was produced in the E. coli cells were approximately 24 kDa and 38 kDa in the Vero cells. The RR2 proteins were soluble. The differences in the molecular weights might be due to modifications in the Vero cells.  相似文献   

16.
1. The distribution of the hydrolyses of phosphatidylcholine by phospholipase A2 and phospholipase A1, and the hydrolysis of lysophosphatidylcholine by lysophospholipase, in subcellular and subsynaptosomal fractions of cerebral cortices of guinea-pig brain, was determined. 2. Noradrenaline stimulated hydrolysis by phospholipase A2 in whole synaptosomes, synaptic membranes and fractions containing synaptic vesicles. 3. Stimulation of hydrolysis by phospholipase A2 in synaptic membranes by noradrenaline was enhanced by CaCl2, and by a mixture of ATP and MgCl2. The optimum concentration of CaCl2, in the presence of ATP and MgCl2, for stimulation by 10 muM-noradrenaline was in the range 1-10muM. The optimum concentration for ATP-2MgCl2 in the presence of 1 muM-CaCl2 was in the range 0.1-1mM. 4. Hydrolysis by phospholipase A2 of synaptic membranes was also stimulated by acetylcholine, carbamoylcholine, 5-hydroxytryptamine, dopamine (3,4-dihydroxyphenethylamine), histamine, psi-aminobutyric acid, glutamic acid and aspartic acid. With appropriate concentrations of cofactors, sigmoidal dose-response curves were obtained, half-maximum stimulations being obtained with concentrations of stimulant in the range 0.1-1muM. 5. Taurine also stimulated hydrolysis of phosphatidylcholine by phospholipase A2. There were only slight stimulations with methylamine, ethylenediamine or spermidine. No stimulation was obtained with glucagon.  相似文献   

17.
庚型肝炎病毒E2区cDNA在毕赤酵母中的表达及抗原性鉴定   总被引:2,自引:1,他引:1  
从含有庚型肝炎病毒(GBVC/HGV)包膜蛋白E2 cDNA(559bp)的质粒pGEX\|E2中,扩增得到能够编码日本血吸虫谷胱甘肽硫转移酶(GST)和GBVC/HGV包膜蛋白E2的融合基因片段。将此长度为1324bp的DNA片段插入到酵母表达载体pPIC9K中,使之位于α因子信号肽下游,且与之同框。通过电激转化将构建的重组表达质粒pPIC9K\|GST\|E2插入到Pichia pastoris GS115菌株染色体中。筛选His\++Mut\+s表型的转化子,震荡培养,用05%甲醇诱导表达5d后,在培养液中得到表达的GSTE2融合蛋白。经过表达条件的优化,GSTE2蛋白可占培养液中总蛋白的50%。通过谷胱甘肽亲和层析柱纯化,GSTE2融合蛋白的纯度可达95%左右。以庚型肝炎病人血清为探针,进行免疫印迹及ELISA实验,结果表明该融合蛋白具有能被庚型肝炎病人血清特异性识别的抗原性。  相似文献   

18.
应用PCR方法从含有1TrVirusORF2的质粒pET-His-TTV2中扩增出606bp的蛋白质编码区,并将其克隆到真核表达载体pEGFP.NI中以表达成GFP—VP2融合蛋白。构建出的重组质粒pEGFPTFV2经过酶切分析和PCR鉴定。用脂质体介导法将pEGFPTTV2质粒DNA转染Cos7细胞,通过RT-PCR分析,证实细胞中存在ORF2基因的转录产物。用共聚焦显微镜结合PI染色技术研究1TTV P2蛋白在细胞中的分布情况。结果表明,1TrVVP2分布在细胞质中和细胞核膜内侧。因此推测VP2作为一种非结构蛋白,功能可能是参与病毒DNA的复制或转录。  相似文献   

19.
The effect of pyocin R1 on the glucose metabolism of sensitive Pseudomonas cells was investigated. Upon treatment with pyocin R1, although the rate of O2 uptake of the sensitive cells for glucose or gluconate was not very much affected at first, the final level of O2 uptake was greatly reduced. When 2-oxogluconate was used as a substrate, O2 uptake was immediately halted by pyocin. By determining the amounts of glucose, gluconate, and 2-oxogluconate before and after the reaction and the amount of O2 consumed, it was concluded that glucose was exclusively metabolized via the following pathway with quantitative accumulation of 2-oxogluconate after pyocin treatment. (Formula: see text). The possible mechanism of this change is discussed.  相似文献   

20.
Anaerobic enrichment cultures with H2S and N2O as substrates which were inoculated with a biofilm sample showed rapid growth and gas formation after 2 to 3 days at 27 degrees C. By using the deep-agar dilution technique, a pure culture was obtained. The strain was tentatively identified as Thiobacillus denitrificans. The isolate was used for batch and gradient culture studies under denitrifying conditions, oxidizing H2S with concomitant reduction of N2O to N2. In batch culture, oxidation of H2S was stepwise, with transient accumulation of elemental sulfur; the final oxidation product was SO4(2-). In gradient culture, there was no notable accumulation of elemental sulfur and microsensor measurements of H2S and N2O showed that H2S was oxidized directly to SO4(2-). In the presence of C2H2, however, oxidation of H2S stopped at the level of elemental sulfur and no SO4(2-) was produced in either batch or gradient cultures. This is a hitherto unknown inhibitory effect of C2H2. The inhibition is suggested to occur at the level of sulfite reductase, which catalyzes the oxidation of elemental sulfur to SO3(2-) in T. denitrificans. However, reduction of N2O in this strain was, surprisingly, not affected by C2H2. The isolate is the first chemolithoautotrophic organism shown to reduce N2O in the presence of C2H2. Denitrification in natural ecosystems is often quantified as N2O accumulation after C2H2 addition. However, the presence of large numbers of similar organisms with C2H2-insensitive N2O reduction could lead to underestimation of in situ rates.  相似文献   

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