Identification and characterization of a GDSL lipase-like protein that catalyzes the ester-forming reaction for pyrethrin biosynthesis in Tanacetum cinerariifolium- a new target for plant protection

Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.     

除虫菊内生真菌的分离及其发酵产物抑菌活性筛选

从除虫菊(Pyrethrumcinerariifolium Trev.)的根、茎、叶和花中分离出128株内生真菌。生长速率法测定结果表明,有56株内生真菌发酵液10倍稀释液至少对供试的番茄灰霉病菌(Botrytis cinerea)、辣椒疫霉病菌(Phytophthora capsici)、苹果炭疽病菌(Colletotrichum gloeosporioides)、玉米大斑病菌(Exserohilumturcicum)、小麦纹枯病菌(Rhizoctonia cerealis)和小麦赤霉病菌(Fusarium graminearum)等6种病原菌中的一种抑菌活性达75%以上;活体组织法测定结果,Y1、Y2、H2共3株菌的发酵液对番茄灰霉病的防效达到50%以上;盆栽试验结果表明,Y1、Y2、Y7、H2、G13共5株菌株发酵液对辣椒疫霉病的防治效果达到50%以上。综合分析认为,Y1、Y2、H2、G13等内生真菌的抑菌作用值得进一步研究。     

阿霉素注射剂量对肾病综合征大鼠脂蛋白脂酶和卵磷脂胆固醇酰基转移酶水平的影响

摘要 目的：探讨阿霉素注射剂量对肾病综合征（nehpmtic syndrome,NS）大鼠脂蛋白脂酶和卵磷脂胆固醇酰基转移酶（Leci-thin cholesterol acyltransferase,LCAT）水平的影响。方法：64只SD大鼠随机平分为四组-对照组、小剂量阿霉素组、中剂量阿霉素组与高剂量阿霉素组,四组大鼠经尾静脉一次性注射阿霉素0 mg/kg、2 mg/kg、4 mg/kg、8 mg/kg,检测造模后不同时间点大鼠肾脏脂蛋白脂酶和卵磷脂胆固醇酰基转移酶水平变化情况。结果：小剂量阿霉素组、中剂量阿霉素组与高剂量阿霉素组造模后7 d、14 d、21 d的体重与每日采食量、血肌酐与尿素氮都低于对照组（P<0.05）,24 h尿蛋白高于对照组（P<0.05）,且存在剂量依赖性,三组间对比差异有统计学意义（P<0.05）。小剂量阿霉素组、中剂量阿霉素组与高剂量阿霉素组造模后21 d、28 d的肾脏脂蛋白脂酶和卵磷脂胆固醇酰基转移酶相对表达水平低于对照组（P<0.05）,且存在剂量依赖性,三组间对比差异有统计学意义（P<0.05）。结论：小剂量阿霉素可抑制大鼠肾脏脂蛋白脂酶和卵磷脂胆固醇酰基转移酶的表达,能快速有效建立肾病综合征大鼠模型,具有很好的模拟造模效果。     

Effects of Alcohol and Buffer Treatments on the Activity and Enantioselectivity of Candida rugosa Lipase

Abstract

Several treatments were employed on Candida rugosa lipase (CRL) to improve its biocatalytic performance. Besides conventional alcohol treatment conditions, the effects of pH of the buffer solution used in the treatment as well as the changes in stirring, dialysis, and centrifugation steps of the treatment procedure were investigated for the first time for the resolution of racemic naproxen methyl ester. The highest enantioselectivity and conversion in S-naproxen production were achieved by CRL treated with pH 7.5 buffer solution. The elimination of the centrifugation step resulted in an increase in the enantioselectivity, whereas alcohol treatment of CRL was found to be inconvenient for S-naproxen production. Higher activity for p-nitrophenyl acetate was achieved when 20% butanol and pH 4 buffer solution were used, and when dialysis and stirring times were shortened.     

棘腹蛙消化道脂肪酶的分布及pH和温度对脂肪酶活力的影响

采用聚乙烯醇橄榄油乳化液水解法,测定了棘腹蛙消化道不同部位的脂肪酶活力,研究了pH和温度对脂肪酶活力的影响。结果显示,棘腹蛙消化道不同部位脂肪酶活力依次为回肠>直肠>十二指肠>胃>食道。pH和温度显著影响脂肪酶的活力,二者对脂肪酶活力影响的关系曲线均呈现为单峰型,食道、胃和肠道脂肪酶活力的最适pH值分别为5.0、5.0和7.5,最适温度均为35℃。     

Restraint Stress-Induced Alterations in the Levels of Biogenic Amines,Amino Acids,and AChE Activity in the Hippocampus

Inspite of large number of studies on the neurochemical changes in the stress, an equivocal case is yet to be made for the role of a specific neurotransmitter in this important neurobiological disorder. The difficulty arises from the fact that there is no single neurotransmitter system appears to be responsible for the stress induced damage to the hippocampal neurons. The present study evaluates the effect of restraint stress on the alterations in the levels of biogenic amines, aminoacids and acetylcholinesterase activity in the hippocampus. Male Wistar rats of 45 days old were subjected to 6 hours of daily restraint stress over a period of 21 days. Immediately after the last session of stress, rats were sacrificed and neurotransmitter levels were estimated in the hippocampus. A significant (p < 0.001) decrease in the levels of noradrenaline, dopamine, 5-hydroxytryptamine and acetylcholinesterase activity in the stressed rats was observed compared to controls. However, levels of glutamate was significantly (p < 0.001) increased in stressed rats. These results indicate that chronic restraint stress decreases aminergic and cholinergic neurotransmission, and increases the glutamatergic transmission in the hippocampus.     

Activity and stability of lipase in Aerosol-OT/isooctane reverse micelles

The stability of Candida rugosa lipase, which catalyzes the hydrolysis reaction of olive oil in AOT/isooctane reverse micelles, decreased with the increase of ₀ (defined as the molar ratio of water to surfactant) and Aerosol-OT concentration. The addition of a non-ionic cosurfactant, tetraethylene glycol dodecyl ether (C₁₂E₄), preserved enzymatic activity. The residual activity of the lipase was 53% after 24 h, while the enzyme completely lost its activity within 6 h in the absence of C₁₂E₄ addition. The stabilizing effect of C₁₂E₄ resulted in the increase of conversion. The enhancement of the activity and stability of lipase in reverse micelles by the addition of C₁₂E₄ may contribute to increase the rigidity of the micellar matrix stabilizing the enzyme structure.     

Changes in Extracellular Amino Acids and Spontaneous Neuronal Activity During Ischemia and Extended Reflow in the CA1 of the Rat Hippocampus

This study addresses the possible involvement of an agonist-induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four-vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple-unit activity. Multiple-unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and gamma-aminobutyric acid increased in both acute and chronic experiments (seven- to 26-fold). Multiple-unit activity recovered to preischemic levels following 4-6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple-unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple-unit activity recovered faster and reached 140% of control at 8 h, paralleled by an increase in extracellular aspartate (5.5-fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.     

Biosynthesis of the irregular monoterpene artemisia ketone, the sesquiterpene germacrene D and other isoprenoids in Tanacetum vulgare L. (Asteraceae)

The incorporation of [1-13C]-labeled glucose into the irregular monoterpene artemisia ketone, the regular monoterpenes camphor and beta-thujone, the sesquiterpene germacrene D, the diterpene trans-phytol and beta-sitosterol and isofucosterol has been studied in axenic cultures of Tanacetum vulgare L. (Asteraceae). Quantitative 13C NMR spectroscopic analysis of the resulting labeling patterns showed that the isoprene units of the monoterpenes and the diterpene are formed via the methylerythritol phosphate (MEP) pathway, whereas the isoprene building blocks of the sesquiterpene and the sterols originate from the mevalonic acid (MVA) pathway.     

Dizocilpine Inhibits Amphetamine-Induced Formation of Nitric Oxide and Amphetamine-Induced Release of Amino Acids and Acetylcholine in the Rat Brain

Glutamate receptor activation participates in mediation of neurotoxic effects in the striatum induced by the psychomotor stimulant amphetamine. The effects of the non-competitive NMDA receptor antagonist dizocilpine (MK-801) on amphetamine-induced toxicity and formation of nitric oxide (NO) in both striatum and cortex and on induced transmitter release in the nucleus accumbens were investigated. Repeated, systemic application of amphetamine elevated striatal and cortical lipid peroxidation and NO production. Moreover, amphetamine caused an immediate release of acetylcholine and aspartate and a delayed release of GABA in the nucleus accumbens. Surprisingly, glutamate release was not affected. Dizocilpine abolished the amphetamine-induced lipid peroxidation and NO production in striatum and cortex and diminished the elevation of neurotransmitter release. These findings suggest that amphetamine evokes neurotoxic effects in both striatal and cortical brain areas that are prevented by inhibiting NMDA receptor activation. The amphetamine-induced acetylcholine, aspartate and GABA release in the nucleus accumbens is also mediated through NMDA receptor-dependent mechanisms. Interestingly, the enhanced aspartate release might contribute to NMDA receptor activation in the nucleus accumbens, while glutamate does not seem to mediate amphetamine-evoked transmitter release in this striatal brain area.     

Effect of Lesions of the Olfactory Bulb on the Levels of Amino Acids and Related Enzymes in the Olfactory Cortex of the Guinea Pig

Abstract: Olfactory bulb removal and consequential degeneration of the lateral olfactory tract led to a decreasein the levels of glutaminase and malate dehydrogenase inthe ipsilateral olfactory cortex. These changes in enzyme activity may account for the well established decrease inthe levels of aspartate and glutamate in the olfactory cortex following ipsilateral bulbectomy. The level of glutamine synthetase, a glial marker enzyme, was slightly-increased while the activities of glutamate decarboxylase, glutamate dehydrogenase, and glutamate oxaloacetic transaminase were unchanged.     

Activity, release and flow of digestive enzymes in the cricket, Gryllus bimaculatus

Abstract.  In the cricket Gryllus bimaculatus , proventricular pressure forces a nutrient fluid from the ground food-brei through a sieve at the base of the caecae, and the combination of secreted enzymes and water causes a rapid inflation of the caecae on the first day after imaginal ecdysis. The ceacal region of the midgut is the primary site for the secretion of digestive enzymes. Proteases and amylase flow from the caecae into the mostly empty crop on day 1, and carbohydrate and protein digestion starts as soon as food is present (6 h). Thereafter, much of the amylase activity (but not protease) in the crop is synthesized and released by the crop tissues themselves. Regurgitating proteases and amylases from the caecae into the crop after day 1 is most likely accomplished by temporarily halting proventricular peristalsis and allowing the caecal muscles to contract, forcing caecal contents, including enzymes, forward. The total activity of digestive enzymes in the caecae is virtually identical in 2-day-old fed and unfed females, indicating little or no secretagogue (prandial) regulation of enzyme secretion. Most of the digestive enzymes in the ventricular endoperitrophic space may originate from the mucus dragged from the caecae. Lipase activity is low in all gut regions in both starved and fed females. Head ligation or injection of trypsin modulating oostatic factor, allatostatin A or B fails to indicate any involvement of nerves or hormones in the release of digestive enzymes in the caecae. Gryllus bimaculatus appears to secrete digestive enzymes continuously, and a considerable loss of enzymes may occur at certain times through egestion.     

Mutational Analysis of the Amino Acid Residues Essential for the cis and trans Cleavage Activity of the Potato Virus Y 50-kDa Protease

To examine, the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234→Tyr, Asp269→Asn, or Cys339→Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339→Ser replacement retained a reduced proteolytic activity.     

The Response of Amino Acid Frequencies to Directional Mutation Pressure in Mitochondrial Genome Sequences Is Related to the Physical Properties of the Amino Acids and to the Structure of the Genetic Code

The frequencies of A, C, G, and T in mitochondrial DNA vary among species due to unequal rates of mutation between the bases. The frequencies of bases at fourfold degenerate sites respond directly to mutation pressure. At first and second positions, selection reduces the degree of frequency variation. Using a simple evolutionary model, we show that first position sites are less constrained by selection than second position sites and, therefore, that the frequencies of bases at first position are more responsive to mutation pressure than those at second position. We define a measure of distance between amino acids that is dependent on eight measured physical properties and a similarity measure that is the inverse of this distance. Columns 1, 2, 3, and 4 of the genetic code correspond to codons with U, C, A, and G in their second position, respectively. The similarity of amino acids in the four columns decreases systematically from column 1 to column 2 to column 3 to column 4. We then show that the responsiveness of first position bases to mutation pressure is dependent on the second position base and follows the same decreasing trend through the four columns. Again, this shows the correlation between physical properties and responsiveness. We determine a proximity measure for each amino acid, which is the average similarity between an amino acid and all others that are accessible via single point mutations in the mitochondrial genetic code structure. We also define a responsiveness for each amino acid, which measures how rapidly an amino acid frequency changes as a result of mutation pressure acting on the base frequencies. We show that there is a strong correlation between responsiveness and proximity, and that both these quantities are also correlated with the mutability of amino acids estimated from the mtREV substitution rate matrix. We also consider the variation of base frequencies between strands and between genes on a strand. These trends are consistent with the patterns expected from analysis of the variation among genomes. [Reviewing Editor: Dr. David Pollock]     

Extracellular Acidic Sulfur-Containing Amino Acids and γ-Glutamyl Peptides in Global Ischemia: Postischemic Recovery of Neuronal Activity Is Paralleled by a Tetrodotoxin-Sensitive Increase in Cysteine Sulfinate in the CA1 of the Rat Hippocampus

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.     

Amino Acids in the Capsid Protein of Tomato Yellow Leaf Curl Virus That Are Crucial for Systemic Infection, Particle Formation, and Insect Transmission

A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sic, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sic, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.     

持续水分胁迫对丹参茎叶酚酸含量及其抗氧化活性的影响北大核心CSCD

丹参种植中产生大量废弃茎叶,造成巨大的资源浪费,干旱显著影响药用植物活性成分的产生和积累。该研究以‘川丹参1号’为材料,采用土壤水分胁迫法,探讨长期水分胁迫对丹参茎叶的总酚酸(TPA)、8种主要酚酸含量和乙醇提取物抗氧化活性的影响,以初步明确丹参茎叶中酚酸及其抗氧化能力对土壤水分胁迫的响应特征,为丹参茎叶的开发利用提供理论依据。结果表明:(1)丹参茎叶TPA对土壤水分响应敏感,8、9、10月份样品(SL-8、SL-9和SL^(-1)0)的TPA含量随着土壤相对含水量(SRWC)降低而增加,且不同月份间差异显著;在相同SRWC下,SL-9的TPA含量最低,仅为SL-8的41.4%~91.1%和SL^(-1)0的24.0%~79.9%。(2)长期土壤水分胁迫下,丹参茎叶中迷迭香酸、丹酚酸B、咖啡酸和原儿茶醛明显积累;干旱增加了TPA和单一酚酸的含量,但严重缺水(SRWC为35%)会降低酚酸含量。(3)丹参茎叶乙醇提取物具有很强的抗氧化活性,其DPPH·和ABTS·+清除活性随着SRWC的降低而增强,与TPA含量变化趋势一致。研究发现,适度干旱胁迫能显著增加丹参茎叶酚酸含量及抗氧化活性,在丹参种植中可以通过科学灌溉技术来增加茎叶中酚酸含量,促进丹参的综合利用。     

PSP94融合蛋白在大肠杆菌中的表达及其抗前列腺癌活性分析

在从成年人正常前列腺组织中获得人９４个氨基酸的前列腺分泌蛋白（ＰＳＰ９４）ｃＤＮＡ基础上,利用ＰＬ表达系统,实现了人ＰＳＰ９４成熟肽Ｎ 末端带有１９个外源氨基酸的融合蛋白在大肠杆菌中的表达。目的蛋白在细胞中主要以包涵体形式存在,表达量约占菌体总蛋白的３０％,分子量约为１６－５ｋＤ。表达产物在人前列腺癌细胞ＰＣ ３上活性分析表明,该融合蛋白能明显抑制前列腺癌细胞的生长。     

Role of Glial Cells for the Basal and Ca2+-Dependent K+-Evoked Release of Transmitter Amino Acids Investigated by Microdialysis

The role of glial cells for the inactivation and synthesis of precursors for amino acid transmitters was studied in the brains of anesthetized rats in vivo using the microdialysis technique. The dialysis probes were inserted stereotactically into each neostriatum. One neostriatum was treated with the gliotoxin fluorocitrate, whereas the contralateral side served as a control. The basal efflux of amino acids, reflecting the extracellular level, was measured as well as the efflux during depolarization with 100 mM K+ in the dialysis stream. The potassium-evoked efflux of transmitter amino acids was calcium dependent and thus considered to reflect release from the transmitter pool. gamma-Aminobutyric acid (GABA) and glutamate release from the treated side was higher than the control value during the first 2-3 h, a result indicating an important role of glial cells in the inactivation of released transmitter. After 6-7 h with fluorocitrate, the release of glutamate was lower than the control value, a result indicating an important role of glial cells in the synthesis of precursors for the releasable pool of glutamate. The role of glutamine for the production of transmitter glutamate and GABA in vivo was further investigated by inhibiting glutamine synthetase with intrastriatally administered methionine sulfoximine. The release of gluatamate into the dialysis probe decreased to 54% of the control value, whereas the release of GABA decreased to 22% of the control value, a result indicating that glutamine may be more important for transmitter GABA than for transmitter glutamate.     

Na2CO3胁迫下星星草幼苗叶绿体GST活性变化及其与相关指标的关系

通过对不同浓度Na2CO3胁迫下星星草幼苗培养基质和叶片渗透势、叶片相对电导率、叶绿体谷胱甘肽转移酶（GST）活性和O2^-产生速率的研究,以探讨星星草幼苗叶绿体GST活性在Na2CO3胁迫下的变化规律及其在抗Na2CO3胁迫中的作用。结果表明,在0～0．4％Na2CO3胁迫范围内,随着其浓度的增加星星草幼苗叶绿体GST活性增强,Na2CO3胁迫浓度继续增加则GST活性急剧减弱。星星草幼苗叶绿体GST活性随培养基质渗透势、叶片渗透势和叶片相对电导率以及叶绿体O2产生速率的变化趋势相似。叶绿体GST活性和Na2CO3胁迫浓度以及叶片渗透势之间、叶绿体GST活性和O2^-产生速率以及叶片渗透势之间、叶绿体GST活性和培养基质渗透势以及叶片渗透势之间、叶绿体GST活性和O2^-产生速率以及叶片相对电导率之间相关关系显著。说明叶绿体GST在星星草幼苗抵抗低强度（浓度）Na2CO3胁迫中起着非常重要的作用。