Effect of abscisic acid on vitellogenesis in Sarcophaga bullata

In the carnivorous dipteran Sarcophaga bullata Parker, vitellogenesis was partially inhibited by injection of two doses of 12 g abscisic acid (ABA). There was no significant difference between injections on day 2 and 4, or on day 4 and 6. Higher and lower doses were less effective. The mixture of isomers inhibited vitellogenesis more than the cis-trans isomer. ABA had no effect on the total lipid concentration of the haemolymph but it inhibited the sharp increase in total protein concentration of the haemolymph and particularly that of vitellogenin, which normally occurred within 24 hr following liver feeding on day 4. Since vitellogenin synthesis is under the control of moulting hormone (MH), the MH activity was measured by radioimmunoassay to find out whether ABA might interact with vitellogenin synthesis via its hormonal inductor. Eight hours after liver feeding, there was a MH peak in the control groups while following ABA treatment this peak occurred after 18 hr. The inhibitory effect of ABA on vitellogenesis could be overruled by feeding sugar impregnated with ecdysterone (5 mg/g), not by topical application of JH. Our results suggest that ABA might interact with a mechanism which phytophagous and non-phytophagous insects share in common. If in phytophagous insects the same amount of ABA per gram weight, as was effective in Sarcophaga (about 200 g/g), is needed to reduce fecundity, it is not probable that this plant hormone plays a role in the seasonal synchronisation of the growth and reproduction of insects with the senescence of their host plants.

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Résumé La vitellogenèse du diptère carnivore, Sarcophaga bullata a été partiellement inhibée par injection de deux doses de 12 g d'acide absisique (ABA). Il n'y avait pas de différence significative entre les injections du deuxième et quatrième jour, ou du quatrième et sixième jour. La mixture d'isomères a plus inhibé la vitellogenèse que le cis-trans isomère.ABA n'a pas eu d'effect sur la concentration totale des lipides de l'hémolymphe, mais il a inhibé l'augmentation brutale de la concentration protéique totale de l'hémolymphe, et particulièrement celle de la vitellogénine qui se produit normalement dans les 24 h après consommation de foie le 4ème jour. La synthèse de la vitellogénine étant sous le contrôle de l'hormone de mue (MH), l'activité MH a été mesurée par radio-immunologie, pour découvrir si l'ABA agissait sur la synthèse de la vitellogénine par intermédiaire de son inducteur hormonal. Huit heures après alimentation sur foie, le taux de MH chez les témoins était élevé tandis qu'après traitement avec ABA ce pic n'apparaissait qu'après 18 h. L'effet inhibiteur de l'ABA sur la vitellogénine a pu être neutralisé en fournissant aux mouches du sucre impregné d'ecdystérone (5mg/g), mais non par application locale d'hormone juvénile. Nos résultats suggèrent qu'ABA agit probablement sur un mécanisme commun aux insectes phytophages et non phytophages.Il est peu probable que l'acide absisique joue un rôle dans la synchronisation saisonnière de la croissance et de la reproduction des insectes avec la sénescence des plantes-hôtes.

     

High temperature pulses decrease indirect chilling injury and elevate ATP levels in the flesh fly, Sarcophaga crassipalpis

Indirect chilling injury commonly occurs during long-term exposure to low temperature in many organisms including insects. A previous study revealed increased rates of survival and reduced cold injury in flesh flies, Sarcophaga crassipalpis, that experienced an intermittent pulse of high temperature during a low-temperature regiment. We extended these studies by determining survival rates and ATP levels for flies that had undergone continuous long-term exposure at 0 °C versus those experiencing a 24-h warming pulse of either 15 or 20 °C. Survival among flies that had undergone a warming pulse was significantly greater than for flies that were maintained continuously at 0 °C. Furthermore, ATP levels of flies that had experienced a warming pulse were significantly higher than those of flies maintained at 0 °C. These data suggest that brief warming pulses during long-term cold storage allow regeneration of energy reserves that promote survival and reduce indirect chilling injury.     

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca²⁺ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl₂ (0.1–2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.     

利用重组大肠杆菌合成几丁寡糖的研究

提取根瘤菌Mesorhizobium.loti基因组,克隆编码N-乙酰氨基葡萄糖转移酶nodC基因,插入质粒pUC19的lac启动子的下游,构建并筛选出能够合成几丁寡糖的重组大肠杆菌DCL-3。利用优化的MMYNG培养基,重组大肠杆菌DCL-3在10L发酵罐中培养26h后,培养液菌体浓度测定OD560=10.8,几丁寡糖得率达到526mg/L。收集重组细菌的细胞并煮沸破碎,利用活性炭的吸附和P4凝胶层析对几丁寡糖产物进行分离纯化。纯化产物的液质分析(LC-ESI-MS)结果表明主要寡糖产物为几丁四糖(m/z,831[M H] )和几丁五糖(m/z,1034[M H] )。     

Transfection of Escherichia coli by Mu DNA

Summary Infectivity of Mu DNA was demonstrated in Ca⁺⁺-treated Escherichia coli cells that lacked the nucleases Exo V and Endo I. The efficiency of transfection is about 10^-7 per phage equivalent. Infectivity is destroyed by denaturation of Mu DNA, and cannot be restored by renaturation.     

Analysis of rpsD mutations in Escherichia coli

Summary A certain proportion of protein S7 exists in an altered form in E. coli rpsD (S4) mutants. Depending on the type of S4 mutation involved, two different forms of the altered S7 can be distinguished. The unusual form is longer than normal S7 by about 500 daltons due to extra material at the carboxyl end of the protein. It is suggested that a mutationally altered S4 might lower the efficiency of termination during translation of the messenger for S7. This results in an increased frequency of translational read-through, which gives the observed longer forms of S7. Data are interpreted to mean that one class of S4 mutants might suppress UGA and UAG whereas another class only suppresses UGA.     

Production of mevalonate by a metabolically-engineered Escherichia coli

Mevalonate is biosynthesized from acetyl-CoA and metabolized to isoprenoid compounds in a wide variety of organisms although certain types of prokaryotes employ another route for isoprenoid biosynthesis (the non-mevalonate pathway). To establish a fermentative process for mevalonate production, enzymes for mevalonate synthesis from Enterococcus faecalis were expressed in Escherichia coli, a non-mevalonate pathway bacterium. Mevalonate was accumulated, indicating a redirection of acetate metabolism by the expressed enzyme. The recombinant E. coli produced 47 g mevalonate l^–1 in 50 h of fed-batch cultivation in a 2 l jar fermenter; this is the highest titer ever reported demonstrating the superiority of E. coli in its ability of acetyl-CoA supply and its inability is degrade mevalonate.     

Biosynthesis of RNA polymerase in Escherichia coli

Summary Effect of temperature-sensitive, assembly-defective mutations in Escherichia coli RNA polymerase (rpoB) or subunit gene (rpoC) was investigated on the expression of wild-type rpoB ⁺C⁺operon, which was introduced by infection of a lambda transducing phage drif ⁺ (rpoB ⁺)-6 after UV-irradiation of the mutant cells. In rpoB2·rpoB7 strain which accumulates assembly-intermediates, free , ₂ complex and premature core, the expression of rpoB ⁺C⁺operon measured by the rate of subunit synthesis was considerably inhibited whereas that of EF(translation elongation factor)-Tu, ribosomal proteins L1 and L7/L12, and some -coded proteins remained unaffected. On the other hand, the expression was enhanced specifically for only rpoB ⁺C⁺operon in either rpoC4 or rpoC1 mutants, which are defective in the association of ₂ complex and subunit or the activation of premature core enzyme, respectively. Upon preincubation of the mutant cells at 42° C prior to phage infection, during which assembly intermediates degraded rapidly, the rate of subunit synthesis relative to other phage-corded proteins increased remarkably in rpoB2·rpoB7 mutant as well as in rpoC4 and rpoC1 mutants. These observations strongly suggested the autogenous regulation for at least (rpoB ⁺C⁺) operon by some trans-active diffusible protein complexes built of RNA polymerase subunits. Nature of the regulatory molecules is discussed.Paper VI in this series is Saitoh and Ishihama (1977)     

Improved mapping of the tyrS locus in Escherichia coli

Summary A tyrosyl-tRNA synthetase mutant of Escherichia coli was isolated and the tyrS gene assigned a map position between man and pdxH at 36.0 min on the chromosome. The tyrS mutant grew badly on broth as did previously described tyrS mutants. This sensitivity to broth was suppressed by tyrR mutations. F-prime factors were found to complement the tyrS mutation.     

Initiation of DNA replication in Escherichia coli

Summary When E. coli F⁺ cells carrying the dna-167 or dnaC2 mutation, which causes the temperature-sensitive initiation of DNA replication, are exposed to a non-permissive temperature to stop the replication of chromosome and F factor, and then transferred back to a permissive temperature with the addition of chloramphenicol, one round of the chromosomal replication occurs, but further replication is inhibited. Under these conditions, F DNA replicates coincidentally with the initiation of the chromosomal replication in both strains. When rifampicin is added to the cells upon lowering of the temperature, the chromosome can not replicate in the F⁺ dna-167 strain, but can do so in the F⁺ dnaC2 strain. F DNA can replicate in both of the mutant strains under these conditions.     

产麦角硫因大肠杆菌工程菌株的构建与优化

麦角硫因(ergothioneine,ERG)是一种天然的抗氧化剂,广泛应用于化妆品、食品以及医药领域.相比于传统植物提取和化学合成方法,微生物发酵合成麦角硫因具有周期短、成本低等优点,因而受到广泛关注.为构建高产麦角硫因的大肠杆菌工程菌株,本研究以大肠杆菌(Escherichia coli) BL21 (DE3)为出...     

Biosynthesis of RNA polymerase in Escherichia coli

Summary In spite of the generally well-coordinated synthesis of RNA polymerase core enzyme subunits (, and ) in Escherichia coli, a situation was found during the growth transition from exponential to stationary phase in which this coordination was broken (the order of differential repression being ; Kawakami et al. (1979)). The present study indicates that, during a certain period of the growth transition, twice as much subunit is synthesized as subunit and the overproduced subunit accumulates as the assembly intermediate ₂ complex, which is rapidly and preferentially degraded.Two independent factors, i.e., carbon source down-shift and oxygen depletion, were examined separately for their influence on the coordinated regulation of the synthesis of RNA polymerase subunits. The depletion of glucose added as a sole carbon source was accompanied by repression of the synthesis of all core enzyme subunits, while under the same conditions the differential rate of subunit synthesis increased. In contrast, the sudden ending of the oxygen supply resulted in specific repression of the synthesis of only and subunits but not of and subunits. The latter result may be explained by the autogenous repression of the rpoBC genes by a temporal increase in the amount of unused cytoplasmic RNA polymerase.Paper XI in this series is Kawakami and Ishihama (1980)     

Introduction of a Micrococcus plasmid in Escherichia coli

A 6-MDa plasmid (pMQV10), carrying cholesterol hydroxylase and streptomycin-resistance genes, from a gram-positive strain of Micrococcus sps., (RJ6) has been successfully transformed in gram-negative Escherichia coli K12 C600. pMQV10 is maintained stably and expresses its drug resistance in the new host.     

大肠杆菌中转录-翻译耦合的机制

在大肠杆菌（Escherichia coli,E. coli）等原核生物中,转录和翻译往往是耦合的,这种耦合通常表现在转录和翻译的互相调控上,如转录极性、转录衰减和转录-翻译速率的同步。间接耦合和物理耦合是耦合的两种模式。由警报素（alarmone）(p)ppGpp维持的间接耦合可能需要DksA和TufA蛋白的辅助。物理耦合分为NusG或RfaH因子介导的耦合和非因子条件下产生的“碰撞”耦合。响应于压力的转录或翻译的变化会引发几种耦合模式间的相互转变。耦合对于基因正常表达是必要的,其解除将引发转录终止、R环形成、复制-转录冲突、mRNA切割等不利的事件。结构生物学的相关技术已经清晰地展示了部分耦合的表达体（expressome）的结构细节和特征,这些技术联合多组学分析等方法将提供关于耦合的更深层次的见解。重要的是,对耦合的研究或许会为靶向抗菌药物的开发带来新的思路。     

Quantitation of RNA polymerase subunits in Escherichia coli during exponential growth and after bacteriophage T4 infection

Summary A method was developed to measure the amounts of RNA polymerase subunits, , , and in crude extracts of Escherichia coli. The proteins were labelled by growing the cells in ³⁵S-sulphate containing media. For measuring and , the cell lysate was electrophoresed on 6% polyacrylamide gels containing SDS and the and bands cut out and counted. For measuring and , the cell lysate was co-electrophoresed with dansylated RNA polymerase on 8% polyacrylamide gels containing SDS. The fluorescent bands were cut out, the proteins eluted, and the and subunits further purified on polyacrylamide gels containing 8 molar urea.The results are: (1) is the subunit of the core RNA polymerase which is present in limiting amount. (2) The core enzyme, as measured by , constitutes a constant fraction of total cellular protein (0.9%), independent of the bacterial growth rate. (3) The subunit is made in excess and is probably regulated independently. (4) The subunit is present in 0.3–0.4 times the amount of the core enzyme. (5) All four subunits are fully conserved after bacteriophage T4 infection.     

The relation of the genes envA and ftsA in Escherichia coli

Summary The sequence of three genes involved in cell division in E. coli has been determined to be ftsA-envA-azi by three-point transduction experiments. An ftsA envA double mutant strain forms filaments at the restrictive temperature of 42° C, and not chains, but, like the chain forming envA parent strain, is hypersensitive to rifampicin.     

Recombinant streptokinase production by fed-batch cultivation of Escherichia coli

Streptokinase (SK) is a specific effective medicine for thrombolytic therapy of acute myocardial infarction. This study established a process for the pilot production of recombinant streptokinase (r-SK). Engineering bacteria were fermented in a 20-l fermentor to produce r-SK. After simple renaturation and purification, 12.9 g of r-SK with 97.8% of purity and about 10⁵ IU mg⁻¹ of specific activity was obtained, the yield of protein and the recovery of activity were 44.9% and 51%, respectively. Finally, the r-SK was made into about 700 doses of injections for clinical applications.