Tyramine from the leaves of wild parsnip: a stimulant and synergist for oviposition by the black swallowtail butterfly

Abstract .Oviposition stimulants from the foliage of wild parsnip, Pastinaca sativa (Apiaceae), were isolated by column chromatography and HPLC and tested in bioassay experiments with hand-held female black swallowtail butterflies, Papilio polyxenes (Lepidoptera: Papilionidae). Two of the stimulants were identified as tyramine and trans -chlorogenic acid. A combination of tyramine, trans -chlorogenic acid and an active neutral fraction was needed to elicit a significant oviposition response. These results are discussed in the context of previous research on the oviposition stimulants of swallowtail butterflies and on the significance of tyramine as a neuromodulator of physiological processes in invertebrates.     

Characterization of Strigolactones, Germination Stimulants for the Root Parasitic Plants Striga and Orobanche, Produced by Maize, Millet and Sorghum

The germination stimulants for root parasitic plants Striga and Orobanche produced by sorghum (Sorghum bicolor (L.) Moench), maize (Zea mays L.), and pearl millet (Pennisetum typhoideum Rich.) were examined. Characterization of strigolactones in the root exudates from the plants grown hydroponically was conducted by comparing retention times of germination stimulants on reverse phase high performance liquid chromatography (HPLC) with those of synthetic standards, and by using HPLC linked with tandem mass spectrometry (LC/MS/MS). All the plants tested, except for a sorghum cultivar Swarna, were found to exude two major stimulants, 5-deoxy-strigol, which is known as a branching factor for arbuscular mycorrhizal (AM) fungi, and an isomer of strigol, tentatively named sorghumol. Swarna was found to exude 5-deoxy-strigol and strigol. These results imply that 5-deoxy-strigol is one of major germination stimulants of gramineous plants and that major stimulants may differ even among cultivars within the same species.     

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.     

A simple and rapid purification of kallikrein from rat submandibular gland.

Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme.     

蛋白的色谱复性及同时纯化

对近年来新发展的用液相色谱(LC)进行蛋白质复性及同时纯化的方法做了评述,详细介绍了蛋白质在4种液相色谱上的复性及同时纯化的方法、设备和影响因素,并对各自的优缺点进行了比较,为色谱法作为研究蛋白质折叠及用于基因工程生产治疗蛋白质的复性及同时纯化技术的进一步应用提供依据。     

Large-scale purification, crystallization and some physicochemical properties of extracellular guanyl-RNases C2 and Pch1]

The purification of RNase C2 from 76.5 1 of Asp. clavatus cultural fluid and RNase Pch1 from 160 1 of Pen. chrysogenum 152 A cultural fluid was described. 1150-fold purification of RNase C2 was attained by precipitation with ammonium sulfate, ion-exchange chromatography and rechromatography on DEAE-cellulose, gel chromatography on Sephadex G-75, and crystallization from diluted acidic buffer. During the preparation of RNase Pch1 additional chromatography on CM-cellulose was used before crystallization, the purification being 2220-fold. It was obtained 600 mg RNase C2 and 900 mg RNase Pch1. Some physico-chemical properties of crystalline RNases were studied.     

Human alpha-fetoprotein /AFP/ I. Isolation of homogenous AFP from cord blood serum

Summary The AFP from human cord blood was isolated by means of affinity chromatography with the use of antibodies as ligands and by gel filtration. The preliminary purification was achieved by affinity chromatography on CNBr-Sepharose 4B coupled with anti AFP-antibody. Further purification was obtained by the use of immunoadsorbent with anti-human serum protein antibodies. Final purification was achieved by gel filtration on Sephadex G-200. Homogeneity of the purified AFP was demonstrated by means of gel filtration, polyacrylamide gel electrophoresis, isoelectric focusing and immunoelectrophoresis.Supported by Polish National Cancer Programm within the project PR 6 0227/02/.     

Application of hydrophobic chromatography to the large scale purification of Desulfovibrio desulfuricans cytochrome C3

Hydrophobic chromatography on Phenyl-Sepharose was employed for large scale purification of cytochrome c3 from Desulfovibrio desulfuricans. This chromatographic procedure minimizes operational volumes and eliminates the lengthy dialysis ordinarily used to remove large amounts of salts. Pure cytochrome c3 was obtained after one additional purification by ion-exchange chromatography.     

基因工程菌发酵合成L－苏氨酸－N~（15）

采用含有稳定同位素１５Ｎ－硫酸铵为主要氮源的专用发酵培养基配方和提取精制条件,在国内、外首次采用基因工程菌ＡＡ７（ｐＴＨ２）（ＡＨＶｒＡＥＣｒ,Ｔｈｒ－Ｎ－,Ｈｏｍｒ,Ａｐｒ）直接发酵方法研制Ｌ－苏氨酸－Ｎ１５高丰度精制产品。每ｍｏｌ１５Ｎ－硫酸铵实际得到０．６３８ｍｏｌＬ－苏氨酸－Ｎ１５,产品Ｎ１５丰度达９９．０９％,仅比原料１５Ｎ－硫酸铵丰度下降０．４２％,提取精制得率高达９２．８３％。     

Production of (+)-5-deoxystrigol by Lotus japonicus root culture

Lotus japonicus roots, cultured in a modified B5 medium, produced and secreted germination stimulants that induced Striga hermonthica seed germination. The germination-inducing activity was detected both in the roots and the culture filtrate. Following bioassay-guided purification procedures, an active compound was isolated from hexane extracts of the roots and the culture filtrate. Based on chromatographic behaviour on HPLC, and 1H NMR, UV, MS and CD spectroscopic analyses, the germination stimulant was identified as (+)-5-deoxystrigol.     

Purification of the Proteinaceous α-Amylase Inhibitor from Phaseolus Vulgaris by Affinity Chromatography with Immobilized Enzyme or Antibody

A protein inhibiting salivary and pancreatic a-amylase of mammalian origin is contained in dry seeds of beans (Phaseolus vulgaris). Starting from a crude bean extract, the amylase inhibitor may be purified about 30fold in one step to apparent homogeneity by chromatography on matrix-bound salivary amylase. Compared with protein obtained by a conventional purification procedure and in similar yield, the amylase inhibitor obtained by affinity chromatography had the same specific activity (4.5 (akat inhibitor units/mg protein). A one step purification from crude extracts to homogenous inhibitor with the same specific activity was achieved by immuno-affinity chromatography on immobilized rabbit antibody raised against pure amylase inhibitor. The yield was 60 % that of a conventional purification. Criteria of purity of the inhibitor protein were thin-layer electrofocussing and immuno-electrophoresis.     

Production of Strigolactones by Arabidopsis thaliana responsible for Orobanche aegyptiaca seed germination

The germination stimulants produced by Arabidopsis thaliana, a host of root parasitic plants Orobanche spp. but not of arbuscular mycorrhizal (AM) fungi were examined. Root exudates from the hydroponically grown A. thaliana plants were subjected to reverse phase high performance liquid chromatography (HPLC) and retention times of germination stimulants inducing O. aegyptiaca seed germination were compared with those of strigolactone standards. In addition, the root exudates were analyzed by using HPLC linked with tandem mass spectrometry (LC/MS/MS). A. thaliana was found to exude at least three different germination stimulants of which one was identified as orobanchol. This is the first report of strigolactone production by a non-mycotrophic plant. These results together with recent knowledge imply that strigolactones have other unrevealed functions in plant growth and development.     

离子交换层析结合反向色谱纯化聚二氨基丙酸

ε-聚赖氨酸生产菌株Streptomyces albulus PD-1可合成一种新型非蛋白质氨基酸均聚物聚二氨基丙酸,采用离子交换层析和反向色谱,对聚二氨基丙酸的分离纯化进行研究。离子交换层析柱选用DEAE-Sepharose Fast Flow填料,50 mmol/L磷酸盐缓冲液(p H 7.5)平衡上样,含0.5 mol/L Na Cl的磷酸盐缓冲液(p H 7.5)洗脱,收集洗脱液用分子筛Sephadex G-25除去磷酸盐缓冲液。然后用C18反相色谱进一步纯化,流动相为V(甲醇)/V(0.1%磷酸)=5/95。经过离子交换层析和反向色谱,纯化得到聚二氨基丙酸纯品,回收率为39.8%,样品纯度达98.4%,为后续的聚二氨基丙酸的深入研究奠定基础。     

层析法纯化破伤风毒素的试验研究

为了获得高纯度的破伤风毒素,用疏水层析和离子交换层析纯化破伤风毒素。破伤风毒素培养滤液经Phenyl Sepharose疏水层析除去大部分杂质,再经DEAE Sephadex离子交换层析进一步纯化。经两步层析纯化后,毒素纯度达到2000Lf/mg PN以上,回收率为52%~73%。用此方法,连续纯化五批毒素,均获得高纯度的破伤风毒素。试验证明破伤风毒素经疏水层析和离子交换层析可得到有效纯化。     

Large-scale purification of recombinant human angiostatin

A process for the purification of recombinant human angiostatin (rhAngiostatin), produced by Pichia pastoris fermentation operated at the 2000-L scale, is reported. rhAngiostatin was recovered and purified directly from crude fermentation broth by cation exchange expanded bed adsorption chromatography. Anion exchange chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography were used for further purification. Full-length rhAngiostatin was separated from rhAngiostatin molecules fragmented by endoproteolysis. On average, 140 g of rhAngiostatin was produced per batch, with an overall yield of 59% (n = 9). The purification process was completed in approximately 48 h and used only inexpensive and nontoxic raw materials. Methods development, process synthesis, and process scale-up data are presented and discussed.     

Development of a purification procedure for the placental protein 14 involving metal-chelate affinity chromatography and hydrophobic interaction chromatography

Placental protein 14 was isolated from the biological material of patients undergoing legal abortions. The major part of ballast protein was removed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose. Albumin was separated by chromatography on Blue-Sepharose. Complete purification was obtained by metal-chelate affinity chromatography on Nickel-Chelate Sepharose and hydrophobic interaction chromatography on Phenyl-Sepharose and Octyl-Sepharose. The protein was not exposed to denaturing agents or extreme pH.     

Purification and partial characterization of a factor in cotton wax stimulating the germination of self-inhibited wheat stem rust uredospores

Filter paper, nonabsorbent cotton, and cotton wax were found to be progressively richer sources of germination-stimulatory activity effective in counteracting the self-inhibition of Puccinia graminis var. tritici Erikss. and E. Henn uredospores. The major stimulatory component of cotton wax was purified and partially characterized. It was catalytically effective in stimulating germination and oxygen consumption of uredospores and appeared to be as active as pelargonaldehyde. Unlike most of the previously reported chemical stimulants, however, it was not active across an air gap.

Although the active compound was not identified, both the ultraviolet spectrum and the nonionic and solubility properties of the active fractions were consistent with the infrared spectrum which indicated a relatively long-chained. α. β unsaturated carbonyl compound such as a ketone or possibly an ester.

The purification procedure involved deionization of ethanolic extracts from cotton or cotton wax on Dowex 50 (H⁺) and Dowex 1 (OH⁻) columns followed by chromatography on neutral alumina using ethylene dichloride as the developing and eluting solvent.

     

A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha

The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.     

Purification of small interfering RNA using nondenaturing anion-exchange chromatography

A manufacturing and purification process for duplex oligonucleotides was established, which shortens and simplifies currently used procedures, yielding a product of higher purity. The reported procedure is based on nondenaturing anion-exchange (AEX) chromatography, which is performed on the annealed duplex rather than the individual single strands. The duplex is formed early in the process by annealing of the crude single strands directly after solid-phase synthesis. Two 30 μmol manufacturing runs using duplex purification were performed on 2 different AEX resins and compared with a manufacturing run of the same scale using conventional single-strand chromatography. The same pooling strategy was employed for all purifications. Content of optimal duplex (duplex exclusively comprising full-length single strands) was 90.5% and 90.2% for the batches obtained by duplex purification and 86.1% for the batch obtained by single-strand purification. Maximum chromatographic recoveries were 67% for the duplex purification and 68% for the single-strand purification. Hence, the manufacture of small interfering RNA (siRNA) using duplex purification was simpler and faster than conventional single-strand purification and provided better purity and similar yield of final siRNA.     

Stabilization and purification of the secondary metabolism specific enzyme, m-hydroxybenzylalcohol dehydrogenase

m-Hydroxybenzylalcohol dehydrogenase (EC 1.1.1.97), a secondary metabolism associated protein from stationary phase cultures of Penicillium urticae, was stabilized in crude extracts prior to purification. Stabilization studies resulted in the formulation of an optimal cell breakage and purification buffer. This buffer increased the enzyme's in vitro half-life at 30 degrees C from 14 to over 800 min which greatly aided purification and enhanced yields. Purification was achieved by salt fractionation, size-exclusion chromatography, affinity chromatography, and ion-exchange chromatography. The 1200-fold purified protein gave only one major band by sodium dodecyl sulphate - polyacrylamide gel electrophoresis.