Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Construction and expression of nonsense suppressor tRNAs which function in plant cells

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.     

Enhancement of CO2 tolerance of Chlorella vulgaris by gradual increase of CO2 concentration

Summary Chlorella vulgaris UTEX259 was cultivated using two different methods of gas supply. In one method the CO₂ concentration in bubbled gas was held constant and in the other method it was increased gradually. Algal growth was almost linear after a short period of lag phase in both methods. With the constant CO₂ concentration, the CO₂ fixation rate in the linear growth phase decreased over 10%(v/v) CO₂, while the rate increased up to 6% CO₂. However, the rate was enhanced by using the latter incremental increase method, especially under a higher concentration of CO₂. The maximum rate of CO₂ fixation was 52 mg CO₂/l·h at 20% CO₂ during the gradual increase of CO₂ concentration.     

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.     

Spectrophotometric determination of bromopyruvate by reaction with 2-nitro-5-thiobenzoic acid

A spectrophotometric method for determination of bromopyruvate, based on the reaction with 2-nitro-5-thiobenzoic acid, is described. The reaction is complete in 30 min at room temperature in 0.1 m Tris-MES, 1 mm EDTA, pH 8.0. The method is sensitive to at least 1 × 10^?5m bromopyruvate. Reagents are stable, easy to prepare, and specific for β-halopyruvate. Bromopyruvate solutions must be prepared fresh daily. Solutions of bromopyruvate at pH 8.0 and 23°C have a half-life of 3 hr.     

抗FLAG标签抗体在植物中的高效表达

植物生物反应器是一种新兴的重组蛋白表达系统,是分子农业的核心内容之一。本研究在本氏烟草(Nicotiana benthamiana)中表达了抗八肽(DYKDDDDK, FLAG)标签抗体,并对其进行纯化与鉴定。通过多次免疫小鼠获得高效价抗FLAG抗体并测出其编码序列,然后亚克隆至植物DNA病毒表达载体,最后通过农杆菌介导转染烟草叶片。经Western blotting检测了转染后2−9 d抗体的表达情况：3 d后FLAG抗体开始在烟草叶片中表达,5 d后表达量达到峰值,每千克鲜叶估计可表达66 mg FLAG抗体。抗体经过分离纯化后浓缩为1 mg/mL,按1:10 000稀释仍可识别1 ng/mL的抗原,表明植物生产的FLAG抗体具有高亲和力。植物生物反应器可用于生产高亲和力抗体,并具有简易、成本低和生产周期短等特点,具有很高的应用价值。     

1.42A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.