巨型引物PCR法对抗CD3改型单链抗体

亲和力是影响改型单链抗体应用于临床的重要因素之一.利用巨型引物PCR定点诱变方法,设计并化学合成出两组含多个突变位点的简并引物,在第一轮PCR中使用简并引物分别扩增出含突变碱基的两条特异性的DNA片段,即巨型引物,将其经琼脂糖凝胶电泳分离纯化后,作为3′和5′的两端引物应用于第二轮PCR反应中.通过改变标准PCR反应条件,调整引物与模板的浓度,扩增出特异性较强的目的DNA条带.PCR产物经回收后,进行DNA测序.测序结果表明利用该方法扩增得到特异的抗CD3改型单链抗体的突变体库.     

The role of RBF in developmentally regulated cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth

During Drosophila eye development, cell proliferation is coordinated with differentiation. Immediately posterior to the morphogenetic furrow, cells enter a synchronous round of S phase called second mitotic wave. We have examined the role of RBF, the Drosophila RB family homolog, in cell cycle progression in the second mitotic wave. RBF-280, a mutant form of RBF that has four putative cdk phosphorylation sites mutated, can no longer be regulated by Cyclin D or Cyclin E. Expression of RBF-280 in the developing eye revealed that RBF-280 does not inhibit G1/S transition in the second mitotic wave, rather it delays the completion of S phase and leads to abnormal eye development. These observations suggest that RB/E2F control the rate of S-phase progression instead of G1/S transition in the second mitotic wave. Characterization of the role of RBF in Cyclin D/Cdk4-mediated cellular growth showed that RBF-280 blocks Cyclin D/Cdk4 induced cellular growth in the proliferating wing disc cells but not in the non-dividing eye disc cells. By contrast, RBF-280 does not block activated Ras-induced cellular growth. These results suggest that the ability of Cyclin D/Cdk4 to drive growth in the proliferating wing cells is distinct from that in the none-dividing eye cells or the ability of activated Ras to induce growth, and that RBF may have a role in regulating growth in the proliferating wing discs.     

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.     

Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme

Particulate methane monooxygenase (pMMO) has been exfoliated and isolated from membranes of the Methylosinus trichosporium IMV 3011. It appears that the stability of pMMO in the exfoliation process is increased with increasing copper concentration in the growth medium, but extensive intracytoplasmic membrane formed under higher copper concentration may inhibit the exfoliation of active pMMO from membrane. The highest total activity of purified pMMO is obtained with an initial concentration of 6 microM Cu in the growth medium. The purified MMO contains only copper and does not utilize NADH as electron donor. Treatment of purified pMMO with EDTA resulted in little change in copper level, suggesting that the copper in the pMMO is tightly bound with pMMO.     

Improved production of (<Emphasis Type="Italic">S</Emphasis>)-ketoprofen ester hydrolase by a mutant of<Emphasis Type="Italic"> Trichosporon brassicae</Emphasis> CGMCC 0574

An efficient screening method following UV mutagenesis yielded a high frequency of improved mutants of Trichosporon brassicae CGMCC 0574, a wild-type esterase-producer capable of enantioselectively hydrolyzing the ethyl ester of ketoprofen [2-(3-benzoylphenyl) propionic acid]. The mutant had an activity 1.8-fold higher than the wild type and was stable in its enzyme production for ten serial transfers. As the best single carbon source, isopropanol improved the specific activity of the enzyme 5-fold; and this did not result from the effect of cell permeabilization. An 18-h culture grown on a medium containing 0.5% glucose plus 0.5% isopropanol produced 3-fold as much esterase as a culture grown on 1% glucose.     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

Determination of glutathione in single human hepatocarcinoma cells by capillary electrophoresis with electrochemical detection

A method for determination of glutathione (GSH) in single human hepatocarcinoma (HH) cells was described by capillary zone electrophoresis with electrochemical detection at a gold/mercury amalgam micro-disk electrode. When HH cells were washed with the running buffer instead of physiological buffer saline, only one electrophoretic peak for GSH is depicted on the electropherograms of single HH cells. When electroosmotic injection of 0.01 mol/l NaOH for lysing the cell introduced into the capillary, the lysis time can be shorten to 5 s. The whole cell injection and no need of derivatization reaction lead more accurate and precise results. The average amount of GSH in an individual HH cell is 22.3+/-5.8 fmol (mean+/-standard deviation), which is consistent with that of its homogenate.     

鹟科五种鸟线粒体DNA序列变化与亲缘关系的研究

首次对鹟科（Muscicapidae）画眉亚科三种鸟----画眉（Garrulax canorus）、红嘴相思鸟（Leiothrix lutea）、棕头雅雀(Paradoxornis webbiana);鸫亚科的乌鸫（Turdinae merula）线粒体DNA的12S rRNA基因片段的DNA序列进行了测定,并与北美画眉亚科弯嘴鹛属（Pomatostomus）的灰冠弯嘴鹛（P.temporalis）同源序列进行分析比较并构建分子进化树。实验结果与传统形态学论述存在一定的差异,与同工酶研究结果相同：画眉与红嘴相思鸟的亲缘关系最近,棕头鸦雀较乌鸫与画眉亚科的关系更近,为将其置于画眉亚科提供了分子证据。 Abstract：The classfication on Muscicapinae disturbed us for a long time.The important thing is to find an appropriate character to resolve it.It is the first time that we testified the mitochondrial 12S rRNA gene fragment sequence of four birds (G.canorus,L.lutea,P.webbianus and T.merula-owing to Muscicapinae) and aligned with the same sequence of P.temporalis.Using the mitochondrial 12S rRNA sequences date,we built phylogenetic trees.The results indicated that that the relationship between Garrulax canorus and Leiothrix lutea in the four birds was closer which is congruent with morphology.According to the results of isoenzyme and DNA sequencing data,we can draw a conclusion that Paradoxornis webbianus has closer relationship to Timaliinae than that of Turdus merula,and that putting Paradoxonithinae to Timaliinae was reasonable.