Identification of a novel C2 domain factor in ovaries of orange-spotted grouper (Epinephelus coioides)

Follicle consists of an oocyte and a lot of surrounding follicular cells, and significant interactions exist between the oocyte and the somatic cells. In this study, a novel cDNA has been screened from a subtractive cDNA library between tail bud embryos and blastula embryos in the protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Its full-length cDNA is 821 bp, and has an ORF of 414 by for encoding a peptide of 137 aa, which shows 38%, 37%, 33%, and 33% homology with 4 putative proteins screened from zebrafish (Danio rerio). Conserved domain search in NCBI reveals a single C2 domain existing in the C2 domain superfamily proteins, and has only 7 beta strands in comparison with 8 beta strands of C2 domains in other C2 domain superfamily proteins. Artificial sex reversal, RT-PCR analysis and Western blot detection demonstrated ovary-specific expression of the C2 domain factor, and therefore the novel gene was designated as E. coioides ovary-specific C2 domain factor, EcOC2 factor. Moreover, predominant expression of EcOC2 factor was further revealed in grouper mature ovary, and its strong immunofluorescence signals were located between granulosa cells and oocyte zona radiata in grouper mature follicles. The data indicate that the novel EcOC2 factor might be a main component that associates between granulosa cells and the oocyte during oocyte maturation, and might play significant roles in regulating oocyte maturation and ovulation. Further studies on its developmental behaviour and physiological functions will elucidate the interactions between oocyte and the surrounding somatic cells and the underlying molecular mechanisms. (C) 2005 Elsevier Inc. All rights reserved.

金黄色葡萄球菌肠毒素C2的结构特征与生物学功能研究

通过PCR技术对Staphylococcal enterotoxin C2(SEC2)的氨基端和羧基端部位进行删除突变。研究结果显示删除羧基端77个氨基酸对SEC2超抗原和致热活性没有显著影响，但更进一步的删除导致SEC2活性显著降低。而少量氨基端残基删除则可导致SEC2超抗原活性显著降低，说明SEC2超抗原活性主要集中于其氨基端部位。以上研究结果也表明SEC2分子的完整性并不是其超抗原活性所必需的。 对二硫环及Zn结合位点配位组氨酸残基在SEC2分子中的生物学功能进行研究。以定点突变技术构建SEC2二硫环突变蛋白，从而干扰二硫键的形成。研究结果表明干扰二硫环形成对SEC2的超抗原、催吐和致热等活性均有显著影响，说明二硫环在维持SEC2生物学活性方面具有重要作用。此外，针对Zn结合位点配位组氨酸残基的研究结果显示突变118位和122位组氨酸后对SEC2的超抗原活性没有有显著影响，而47位组氨酸的突变导致SEC2分子的超抗原活性显著降低。动物实验结果显示分别突变118位和122位组氨酸残基后均导致SEC2分子催吐及热源活性显著降低，而47位组氨酸突变仍具有较高的催吐和致热活性。说明Zn结合位点配位组氨酸残基在SEC2的催吐、致热和超抗原活性中扮演了重要的作用，并进一步揭示肠毒素诱导的超抗原和致热活性之间没有明显的相关性。 以Leu及Glu分别替代20和22位氨基酸残基后SEC2超抗原活性有显著提高，并将具有减毒作用的组氨酸位点突变引入上述活性增强的SEC2突变，从而构建减毒突变蛋白。结果显示引入118和122位组氨酸双突变后突变蛋白保留了与SEC2相当的超抗原和抗肿瘤活性，但与SEC2相比致热活性有显著下降，为减毒SEC2超抗原药物的开发提供了可能性。 为探索金黄色葡萄球菌的体内基因突变方法，本研究对金黄色葡萄球菌基因组中的α-溶血毒素基因进行敲除。首先构建同源重组质粒pMHL-α，经金黄色葡萄球菌RN4220修饰后再通过原生质体转入金黄色葡萄球菌SM-01。含重组质粒pMHL-α的金黄色葡萄球菌SM-01在42℃诱导条件下培养多代，最终筛选出α-溶血毒素基因缺失菌株。经序列分析和血平板溶血实验结果证实最终获得产SEB金黄色葡萄球菌α-HL缺失菌株。为构建产减毒肠毒素的金黄色葡萄球菌基因工程菌株提供了一定的理论基础和方法。

Cluster assistant generation of C2+ and C3+ ions in nanosecond laser ionization of seeded benzene beam

Cluster assisted photoionization processes of benzene, which was seeded in argon, induced by an intense 25 ns Nd-YAG laser has been studied by means of time-of-flight mass spectrometry. At the laser intensity of 10(11) W/cm(2), multicharged ions Cq+ (q = 2-3) with kinetic energy up to 150 eV were observed in the mass spectra. Strong evidences Support that these ions are formed in the Coulomb explosion of multicharged benzene cluster ions. (C) 2004 Elsevier B.V. All rights reserved.