Metabolism of Storage Lipids and the Role of Lipid Droplets in the Yeast Schizosaccharomyces pombe

Storage lipids, triacylglycerols (TAG), and steryl esters (SE), are predominant constituents of lipid droplets (LD) in fungi. In several yeast species, metabolism of TAG and SE is linked to various cellular processes, including cell division, sporulation, apoptosis, response to stress, and lipotoxicity. In addition, TAG are an important source for the generation of value-added lipids for industrial and biomedical applications. The fission yeast Schizosaccharomyces pombe is a widely used unicellular eukaryotic model organism. It is a powerful tractable system used to study various aspects of eukaryotic cellular and molecular biology. However, the knowledge of S. pombe neutral lipids metabolism is quite limited. In this review, we summarize and discuss the current knowledge of the homeostasis of storage lipids and of the role of LD in the fission yeast S. pombe with the aim to stimulate research of lipid metabolism and its connection with other essential cellular processes. We also discuss the advantages and disadvantages of fission yeast in lipid biotechnology and recent achievements in the use of S. pombe in the biotechnological production of valuable lipid compounds.     

Yeast sequencing reports. Isolation and sequence analysis of a gene from the linear DNA plasmid pPAC1–2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis

The toxin-encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in function and structural organization. By Southern hybridization, a linear plasmid of P. acaciae, pPac1–2, was found to hybridize to the second open reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the α and β subunits of the K. lactis toxin. A 1·7 kbp segment of pPac1–2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1·7 kbp fragment also contained an ORF of 1473 bp that could encode a protein of ～ 55·8 kDa. Like the α subunit gene of K. lactis ORF2, a very hydrophobic region occurs at the N-terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and lacks a recognizable domain common to chitinases. The structure of a toxin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream conserved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of the four K. lactis pGKL1 ORFs, was found just upstream of the presumed start codon. The similarity of the promoter-like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of the existence of a unique, but highly conserved, expression system for these novel plasmids. The sequence has been deposited in the GenBank data library under Accession Number U02596.     

The complete sequence of a 10.8 kb segment distal of SUF2 on the right arm of chromosome III from Saccharomyces cerevisiae reveals seven open reading frames including the RVS161, ADP1 and PGK genes.

We have entirely sequenced a 10,835 bp segment of the right arm from chromosome III contained in the J11D and J11D-K3B GF clones. The segment contains seven open reading frames longer then 100 amino acids. Three of them, RVS161 (Urdaci et al., 1990; Crouzet et al., 1991), ADP1 (Purnelle et al., 1991) and PGK1 (Hitzeman et al., 1982) have been described previously. YCR10C encodes a putative membrane protein. YCR8W (encoding a putative protein kinase) and YCR14C extend inside the D10H (Skala et al., 1991) and 62B5-2D clones respectively. Four ARS elements previously reported by Palzkill et al. (1986) are located between RVS161 and YCR10C.     

甘蔗糖蜜生产核苷酸的分离方法研究

本文介绍了利用离子交换层析技术分离甘蔗糖蜜生产的核苷酸之现实性与可行性。探讨了分离核苷酸的最佳工艺条件。     

聚乙二醇化重组人生长激素的性质及活性

目的　研究聚乙二醇化重组人生长激素的性质及活性。方法　通过分子筛高效液相 (SizeexclusionHPLC)、毛细管等电聚焦 (CIEF)、圆二色谱 (CD)以及荧光光谱等方法检测单聚PEG- GH的纯度、等电点、二级结构以及分子构象 ;采用妊娠兔肝细胞放射受体分析法 (RRA)检测单聚PEG GH的体外受体亲和力 ,采用未成年去垂体大鼠体重增加法检测体内生物活性。结果　分子筛高效液相图谱上仅出现一个峰 ,表明样品纯度很高 ,等电点范围为 5 .1～ 5 .2 ,与未修饰的rhGH的等电点相一致 ;CD谱与荧光光谱均表明单个mPEG ALD的修饰对rhGH的二级结构以及分子构象没有明显影响 ;与未修饰的rhGH相比 ,单聚PEG GH体外受体亲和力明显降低 ;但体内作用时间更长 ,且更加有效。结论　此结果为进一步研究和开发长效重组人生长激素提供了理论依据。     

重组人EC-SOD包涵体的稀释复性及重折叠后蛋白的纯化

目的确定重组人ECSOD包涵体体外稀释复性和复性后蛋白纯化的适宜方法。方法通过对pH、温度、包涵体蛋白浓度、尿素和精氨酸浓度及氧化还原对比率的定性定量分析,研究重组人ECSOD包涵体体外稀释复性的基本条件。采用金属离子亲和柱(IMAC)和肝素亲和柱(Heparinsepharose)对复性蛋白进行纯化,比较纯化效率的差异。结果最佳复性条件为pH8.5,尿素浓度为1.5molL,精氨酸浓度大于0.3molL,GSH∶GSSG=4∶1;复性温度及包涵体蛋白终浓度越高,复性效率越低。IMAC和Heparinsepharose亲和柱均可用来纯化复性蛋白溶液,但Heparinsepharose纯化效率较高。结论确定了重组人ECSOD包涵体体外稀释复性的最佳条件及复性后蛋白纯化的方法。     

重组人白细胞介素-10在大肠杆菌中的表达及生物学活性分析

目的在大肠杆菌中表达重组人白细胞介素-10(Interleukin-10,IL-10),并检测其生物学活性。方法将IL-10基因重组到质粒pET11c中,转化BL21(DE),提取质粒,经酶切鉴定和测序分析;在25℃用低浓度的IPTG诱导表达,对包涵体IL-10稀释复性;经ELISA检测其含量,MC/9细胞增殖法检测其生物学活性。结果工程菌IL-10/pET11c/BL21诱导表达的目的蛋白以可溶性和包涵体两种形式存在,Westernblot鉴定证实为IL-10蛋白,两种形式的IL-10均具有一定的生物学活性。结论已成功地在大肠杆菌中表达了IL-10,为进一步纯化和制备IL-10的基因工程药物打下了基础。     

应用免疫策略对蒙脱石HIV DNA疫苗免疫作用的研究

目的运用Prime—Boost策略,研究蒙脱石(MMT)DAN疫苗对小鼠免疫功能的影响。方法选用3H-TdR掺入法测定MMT和DNA联合免疫小鼠脾淋巴细胞的增殖反应;采用ELISA试剂盒检测血清中的抗体和细胞因子。结果使用痘苗病毒和/或加入MMT加强免疫DNA疫苗后,均可以提高其体液和细胞免疫功能。结论“Prime-boost-boost”免疫方案在小动物模型中取得了较好的免疫效果。     

重组人干扰素α2b阴道泡腾片抗豚鼠单纯疱疹病毒性阴道炎的疗效

目的观察重组人干扰素α2b阴道泡腾片对单纯疱疹病毒性阴道炎动物模型的疗效。方法采用单纯疱疹病毒感染建立豚鼠实验性阴道炎模型,并分别用重组人干扰素α2b阴道泡腾片600、3 000和15 000 IU/只进行治疗。以阿昔洛韦(无环鸟苷)和干扰素α2b栓剂15 000 IU/只作为对照。对治疗前后豚鼠阴道外观及组织切片进行评分。结果应用重组人干扰素α2b阴道泡腾片对豚鼠实验性单纯疱疹病毒性阴道炎进行治疗后,豚鼠阴道外观病变与组织切片病变评分均显著降低,其中15 000 IU/只效果最好。同样剂量的α2b泡腾片药效优于栓剂。结论重组人干扰素α2b阴道泡腾片对实验性单纯疱疹病毒性阴道炎有明显的疗效。     

重组人干扰素α2b内毒素检查法的研究

目的研究鲎试剂法与家兔法检测重组人干扰素α2b内毒素结果的相关性,并参照家兔热原试验的阈值量,来确定本制品质量标准中内毒素的限量标准。方法分别用不同浓度的内毒素溶液和含不同浓度内毒素的供试品溶液进行家兔热原质试验,确定供试品内毒素限值,并在此限值下进行内毒素检测,方法均参照2000年版《中国药典》和2000年版《中国生物制品规程》进行。结果在含不同浓度内毒素溶液的家兔热原质试验中,致热阈值小于5.0EUml。在含不同浓度内毒素供试品溶液的家兔热原质试验中,致热阈值小于1.25EUml。以此确定本品内毒素限值,检测该制品的内毒素含量,结果均符合规定。结论本品在内毒素含量小于3.5EU500万单位dose的范围内,根据具体的对照实验及制品的用途来规定细菌内毒素限值,可用鲎试剂法代替家兔法检测注射用重组人干扰素α2b冻干制剂的热原质。