人αA干扰素在克鲁氏乳酸酵母中的表达和分泌

pE1是由酵母天然质粒pKD1衍生出来的重组穿梭质粒,在克鲁氏乳酸酵母中具有高拷贝,高稳定性等特点。把人αA干扰素分泌表达单元克隆到pE1载体中,得到分泌型表达质粒pE-IFN1。pE-IFN1在克鲁氏乳酸酵母中相当稳定,在非选择性培养基中生长50世代后,大多数酵母细胞仍带有质粒。结果表明,分泌表达单元中的酿酒酵母α因子的分泌信号肽能被克鲁氏乳酸酵母的蛋白质分泌系统所识别,人αA干扰素被分泌到细胞外。在摇瓶培养条件下每升发酵液中含有1—2毫克αA干扰素。     

An infrared spectroscopic study of the conformational transition of elastin-like polypeptides

The infrared spectroscopy of elastin-like polypeptides and the relation to the inverse thermal transition are discussed. To correlate the spectroscopic observations with structure a density function theory model was created that captures the essential hydrogen bonding and packing of the beta-spiral structure proposed for elastin and elastin-like polypeptides. The infrared spectrum was calculated using periodic boundary conditions and a method for estimating the difference dipole moment permits both frequencies and intensities to be obtained for the modeling of spectra. The two observed amide I bands at 1615 cm(-1) and 1656 cm(-1) are shown to arise from the beta-spiral structure. The increase in intensity of these bands with increasing salt concentration and temperature is assigned to the closer association of strands of the beta-spiral. The sharp inverse temperature transition is observed within 1 degrees C and involves a change in secondary structure that involves formation of interstrand beta-sheets for approximately 25% of the amino acids. This conclusion is consistent with available data and simulations that have been reported to date.     

Vermicompost can suppress Fusarium oxysporum f. sp. lycopersici via generation of beneficial bacteria in a long-term tomato monoculture soil

Plant and Soil - Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol) has severely decreased global tomato production. Organic amendments are widely applied to suppress Fol all over...     

Targeting p53 by PTD-mediated transduction

p53 is a major target for tumor therapy. Attempts have been made to restore or enhance p53 activity in tumor cells, including overexpression of exogenous p53 and small molecules that can rescue mutant p53. Notably, p53 peptides corresponding to the p53 carboxyl terminus can trigger a p53 response in both wild-type or mutant p53-containing cells. The recent protein transduction domain (PTD)-mediated cell entry might solve the obstacle of efficient delivery of peptides or large molecular biological cargos into cells. PTD-mediated transfer through the cell membrane occurs through a kind of endocytosis, macropinocytosis. Destabilization of macropinocytosomes by the influenza virus hemagglutinin protein (HA2) helps the escape of the PTD-cargo from macropinocytosomes and therefore significantly enhances the functional impact of transduced cargo.     

Evidence that the pre-mRNA splicing factor Clf1p plays a role in DNA replication in Saccharomyces cerevisiae

Clf1p is an essential, highly conserved protein in S. cerevisiae that has been implicated in pre-mRNA splicing. Clf1p's ortholog in Drosophila, Crn, is required for normal cell proliferation. Cells depleted of Clf1p arrest primarily with large buds, a single nucleus, a 2C DNA content, and a short, intact mitotic spindle. We isolated temperature-sensitive clf1 mutants that exhibit similar mitotic defects when released to the restrictive temperature from an early S-phase block. While these mutants also accumulate unspliced pre-mRNA at the restrictive temperature, the mitotic arrest does not appear to result from a failure to splice tubulin pre-mRNA. Moreover, the same mutants exhibit a delayed entry into S phase when released to the restrictive temperature from a G1 phase block. This delay could not be suppressed by disruption of the S-phase CDK inhibitor SIC1, suggesting that Clf1p is involved in DNA replication. Consistent with this possibility, we find that Clf1p (but not the mutant clf1p) interacts with the DNA replication initiation protein Orc2p in two-hybrid and co-immunoprecipitation assays, that Clf1p preferentially associates with origins of DNA replication, and that this association is Orc2p dependent. These observations suggest that Clf1p plays a direct role in the initiation of DNA replication.     

有基因转录活性的血管紧张素Ⅱ1B受体的组织分布

我们首次利用基因同源重组技术,用包含编码β半乳糖苷酶（ＬａｃＺ基因）基因的ＡＴ１Ｂ同源ＤＮＡ片段,置换ＡＴ１Ｂ受体基因的外显子序列,通过显色示踪基因ＬａｃＺ产生的β半乳糖苷酶,观察了有基因转录活性的ＡＴ１Ｂ受体的组织分布。结果显示ＡＴ１Ｂ受体主要分布在睾丸、肾上腺皮质的球状带和蛛网膜下腔血管及侧脑室脉络膜血管上,在垂体前叶组织中也有少量的存在。为进一步研究ＡＴ１Ｂ受体的生理和生化功能确定了组织学分布范围     

TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis

In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutant^cas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1^cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1^cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1^cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.     

Design and synthesis of quinolin-2(1H)-one derivatives as potent CDK5 inhibitors

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine protein kinase and its deregulation is implicated in a number of neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and ischemic stroke. Using active site homology modeling between CDK5 and CDK2, we explored several different chemical series of potent CDK5 inhibitors. In this report, we describe the design, synthesis, and CDK5 inhibitory activities of quinolin-2(1H)-one derivatives.