Gene cloning and heterologous expression of a serine protease from Streptomyces fradiae var.k11

The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 degrees C for its caseinolytic activity and pH 9 and 62 degrees C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.     

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP's regulatory functions in controlling proinflammatory cytokines.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

Rab39, a novel Golgi-associated Rab GTPase from human dendritic cells involved in cellular endocytosis

Rab GTPases are Ras-like small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. Here we report that Rab39, a novel Rab protein, is a Golgi-associated protein involved in endocytosis of HeLa cells. Full-length cDNA of Rab39 contains 1251bp with an open reading frame (ORF) of 636bp, which is predicted to encode a 211 aa protein. By blast analysis of Rab39 cDNA and protein sequence with homologues, we find that Rab39 may be a short variant of Rab34. Rab39 contains conserved motifs involved in phosphate/guanosine binding and a microbody C-terminal targeting signal. RT-PCR analysis indicates that Rab39 is mainly detected in epithelial cell lines, and Northern blot analysis shows that Rab39 is expressed ubiquitously in human tissues. By using FITC-BSA as an endocytic tracer, we show that Rab39 can facilitate endocytosis in HeLa cells when expressed either transiently or stably. Confocal microscopy examination of Rab39 subcellular localization suggests that Rab39 is associated with Golgi-associated organelles. Our findings demonstrate that Rab39 is a novel Rab GTPase involved in cellular endocytosis.     

STAT-3-dependent cytosolic phospholipase A2 expression is required for thrombin-induced vascular smooth muscle cell motility

Vascular smooth muscle cell (VSMC) migration from media to intima and its multiplication in intima is a contributing factor in the pathogenesis of atherosclerosis and restenosis after angioplasty. Previously, we have demonstrated that STAT-3-dependent cytosolic phospholipase A(2) (cPLA(2)) expression is needed for VSMC motility induced by platelet-derived growth factor-BB, a receptor tyrosine kinase agonist (Neeli et al. (2005) J. Biol. Chem. 279, 46122-46128). In order to learn more about the STAT-3-cPLA(2) axis in motogenic signaling, here we have studied its role in VSMC motility in response to a G protein-coupled receptor (GPCR) agonist, thrombin. Thrombin induced VSMC motility in a dose-dependent manner with a maximum effect at 0.5 units/ml. Thrombin activated STAT-3 as measured by its tyrosine phosphorylation and translocation from the cytoplasm to the nucleus. Forced expression of a dominant negative mutant of STAT-3 reduced thrombin-induced STAT-3 tyrosine phosphorylation and its translocation from the cytoplasm to the nucleus. Thrombin stimulated STAT-3-DNA binding and reporter gene activities in VSMC, and these responses were blocked by FS3DM, a dominant negative mutant of STAT-3. FS3DM also attenuated thrombin-induced VSMC motility. Thrombin induced the expression of cPLA(2) in a time- and STAT-3-dependent manner. In addition, pharmacological inhibition of cPLA(2) blocked thrombin-induced VSMC motility. Furthermore, exogenous addition of arachidonic acid rescued thrombin-induced VSMC motility from inhibition by blockade of STAT-3 activation. Forced expression of cPLA(2) also surpassed the inhibitory effect of dominant negative STAT-3 on thrombin-induced VSMC motility. Together, these results show that thrombin-induced VSMC motility requires STAT-3-dependent induction of expression of cPLA(2).     

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.     

秸秆还田条件下灌水模式对冬小麦产量和水肥利用效率的影响

于2008-2010年,在山西省临汾市尧都区半干旱、半湿润季风气候区,通过大田试验研究了玉米秸秆连续还田条件下灌水模式对冬小麦籽粒产量、干物质转移及水肥利用效率的影响.结果表明:浇越冬水可促进小麦分蘖;浇拔节水可提高分蘖成穗率,增加成穗数;浇孕穗水可促进穗部干物质积累,提高千粒重.浇2水时,推迟第2次浇水时期使叶片干物质转移量和穗粒数增加;浇2水比浇l水的肥料表观利用率高,可促进穗部干物质积累.越冬水灌水量和总灌水量对分蘖、穗部干物质积累的影响较小;拔节期或孕穗期增加灌水量则更有利于养分吸收及干物质积累与转移,提高籽粒水分利用效率,产量构成因素协调,增产效果明显.因此,确保越冬水可实现稳产,在越冬水基础上,拔节期增量灌水(900 m3·hm-2)可满足冬小麦中后期生长发育的需要,提高籽粒水分利用效率,实现节水高产栽培.     

提高榨菜离体培养植株再生频率

采用榨菜“浙桐1号”品种为材料,以MS为基本培养基,通过对不同植物生长调节剂的组合和不同外植体等主要因素的筛选,大幅度提高了榨菜离体培养植株再生频率。结果表明,2mg／L6．BA 0．2mg／L2,4-D的组合较为适宜,其不定芽再生频率可达50％,且外植体以下胚轴为好：而CPPU和2,4-D的适宜组合为1．5mg／L 0．2mg／L,其不定芽再生频率高达66．67％,最适外植体为带柄子叶。同时,研究结果显示,添加0．25～1mg／L的GA,对榨菜已分化的不定芽的伸长有抑制作用;子叶柄和下胚轴外植体的分化具有极性现象。