Solvent-free enzymatic synthesis of 1,3-diconjugated linoleoyl glycerol optimized by response surface methodology

An operation mode with N(2) bubbling under vacuum was employed for the solvent-free synthesis of 1,3-diconjugated linoleoyl glycerol (1,3-dCLG) from conjugated linoleic acid (CLA) catalyzed by Novozym 435. The response surface methodology (RSM) was adopted for the optimization of the reaction conditions with five major factors (incubation time, temperature, enzyme load, substrate mole ratio, and system vacuum) and three responses (CLA conversion, 1,3-dCLG yield, and acyl migration). Two sets of optimal conditions were recommended. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of 1,3-dCLG yield. Under the optimal conditions, the yield of 1,3-dCLG up to 93% was obtained. The reaction was scaled up to a production level of 100 g of 1,3-dCLG at a yield of 90.7%, indicating a promising feature of the technology in industrial applications.     

染色体步移技术研究进展

染色体步移技术是一种常用的克隆已知片段旁侧序列的技术.综述了近年来染色体步移技术的发展情况,介绍了结合基因组文库的染色体步移技术和基于PCR的染色体步移技术.同时总结了物理剪切法和限制性内切酶法构建亚克隆文库的优化步骤,以及连接成环PCR法、外源接头介导PCR法和半随机引物PCR法的原理,并且比较分析了他们之间的优缺点,以期对实际操作起到借鉴作用.     

施肥措施对鳄嘴花(Clinacanthus nutans)生物量的影响

通过施肥措施对鳄嘴花[Clinacanthus nutans(Burm. f.) Lindau]生物量分配的影响研究得到,鳄嘴花各构件生物量与各构件生物量的分配比例并不总是一致。N肥处理组根茎叶以及总生物量均较K低。P肥则为处理4根茎叶以及总生物量较大。而K肥根茎生物量较大的均为处理5,叶和总生物量较大的则为处理4。有机肥的施肥效果对鳄嘴花各构件生物量的促进效果均较差。施肥配比中,各个施肥配比对根、茎生物量的增加效果均较差。叶和总生物量中,则为N1∶P1∶K1效果较好。总体来看,合理施肥对鳄嘴花生物量的增加有促进作用,对生物量的分配产生一定的影响。     

壳聚糖固定化AS1．398中性蛋白酶稳定性的研究

对以壳聚糖为载体,由戊二醛作交联剂。制备的固定化ＡＳ１．３９８中性蛋白酶的稳定性进行了研究。实验结果表明,固定化ＡＳ１．３９８中性蛋白酶对热、乙醇、尿素以及ｐＨ值的稳定性均有明显的提高。     

The lipid-droplet proteome reveals that droplets are a protein-storage depot

BACKGROUND: Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. RESULTS: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. CONCLUSIONS: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.     

Fe- and S-Metabolizing Microbial Communities Dominate an AMD-Contaminated River Ecosystem and Play Important Roles in Fe and S Cycling

Indigenous Fe- and S-metabolizing bacteria play important roles both in the formation and the natural attenuation of acid mine drainage (AMD). Due to its low pH and Fe-S-rich waters, a river located in the Dabaoshan Mine area provides an ideal opportunity to study indigenous Fe- and S-metabolizing microbial communities and their roles in biogeochemical Fe and S cycling. In this work, water and sediment samples were collected from the river for physicochemical, mineralogical, and microbiological analyses. Illumina MiSeq sequencing indicated higher species richness in the sediment than in the water. Sequencing also found that Fe- and S-metabolizing bacteria were the dominant microorganisms in the heavily and moderately contaminated areas. Fe- and S-metabolizing bacteria found in the water were aerobes or facultative anaerobes, including Acidithiobacillus, Acidiphilium, Thiomonas, Gallionella, and Leptospirillum. Fe- and S-metabolizing bacteria found in the sediment belong to microaerobes, facultative anaerobes, or obligatory anaerobes, including Acidithiobacillus, Sulfobacillus, Thiomonas, Gallionella, Geobacter, Geothrix, and Clostridium. Among the dominant genera in the sediment, Geobacter and Geothrix were rarely detected in AMD-contaminated natural environments. Canonical correspondence analysis indicated that pH, S, and Fe concentration gradients were the most important factors in structuring the river microbial community. Moreover, a scheme explaining the biogeochemical Fe and S cycling is advanced in light of the Fe and S species distribution and the identified Fe- and S-metabolizing bacteria.     

Interaction with magnesium and ADP stabilizes both components of nitrogenase from Klebsiella pneumoniae against urea denaturation

The nitrogenase enzyme of Klebsiella pneumoniae consists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild-type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2 and ADP greatly increases the stability of the smaller Fe protein (from <4 to >6 M for full unfolding), an effect directly analogous to stabilization in p21ras induced by Mg+2 and GDP. Stabilization by Mg+2 is slight for the holo MoFe protein (from approximately 1.5 to approximately 2.4 M) but more dramatic for the apo protein form of the MoFe protein accumulated by certain Fe protein (nifH gene) mutants. The potent product inhibitor of nitrogenase function, MgADP, increases stability of the MoFe protein more than Mg+2 alone, to approximately 3.6 M, showing that nucleotides interact with the MoFe protein. Mutations of the nifM gene result in slower accumulation of less stable Fe protein, indicating that NifM is involved in correct folding of the Fe protein. Mutationally altered proteins are often difficult to purify for study because of their inherent instability, low expression level, or oxygen lability. Crude extracts of 11 different mutants of Fe protein (nifH gene) were examined by transverse urea gradient gels to rapidly screen for stabilizing interactions in the presence or absence of substrate or inhibitor analogs. Amino acid alterations D44N and R188C, at the interface of the dimer, in the vicinity of the nucleotide binding site(s), have significantly lower stability than the wild-type enzyme in the absence of Mg+2 but comparable stability in its presence, showing the importance of Mg+2 in the subunit interactions. Mutations N163S and E266K, in which residues normally involved in hydrogen bonding far from the active site were altered, are more labile than the wild-type even with Mg+2 added. Seven other mutants, though nonfunctional, did not appear altered in stability compared to the wild-type.     

IKBKE protein activates Akt independent of phosphatidylinositol 3-kinase/PDK1/mTORC2 and the pleckstrin homology domain to sustain malignant transformation

Serine/threonine kinase Akt regulates key cellular processes such as cell growth, proliferation, and survival. Activation of Akt by mitogenic factor depends on phosphatidylinositol 3-kinase (PI3K). Here, we report that IKBKE (also known as IKKε and IKKi) activates Akt through a PI3K-independent pathway. IKBKE directly phosphorylates Akt-Thr308 and Ser473 independent of the pleckstrin homology (PH) domain. IKBKE activation of Akt was not affected by inhibition of PI3K, knockdown of PDK1 or mTORC2 complex. Further, this activation could be inhibited by Akt inhibitors MK-2206 and GSK690693 but not the compounds (perifosine and triciribine) targeting the PH domain of Akt. Expression of IKBKE largely correlates with activation of Akt in breast cancer. Moreover, inhibition of Akt suppresses IKBKE oncogenic transformation. These findings indicate that IKBKE is an Akt-Thr308 and -Ser473 kinase and directly activates Akt independent of PI3K, PDK1, and mTORC2 as well as PH domain. Our data also suggest that Akt inhibitors targeting the PH domain have no effect on the tumors in which hyperactive Akt resulted from elevated IKBKE.     

Preliminary results on nematicidal activity from culture filtrates of Basidiomycetes against the pine wood nematode,Bursaphelenchus xylophilus (Aphelenchoididae)

One hundred and eighty one fungal species that were isolated from the fresh fruiting bodies collected in the Mountains of Pu Er County of Yunnan Province, China were tested on the pine wood nematode,Bursaphelenchus xylophilus in vitro. Each fungal species was grown in Czapek broth and potato dextrose broth (PDB). Fifteen filtrates fromAmauroderma austrosinense, Amauroderma macer, Filoboletus sp.,Laccaria tortilis, Lactarius gerardii, Lentinula edodes, Oudemansiella longipes, Oudemansiella mucida, Peziza sp.,Pleurotus sp.,Sinotermitomyces carnosus (two strains),Strobilomyces floccopus, Termitomyces albuminosus, Tylopilus striatulus grown on PDB were found to be pathogenic to the tested nematodes. Eleven filtrates fromAmanita junguillea, Amanita sp.,Daedalea sepiaria, Fistulina hepatica, Omphalotus olearius, Oudemansiella mucida, Peziza sp.,Pleurotus pulmatus, Ramaria sp.,Tricholoma conglobatum, Tylopilus striatulus grown on Czapek broth were also pathogenic to the nematodes. When screening for nematicidal potential of fungi, it is important to study the growth medium conditions necessary to obtain the optimal nematicidal effect as fungal filtrates growing on different liquid media showed a very inconsistent toxicity towards nematodes.