瓦氏马尾藻规模化繁育技术研究

研究了瓦氏马尾藻(Sargassum vachellianum)规模化育苗及海区养殖技术。采集海区成熟藻体, 室内催熟放散受精卵, 收集并喷洒于水泥板、棕绳、木板3种附着基, 进行受精卵附着、萌发及苗体生长等实验, 发现棕绳育苗效果最佳。受精卵喷洒10d后, 3种附着基的出苗率分别为 85.5%、80.2%和91.3%, 平均株高约1.3 mm。前20天木板幼苗密度最高, 达8.2株/cm2, 但幼苗均生长缓慢。第30天, 幼苗出现明显分枝, 生长率增大到前20天的1.6倍, 其中棕绳幼苗株高最大, 幼苗存活率分别为83.6%、79.7%和75.6%。第60天棕绳幼苗密度及平均株高均最大。将附有幼苗的水泥板(藻礁)、棕绳放入海区进行养殖, 发现幼苗在浪大流急海区生长较快, 4周后平均株高分别达98.7和103.1 mm, 是对照组的1.5倍, 但棕绳脱苗严重。藻礁较适合海区规模化投放养殖, 是藻场修复和重建的理想材料。     

一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 *

羟基化氨基酸是一种新型氨基酸衍生物,可广泛用作化工材料的前体物及医药合成的中间体。将来源于Nostoc minutum的新型L-亮氨酸5-羟化酶 (NmLEH) 通过重组质粒在大肠杆菌中异源表达。结果表明,在BL21(DE3) 宿主细胞中,诱导温度为25℃,IPTG诱导浓度为0.5mmol/L,诱导10h时,蛋白质表达量最高 (0.45mg/ml);通过Ni-亲和层析和凝胶过滤层析两步分离纯化获得了高度纯化的重组NmLEH蛋白;对NmLEH的酶学性质进行了表征,该酶的最适反应温度为25℃,最适pH 为7.5,在pH 7.0~9.0较为稳定,最适底物为亮氨酸和甲硫氨酸;同源序列分析表明NmLEH属于亚铁和α-酮戊二酸依赖性双加氧酶家族[Fe(II)/αKG-Dos],并预测了该酶的保守催化活性位点(H150、D152、H236);通过同源建模得到了该蛋白质的模拟结构,分析了该蛋白质催化活性中心的形成机制。     

Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics

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Highlights

• •Natural substrates of FAP were identified using degradomic and proteomic techniques and FAP gene knockout mouse derived embryonic fibroblasts stably transduced with enzymatically active or inactive FAP.
• •Terminal amine isotopic labelling of substrates (TAILS) based degradomics identified cleavage sites in collagens, and many other extracellular matrix (ECM) and associated proteins.
• •Cleavages of lysyl oxidase-like-1, CXCL-5, CSF-1 and C1qT6 by FAP were confirmed in vitro.
• •Differential metabolic labelling coupled with quantitative proteomic analysis implicated FAP in regulating proteins that are associated with ECM, ECM-cell interactions, coagulation, metabolism and wound healing.

     

High-throughput Identification of FLT3 Wild-type and Mutant Kinase Substrate Preferences and Application to Design of Sensitive In Vitro Kinase Assay Substrates

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Highlights

• •Developed a data processing pipeline to format phosphopeptide identifications.
• •Identified the preferred substrate motif for FLT3 and mutant kinases.
• •Designed and validated a panel of pan-FTL3 artificial substrates.
• •Monitored FLT3 and mutant kinase activity through FAStide phosphorylation.

     

Structural and functional insights into the role of a cupin superfamily isomerase in the biosynthesis of Choi moiety of aeruginosin

The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is a hallmark of aeruginosins, a class of cyanobacterial derived bioactive linear tetrapeptides that possess antithrombotic activity. The biosynthetic pathway of Choi has yet to be resolved. AerE is a cupin superfamily enzyme that was shown to be involved in the biosynthesis of Choi, but its exact role remains unclear. This study reports the functional characterization and structural analyses of AerE. Enzymatic observation reveals that AerE can dramatically accelerate 1,3-allylic isomerization of the non-aromatic decarboxylation product of prephenate, dihydro-4-hydroxyphenylpyruvate (H₂HPP). This olefin isomerization reaction can occur non-enzymatically and is the second step of the biosynthetic pathway from prephenate to Choi. The results of comparative structural analysis and substrate analogue binding geometry analysis combined with the results of mutational studies suggest that AerE employs an induced fit strategy to bind and stabilize the substrate in a particular conformation that is possibly favorable for 1,3-allylic isomerization of H₂HPP through coordinate bonds, hydrogen bonds, π–π conjugation interaction and hydrophobic interactions. All of these interactions are critical for the catalytic efficiency.     

Isolation of a native osteoblast matrix with a specific affinity for BMP2

During their commitment and differentiation toward the osteoblast lineage, mesenchymal stem cells secrete a unique extracellular matrix (ECM) that contains large quantities of glycosaminoglycans (GAGs). Proteoglycans (PGs) are major structural and functional components of the ECM and are composed of a core protein to which one or more glycosaminoglycan sugar chains (GAGs) attach. The association of BMP2, a member of the TGF-β super-family of growth factors, and a known heparin-binding protein, with GAGs has been implicated as playing a significant role in modulating the growth factor’s in vitro bioactivity. Here we have characterised an osteoblast-derived matrix (MX) obtained from decellularised MC3T3-E1 cell monolayers for its structural attributes, using SEM and histology, and for its functional ability to maintain cell growth and viability. Using a combination of histology and anion exchange chromatography, we first confirmed the retention of GAGs within MX following the decellularisation process. Then the binding specificity of the retained GAG species within the MX for BMP2 was examined using a BMP2-HBP/EGFP (BMP2 Heparin-Binding Peptide/Enhanced Green Fluorescent Protein) fusion protein. The results of this study provide further evidence for a central role of the ECM in the regulation of BMP2 bioactivity, hence on mesenchymal stem cell commitment to the osteoblast lineage.     

Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

Background

Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site.

Cyclin-dependent kinases: inhibition and substrate recognition

Four unresolved issues of cyclin-dependent kinase (CDK) regulation have been addressed by structural studies this year - the mechanism of CDK inhibition by members of the INK4 family of CDK inhibitors, consensus substrate sequence recognition by CDKs, the role of the cyclin subunit in substrate recognition and the structural mechanism underlying CDK inhibition by phosphorylation.     

Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and<Emphasis Type="Italic"> m</Emphasis>-xylene metabolism

The anaerobic degradation pathways of toluene and m-xylene are initiated by addition of a fumarate cosubstrate to the methyl group of the hydrocarbon, yielding (R)-benzylsuccinate and (3-methylbenzyl)succinate, respectively, as first intermediates. These reactions are catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase. Substrate specificities of benzylsuccinate synthases were analyzed in Azoarcus sp. strain T and Thauera aromatica strain K172. The enzyme of Azoarcus sp. strain T converts toluene, but also all xylene and cresol isomers, to the corresponding succinate adducts, whereas the enzyme of T. aromatica is active with toluene and all cresols, but not with any xylene isomer. This corresponds to the capabilities of Azoarcus sp. strain T to grow on either toluene or m-xylene, and of T. aromatica to grow on toluene as sole hydrocarbon substrate. Thus, differences in the substrate spectra of the respective benzylsuccinate synthases of the two strains contribute to utilization of different aromatic hydrocarbons, although growth on different substrates also depends on additional determinants. We also provide direct evidence by electron paramagnetic resonance (EPR) spectroscopy that glycyl radical enzymes corresponding to substrate-induced benzylsuccinate synthases are specifically detectable in anoxically prepared extracts of toluene- or m-xylene-grown cells. The presence of the EPR signals and the determined amount of the radical are consistent with the respective benzylsuccinate synthase activities. The properties of the EPR signals are highly similar to those of the prototype glycyl radical enzyme pyruvate formate lyase, but differ slightly from previously reported parameters for partially purified benzylsuccinate synthase.     

Counting on mitogen-activated protein kinases--ERKs 3, 4, 5, 6, 7 and 8

Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on three subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the p38 MAPKs. However, the ERK family is broader than the ERK1 and ERK2 proteins that have been the subject of most studies in this area. Here we overview the work on ERKs 3 to 8, emphasising where possible their biological activities as well as distinctive biochemical properties. It is clear from these studies that these additional ERKs show similarities to ERK1 and ERK2, but with some interesting differences that challenge the paradigm of the archetypical ERK1/2 MAPK pathway.