Synthesis and characterization of three group 10 complexes containing nido-carborane diphosphine: [MCl(PPh3){7,8-(PPh2)2-7,8-C2B9H10}] (M = Ni, Pd, Pt)

Three group 10 complexes containing nido-carborane diphosphine, [NiCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] (1), [PdCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] · 1.25CH₂Cl₂ (2) and [PtCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] · 2.5CH₂Cl₂ (3) have been synthesized by the reactions of [M(PPh₃)₂Cl₂] (M = Ni, Pd, Pt) with closo carborane diphosphine 1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀ in ethanol. For complex 3, it could also be obtained under solvothermal condition. All three complexes were characterized by elemental analysis, FT-IR, ¹H and ¹³C NMR spectroscopy and X-ray structure determination. Single crystal structures show that their structures are similar to each other. In each complex, the nido [7,8-(PPh₂)₂-7,8-C₂B₉H₁₀]⁻, which resulted from the degradation of the initial closo ligand 1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀ during the reaction process, was coordinated bidentately through the P atoms to M(II) ion, and this resulted in a stable five-membered chelating ring between the bis-diphosphine ligand and the metal. The coordination mode of the metal can be described as a slightly distorted square-planar, in which the remaining two positions were occupied by one Cl⁻ and one PPh₃ group.     

A New Screening Method of Viral-neuraminidase Inhibitors

A new and practical method for the screening of neuraminidase inhibitors (NI) by means of the viral hemagglutination (HA)-dehemagglutination(deHA) reactions was suggested. The best conditions for the HA and deHA reactions were investigated. Existence of strong inhibition activity on the viral deHA has been recognized in the culture filtrates of some strains of actinomycetes. All of these deHA inhibitors showed NI activity that is not specified to the strain of the test viruses. About 0.25 mg/ml of the preparation obtained from the culture filtrate of the strongest actinomycetes, No. 289, inhibited the liberation of neuraminic acid from bovine submaxillary mucin by 80 HA units/ml of influenza A Fukuoka/1/70 (H₃N₂) virus up to 80%.     

小立碗藓GPX家族的功能分化与催化特性研究

谷胱甘肽过氧化物酶(GPX)在植物抵抗氧化胁迫中发挥重要作用。该研究从小立碗藓(Physcomitrella patens)基因组中挖掘到3个GPX基因,分别命名为PpGPX1、PpGPX2和PpGPX3。其中PpGPX1和PpGPX3只含有1个外显子,而PpGPX2含有6个外显子。表达模式分析发现PpGPX1和PpGPX2在检测的所有条件下均表达,而PpGPX3在检测的所有条件下均不表达。蛋白亚细胞定位分析发现,PpGPX1蛋白定位在细胞质,而PpGPX2蛋白定位在叶绿体。在大肠杆菌中表达并纯化了PpGPX1和PpGPX2蛋白,酶学性质分析发现,PpGPX1和PpGPX2蛋白均只能利用Trx电子供体系统,而不能利用GSH电子供体系统;PpGPX2蛋白对过氧化物底物的催化活性和催化效率均高于PpGPX1。基因结构、表达模式、亚细胞定位和蛋白酶学性质的差异预示小立碗藓GPX基因家族成员发生了功能分化,将PpGPX2蛋白的Pro158、Phe167和Phe172氨基酸残基均突变为Ala,发现突变体蛋白对底物催化活性降低,说明这3个氨基酸位点对PpGPX2蛋白具有重要催化活性。     

Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo

BACKGROUND AND AIMS: The aim of this study was to develop species-specific molecular markers for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. METHODS: Two putative, species-specific RAPD markers, Bb836 for B. balcooa and Bt609 for B. tulda were generated using a PCR-based RAPD technique. Species-specificity of these two markers was confirmed through Southern hybridization in which RAPD gels were blotted and hybridized with radiolabelled cloned RAPD markers. Southern hybridization analyses were also performed to validate homology of the co-migrating Bb836 and Bt609 marker bands amplified from 16 different populations of B. balcooa and B. tulda, respectively. Sequence-characterized amplified region (SCAR) markers were developed from Bb836 and Bt609 sequences, using 20-mer oligonucleotide primers designed from both the flanking ends of the respective RAPD primers. KEY RESULTS: As anticipated, Bb836 hybridized with an amplified band from B. balcooa and Bt609 hybridized only with an amplified product from B. tulda; the two markers did not hybridize with the amplified products of any of the other 14 bamboo species studied. The two pairs of SCAR primers amplified the target sequences only in the respective species. The species-specific SCAR fragments were named as 'Balco836' for B. balcooa and 'Tuldo609' for B. tulda. The species-specific 'Balco836' was amplified from the genomic DNA of 80 individuals of 16 populations of B. balcooa studied. Similarly, the presence of 'Tuldo609' was noted in all the 80 individuals representing 16 populations of B. tulda assessed. These SCAR fragments contained no obvious repetitive sequence beyond the primers. CONCLUSION: These two molecular markers are potentially useful for regulatory agencies to establish sovereign rights of the germplasms of B. balcooa and B. tulda. In addition, this is the first report of species-specific SCAR marker development in bamboo.     

Expression and characterization of the intact N-terminal domain of streptokinase

Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero-Lara F et al., 1996, Protein Sci 5:2583-2591). The N-terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N-terminal region of the protein, residues 1-146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein.     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37 ℃下在pH8～10的缓冲液中保温1 h酶活几乎不改变。重组酶反应的最适温度为75 ℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的K_m^KG为7.559mmol/L,V_max^KG为0.086mmol/(L·min),K_m^Asp为2.031mmol/L,V_max^Asp为0.024mmol/(L·min)。Ca²⁺、Fe³⁺、Mn²⁺等金属离子对酶活性有微弱抑制作用。     

The hydrodynamic characterization of waxy maize amylopectin in 90% dimethyl sulfoxide–water by analytical ultracentrifugation, dynamic, and static light scattering

Static light scattering of high amylopectin waxy maize starch gently dispersed in 90% dimethyl sulfoxide–water yielded a weight average molecular weight M_w and radius of gyration R_g of 560×10⁶ g/mol and 342 nm, respectively. To obtain an independent hydrodynamic characterization of these solutions, we measured the sedimentation coefficient for the main component in an analytical ultracentrifuge. The value of s⁰, the infinite dilution sedimentation coefficient, was 199 S. The translational diffusion coefficient D⁰ in very dilute solutions was measured by dynamic light scattering at 90° and found to be 2.33×10⁻⁹ cm²/s. An effective hydrodynamic radius R_h was calculated from this diffusion constant using the Stokes–Einstein equation and found to be 348 nm. The structure-related parameter ρ=R_g/R_h was calculated to be 0.98. The weight average molecular weight calculated from the Svedberg equation using the values measured for s⁰ and D⁰ was 593×10⁶ g/mol. This result is in reasonable agreement with the light scattering results. As light scattering results are subject to experimental errors due to the possibility of dust contamination, the presence of microgel or aggregates, and the questionable applicability of light scattering theory to interpret results for macromolecular sizes approaching the wave length of light used as a source for scattering, it is advisable to have corroborating hydrodynamic data when possible to further validate light scattering results in this very high molecular weight range.     

Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization

It is often essential to focus the study on the small-size domains of large proteins in eukaryotic cells in the post-genomic era, but the low expression level, insolubility, and instability of the domains have been continuing to hinder the massive purification of domain peptides for structural and biological investigation. In this work, a highly efficient expression and purification system based on a small-size fusion partner GB1 and histidine tag was utilized to solve these problems. Two vectors, namely pGBTNH and pGBH, were constructed to improve expression and facilitate purification. The linker and thrombin cleavage site have been optimized for minimal degradation during purification process. This system has been tested for eight domain peptides varying in size, linker, hydrophobicity, and predicted secondary structure. The results indicate that this system is achievable to produce these domain peptides with high solubility and stability for further biochemical characterization. Moreover, the fusion protein without the linker and thrombin cleavage site is also suitable for spectroscopic studies especially for NMR structural elucidation, if the target peptide is prone to precipitation or easily degraded during purification. This system will be beneficial to the research field of structure and function of small domain and peptide fragment.     

分离自冬虫夏草可培养真菌的多样性研究

冬虫夏草是生长于青藏高原的一种名贵中药材。天然冬虫夏草及其微环境中生活着多种真菌。作者使用常规分离培养方法对冬虫夏草的真菌区系进行研究。从天然冬虫夏草的子座、菌核和菌膜3个部位共分离到572个真菌菌株,并根据形态特征将大部分菌株鉴定到37个不同的属。这些菌株经SSCP(single-strand conformation polymorphism)分析后,再根据nrDNAITS序列的相似性(以97%为阈值)共区分出92种不同的分类单元(operational taxonomic unit,OTU)。其中,属于子囊菌的菌株数及OTU数均比接合菌和担子菌多。从菌膜分离的菌株数及OTU数都明显多于子座和菌核。分离自子座的优势真菌是产黄青霉Penicillium chrysogenum,而分离自菌核和菌膜的优势真菌均为玫红假裸囊菌Pseudogymnoascus roseus。尚未最终鉴定的部分真菌可能为新的真菌物种。