单分子水平的酶催化与基因表达研究

近半个多世纪以来生命科学取得了非凡的进展, 从DNA双螺旋结构的提出, 到第一个酶晶体结构的被解析, 都得益于像X射线衍射、核磁共振、质谱这样的物理化学工具的发展. 如今, 在深入细致地定量研究生物活体系统中我们正面临新的挑战, 例如:了解酶及其他大分子复合物在体内是如何实时工作的, 它们在分子数很少时是怎样工作的, 在活细胞中大分子复合物是如何协调工作的, 以及不同的基因在活细胞中分子数很少的情况下是如何实现表达和不表达的等等. 近十多年来, 单分子成像, 超高分辨率显微镜和单分子操纵技术在世界范围内被广泛地运用于生物医学研究, 对生物化学和分子生物学的发展产生着深远的影响, 因为运用这些单分子、超高分辨技术, 使很多如上述的令人感兴趣的生物学问题实现了单分子层面上的研究和理解. 本文拟就近年来相关的物理化学方法特别是单分子方法和技术在生物医学中的应用做一个简要介绍.     

BL Lac天体的γ射线和射电辐射的统计特性

给出了一个带有射电5.0GHz,8.4GHz 和γ射线辐射流量密度的22个γ噪BL Lac天体的样本,研究了它们在1GeV处的γ射线辐射流量密度最大值、平均值及最小值与射电5.0GHz,8.4GHz辐射流量之间的可能关系.结果表明：1）射电5.0GHz,8.4GHz辐射与γ射线辐射在低态时没有相关,但在高态和平均态时都存在较强的相关,最大相关系数r=0.85,置信度均好于10^-4;2）γ射线谱指数和射电谱指数之间也有一个弱相关关系存在.因此,认为γ射线的辐射主要是同步自康普顿辐射. 关键词： BL Lac天体 γ射线 射电辐射 谱指数     

不同类型紫胶的红外光谱鉴别

利用傅里叶红外光谱分别对紫胶原胶、漂白紫胶、掺有松香的紫胶、接枝共聚紫胶进行了鉴别研究。结果表明：紫胶原胶和掺有松香的紫胶, 红外光谱较为相似, 只要根据3 500～2 500 cm-1之间吸收峰的位置和峰形、1 700 cm-1附近CO振动峰的不同,以及1 200～1 400 cm-1之间松香的特征峰、其他振动峰的数目和位置的变化即可将它们区分开来;而漂白紫胶和接枝共聚紫胶是经过化学处理的改性紫胶,接枝共聚紫胶是在漂白紫胶的基础上,采用高锰酸钾为引发剂,接枝甲基丙烯酸甲脂制取的接枝共聚物,所以它们的区别主要是： 1 700 cm-1附近CO振动峰的变化、1 558 cm-1出现一个尖锐的小峰,681 cm-1出现一个中等强度的峰,以及1 151 cm-1分裂为两个小峰。实践证明,红外光谱法比传统的鉴别法科学、省时。     

BL Lac天体S5 0716+714的光变周期特性研究

从大量文献资料中, 收集了BL Lac天体S5 0716+714光学B，V，R，I波段的有效观测数据点共16818个， 获得了从1994年至2006年的长期光变曲线，光变曲线表明S5 0716+714天体存在完整的非正弦变化并且它的活动非常剧烈. 用功率谱方法分析了S5 0716+714天体的光变周期, 结果表明S5 0716+714天体存在的长光变周期为1.1年，Raiteri等人发现的3.3年周期可能是1.1年周期的叠合. 预期在2007年2月应该为S5 0716+714天体的再次爆发期. 关键词： BL Lac天体S5 0716+714 光变周期 功率谱方法     

Analysis of mutations induced by 4′-hydroxymethyl-4,5′,8-trimethylpsoralen and UVA in Escherichia coli lac Z gene and its regulatory region

The mutagenic consequences of covalent adducts induced in M13mp19 DNA by 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) and UVA have been determined in a forward mutational system capable of detecting all classes of mutagenic events. The photoreaction mediated by HMT has been carried out at a very low molar ratio of HMT to DNA which favours the induction of cross-links between high affinity reaction sites. When damaged M13mp19 DNA is used to transfect competent Escherichia coli K12 JM105 cells, a five-fold increase in mutation frequency is observed at 3.5% survivors when measured as a loss of β-galactosidase -complementation. The enhanced mutation frequency is largely due to base substitutions, frameshift events and large deletions. The single nucleotide substitutions occur both in the lac Z coding sequence and in its regulatory region. Transversion and transition have been detected with a predominent form consisting of A.T-to-G.C transversion at position + 159. Frameshift mutations have been observed at five positions while three large deletions removing either part of the coding sequence or both the coding and the regulatory regions have been detected with a higher frequency. The spectrum of base substitutions detected between the M13 lac Z⁻ phages surviving to the treatment is totally different from those appearing spontaneously whereas several frameshift events or deletions can already be detected between the spontaneous mutations. Despite the presence of these spontaneous hot spots, the spectrum of mutations recovered after HMT photoaddition appears to be unique and a detailed analysis of the different classes of mutations indicates an important role of cross-links in the production of mutations.     

Looping mechanics of rods and DNA with non-homogeneous and discontinuous stiffness

DNA molecules may form loops in response to binding with regulatory proteins that control the expression of genes. While DNA looping is a widely accepted gene regulatory mechanism, basic questions regarding the mechanics of the looping process remain open. The present paper contributes a computational rod model that accounts for non-homogeneous and discontinuous changes in stiffness to support the analysis of DNA looping. We pursue this objective in two steps. First, we illustrate the effects of non-homogeneous stiffness on the looping of generic rods under pure torsion. Results computed for this idealized case support our intuition that elastic deformation and strain energy localize in ‘soft’ regions and that equilibrium bifurcations are sensitive to non-homogenous stiffness. Next, we extend the formulation to describe the combined bending, torsion and compression induced on DNA by the LacR protein. We demonstrate that while moderate stiffness variations have only modest influence on LacR-mediated DNA looping, highly localized softening (e.g., ‘kinkable’ or ‘melted’ subdomains) may substantially reduce the energetic cost of looping and profoundly affect loop geometry.     

Dual affinity method for plasmid DNA purification in aqueous two-phase systems

The DNA binding fusion protein, LacI–His₆–GFP, together with the conjugate PEG–IDA–Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600–DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG–IDA–Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG–dextran system as a second extraction system, with 80–90% of pDNA partitioning to the bottom phase. This represents about 7.4 μg of pDNA extracted per 1 mL of pUC19 desalted lysate.     

Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor

We constructed a biosensor by electrodeposition of gold nano-particles (AuNPs) on glassy carbon (GC) and subsequent formation of a 4-mercaptobenzoic acid self-assembled monolayer (SAM). The enzyme horseradish peroxidase (HRP) was then covalently immobilized onto the SAM. Two forms of HRP were employed: non-modified and chemically glycosylated with lactose. Circular dichroism (CD) spectra showed that chemical glycosylation did neither change the tertiary structure of HRP nor the heme environment. The highest sensitivity of the biosensor to hydroquinone was obtained for the biosensor with HRP-lactose (414 nA μM⁻¹) compared to 378 nA μM⁻¹ for the one employing non-modified HRP. The chemically glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly than the native form of the enzyme. The sensor employing lactose-modified HRP also had a lower limit of detection (74 μM) than the HRP biosensor (83 μM). However, most importantly, chemically glycosylation improved the long-term stability of the biosensor, which retained 60% of its activity over a four-month storage period compared to only 10% for HRP. These results highlight improvements by an innovative stabilization method when compared to previously reported enzyme-based biosensors.