极大节旋藻(Arthrospira maxima)生物钟基因kaiC的克隆及分析

以kaiC基因簇部分已知序列为引物设计位点,采用PCR反应池法从节旋藻基因组fosmid文库中筛选到kaiC基因克隆,通过步移测序获得了kaiC基因全长序列。kaiC基因编码区长1554bo,基因GC碱基含量平均为41．76％,密码子第三位明显偏向于U。在基因上游385bp范围内发现一个可能的启动子序列和一些基因调控元件。KaiC蛋白分析发现了Walker A、DXXG、不完整的Walker B等重要模体和具有催化作用的功能位点。Southem杂交的结果证明kaiC基因在极大节旋藻中为单拷贝。极大节旋藻kaiC基因的特殊结构特征为研究kai基因簇的进化提供了重要的启示。     

The HIV-1 Gag Protein Displays Extensive Functional and Structural Roles in Virus Replication and Infectivity

Once merely thought of as the protein responsible for the overall physical nature of the human immunodeficiency virus type 1 (HIV-1), the Gag polyprotein has since been elucidated to have several roles in viral replication and functionality. Over the years, extensive research into the polyproteins’ structure has revealed that Gag can mediate its own trafficking to the plasma membrane, it can interact with several host factors and can even aid in viral genome packaging. Not surprisingly, Gag has also been associated with HIV-1 drug resistance and even treatment failure. Therefore, this review provides an extensive overview of the structural and functional roles of the HIV-1 Gag domains in virion integrity, functionality and infectivity.     

Shape Transformations and Self-Assembly of Hairy Particles under Confinement

Molecular dynamics simulations are used to investigate the behavior of polymer-tethered nanoparticles between two inert or attractive walls. The confinement in pores creates new possibilities for controlling the shape transformation of individual hairy particles and their self-organization. We introduce a minimalistic model of the system; only chain-wall interactions are assumed to be attractive, while the others are softly repulsive. We show how the shape of isolated particles can be controlled by changing the wall separation and the strength of the interaction with the surfaces. For attractive walls, we found two types of structures, “bridges” and “mounds”. The first structures are similar to flanged spools in which the chains are connected with both walls and form bridges between them. We observed various bridges, symmetrical and asymmetrical spools, hourglasses, and pillars. The bridge-like structures can be “nano-oscillators” in which the cores jump from one wall to the other. We also study the self-assembly of a dense fluid of hairy particles in slit-like pores and analyze how the system morphology depends on interactions with the surfaces and the wall separation. The hairy particles form layers parallel to the walls. Different ordered structures, resembling two-dimensional crystalline lattices, are reported. We demonstrate that hairy particles are a versatile soft component forming a variety of structures in the slits.     

Molecular Weight Measurement of UG8 Asphaltene Using APCI Mass Spectroscopy

Abstract

Asphaltene molecular weight has been a controversial issue in the past several decades and continues on nowadays. From industrial application point of view, asphaltene molecular weight is important for setting up a heavy oil refining strategy so that the process is efficient and economically viable. If the measured average molecular weight of asphaltene is high and is the true molecular weight, then substantial amount of energy will be needed, in order to break the molecule into light products during refining process. This is likely not an economical option. On the other, if the measured high molecular weight is due to self-association and the true molecular weight is low (e.g., less than 1500 Da), it will be energetically attractive to refiners to develop heavy oil cracking technology. Vapor pressure osmometry (VPO) has been routinely used for measuring molecular weight. However, it measures the apparent molecular weight and is likely not the true molecular weight. In order to unambiguously measure the molecular weight, it is necessary to develop a convincing technology and a reliable experimental procedure that allows one to measure the molecular weight accurately and consistently. We chose the Atmospheric Pressure Chemical Ionization (APCI) technique and Atmospheric Pressure Photo Ionization (APPI) to measure UG8 asphaltene. Both APCI and APPI have mild ionization processes and have been applied to many unstable drug compounds such as proteins and peptides with reliable outcomes. In addition, we measured the sample on two APPI instruments to compare the results. We also demonstrated how one can choose wrong set of operating parameters and lead to erroneous results. The relevant parameters for APCI and APPI are temperature, voltage, and sample concentration. We chose 0.01 mg/mL as the concentration, much below any known critical aggregation concentration. As for temperature and ionization voltage, we varied systematically varied (T = 300–600°C; V = 30–150 V) in order to demonstrate the consistency of the methods and how one can easily make mistake. Through these measurements, an average molecular weight of 400 to 900 Da was obtained for UG8 asphaltene.     

拓扑集成指数与TIBO类衍生物构效关系的研究

4H-甲基咪唑苯二氮(?)酮(TIBO)类衍生物是抗爱滋病的一种新药,分子连接性指数是经证明应用广泛、较为成功的一种指数,本文定义并计算了TIBO类衍生物原子的特征值δi,利用量子化学计算方法,建构新的拓扑集成指数G和分子连接性指数mX,基于多元回归技术建立的对TIBO类衍生物药物的油水分配系数,作出精确估算和预测的定量结构-活性相关关系,得到的多元回归方程为:logP=0.782 G-0.1430X 0.2312X-3.829,估算的平均相对误差为2.53%。为了检验模型的稳定性和预测能力,做了留一法交互校验,预测平均相对误差为3.40%。该模型相关系数高,稳定性好,预测能力强。     

Consecutive Aromatic Residues Are Required for Improved Efficacy of β-Sheet Breakers

Alzheimer’s disease is a fatal neurodegenerative malady which up to very recently did not have approved therapy modifying its course. After controversial approval of aducanumab (monoclonal antibody clearing β-amyloid plaques) by FDA for use in very early stages of disease, possibly new avenue opened for the treatment of patients. In line with this approach is search for compounds blocking aggregation into amyloid oligomers subsequently forming fibrils or compounds helping in getting rid of plaques formed by β-amyloid fibrils. Here we present in silico work on 627 sixtapeptide β-sheet breakers (BSBs) containing consecutive three aromatic residues. Three of these BSBs caused dissociation of one or two β-amyloid chains from U-shaped β-amyloid protofibril model 2BEG after docking and subsequent molecular dynamics simulations. Thorough analysis of our results let us postulate that the first steps of binding these successful BSBs involve π–π interactions with stacked chains of F19 and later also with F20 (F3 and F4 in 2BEG model of protofibril). The consecutive location of aromatic residues in BSBs makes them more attractive for chains of stacked F3 and F4 within the 2BEG model. Spotted by us, BSBs may be prospective lead compounds for an anti-Alzheimer’s therapy.     

Sulfated Hyaluronan Binds to Heparanase and Blocks Its Enzymatic and Cellular Actions in Carcinoma Cells

We examined whether sulfated hyaluronan exerts inhibitory effects on enzymatic and biological actions of heparanase, a sole endo-beta-glucuronidase implicated in cancer malignancy and inflammation. Degradation of heparan sulfate by human and mouse heparanase was inhibited by sulfated hyaluronan. In particular, high-sulfated hyaluronan modified with approximately 2.5 sulfate groups per disaccharide unit effectively inhibited the enzymatic activity at a lower concentration than heparin. Human and mouse heparanase bound to immobilized sulfated hyaluronan. Invasion of heparanase-positive colon-26 cells and 4T1 cells under 3D culture conditions was significantly suppressed in the presence of high-sulfated hyaluronan. Heparanase-induced release of CCL2 from colon-26 cells was suppressed in the presence of sulfated hyaluronan via blocking of cell surface binding and subsequent intracellular NF-κB-dependent signaling. The inhibitory effect of sulfated hyaluronan is likely due to competitive binding to the heparanase molecule, which antagonizes the heparanase-substrate interaction. Fragment molecular orbital calculation revealed a strong binding of sulfated hyaluronan tetrasaccharide to the heparanase molecule based on electrostatic interactions, particularly characterized by interactions of (−1)- and (−2)-positioned sulfated sugar residues with basic amino acid residues composing the heparin-binding domain-1 of heparanase. These results propose a relevance for sulfated hyaluronan in the blocking of heparanase-mediated enzymatic and cellular actions.     

NMR Characterization of Long-Chain Fatty Acylcarnitine Binding to the Mitochondrial Carnitine/Acylcarnitine Carrier

The mitochondrial carnitine/acylcarnitine carrier (CAC) transports short-, medium- and long-carbon chain acylcarnitines across the mitochondrial inner membrane in exchange for carnitine. How CAC recognizes the substrates with various fatty acyl groups, especially long-chain fatty acyl groups, remains unclear. Here, using nuclear magnetic resonance (NMR) technology, we have shown that the CAC protein reconstituted into a micelle system exhibits a typical six transmembrane structure of the mitochondrial carrier family. The chemical shift perturbation patterns of different fatty acylcarnitines suggested that the segment A76–G81 in CAC specifically responds to the long-chain fatty acylcarnitine. Molecular dynamics (MD) simulations of palmitoyl-L-carnitine inside the CAC channel showed the respective interaction and motion of the long-chain acylcarnitine in CAC at the cytosol-open state and matrix-open state. Our data provided a molecular-based understanding of CAC structure and transport mechanism.