植物甘油脂合成途径第一步酰化反应的研究进展

甘油脂是生物膜的主要组成成分,参与能量与信号转导、蛋白转运等一系列生物学过程,在植物的生长发育过程中发挥着重要作用。3-磷酸甘油酰基转移酶（Glycerol-3-phosphate acyltransferase, GPAT）催化磷脂酸从头生物合成的第一步关键反应,磷脂酸不仅是膜脂与中性三酰甘油的合成前体,同时还是一个重要的信号分子。然而,目前仍不清楚植物中GPAT酶究竟由多少基因编码的,造成这种现象的一个主要原因是缺乏可简易且有效地鉴定此酶的方法。本文分析总结甘油脂生物合成及GPAT基因克隆与鉴定的研究进展;随后介绍GPATs的鉴定方法,特别是酵母遗传互补法的建立与运用;最后对甘油脂合成途径第一步反应的未来研究进行展望。     

甘蔗黄叶病毒与花叶病毒CP基因RNAi载体构建与转化甘蔗研究

甘蔗黄叶病和甘蔗花叶病是我国甘蔗最主要的病毒病,感病品种产量下降和品质变劣,传统方法难以防治。RNA干扰技术使培育抗病毒甘蔗品种成为可能。本研究根据甘蔗黄叶病毒和侵染甘蔗的高粱花叶病毒结构与功能特性,广泛收集不同来源病毒分离物CP基因序列,经过比对选取保守序列作为干扰序列。通过在序列两端添加酶切位点,合成并连接成双价甘蔗黄叶病和花叶病毒干扰序列,然后构建到中间载体p HANNIBAL上,形成双价RNAi干扰结构,最后插入到p ART27上形成干扰表达载体。利用基因枪轰击甘蔗品种福农15号愈伤组织进行转化,经G418筛选获得抗性再生植株。通过提取DNA和RNA分析,证实双价RNAi干扰结构不仅以不同拷贝整合到甘蔗基因组中,而且得到了转录表达。     

Mechanism of Action and Therapeutic Potential of the β-Hairpin Antimicrobial Peptide Capitellacin from the Marine Polychaeta Capitella teleta

Among the most potent and proteolytically resistant antimicrobial peptides (AMPs) of animal origin are molecules forming a β-hairpin structure stabilized by disulfide bonds. In this study, we investigated the mechanism of action and therapeutic potential of the β-hairpin AMP from the marine polychaeta Capitella teleta, named capitellacin. The peptide exhibits a low cytotoxicity toward mammalian cells and a pronounced activity against a wide range of bacterial pathogens including multi-resistant bacteria, but the mechanism of its antibacterial action is still obscure. In view of this, we obtained analogs of capitellacin and tachyplesin-inspired chimeric variants to identify amino acid residues important for biological activities. A low hydrophobicity of the β-turn region in capitellacin determines its modest membranotropic activity and slow membrane permeabilization. Electrochemical measurements in planar lipid bilayers mimicking the E. coli membrane were consistent with the detergent-like mechanism of action rather than with binding to a specific molecular target in the cell. The peptide did not induce bacterial resistance after a 21-day selection experiment, which also pointed at a membranotropic mechanism of action. We also found that capitellacin can both prevent E. coli biofilm formation and destroy preformed mature biofilms. The marked antibacterial and antibiofilm activity of capitellacin along with its moderate adverse effects on mammalian cells make this peptide a promising scaffold for the development of drugs for the treatment of chronic E. coli infections, in particular those caused by the formation of biofilms.     

Antiviral activities and putative identification of compounds in microbial extracts from the Hawaiian coastal waters

Marine environments are a rich source of significant bioactive compounds. The Hawaiian archipelago, located in the middle of the Pacific Ocean, hosts diverse microorganisms, including many endemic species. Thirty-eight microbial extracts from Hawaiian coastal waters were evaluated for their antiviral activity against four mammalian viruses including herpes simplex virus type one (HSV-1), vesicular stomatitis virus (VSV), vaccinia virus and poliovirus type one (poliovirus-1) using in vitro cell culture assay. Nine of the 38 microbial crude extracts showed antiviral potencies and three of these nine microbial extracts exhibited significant activity against the enveloped viruses. A secosteroid, 5α(H),17α(H),(20R)-beta-acetoxyergost-8(14)-ene was putatively identified and confirmed to be the active compound in these marine microbial extracts. These results warrant future in-depth tests on the isolation of these active elements in order to explore and validate their antiviral potential as important therapeutic remedies.     

Optimization of a Chemiluminescent Dot-Blot Immunoassay for Detection of Potato Viruses

Vegetative propagation of potato leads to virus accumulation, resulting in significant yield losses and reduced quality. Virus identification is critical for developing disease management strategies and measuring seed lot health. The most widely used method of virus diagnosis in seed potatoes is a post-harvest test, for which the enzyme-linked immunosorbent assay (ELISA) is often used. ELISA was previously modified by substituting microtiter plates with membranes to develop a more flexible and inexpensive assay. We optimized a dot-blot immunoassay with viral proteins bound to a polyvinylidene fluoride (PVDF) membrane and detection of the proteins with alkaline phosphatase-labeled antibodies and a chemiluminescence reagent. The assay was tested for detection of viruses of seven genera. We have also altered the assay by spotting an antibody array onto a PVDF membrane and tested it for its potential uses as a diagnostic tool for Potato virus Y, Hosta virus X, and Potato leafroll virus.     

Dietary Peptides from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. Reduced AOM/DSS-Induced Colitis-Associated Colon Carcinogenesis in Balb/c Mice

The aim was to evaluate the antineoplastic potential of a previously characterized peptide extract from the non-digestible fraction of common bean cv. Azufrado Higuera (AH) and its most abundant pure peptide GLTSK, in an azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colitis-associated colon carcinogenesis Balb/c mice model. The healthy control (C-) had no induction and no treatment, and the induced control (C+) had induction but no treatment. Groups AH and GLTSK were administered 50 mg/kg-bw of AH or GLTSK, respectively. The administration of AH and GLTSK decreased (p < 0.05) the disease activity index (DAI) compared to C+ (5.8, 9.1, 11.8, respectively). Furthermore, AH reduced the number of evident neoplasms compared to group C+ (1.8, 5.9 neoplasms/mice, respectively). The results suggest that peptides from common bean cv. Azufrado Higuera could prevent colitis-associated colon carcinogenesis.     

pH-Dependent Solution Structure and Activity of a Reduced Form of the Host-Defense Peptide Myticin C (Myt C) from the Mussel Mytilus galloprovincialis

Myticin C (Myt C) is a highly variable host-defense peptide (HDP) associated to the immune response in the mediterranean mussel (Mytilus galloprovincialis), which has shown to be active across species due to its strong antiviral activity against a fish rhabdovirus found in fish cells overexpressing this HDP. However, the potential antimicrobial properties of any synthetic analogue of Myt C has not yet been analysed. Thus, in this work we have synthesised the sequence of the mature peptide of Myt C variant c and analysed the structure activity relationships of its reduced (non-oxidized) form (red-MytCc). In contrast to results previously reported for oxidized isoforms of mussel myticins, red-MytCc was not active against bacteria at physiological pH and showed a moderate antiviral activity against the viral haemorrhagic septicaemia (VHS) rhabdovirus. However, its chemotactic properties remained active. Structure/function studies in neutral and acid environments by means of infrared spectroscopy indicated that the structure of red-MytCc is pH dependent, with acid media increasing its alpha-helical content. Furthermore, red-MytCc was able to efficiently aggregate artificial phospholipid membranes at low pH, as well as to inhibit the Escherichia coli growth, suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of red-Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial/immunomodulatory activities. On the other hand, the pH-dependent direct antimicrobial activity of Myt C suggests that this HDP may be a suitable template for the development of antimicrobial agents that would function selectively in specific pH environments, which are sorely needed in this “antibiotic-resistance era”.     

The Effect of Cholesterol on the Long-Range Network of Interactions Established among Sea Anemone Sticholysin II Residues at the Water-Membrane Interface

Actinoporins are α-pore forming proteins with therapeutic potential, produced by sea anemones. Sticholysin II (StnII) from Stichodactyla helianthus is one of its most extensively characterized members. These proteins remain stably folded in water, but upon interaction with lipid bilayers, they oligomerize to form a pore. This event is triggered by the presence of sphingomyelin (SM), but cholesterol (Chol) facilitates pore formation. Membrane attachment and pore formation require changes involving long-distance rearrangements of residues located at the protein-membrane interface. The influence of Chol on membrane recognition, oligomerization, and/or pore formation is now studied using StnII variants, which are characterized in terms of their ability to interact with model membranes in the presence or absence of Chol. The results obtained frame Chol not only as an important partner for SM for functional membrane recognition but also as a molecule which significantly reduces the structural requirements for the mentioned conformational rearrangements to occur. However, given that the DOPC:SM:Chol vesicles employed display phase coexistence and have domain boundaries, the observed effects could be also due to the presence of these different phases on the membrane. In addition, it is also shown that the Arg51 guanidinium group is strictly required for membrane recognition, independently of the presence of Chol.     

茶的抗病毒作用研究进展

茶是世界公认的健康饮料,其消费群体在不断扩大。近30年来,茶的抗病毒研究成果不断涌现。综述了茶叶及其功能成分(尤其是茶多酚)对流感病毒、冠状病毒、肝炎病毒、人类免疫缺陷病毒等病毒的干预作用,并简要阐述了其作用机制。茶叶及其功能成分的抗病毒作用主要集中于体外生化研究和细胞试验,比较缺乏动物试验、人体临床试验,以及人体流行病学调查等研究。人们日常饮茶过程中,茶叶及其功能成分能否发挥抗病毒作用尚不明确。因此,研究人员需要开展更多的大规模随机化干预试验和人群流行病学调查等研究,进一步探讨茶叶及其功能成分的抗病毒作用。     

Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 μg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 μg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.     

酵母双杂交系统筛选与水稻条纹病毒Pc2互作的寄主蛋白基因片段

水稻条纹病毒编码的Pc2蛋白在病毒侵染及宿主症状发生中起作用。研究其与寄主间的分子互作有助于揭示病毒侵染与致病的分子机制。分别将编码Pc2 N端（Pc2-N）与Pc2 C端（Pc2-C）的基因片段克隆到酵母双杂交诱饵载体pGBKT7上，并构建水稻叶片cDNA文库，然后分别用Pc2-N和Pc2-C为诱饵筛选文库寻找与其互作的寄主因子。结果表明，诱饵载体中插入的Pc2-N和Pc2-C编码框和氨基酸序列均正确。Pc2-N对酵母菌株AH109无毒性和自主激活能力，但Pc2-C对酵母具有细胞毒性。文库滴度为4×107 CFU/mL，且大多数插入片段在500~2 000 bp之间。利用Pc2-N为诱饵筛选水稻cDNA文库，获得有8个候选阳性克隆。经序列测定和BLAST分析结果表明，这些克隆共编码5种蛋白，主要为胁迫相关蛋白与光系统相关蛋白。     

Preparation and Characterization of Tilapia Collagen-Thermoplastic Polyurethane Composite Nanofiber Membranes

Marine collagen is an ideal material for tissue engineering due to its excellent biological properties. However, the limited mechanical properties and poor stability of marine collagen limit its application in tissue engineering. Here, collagen was extracted from the skin of tilapia (Oreochromis nilotica). Collagen-thermoplastic polyurethane (Col-TPU) fibrous membranes were prepared using tilapia collagen as a foundational material, and their physicochemical and biocompatibility were investigated. Fourier transform infrared spectroscopy results showed that thermoplastic polyurethane was successfully combined with collagen, and the triple helix structure of collagen was retained. X-ray diffraction and differential scanning calorimetry results showed relatively good compatibility between collagen and TPU.SEM results showed that the average diameter of the composite nanofiber membrane decreased with increasing thermoplastic polyurethane proportion. The mechanical evaluation and thermogravimetric analysis showed that the thermal stability and tensile properties of Col-TPU fibrous membranes were significantly improved with increasing TPU. Cytotoxicity experiments confirmed that fibrous membranes with different ratios of thermoplastic polyurethane content showed no significant toxicity to fibroblasts; Col-TPU fibrous membranes were conducive to the migration and adhesion of cells. Thus, these Col-TPU composite nanofiber membranes might be used as a potential biomaterial in tissue regeneration.     

大麦黄矮病毒运动蛋白及其介导的小麦抗病性研究进展

麦黄矮病毒依靠介体蚜虫传播引起小麦黄矮病,对我国麦类作物生产造成严重威胁。本文结合本课题组的研究,对大麦黄矮病毒编码的运动蛋白的生化特性、生物学功能及其介导小麦的抗病性等方面的研究进展进行了综述。该运动蛋白在病毒构成中能够结合病毒核酸,并与宿主细胞膜系统相互作用,是一个与黄矮病毒系统侵染相关的致病因子。     

Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC₅₀ values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC₅₀ values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.     

Characteristics of Marine Biomaterials and Their Applications in Biomedicine

Oceans have vast potential to develop high-value bioactive substances and biomaterials. In the past decades, many biomaterials have come from marine organisms, but due to the wide variety of organisms living in the oceans, the great diversity of marine-derived materials remains explored. The marine biomaterials that have been found and studied have excellent biological activity, unique chemical structure, good biocompatibility, low toxicity, and suitable degradation, and can be used as attractive tissue material engineering and regenerative medicine applications. In this review, we give an overview of the extraction and processing methods and chemical and biological characteristics of common marine polysaccharides and proteins. This review also briefly explains their important applications in anticancer, antiviral, drug delivery, tissue engineering, and other fields.     

Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.     

SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.     

Marine bacterial sialyltransferases

Sialyltransferases transfer N-acetylneuraminic acid (Neu5Ac) from the common donor substrate of these enzymes, cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), to acceptor substrates. The enzymatic reaction products including sialyl-glycoproteins, sialyl-glycolipids and sialyl-oligosaccharides are important molecules in various biological and physiological processes, such as cell-cell recognition, cancer metastasis, and virus infection. Thus, sialyltransferases are thought to be important enzymes in the field of glycobiology. To date, many sialyltransferases and the genes encoding them have been obtained from various sources including mammalian, bacterial and viral sources. During the course of our research, we have detected over 20 bacteria that produce sialyltransferases. Many of the bacteria we isolated from marine environments are classified in the genus Photobacterium or the closely related genus Vibrio. The paper reviews the sialyltransferases obtained mainly from marine bacteria.     

Antiviral lead compounds from marine sponges

Marine sponges are currently one of the richest sources of pharmacologically active compounds found in the marine environment. These bioactive molecules are often secondary metabolites, whose main function is to enable and/or modulate cellular communication and defense. They are usually produced by functional enzyme clusters in sponges and/or their associated symbiotic microorganisms. Natural product lead compounds from sponges have often been found to be promising pharmaceutical agents. Several of them have successfully been approved as antiviral agents for clinical use or have been advanced to the late stages of clinical trials. Most of these drugs are used for the treatment of human immunodeficiency virus (HIV) and herpes simplex virus (HSV). The most important antiviral lead of marine origin reported thus far is nucleoside Ara-A (vidarabine) isolated from sponge Tethya crypta. It inhibits viral DNA polymerase and DNA synthesis of herpes, vaccinica and varicella zoster viruses. However due to the discovery of new types of viruses and emergence of drug resistant strains, it is necessary to develop new antiviral lead compounds continuously. Several sponge derived antiviral lead compounds which are hoped to be developed as future drugs are discussed in this review. Supply problems are usually the major bottleneck to the development of these compounds as drugs during clinical trials. However advances in the field of metagenomics and high throughput microbial cultivation has raised the possibility that these techniques could lead to the cost-effective large scale production of such compounds. Perspectives on biotechnological methods with respect to marine drug development are also discussed.