Homology modelling and molecular dynamics studies of human placental tissue protein 13 (galectin-13)

The primary structure of the newly sequence analysed placentaltissue protein 13 (PP13) was highly homologous to several membersof the ß-galactoside-binding S-type lectin (galectin)family. By homology modelling, the three-dimensional structureof PP13 was built based on high-resolution crystal structuresof homologues and also their characteristic `jellyroll' foldwas found in the case of PP13. Our model has been depositedin the Brookhaven Protein Data Bank. By multiple sequence alignmentand structure-based secondary structure prediction, we underlinedthe structural similarity of PP13 with its homologues. The secondarystructure of PP13 was identical with `proto-type' galectinsconsisting of a five- and a six-stranded ß-sheet,joined by two     

Homology modelling of transferrin-binding protein A from Neisseria meningitidis

Neisseria meningitidis, a causative agent of bacterial meningitis, obtains transferrin-bound iron by expressing two outer membrane located transferrin-binding proteins, TbpA and TbpB. TbpA is thought to be an integral outer membrane pore that facilitates iron uptake. Evidence suggests that TbpA is a useful antigen for inclusion in a vaccine effective against meningococcal disease, hence the identification of regions involved in ligand binding is of paramount importance to design strategies to block uptake of iron. The protein shares sequence and functional similarities to the Escherichia coli siderophore receptors FepA and FhuA, whose structures have been determined. These receptors are composed of two domains, a 22-stranded beta-barrel and an N-terminal plug region that sits within the barrel and occludes the transmembrane pore. A three-dimensional TbpA model was constructed using FepA and FhuA structural templates, hydrophobicity analysis and homology modelling. TbpA was found to possess a similar architecture to the siderophore receptors. In addition to providing insights into the highly immunogenic nature of TbpA and allowing the prediction of potentially important ligand-binding epitopes, the model also reveals a narrow channel through its entire length. The relevance of this channel and the spatial arrangement of external loops, to the mechanism of iron translocation employed by TbpA is discussed.     

Inhibitor(s) of platelet-activating factor (PAF) in human saliva

Quantitation of platelet-activating factor (PAF) in human saliva samples by radioimmunoassay indicated there was, at times, sufficient PAF present to aggregate platelets. However, in certain samples, we observed little or no aggregation, and furthermore, these samples were found to inhibit aggregation induced by PAF (200 pg). Chromatographic fractionation of pooled saliva increased the PAF activity 4-fold, and the observed inhibitory activity was found to co-migrate with the fatty acids. The inhibitory fraction was found to be active against platelet aggregation induced by arachidonic acid (3.4 nmole) as well as PAF (25 pg), but not thrombin (20 mU). These results indicate the existence of a PAF inhibitor in saliva, which may explain why potentially toxic levels of PAF can occur in the saliva of normal, healthy individuals. These findings also highlight an important advantage of the radioimmunoassay over platelet aggregation for the quantitation of PAF in, at least, some biological fluids. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Homology modelling of two subtilisin-like proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri

The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 20426-20431). Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri. Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures. Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e. subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization. Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. These highly thermostable proteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues. The latter are often present in putative N-glycosylation sites. Results from modelling of known substrates in the substrate- binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.      

Homology modeling study of the human interleukin-7 receptor complex

Following a recent model of human interleukin-7 (IL-7), we presenthere a modeling study of the extracellular part of the humanIL-7 receptor complex, including the IL-7 specific (IL-7R) andthe common gamma (_c) chains. The investigation is based on structuralhomology to the complex of human growth hormone (hGH) boundto its receptor (hGHR). For domain 1 of IL-7R two differentmodels are presented which differ in the alignment to hGHR inthree regions. However, these differences affect binding toIL-7 in only one region, at the interface between loop EF ofdomain 1 of IL-7R and helix C of IL-7. The disulfide patternin domain 1 of IL-7R is predicted to deviate from that observedin hGHR in that the C'–E disulfide (hGHR) is replacedby a C-C' cross-link. The prediction for the _c chain is comparedwith two previous studies. The models of the complex provideinsight into the binding of IL-7 to its receptor and have implicationsfor the suggestion of mutagenesis experiments and the designof (ant)agonists.     

人肝细胞生长因子基因真核表达质粒的构建及其在人骨髓间充质干细胞中的表达

目的构建人肝细胞生长因子(Human hepatocyte growth factor,hHGF)真核表达质粒,并检测其在人骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)中的表达及其对细胞生长的影响。方法采用密度梯度法从人骨髓中分离BMSCs,流式细胞术检测细胞表型,成脂及成骨诱导其分化。PCR扩增hHGF基因,定向克隆至pEGFP-N1载体中,构建重组表达质粒pEGFP-N1-hHGF,通过电穿孔法转染BMSCs,荧光显微镜下观察增强型绿色荧光蛋白的表达,RT-PCR及Western blot法检测hHGF基因mRNA的转录及蛋白的表达,MTT法检测hHGF对BMSCs增殖活力的影响。结果 BMSCs高表达CD29和CD44,不表达CD34和CD45;BMSCs在体外有向成脂及成骨细胞诱导分化的能力。酶切及基因测序证实,重组表达质粒pEGFP-N1-hHGF构建正确;转染后48 h观察到转染细胞中有绿色荧光蛋白表达;RT-PCR法和Western blot检测到hHGF基因在BMSCs中表达;转染hHGF基因的BMSCs增殖活力明显高于空白对照组和空载体转染组(P<0.05)。结论成功构建了hHGF基因真核表达质粒,转染人BMSCs后获得表达,表达的hHGF可促进BMSCs增殖。     

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

A model is presented for the 3-D structure of the catalyticdomain of the putative leader peptidase NisP of Lactococcuslactis, and the interaction with its specific substrate, theprecursor of the lantibiotic nisin. This homology model is basedon the crystal structures of subtilisin BPN' and thermitasein complex with the inhibitor eglin. Predictions are made ofthe general protein fold, inserted loops, Ca²⁺ binding sites,aromatic interactions and electrostatic interactions of NisP.Cleavage of the leader peptide from precursor nisin by NisPis the last step in maturation of nisin. A detailed predictionof the substrate binding site attempts to explain the basisof specificity of NisP for precursor nisin. Specific acidicresidues in the SI subsite of the substrate binding region ofNisP appear to be of particular importance for electrostaticinteraction with the PI Arg residue of precursor nisin afterwhich cleavage occurs. The hydrophobic S4 subsite of NisP mayalso contribute to substrate binding as it does in subtilisins.Predictions of enzyme-substrate interaction were tested by proteinengineering of precursor nisin and determining susceptibilityof mutant precursors to cleavage by NisP. An unusual propertyof NisP predicted from this catalytic domain model is a surfacepatch near the substrate binding region which is extremely richin aromatic residues. It may be involved in binding to the cellmembrane or to hydrophobic membrane proteins, or it may serveas the recognition and binding region for the modified, hydrophobicC-terminal segment of precursor nisin. Similar predictions forthe tertiary structure and substrate binding are made for thehighly homologous protein EpiP, the putative leader peptidasefor the lantibiotic epidermin from Staphylococcus epidermidis,but EpiP lacks the aromatic patch. Based on these models, proteinengineering can be employed not only to test the predicted enzyme-substrateinteractions, but also to design lantibiotic leader peptidaseswith a desired specificity.     

Spectroscopic studies and first-principles modelling of 2,2,4-trifluoro-5-trifluoromethoxy-1,3-dioxole (TTD) and TTD-TFE copolymers (Hyflon AD)

Amorphous fluorinated optical polymers, characterized by high transparency in the visible and near infrared spectra, high glass transition temperature and very good resistance to chemical environment, have been developed by co-polymerisation of tetrafluoroethylene (TFE) and 2,2,4-trifluoro-5-trifluoromethoxy-1,3-dioxole (TTD). In this work we study at molecular level the effect of the introduction of the bulky TTD unit in a perfluoroalkyl chain.In particular the effect on the molecular structure and chain flexibility is investigated and spectroscopic markers correlated to chemical and structural defects are identified. The study includes a thorough experimental spectroscopic analysis (infrared and Raman spectra) of several different copolymer samples and a modelling based on Density Functional Theory calculations and semiempirical calculations on suitable model molecules.     

Selecting the forgetting factor in subset autoregressive modelling

Abstract. Conventional methods to determine the forgetting factors in autoregressive (AR) models are mostly based on arbitrary or personal choices. In this paper, we present two procedures which can be used to select the forgetting factor in subset AR modelling. The first procedure uses the bootstrap to determine the value of a fixed forgetting factor. The second procedure starts from this base and applies the time-recursive maximum likelihood estimation to a variable forgetting factor. In one illustration using real exchange rates, we demonstrate the effect of the forgetting factor in subset AR modelling on ex ante forecasting of non-stationary time series. In a second illustration, these two procedures are applied to time-update forecasts for a stock market index. Subset AR models not including a forgetting factor act as a set of benchmarks for assessing ex ante forecasting performance, and consistently improved forecasting performance is demonstrated for these proposed procedures.     

Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5-RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as N(alpha)-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function.     

Pore initiation and growth on n-InP(100)

Pore growth on n-type InP(100) can be electrochemically initiated in HCl and HF solutions by polarizing the material anodic to a critical potential value—the pore formation potential (PFP). At surface defects however, the PFP is significantly lower (shifted cathodically). This is evident from simple scratch experiments as well as experiments using focused ion beam (FIB) implantation of Si²⁺ to write defined surface damage/implant patterns into n-type InP(100) substrates. These implant sites represent initiation sites for the onset of dissolution processes. Selective pore formation within the implant patterns is achieved if polarization of the n-type material is carried out anodic to the PFP corresponding to defects but cathodic to the PFP of the unimplanted surface. Furthermore, it is shown that the pore morphology depends strongly on the type of halogen acid present in the electrolyte. Scanning AES images show that the composition of the porous layers is strongly altered from the bulk composition.     

Role of platelet-activating factor (PAF) in superoxide production by human polymorphonuclear leukocytes

The effect of platelet-activating factor (PAF) in superoxide production by human polymorphonuclear leukocytes (PMN) was studied. Cypridina luciferin analog (CLA) dependent chemiluminescence was used to detect superoxide anion radicals. PAF induced superoxide generation in human PMN in a dose-dependent manner. Preincubation with a small amount of PAF (5 x 10⁻⁹ M) enhanced PMN superoxide release induced by various stimuli, such as phorbol myristate acetate (PMA), opsonized zymosan (OZ), calcium ionophore (A23187) andN-formyl-methionyl-leucyl-phenylalanine (FMLP). The PAF antagonist, CV-6209, inhibited superoxide production induced by PAF, but not that induced by other stimuli. These findings would indicate that PAF may play an important role at inflammatory reaction sites and that CV-6209 may inhibit excessive inflammatory reaction.     

A growth inhibitory factor from lambsquarters (Chenopodium album)

Aqueous extract of air-dried lambsquarters (Chenopodium album) at 25 mg/ml significantly inhibited germination and growth of radish and wheat seeds. Soybean seed germination was not inhibited; however, hypocotyl growth was significantly reduced. Germination of radish seeds in sand amended with pulverized lambsquarters shoots at 2 and 4 mg/g was reduced 40 and 95%, respectively. Shoot dry weight and plant height were also reduced 30 and 9%, respectively, at 4 mg/g, but not at 2 mg/g concentration. Residues after extraction with water incorporated in sand were not inhibitory, indicating water solubility of the inhibitor(s). Aqueous extract of shoots decomposed for five days lost nearly 40% of its inhibitory effect; 20% of it still persisted in the extract of shoots decomposed for 30 days. The filtrate from ultrafiltration of aqueous extract through a pad of molecular-weight cutoff 1000 inhibited radish seeds germination and growth, indicating that the molecular weight of the inhibitor(s) was less than 1000. Partitioning of the aqueous extract by a series of solvents resulted in isolation of an inhibitor(s) in the butanol fraction. Seven phenolics were identified in this fraction using high-performance liquid chromatography (HPLC). Paper chromatographic analysis of the butanol fraction revealed six bands, of which one band withR _f =0.83 inhibited germination and growth of radish seeds. Chlorogenic acid identified by HPLC appeared to be the principal component of the phytotoxin.     

一种简便的松果腺素的合成方法

以邻苯二甲酰亚胺钾、１,３二溴丙烷、对甲氧基苯胺为主要原料,经５步化学反应合成了松果腺素：邻苯二甲酰亚胺钾和１,３－二溴丙烷在１５５～１６０℃下反应６ｈ,得到Ｎ－（３－溴丙基）,邻苯二甲酸亚胺（Ⅱ）,产率７７％;Ⅱ在乙醇钠存在下和乙酰乙酸乙酯作用得到２－乙酸基－５－邻苯二甲酰亚氨基戊酸乙酯（Ⅲ）,产率７２％;Ⅲ与对甲氧基苯胺重氮盐偶联后发生环化作用,生成Ｔ２－羧乙基－３－（２－邻苯二甲酰亚氨乙基）－５－甲氧基吲哚（Ⅳ）,产率７４％;Ⅳ经碱皂化、水解、脱羧反萃取纯化,得到５．甲氧基色胺（Ｖ）,产率６６％;Ｖ经乙酸化后得到松果腺素（Ⅵ）,产率８２％。     

(Z)-5-溴甲叉基-2(5H)-呋喃酮的合成

以呋喃酮和顺丁烯二酸酐为原料,经Diels-Alder反应和Wittig反应得到(E)-2-(2-(5H)-呋喃酮-5-叉)-乙酸叔丁酯,酸解脱去叔丁基得(E)-2-[2-(5H)-呋喃酮-5-叉]-乙酸,利用溴化锂和高碘酸钠反应原位生成溴进行溴代脱羧反应生成目标化合物。     

Carbon Nanoparticles on Magnetite: A New Heterogeneous Catalyst for the Oxidation of 5-Hydroxymethylfurfural (5-HMF) to 2,5-Diformoylfuran (DFF)

Development of new materials for catalytic applications is one of the rapidly growing research areas. The selective oxidation of 5-hydroxymethyl furfural (HMF) to 2,5-diformylfuran (DFF) is one of the rapidly growing research area due to having its own importance in the fuel technology. We report a new heterogeneous catalyst for the oxidation of HMF to DFF in aqueous medium using a carbon soot (from candle light) deposited on magnetite (Fe₃O₄@C). Further, the potentiality of our catalyst has also been realized in direct production of DFF from biomass (using either fructose or glucose) with high conversions via two consecutive steps involving dehydration and oxidation. Further, proved that Fe₃O₄@C plays a major role in the dehydration of sugar molecules whereas Fe₃O₄ alone could not.

     

4-氯-2-氰基-5-(4-甲基苯基)咪唑的合成研究

以对甲基苯乙酮为起始原料,经二氧化硒氧化制得4-甲基苯基乙二醛,在水与甲醇的混合溶剂中,与盐酸羟胺、乙二醛水溶液环化反应得4-羟基-5-(4-甲基苯基)-2-肟基次甲基咪唑-3-氧,再以N,N-二甲基甲酰胺为溶剂,与二氯亚砜反应脱水氯化得标题化合物,总收率69.0%。