Identification and genetic characterization of Anisakis larvae from marine fishes in the South China Sea using an electrophoretic‐guided approach

From 35 species of marine fishes (n = 327) from the South China Sea, 237 nematode larvae were collected and identified morphologically as Anisakis. Genomic DNA was isolated from each larva and subjected to PCR‐based RFLP and targeted sequencing of a nuclear ribosomal DNA region between the 3′‐end of the small subunit and 5′‐end of the large subunit of the rRNA genes (= internal transcribed spacers, ITS+). Four different RFLP profile combinations (sets) were detected for all restriction endonucleases (HinfI, HhaI, and TaqI), of which three were characteristic of Anisakis typica, A. pegreffii, and A. physeteris, respectively. One profile set (for sample CA‐2012) was linked to an ITS+ sequence that was identical to a previously published sequence of Anisakis sp. (sample HC‐2005; originating from the African shelf) and another sequence (PH‐2010; Madeira, Portugal). Phylogenetic analysis was carried out using the ITS+ sequence data from this study and reference sequences from the GenBank database. Neighbor joining and maximum parsimony trees displayed three clades. Clades I and II included nine described species of Anisakis, including all type I and type II larvae; clade III represented some undescribed species of Anisakis. Morphological comparison showed that Anisakis sp. CA‐2012 was distinct from type I and type II larvae based on its tail shape and ratio of tail length to body length. The phylogenetic analysis and morphological characters suggest that Anisakis sp. CA‐2012 represents a new record, now called Anisakis type III larvae.     

Separation and quantification of histone H1 subtypes and high-mobility-group proteins by reversed-phase liquid chromatography: protein levels in rat tissues during postnatal development

The rapid separation and quantification of histone H1 subtypes and high-mobility-group (HMG) chromatin proteins by reversed-phase liquid chromatography on a butylsilica-based column is described. The proteins were fractionated by means of a multi-step acetonitrile gradient containing 0.1% trifluoroacetic acid. This system is capable of resolving the four main HMG proteins (1, 2, 14 and 17), HMG I, protein P1 with HMG 18 and HMG 19 (in one peak) and five histone H1 subtypes in a single 33-min analysis. This method was used to study levels of these chromosomal proteins in nuclei of rat liver, spleen, testis and thymus during postnatal development from 1 to 20 weeks of age. Although no clear tissue specificity of the HMG proteins was apparent, there were significant differences in the relative amounts of these proteins in different tissues. The relative amount of HMG 1 increased from 1 to 12 weeks of age and decreased thereafter, whereas those of HMG 14 and HMG 17 remained almost unchanged. Marked quantitative differences were observed in the five histone H1 subtypes in different tissues. The largest changes in their levels during development were found in the liver and the smallest changes in the thymus. The changes in the spleen and testis were intermediate. These results suggest that the changes in the relative amounts of histone H1 subtypes and HMG proteins observed during postnatal development of the rat may result from differences in the structure of chromatin in these tissues and thus reflect the activity of molecular mechanisms involved in replication and differentiation of the cells.     

In silico structure-based design of enhanced peptide inhibitors targeting RNA polymerase PAN-PB1C interaction

Developing antivirals for influenza A virus (FluA) has become more challenging due to high range of antigenic mutation and increasing numbers of drug-resistant viruses. Finding a selective inhibitor to target highly conserved region of protein-protein interactions interface, thereby increasing its efficiency against drug resistant virus could be highly beneficial. In this study, we used in silico approach to derive FluAPep1 from highly conserved region, PA_N-PB1_C interface and generated 121 FluAPep1 analogues. Interestingly, we found that the FluAPep1 interaction region in the PA_N domain are highly conserved in many FluA subtypes. Especially, FluAPep1 targets two pandemic FluA strains, H1N1/avian/2009 and H3N2/Victoria/1975. All of these FluA subtypes PA_N domain (H1N1/H3N2CAN/H3N2VIC/H7N1/H7N2) were superimposed with PA_N domain from H17N10 and the calculated root mean standards deviations were less than 3 Å. FlexPepDock analysis revealed that FluAPep1 exhibited higher binding affinity (score -246.155) with the PA_N domain. In addition, around 86% of non-hot spot mutated peptides (FluAPep28-122) showed enhanced binding affinity with PA_N domain. ToxinPred analysis confirmed that designed peptides were non-toxic. Thus, FluAPep1 and its analogues has potential to be further developed into an antiviral treatment against FluA infection.     

Redesign of protein domains using one-bead-one-compound combinatorial chemistry

A novel combinatorial strategy for the redesign of proteins based on the strength and specificity of intra- and interprotein interactions is described. The strategy has been used to redesign the hydrophobic core of the B domain of protein A. Using one-bead-one-compound combinatorial chemistry, 300 analogues of the C-terminal alpha-helix of the B domain, H3x, have been synthesized using a biocompatible resin and the HMFS linker, allowing the screening to occur while the peptides were bound to the resin. The screening was based on the ability of the H3x analogues to interact with the N-terminal helices of the B domain, H1-H2, and retain the native B domain activity, that is binding to IgG. Eight different analogues containing some nonconservative mutations were obtained from the library, the two most frequent of which, H3P1 and H3P2, were studied in detail. CD analysis revealed that the active analogues interact with H1-H2. To validate the redesign strategy the covalent modified domains H1-H2-H3P1 and H1-H2-H3P2 were synthesized and characterized. CD and NMR analysis revealed that they had a unique, stable, and well-defined three-dimensional structure similar to that for the wild-type B domain. This combinatorial strategy allows us to select for redesigned proteins with the desired activity or the desired physicochemical properties provided the right screening test is used. Furthermore, it is rich in potential for the chemical modification of proteins overcoming the drawbacks associated with the total synthesis of large protein domains.     

Phylogenetic Analysis of Putative Genes Involved in the Tryptophan-Dependent Pathway of Auxin Biosynthesis in Rice

Plant proteome databases were mined for a flavin monooxygenase (YUCCA), tryptophan decarboxylase (TDC), nitrilase (NIT), and aldehyde oxidase (AO) enzymes that could be involved in the tryptophan-dependent pathway of auxin biosynthesis. Phylogenetic trees for enzyme sequences obtained were constructed. The YUCCA and TDC trees showed that these enzymes were conserved across the plant kingdom and therefore could be involved in auxin synthesis. YUCCAs branched into two clades. Most experimentally studied YUCCAs were found in the first clade. The second clade which has representatives from only seed plants contained Arabidopsis sequences linked to embryonic development. Therefore, sequences in this clade were suggested to be evolved with seed development. Examination of TDC activity and expression had previously linked this enzyme to secondary products synthesis. However, the phylogenetic finding of a conserved TDC clade across land plants suggested its essential role in plant growth. Phylogenetic analysis of AOs showed that plants inherited one AO. Recent gene duplication was suggested as AO sequences from each species were similar to each other rather than to AO from other species. Taken together and based on the experimental support of the involvement of AO in abscisic synthesis, AO was excluded as an intermediate in IAA production. Phylogenetic tree for NIT showed that the first clade contained sequences from species across the plant kingdom whereas the second branch contained sequences from only Brassicaceae. Even though NIT4 orthologues were conserved in the second clade, their major role seems to be detoxification of hydrogen cyanide rather than producing IAA.     

Correlation between 2H NMR side-chain order parameters and sequence conservation in globular proteins

Side-chain 2H NMR relaxation data have been collected for the SH3 domain from the Fyn tyrosine kinase and analyzed with respect to sequence preference and per-residue solvent accessibility. Residues that are highly preferred at a given position show a tendency to be less mobile than average with a coefficient of correlation that is greater than that obtained when side-chain flexibility and solvent accessibility are compared. The same trend is observed for five of six additional proteins considered. This provides evidence for the existence of conserved structural features other than hydrophobic burial that govern side-chain motions. Through examination of an SH3 domain structural alignment, we identify side-chain hydrogen bonding of threonine residues and a specific secondary structural element as potential determinants of protein internal dynamics.     

Mass spectrometric sequencing and acylation character analysis of C-terminal anchoring segment from Influenza A hemagglutinin

Influenza A virus hemagglutinin (HA) is a major envelope glycoprotein mediating viral and cell membrane fusion. HA is anchored in the viral envelope by a light HA(2) chain containing one transmembrane domain and a cytoplasmic tail. Three cysteine residues in the C-terminal region, one in the transmembrane domain and two in the cytoplasmic tail, are highly conserved and potentially palmitoylated in all HA subtypes. The HA(2) C- terminal anchoring segments were extracted to organic phase from the bromelain-digested viruses (subviral particles) of three strains: A/X-31 (H3 subtype), A/Puerto Rico/8/34 (H1 subtype) and A/FPV/Weybridge/34 (H7 subtype). Their primary structures were assessed by matrix-assisted laser desorption/ionization time-of-flight time-of- flight mass spectrometry (MALDI-ToF-ToF MS). Trypsin-type protease-cleaved peptides prevailed over bromelain- cleaved ones in the peptide mixtures. All of them included transmembrane domains. Several distinctive features of the C-terminal HA(2) peptides acylation character were discovered by MALDI-ToF MS: 1) the peptides isolated from the viruses, which were digested by bromelain in the absence of beta-mercaptoethanol, were predominantly triply acylated; 2) the peptides were acylated not only by palmitic, but also by stearic acid residues; 3) the palmitate/stearate ratio was different for the three strains studied; 4) the A/FPV/Weybridge/34 strain has a priority to stearate binding. This fatty acid residue was discovered at the first of three conservative cysteine residues located in the transmembrane domain. It was found that presence of thiol reagent during preparation of subviral particles led to the appearence of the C-terminal HA(2) peptides acylated to different degrees. Triply, doubly, mono- and even unacylated peptides were detected. It was demonstrated that the thioester bond in the isolated acylpeptides was extremely sensitive to thiol reagents.     

原位合成氧化镍硼/石墨炔杂化材料用于提高光/电催化产氢性能

当今世界面临严峻的能源紧缺和环境污染问题,发展高效无污染的清洁能源替代传统化石能源成为近几十年科研工作者的研究热点.其中,氢能由于具有高燃烧值和产物无污染等优点成为理想的替代能源.光/电催化水分解产生氢气是最有效的制氢方法之一.目前,高活性的产氢催化剂仍以贵金属为主,但贵金属价格高昂和稀缺性等限制了其大规模应用,因此,开发和设计廉价、高效的非贵金属产氢催化剂变得尤为重要.为了提高非贵金属催化剂的催化活性,基于非贵金属的复合材料的构建被广泛研究.例如,通过非贵金属和碳材料的复合,能够提高比表面积和电子传输速率,优化活性位点的电子结构,从而提高催化活性.石墨炔(GDY)作为一种新兴碳材料,由sp²-和sp-杂化碳共同组成.由于GDY具有高度π共轭结构,大的比表面积和独特的双炔键,可作为载体与非贵金属离子相互作用形成复合材料,制备高效产氢催化剂.基于此,本文在室温下原位合成了强耦合相互作用的氧化镍硼/石墨炔(NiBi/GDY)催化剂,并将其应用于光/电催化产氢反应.在光催化产氢反应中,采用曙红为光敏剂,三乙醇胺为牺牲剂,可见光下NiBi/GDY的光催化产氢速率可达4.54 mmol g^-1h^-1,产氢速率分别是氧化镍硼/石墨烯(NiBi/graphene)和NiBi的2.9倍和4.5倍.此外,NiBi/GDY在1.0 M KOH溶液中也表现出良好电催化产氢性能,电流密度为400 mA/cm²时其过电位为478.0 mV,低于商业铂碳(505.3 mV@400 mA/cm2).NiBi/GDY在光/电催化产氢实验中表现出的较好催化性能可归因于NiBi和GDY之间强耦合相互作用对NiBi电子结构的优化.上述研究结果表明,石墨炔可作为理想载体制备高效的光/电催化剂,同时本文为设计高效稳定的非贵金属产氢催化剂提供了一定的借鉴意义.     

Multiple Effects Tailoring the Self-organization Behaviors of Triphenylene Side-chain Liquid Crystalline Polymers <Emphasis Type="Italic">via</Emphasis> Changing the Spacer Length

Long-alkyl tail triphenylene (TP) side-chain liquid crystalline polymers (SCLCPs) with different spacer length (P-m-TP,m =2,3,4,6,8,which is the number of carbon atom in the flexible alkyl spacers) have been successfully synthesized via free radical polymerization.The differential scanning calorimetry (DSC),polarized light microscopy (POM),ultraviolet-visible spectroscopy (UV-Vis),wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS) measurements were performed to investigate the influence of multiple effects on the self-organization behaviors of P-m-TP,including steric effect,decoupling effect and π-π stacking effect.The experimental results revealed that P-m-TP (m =2,3,4) formed the columnar phase which was developed by the TP moieties and the main chain as a whole,suggesting that the side-chains had strong steric effect even though the number of spacer length (m)exceeded 4.In addition,the clearing points (Tis) of the polymers were above 300 ℃.When m =6 and 8,the polymers displayed hexagonal columnar phase and exhibited the low Tis (91 and 80 ℃ respectively),originating from the self-assembly of triphenylene due to the decoupling effect and π-π stacking effect.This work offers a viable and inspiring pathway to control the phase transition temperature and phase structure of TP SCLCPs via simply tailoring the spacer length and increasing the alkyl tail length of TP.     

Gemini表面活性剂联接基团对合成硅基介孔材料结构的影响

考察了Gemini表面活性剂不同联接基团对介孔结构的影响, 通过改变联接基团碳链的长度和在联接基团中加入苯基和羟基改变链的柔性和亲水性, 可改变表面活性剂在两相界面的头基面积和电荷分布, 形成不同的表面活性剂溶液结构, 并通过自组装过程控制生成不同介孔材料的孔结构. 当联接基团链长为4~8个碳时, 得到六方相孔道结构, 而当碳链长度增加为10~12个碳时, 形成立方相孔道结构, 其中以GEM16-6-16为模板, 形成了高度有序的MCM-41, 以GEM16-12-16为模板, 则得到高度有序的MCM-48. GEM16-3(OH)-16可合成出层状结构, 但有序性较低. GEM16-(1-Ar-1)-16的cmc较低, 在水溶液中溶解度极低, 当加入共溶剂乙醇时, 得到了空心球结构.     

Crystallographic proof for an extended hydrogen-bonding network in small prolyl isomerases

Parvulins compose a family of small peptidyl-prolyl isomerases (PPIases) involved in protein folding and protein quality control. A number of amino acids in the catalytic cavity are highly conserved, but their precise role within the catalytic mechanism is unknown. The 0.8 ? crystal structure of the prolyl isomerase domain of parvulin Par14 shows the electron density of hydrogen atoms between the D74, H42, H123, and T118 side chains. This threonine residue has previously not been associated with catalysis, but a corresponding T152A mutant of Pin1 shows a dramatic reduction of catalytic activity without compromising protein stability. The observed catalytic tetrad is strikingly conserved in Pin1- and parvulin-type proteins and hence constitutes a common feature of small peptidyl prolyl isomerases.     

含聚乙二醇柔性间隔段的主链型液晶聚酯的合成

分子结构与液晶行为间的关系是液晶性高分子物理问题研究的核心,通常的液晶性高分子中除液晶性基元外还引入一些相对来说很柔顺的链段,称作柔性间隔段．这是因为人们认识到高分子的液晶行为不只决定于液晶性基元的结构,也受到这些基元间以及基元和间隔段间相互作用的影...     

Recognition of major DNA adducts of enantiomeric cisplatin analogs by HMG box proteins and nucleotide excision repair of these adducts

We examined HMG domain protein recognition of major 1,2-GG intrastrand DNA crosslinks, formed by two bifunctional enantiomeric analogs of antitumor cis-diamminedichloroplatinum(II) (cisplatin), and removal of these crosslinks during in vitro nucleotide excision repair (NER) reactions. Electrophoretic mobility shift assays show that domains A and B of HMGB1 protein bind to (2R,3R)-diaminobutanedichloroplatinum(II)-generated crosslinks with a higher affinity than to those generated by (2S,3S)-diaminobutanedichloroplatinum(II). The crosslinks of both enantiomers are removed by NER with a similar efficiency; however, HMG1B protein significantly inhibits removal of the (2R,3R)-diaminobutaneplatinum(II) adduct, but not that of the (2S,3S) enantiomer. Thus, HMG domain proteins discriminate among different conformations of the 1,2-GG intrastrand crosslinks of the two enantiomeric analogs of cisplatin, which results in different NER of these crosslinks. This observation may provide insight into the mechanisms underlying antitumor activity of cisplatin and its analogs.     

Separation of friend erythroleukaemic cell histones and high-mobility-group proteins by reversed-phase high-performance liquid chromatography

A procedure for the rapid separation of histones and high-mobility-group (HMG) proteins from Friend erythroleukaemic cells (line F4N) by reversed-phase high-performance liquid chromatography is reported. By using a Nucleosil 300-5 C₄, column and a multistep water—acetonitrile gradient containing 0.1% trifluoroacetic acid, the HMG-1 and HMG-2 proteins, several his subtractions including H1⁰, H4, H2B, two H2A variants and two H3 subtractions were separated. Under changed conditions, by applying a varied acetonitrile gradient system, even two H2B variants were fractionated. The methods described seem to be a real alternative to the time-consuming polyacrylamide gel electrophoresis.     

Liquid crystalline side group polymers with azo-chromophores and fluorinated tails of varying length

A series of side group liquid crystalline polymethacrylates with 4'-(1H,1H-perfluoroalkoxy)-azobenzene mesogenic units was synthesized and characterized by differential scanning calorimetry, polarizing optical microscopy and X-ray diffraction methods depending on the tail length. The phase behaviour is discussed as a function of the length of the tail groups. Very high clearing temperatures up to 290°C were observed for the polymers with long tail groups.     

Effect of the tail‐group length and degree of polymerization on the order parameter of side‐chain liquid‐crystalline polymethacrylates containing phenylbenzoate mesogenic groups

The orientation of the side‐chain liquid‐crystalline polymers (LCP) containing phenylbenzoate mesogenic groups in the magnetic field was examined with ²H NMR spectroscopy. The influence of the degree of polymerization as well as the length of the methylene tail group (n = 1–4) have been established. The decrease of the order parameter S of the LCPs with an increased length of the tail group was found. The order parameter S of LCPs does not depend on the degree of polymerization. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 2044–2048, 2002     

Modification of polyurethane surface with an antithrombin-heparin complex for blood contact: influence of molecular weight of polyethylene oxide used as a linker/spacer

Polyurethane (PU) was modified using isocyanate chemistry to graft polyethylene oxide (PEO) of various molecular weights (range 300-4600). An antithrombin-heparin (ATH) covalent complex was subsequently attached to the free PEO chain ends, which had been functionalized with N-hydroxysuccinimide (NHS) groups. Surfaces were characterized by water contact angle and X-ray photoelectron spectroscopy (XPS) to confirm the modifications. Adsorption of fibrinogen from buffer was found to decrease by ~80% for the PEO-modified surfaces compared to the unmodified PU. The surfaces with ATH attached to the distal chain end of the grafted PEO were equally protein resistant, and when the data were normalized to the ATH surface density, PEO in the lower MW range showed greater protein resistance. Western blots of proteins eluted from the surfaces after plasma contact confirmed these trends. The uptake of ATH on the PEO-modified surfaces was greatest for the PEO of lower MW (300 and 600), and antithrombin binding from plasma (an indicator of heparin anticoagulant activity) was highest for these same surfaces. The PEO-ATH- and PEO-modified surfaces also showed low platelet adhesion from flowing whole blood. It is concluded that for the PEO-ATH surfaces, PEO in the low MW range, specifically MW 600, may be optimal for achieving an appropriate balance between resistance to nonspecific protein adsorption and the ability to take up ATH and bind antithrombin in subsequent blood contact.     

FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo

BACKGROUND: Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity. RESULTS: Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that the drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent. CONCLUSIONS: We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.     

Influence factors studies on the Rh-Catalyzed asymmetric transfer hydrogenation of ketones with surfactant-type ligand in water

The influence factors of surfactant-type catalysts system on the asymmetric transfer hydrogenation (ATH) of ketones in water, including the length of aliphatic tail, the concentration of catalysts and the solvent, have been investigated. These factors played significant roles on reaction activity and stereo control. With single-chain surfactant-type catalyst Rh-L4, α, β-unsaturated ketones especially the aliphatic derivatives affords excellent regioselectivities and enantioselectivities.