A diepitopic sequential oligopeptide carrier (SOCn) as mimic of the sm autoantigen: synthesis, conformation and biological assays.

Anti-Sm (Sm: U1-U6 RNA-protein complex) antibodies are usually considered highly specific for systemic lupus erythematosus (SLE), while anti-U1RNP (U1RNP: U1RNA-protein complex) are thought of as diagnostic criteria for the mixed connective tissue disease (MCTD). However, both antibody specificities coexist in SLE and MCTD, in varying percentages. Although the anti-Sm/anti-U1RNP immunological cross-reactivity has been initially attributed to a common motif, PPXY(Z)PP (where X, Y, Z are various amino acids), found in the Sm, U1-A and U1-C autoantigens, it appears that the conformational features of the Sm epitopes also play an important role in the immunoreactivity. The PPGMRPP and PPGIRGP main epitopes of the Sm antigen were coupled in duplicate to the tetrameric Ac-(Lys-Aib-Gly)4-OH, SOC4, carrier to form the [(PPGMRPP)2, (PPGIRGP)2]-SOC4 construct as a mimic of the native Sm. It was found that: (i) the 3(10) helical structure of SOC4 allows the epitopes to adopt an exposed orientation, similar to their free forms, that facilitates their recognition from the anti-Sm antibodies, and (ii) the U1-RNP cross-reactivity is minimized.     

A new type porous carrier and its application to culture of suspension cells

A new type porous carrier was fabricated from a mixture of sodium alginate, bovine serum albumin and sodium bicarbonate. The porous space of the carrier is an assembly of void spaces. The carrier was successfully applied to the cultivation of suspension animal cells. In the culture, while both cells and carriers were held in suspension, the cells were entrapped hydrodynamically into the void spaces in the carriers. A culture of hybridoma cells using this carrier resulted in a cell density up to 5.7×10⁷ cells per ml-carrier.     

Solution structure of Asl1650, an acyl carrier protein from Anabaena sp. PCC 7120 with a variant phosphopantetheinylation-site sequence

Cyanobacteria, such as Anabaena, produce a variety of bioactive natural products via polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), and hybrid peptide/polyketide pathways. The protein Asl1650, which is a member of the acyl carrier protein family from the cyanobacterium Anabaena sp. PCC 7120, is encoded in a region of the Anabaena genome that is rich in PKS and NRPS genes. To gain new insight into the physiological role of acyl carriers in Anabaena, the solution structure of Asl1650 has been solved by NMR spectroscopy. The protein adopts a twisted antiparallel four-helix bundle fold, with a variant phosphopantetheine-attachment motif positioned at the start of the second helix. Structure comparisons with proteins from other organisms suggest a likely physiological function as a discrete peptidyl carrier protein.     

新型载体蛋白P64K在大肠杆菌中的可溶性表达与纯化

用常规PCR方法从脑膜炎球菌中克隆出P64K基因,构建重组质粒pET28a-P64K转化BL21(DE3)宿主菌,经眦诱导表达筛选P64K蛋白的高表达菌株。结果证实P64K蛋白为胞内可溶性表达,表达量约占胞内总蛋白的30％。重组P64K蛋白经Butyl疏水柱、G200分子筛柱和Q阴离子柱三步层析后纯度达到95％以上,为今后进一步的功能和应用研究打下了良好的基础。     

All-D-cecropin B: Synthesis,conformation, lipopolysaccharide binding,and antibacterial activity

Cecropin B (LCB) is a natural peptide with antibacterial and antifungal properties. The enantiomer of LCB, containing all-D amino acids (DCB), was synthesized to examine its antibacterial and binding properties. The conformation of DCB was compared to its enantiomer by circular dichroism. Both the L- and D-peptides showed an identical induction of -helical secondary structure. However, binding studies between Lipopolysaccharide (LPS) and DCB or LCB were studied with a dimethylmethylene blue spectrophotometric assay, showing the two enantiomeric peptides differed in their interaction with LPS. Antibacterial activity of DCB was determined against three Gram-negative bacteria, Pantoea agglomerans (ATCC 27996), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 17648), giving comparable results to LCB.     

一种新型还原敏感型阳离子寡肽脂质的合成及其在模拟细胞质还原性环境中的降解动力学

目的：设计合成一种新型还原敏感型阳离子寡肽脂质材料,以期由其制得的脂质体可用作核酸药物载体在细胞质还原性环境促发下降解而释放药物。方法：以胱胺为原料引入二硫键,合成还原敏感型阳离子寡肽脂质材料H-SS-E2C14,并用其制备还原敏感型阳离子寡肽脂质体H-SS-E2C14-L;同时,以半胱胺为原料合成H-SS-E2C14 的降解产物CS-E2C14,且采用HPLC 法定量考察H-SS-E2C14在模拟细胞质还原性环境中的降解动力学。结果：合成的H-SS-E2C14 在模拟细胞质还原性环境中的24 h 累积降解率约为70%。结论：H-SS-E2C14-L 有望成为一种理想的基因药物载体。     

Synthesis of new morpholine-connected pyrazolidine derivatives and their antimicrobial,antioxidant, and cytotoxic activities

A simple and convenient one-pot four-component synthesis of morpholine-connected pyrazolidine derivatives 2a–f and 4a–f was developed using direct metal-free catalysis, with the identities of the synthesized compounds confirmed by IR, NMR (¹H and ¹³C), mass spectrometry, and elemental analysis. The prepared compounds were inspected for antimicrobial, antioxidant, and cytotoxic activities.Antimicrobial and antifungal activities against five bacterial and four fungal pathogens, respectively, were investigated using the disc diffusion technique. In antibacterial activity, compounds 2d and 2f (MIC = 2 μg/mL) exhibited significantly higher activity than the standard ciprofloxacin. The results of antifungal assay showed that the activity of compound 4a (MIC = 0.5 μg/mL) was significantly higher than the standard clotrimazole. Antioxidant activity was screened based on ABTS⁺ radical scavenging and linoleic acid peroxidation performance. Compound 4a showed substantial antioxidant (91.3%) activities, as compared with the Trolox standard. Cytotoxicity was evaluated using HepG2 (liver), HeLa (cervical), and MCF-7 (breast) cancer cell lines, with high toxicities observed for 2b (GI₅₀ = 12.2 μm) and 4a (GI₅₀ = 07.8 μm).     

Synthesis,antimicrobial activity and cytotoxicity of some new carbazole derivatives

In this work, some N-(9-Ethyl-9H-carbazole-3-yl)-2-(phenoxy)acetamide derivatives were synthesised and evaluated for their antimicrobial activity and cytotoxicity. The structural elucidation of the compounds was performed by IR, ¹H-NMR, ¹³C-NMR and FAB⁺-MS spectral data and elemental analyses. The title compounds were obtained by reacting 2-chloro-N-(9-ethyl-9H-carbazole-3-yl)acetamide with some substituted phenols. The synthesised compounds were investigated for their antibacterial and antifungal activities against Micrococcus luteus, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Candida albicans. The compounds N-(9-Ethyl-9H-carbazole-3-yl)-2-(4-ethylphenoxy)acetamide (2c) and N-(9-Ethyl-9H-carbazole-3-yl)-2-(quinolin-8-yloxy)acetamide (2n) showed notable antimicrobial activity. The compounds were also studied for their cytotoxic effects using MTT assay, and it was seen that 2n had the lowest cytotoxic activity against NIH/3T3 cells.     

Effect of Sulfated Modification on the Molecular Characteristics and Biological Activities of Polysaccharides from Hypsizigus marmoreus

The effect of sulfated modification on polysaccharides from Hypsizigus marmoreus was examined by determining their molecular structures and bioactivities. The sulfation, which was implemented by using an orthogonal array design, produced polysaccharides with varying degrees of substitution (DS) ranging from 0.11 to 1.06. The sulfated polysaccharides exhibited a lower average molecular weight (M _w) and considerably higher radius of gyration (R _g) than those of native polysaccharide, suggesting that the conformation of the sulfated polysaccharides had been changed towards a more extended type. The inhibitory activity toward cancer cell growth was enhanced by treating with the sulfated polysaccharides by up to 34%, as compared to the native polysaccharide. In addition, treating with the sulfated polysaccharides increased the nitric oxide (NO) and cytokine (IL-1β and TNF-α) release to levels comparable to those detected in the positive control, lipopolysaccharide (LPS), suggesting that the sulfated polysaccharides might have strong immunomodulatory activity.     

Synthesis,conformation, and biological activity of two fMLP-OMe analogues containing the new 2-[2′-(methylthio) ethyl] methionine residue

The new C^α-tetrasubstituted α-amino acid residue 2-[2′-(methylthio) ethyl]methionine (Dmt) has been introduced into the reference chemotactic tripeptide HCO-Met-Leu-Phe-OMe (fMLP-OMe) in place of the leucine or methionine, respectively. The biological activity of the new analogues [Dmt²] fMLP-OMe (2) and [Dmt¹] fMLP-OMe (3) has been determined; whereas 2 is active toward human neutrophils, stimulating directed migration, superoxide anion generation, and lysozyme release, 3 results practically inactive in all tested assays. A conformational analysis on 2 and 3 has been performed in solution by using ir absorption and ¹H-nmr. The conformation of 2 was also examined in the crystal by x-ray diffraction methods. Both 2 and 3 adopt fully extended conformation in correspondence with the Dmt residue. Biological and conformational results are discussed and compared with related previously studied models. © 1997 John Wiley & Sons, Inc. Biopoly 42: 415–426, 1997     

Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein

The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.     

WSE, a new sequence distance measure based on word frequencies

In this article, we present a new distance metric, the Weighted Sequence Entropy (WSE), based on the short word composition of biological sequences. As a revision of the classical relative entropy (RE), our metric (1) works equivalently with RE in the case of small k, (2) avoids the degeneracy when some word types are absent in one sequence but not in the other. Experiments on 25 viruses including SARS-CoVs show that our method and RE give exactly the same phylogenetic tree when word length k3. When k>3, our method still works and gets convergent phylogenetic topology but the RE gives degenerate results.     

The aims of expanded universal carrier screening: Autonomy,prevention, and responsible parenthood

Expanded universal carrier screening (EUCS) entails a population‐wide screening offer for multiple disease‐causing mutations simultaneously. Although there is much debate about the conditions under which EUCS can responsibly be introduced, there seems to be little discussion about its aim: providing carrier couples with options for autonomous reproductive choice. While this links in with current accounts of the aim of foetal anomaly screening, it is different from how the aim of ancestry‐based carrier screening has traditionally been understood: reducing the disease burden in the population. The reasons why the aim of EUCS is presented in terms of ‘autonomy’ rather than ‘prevention’ have not been spelled out in the literature. This paper seeks to fill this gap by considering the morally relevant similarities and dissimilarities between foetal anomaly screening, ancestry‐based carrier screening and EUCS. When carrier screening is performed in the prenatal period, enhancing autonomy appears the most appropriate aim of EUCS, as the alternative of ‘prevention through selective abortion’ would urge women to terminate wanted pregnancies. However, when screening is conducted in the preconception period, carrier couples can avoid the birth of affected children by other means than selective abortion, for instance preimplantation genetic diagnosis. To the extent that this increased control over passing on a genetic disorder raises questions of parental responsibility, it seems necessary that the account of the aims of EUCS is wider than only in terms of enhancing reproductive autonomy.     

Synthesis and biological evaluation of new active For‐Met‐Leu‐Phe‐OMe analogues containing para‐substituted Phe residues

In the present study, we report synthesis and biological evaluation of the N‐Boc‐protected tripeptides 4a–l and N‐For protected tripeptides 5a–l as new For‐Met‐Leu‐Phe‐OMe (fMLF‐OMe) analogues. All the new ligands are characterized by the C‐terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF‐OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with ‐CH₃ and ‐C(CH₃)₃, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i , containing ‐F and ‐I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Leakage and slow allostery limit performance of single drug-sensing aptazyme molecules based on the hammerhead ribozyme

Engineered “aptazymes” fuse in vitro selected aptamers with ribozymes to create allosteric enzymes as biosensing components and artificial gene regulatory switches through ligand-induced conformational rearrangement and activation. By contrast, activating ligand is employed as an enzymatic cofactor in the only known natural aptazyme, the glmS ribozyme, which is devoid of any detectable conformational rearrangements. To better understand this difference in biosensing strategy, we monitored by single molecule fluorescence resonance energy transfer (FRET) and 2-aminopurine (AP) fluorescence the global conformational dynamics and local base (un)stacking, respectively, of a prototypical drug-sensing aptazyme, built from a theophylline aptamer and the hammerhead ribozyme. Single molecule FRET reveals that a catalytically active state with distal Stems I and III of the hammerhead ribozyme is accessed both in the theophylline-bound and, if less frequently, in the ligand-free state. The resultant residual activity (leakage) in the absence of theophylline contributes to a limited dynamic range of the aptazyme. In addition, site-specific AP labeling shows that rapid local theophylline binding to the aptamer domain leads to only slow allosteric signal transduction into the ribozyme core. Our findings allow us to rationalize the suboptimal biosensing performance of the engineered compared to the natural aptazyme and to suggest improvement strategies. Our single molecule FRET approach also monitors in real time the previously elusive equilibrium docking dynamics of the hammerhead ribozyme between several inactive conformations and the active, long-lived, Y-shaped conformer.     

Synthesis, conformation, and immunogenicity of monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178

Several monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178 were synthesized. Conformational studies showed that multivalent assembly enhanced the alpha-helical content of the peptide. Therefore, 2-, 3-, or 4-alpha-helix bundles of peptide DP178 could be obtained by assembling the peptide on a suitable bi-, tri-, or tetravalent template. Immunization studies indicated that while peptide DP178 alone was poorly immunogenic, the tetravalent peptide MVP-1 raised high titers of antibodies in mice that recognize not only peptide DP178 but also the native HIV-1 glycoprotein gp41, even in the absence of a carrier protein or adjuvant. The study suggests that carbohydrate-centered multivalent peptides provide not only a model for mimicking protein alpha-helix-bundle structure, but also an effective immunogen for raising high-titer antibodies against HIV-1 envelope glycoprotein gp41.     

Synthesis and biological evaluation of new 4-thiazolidinone derivatives

A series of new 2-aryl-4-thiazolidinones (3 and 4) was synthesized from 2-hydroxy-2,2-diphenyl-N’-[(substituted phenyl)methylene]acetohydrazides (2) and mercaptoacetic acid or 2-mercaptopropionic acid. The antimycobacterial activity of these compounds was determined and several leads with 95–99% inhibition at 6.25 μg/mL test concentration were identified. In addition, antitumor activities were measured against several tumor cell lines, and significant growth inhibition was observed for compound 4p. Taken together, 2-aryl-4-thiazolidinones were shown to be promising scaffolds for both antimycobacterial and tumor-targeting compounds.     

Synthesis of a mesoporous functional copolymer bead carrier and its properties for glucoamylase immobilization

A series of mesoporous and hydrophilic novel bead carriers containing epoxy groups were synthesized by modified inverse suspension polymerization. Glycidyl methacrylate and acryloyloxyethyl trimethyl ammonium chloride were used as the monomers, and divinyl benzene, allyl methacrylate, and ethylene glycol dimethacrylate as crosslinking agents, respectively. The resulting carriers were employed in the immobilization of glucoamylase (Glu) with covalent bond between epoxy groups and enzymes. The activity recovery of the three series of immobilized Glus could reach 76%, 79%, and 86%, respectively. The immobilized Glus exhibit excellent stability and reusability than that of the free ones.