Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7 Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600 Å³). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.     

红腹锦鸡肺的组织结构与微血管构筑

为了了解红腹锦鸡(Chroysolophus pictus)肺的微细结构和微血管构筑特征,为呼吸生物学研究提供形态学依据,用组织学方法和微血管铸型技术在光镜和扫描电镜下观察研究了红腹锦鸡肺的组织结构与微血管构筑情况。结果表明,红腹锦鸡肺主要由各级支气管构成,从三级支气管上呈辅射状分出许多呼吸毛细管(微气管),并相互吻合成网状,呼吸毛细管外面包围有丰富的毛细血管;红腹锦鸡肺毛细血管垂直围绕在各微气管外,并相互吻合成密集的立体微血管网;毛细血管管径4.5~7.0μm,微气管直径11~50μm。并对肺微血管构筑情况与呼吸效率的关系作了探讨。     

Olive Phenolics as c-Met Inhibitors: (-)-Oleocanthal Attenuates Cell Proliferation,Invasiveness, and Tumor Growth in Breast Cancer Models

Dysregulation of the Hepatocyte growth factor (HGF)/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. (-)-Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (-)-oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (-)-oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (-)-oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (-)-oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (-)-oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (-)-oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity.     

Recombinant expression, synthesis, purification, and solution structure of arenicin

Arenicins are 21-residue cationic antimicrobial peptides, isolated from marine polychaeta Arenicola marina. In order to determine a high-resolution three-dimensional structure of arenicin-2, the recombinant peptide was overexpressed as a fused form in Escherichia coli. Both arenicin isoforms were synthesized using the Fmoc-based solid-phase strategy. Recombinant and synthetic arenicins were purified, and their antimicrobial and spectroscopic properties were analyzed. NMR investigation shows that in water solution arenicin-2 displays a prolonged beta-hairpin, formed by two antiparallel beta-strands and stabilized by one disulfide and nine hydrogen bonds. A significant right-handed twist in the beta-sheet is deprived the peptide surface of amphipathicity. CD spectroscopic analysis indicates that arenicin-2 binds to the SDS and DPC micelles, and conformation of the peptide is significantly changed upon binding. Arenicin strongly binds to anionic lipid (POPE/POPG) vesicles in contrast with zwitterionic (POPC) ones. These results suggest that arenicins are membrane active peptides and point to possible mechanism of their selectivity toward bacterial cells.     

Heterologous expression and solution structure of defensin from lentil Lens culinaris

A new defensin Lc-def, isolated from germinated seeds of the lentil Lens culinaris, has molecular mass 5440.4 Da and consists of 47 amino acid residues. Lc-def and its ¹⁵N-labeled analog were overexpressed in Escherichia coli. Antimicrobial activity of the recombinant protein was examined, and its spatial structure, dynamics, and interaction with lipid vesicles were studied by NMR spectroscopy. It was shown that Lc-def is active against fungi, but does not inhibit the growth of Gram-positive and Gram-negative bacteria. The peptide is monomeric in aqueous solution and contains one α-helix and triple-stranded β-sheet, which form cysteine-stabilized αβ motif (CSαβ) previously found in other plant defensins. The sterically neighboring loop1 and loop3 protrude from the defensin core and demonstrate significant mobility on the μs–ms timescale. Lc-def does not bind to the zwitterionic lipid (POPC) vesicles but interacts with the partially anionic (POPC/DOPG, 7:3) membranes under low-salt conditions. The Lc-def antifungal activity might be mediated through electrostatic interaction with anionic lipid components of fungal membranes.     

A novel defensin from the lentil Lens culinaris seeds

A novel 47-residue plant defensin was purified from germinated seeds of the lentil Lens culinaris by ammonium sulfate precipitation, gel filtration, chromatography, and RP-HPLC. The molecular mass (5440.41 Da) and complete amino acid sequence (KTCENLSDSFKGPCIPDGNCNKHCKEKEHLLSGRCRDDFRCWCTRNC)¹ of defensin, termed Lc-def, were determined. Lc-def has eight cysteines forming four disulfide bonds. The total RNA was isolated from lentil germinated seeds, RT-PCR and subsequent cloning were performed, and cDNA was sequenced. A 74-residue predefensin contains a putative signal peptide (27 amino acid) and a mature protein. Lc-def shows high sequence homology with legumes defensins, exhibits an activity against Aspergillus niger, but does not inhibit proteolytic enzymes.     

POTENTIATION OF CAFFEINEINDUCED CONTRACTURE BY RAISING EXTRACELLULAR POTASSIUM IN FROG SKELETAL MUSCLE

用蛙胫前肌小束为材料,研究了提高胞外钾［Ｋ＋］Ｏ对咖啡因挛缩的作用。［Ｋ＋］Ｏ从２ｍｍｏｌ／Ｌ提高到１０或２５ｍｍｏｌ／Ｌ,由３ｍｍｏｌ／Ｌ咖啡因引起的挛缩明显增强。以ＰＫＣ／ＰＣ（ＰＫＣ和ＰＣ分别为在高钾和正常钾条件下的咖啡因挛缩）表示的咖啡因挛缩增强,依赖［Ｋ＋］Ｏ和高钾作用时间。随着１０ｍｍｏｌ／Ｌ［Ｋ＋］Ｏ作用时间延长,直至１０ｍｉｎ,增强逐渐增加。但是,２５ｍｍｏｌ／Ｌ［Ｋ＋］Ｏ作用１ｍｉｎ时增强达到最大,然后下降到对照。ＰＫＣ／ＰＣ变化时程不能用高钾引起的去极化解释,而与由相似［Ｋ＋］Ｏ引起的胞浆自由钙变化时程相符。提示,至少在蛙骨骼肌,高钾引起的咖啡因挛缩增强主要是由胞浆自由钙升高引起的。