Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR

Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this review, we will summarize high-resolution structural and dynamic findings towards the understanding of the structure–activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane.     

Metabolism of O6-Propyl and N6-Propyl-carbovir in CEM Cells

Abstract

The metabolism of O⁶-propyl-carbovir and N⁶-propyl-carbovir, two selective inhibitors of HIV replication, has been evaluated in CEM cells. Both compounds were phosphorylated in intact cells to carbovir-5′-triphosphate. The metabolism of these two agents was inhibited by deoxycoformycin and mycophenolic acid, but not erythro-9-(2-hydroxy-3-nonyl)adenine. No evidence of the 5′-triphosphate of either compound was detected in CEM cells.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).     

长期不同施氮水平对草原植物群落结构的影响

为探讨施氮对贝加尔针茅草原植物群落结构的影响,该研究于内蒙古贝加尔针茅草原设置不同施氮水平0(N0)、15(N15)、30(N30)、50(N50)、100(N100)、150(N150)、200(N200)和300(N300)kg·hm-2·a-1,研究连续施氮9年后贝加尔针茅草原群落物种组成、重要值、多样性及生物量等指标对不同施氮水平的响应。结果表明:(1)长期施氮导致植物群落物种组成发生了明显的变化,在高浓度氮素添加量下群落物种数最低,但可显著提高优势种在群落中的重要值(P<0.05)。(2)长期施氮使贝加尔针茅草原植物多样性指数不同程度地降低,使优势度指数增加,其中N300添加水平效果最为明显(P<0.05)。(3)长期施氮使草原植被生产力(地上、地下生物量)整体呈先上升后下降的趋势,地上生物量在N100水平时出现峰值,地下生物量在N50水平时出现最大值(P<0.05),且根系分布具有明显的表聚特征(0~10 cm)。(4)RDA分析表明,寸草苔和狭叶柴胡地上生物量与土壤pH呈显著正相关关系(P<0.05);展枝唐松草和二裂委陵菜地上生物量与土壤有机质含量呈显著正相关关系(P<0.05)。研究认为,长期不同施氮水平均可改变贝加尔针茅草原群落物种组成,降低物种多样性,对植物群落结构有着明显的影响。     

Highly sensitive chemiluminescence immunoassay on chitosan membrane modified paper platform using TiO2 nanoparticles/multiwalled carbon nanotubes as label

A highly sensitive chemiluminescence (CL) immunoassay was incorporated into a low‐cost microfluidic paper‐based analytical device (μ‐PAD) to fabricate a facile paper‐based CL immunodevice (denoted as μ‐PCLI). This μ‐PCLI was constructed by covalently immobilizing capture antibody on a chitosan membrane modified μ‐PADs, which was developed by simple wax printing methodology. TiO₂ nanoparticles coated multiwalled carbon nanotubes (TiO₂/MWCNTs) were synthesized as an amplification catalyst tag to label signal antibody (Ab₂). After sandwich‐type immunoreactions, the TiO₂/MWCNTs were captured on the surface of μ‐PADs to catalyze the luminol‐p‐iodophenol‐H₂O₂ CL system, which produced an enhanced CL emission. Using prostate‐specific antigen as a model analyte, the approach provided a good linear response range from 0.001 to 20 ng/mL with a low detection limit of 0.8 pg/mL under optimal conditions. This μ‐PCLI showed good reproducibility, selectivity and stability. The assay results of prostate‐specific antigen in clinical serum samples were in good agreement with that obtained by commercially used electrochemiluminescence methods at the Cancer Research Center of Shandong Tumor Hospital (Jinan, Shandong Province, China). This μ‐PCLI could be very useful to realize highly sensitive, qualitative point‐of‐care testing in developing or developed countries. Copyright © 2013 John Wiley & Sons, Ltd.     

The Influence of Antimicrobial Peptides and Mucolytics on the Integrity of Biofilms Consisting of Bacteria and Yeasts as Affecting Voice Prosthetic Air Flow Resistances

The integrity of biofilms on voice prostheses used to rehabilitate speech in laryngectomized patients causes unwanted increases in airflow resistance, impeding speech. Biofilm integrity is ensured by extracellular polymeric substances (EPS). This study aimed to determine whether synthetic salivary peptides or mucolytics, including N-acetylcysteine and ascorbic acid, influence the integrity of voice prosthetic biofilms. Biofilms were grown on voice prostheses in an artificial throat model and exposed to synthetic salivary peptides, mucolytics and two different antiseptics (chlorhexidine and Triclosan). Synthetic salivary peptides did not reduce the air flow resistance of voice prostheses after biofilm formation. Although both chlorhexidine and Triclosan reduced microbial numbers on the prostheses, only the Triclosan-containing positive control reduced the air flow resistance. Unlike ascorbic acid, the mucolytic N-acetylcysteine removed most EPS from the biofilms and induced a decrease in air flow resistance.     

Reactive oxygen species play a role in P2X7 receptor-mediated IL-6 production in spinal astrocytes

Purinergic Signalling - Astrocytes mediate a remarkable variety of cellular functions, including gliotransmitter release. Under pathological conditions, high concentrations of the purinergic...     

独行菜族8属(十字花科)植物果实及种子微形态研究

采用GMA(Glycol methacrylate,乙二醇甲基丙烯酸酯)半薄切片法,利用比较解剖学对独行菜族中代表植物8属25种果实及种子微形态结构进行观察分析。同时,以菥蓂属菥蓂为例详细介绍了假隔膜的形成过程。结果显示独行菜族果实及种子微形态特征明显,果实均为短角果,除厚壁荠属和沙芥属果实为背腹压扁外,其他属种均为两侧压扁;果实边缘有翅为独行菜族的典型特征,可分为周翅、微翅、宽翅及披针形翅;部分果皮细胞有纤维层,偶有木化,除菘蓝属、厚壁荠属、沙芥属无假隔膜结构外,其他属种均具有明显的假隔膜。种子大小为(0.8~10)mm×(0.5~2.8)mm,种皮纹饰丰富,遇水或潮湿环境可形成粘液种子。种皮通常由薄壁细胞构成,偶有木化,具内含物;除高河菜属和菥蓂属子叶与胚根的排列方式为子叶缘倚,其他属种均为子叶背倚。假隔膜为内果皮细胞向内延伸连接而成。本文完善了独行菜族果实及种子微形态结构信息,为分子系统学等其他相关研究提供基础资料。     

Construction of photodynamic‐effect immunofluorescence probes by a complex of quantum dots,immunoglobulin G and chlorin e6 and their application in HepG2 cell killing

In this study, tri‐functional immunofluorescent probes (Ce6–IgG–QDs) based on covalent combinations of quantum dots (QDs), immunoglobulin G (IgG) and chlorin e6 (Ce6) were developed and their photodynamic ability to induce the death of cancer cells was demonstrated. Strategically, one type of second‐generation photosensitizer, Ce6, was first coupled with anti‐IgG antibody using the EDC/NHS cross‐linking method to construct the photosensitive immunoconjugate Ce6–IgG. Then, a complex of Ce6–IgG–QDs immunofluorescent probes was obtained in succession by covalently coupling Ce6–IgG to water soluble CdTe QDs. The as‐manufactured Ce6–IgG–QDs maintained the bio‐activities of both the antigen–antibody‐based tumour targeting effects of IgG and the photodynamic‐related anticancer activities of Ce6. By way of polyclonal antibody interaction with rabbit anti‐human epidermal growth factor receptor (anti‐EGFR antibody, N‐terminus), Ce6–IgG–QDs were labelled indirectly onto the surface of human hepatocarcinoma (HepG2) cells in cell recognition and killing experiments. The results indicated that the Ce6–IgG–QDs probes have excellent tumour cell selectivity and higher photosensitivity in photodynamic therapy (PDT) compared with Ce6 alone, due to their antibody‐based specific recognition and location of HepG2 cells and the photodynamic effects of Ce6 killed cells based on efficient fluorescence resonance energy transfer between QDs and Ce6. Copyright © 2015 John Wiley & Sons, Ltd.