Reconstitution of caspase-8 sensitizes JB6 cells to TRAIL

TRAIL induces apoptosis in various tumor cells. We report here that caspase-8 is required in TRAIL-induced cell death. Western blot analyses and enzyme assays showed that exposing Jurkat cells to TRAIL resulted in activation of caspases-8 followed by caspase-3 and -9. Acetyl-IETD-fluoromethylketone, a caspase-8 inhibitor, potently suppressed TRAIL-induced cell death compared to acetyl-DEVD-fluoromethylketone and acetyl-LEHD-fluoromethylketone, inhibitors of caspase-3 and caspase-9, respectively. JB6 cells, a caspase-8-deficient Jurkat variant, were completely resistant to TRAIL. However, reconstitution with a caspase-8, but not with caspase-2 or -3, sensitized JB6 cells to subsequent exposure to TRAIL. These results are indicative of the crucial function of caspase-8 in TRAIL-induced apoptosis in Jurkat cells.     

Characterization of S-Triazine Herbicide Metabolism by a Nocardioides sp. Isolated from Agricultural Soils

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Nine gram-positive bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from four farms in central Canada. The strains were divided into two groups based on repetitive extragenic palindromic (rep)-PCR genomic fingerprinting with ERIC and BOXA1R primers. Based on 16S ribosomal DNA sequence analysis, both groups were identified as Nocardioides sp. strains. None of the isolates mineralized [ring-U-¹⁴C]atrazine. There was no hybridization to genomic DNA from these strains using atzABC cloned from Pseudomonas sp. strain ADP or trzA cloned from Rhodococcus corallinus. S-Triazine degradation was studied in detail in Nocardioides sp. strain C190. Oxygen was not required for atrazine degradation by whole cells or cell extracts. Based on high-pressure liquid chromatography and mass spectrometric analyses of products formed from atrazine in incubations of whole cells with H₂¹⁸O, sequential hydrolytic reactions converted atrazine to hydroxyatrazine and then to the end product N-ethylammelide. Isopropylamine, the putative product of the second hydrolytic reaction, supported growth as the sole carbon and nitrogen source. The triazine hydrolase from strain C190 was isolated and purified and found to have a K_m for atrazine of 25 μM and a V_max of 31 μmol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 μM EDTA but was inhibited by 100 μM Mg, Cu, Co, or Zn. Whole cells and purified triazine hydrolase converted a range of chlorine or methylthio-substituted herbicides to the corresponding hydroxy derivatives. In summary, an atrazine-metabolizing Nocardioides sp. widely distributed in agricultural soils degrades a range of s-triazine herbicides by means of a novel s-triazine hydrolase.     

Segmental territories along the cardinal veins generate lymph sacs via a ballooning mechanism during embryonic lymphangiogenesis in mice

During lymphangiogenesis in the mammalian embryo, a subset of vascular endothelial cells in the cardinal veins is reprogrammed to adopt a lymphatic endothelial fate. The prevailing model of lymphangiogenesis contends that these lymphatic precursor cells migrate away from the cardinal veins and reassemble peripherally as lymph sacs from which a lymphatic vasculature is generated. However, this model fails to account for a number of observations that, as a result, have remained anecdotal. Here, we use optical projection tomography, confocal microscopy and in vivo live imaging to uncover three key stages of lymphatic vascular morphogenesis in the mouse embryo at high resolution. First, we define territories or "pre-lymphatic clusters" of Prox1-positive lymphatic endothelial progenitor cells along the antero-posterior axis of the cardinal veins. Second, these pre-lymphatic clusters undergo progressive extrusion ("ballooning") to generate primitive lymph sacs. Third, lymphatic vessels emerge by a combination of mechanisms including sprouting from the lymph sacs and direct delamination of streams of cells from the cardinal veins. Our data support a new model for lymphatic vascular patterning and morphogenesis, as a basis for identifying the molecular cues governing these processes.     

Fragrant Volatile Sesquiterpenoids Isolated from the Essential Oil of Laggera pterodonta by Using Olfactory‐Guided Fractionation

Chemical composition of the essential oil from Laggera pterodonta (Compositae) was inverstigated. GC/MS Analyses led to the identification of 68 components, representing more than 96% of the total oil. By focusing on the woody note fraction of the essential oil, one new bisabolane‐type sesquiterpenoid, bisabola‐2,7(14),11‐trien‐10‐ol ( 1 ), together with ten known compounds, bisabolol oxide B ( 2 ), ylangenol ( 3 ), copaborneol ( 4 ), guai‐11‐en‐10‐ol ( 5 ), spathulenol ( 6 ), aromadendran‐10‐ol ( 7 ), caryophyllenol ( 8 ), 5α,7α‐eudesm‐11(13)‐en‐4α‐ol ( 9 ), γ‐costic acid ( 10 ), and eudesma‐4(15),11(13)‐diene‐12,5β‐olide ( 11 ), were isolated by using olfactory‐guided fractionation. The structures of the eleven compounds were determined by NMR and MS analyses. All the volatile compounds reported here were isolated for the first time from this plant. On the basis of preliminary odor assessment, the odor of the woody‐note fractions of the essential oil was assumed to be due to these isolated sesquiterpenoids.     

Cochlioquinone Derivatives with Apoptosis‐Inducing Effects on HCT116 Colon Cancer Cells from the Phytopathogenic Fungus Bipolaris luttrellii L439

A new cochlioquinone derivative, cochlioquinone F ( 1 ), as well as three known compounds, anhydrocochlioquinone A ( 2 ), isocochlioquinone A ( 3 ), and isocochlioquinone C ( 4 ), were isolated from the PDB (potato dextrose broth) culture of the phytopathogenic fungus Bipolaris luttrellii. The structure of 1 was elucidated on the basis of NMR techniques. The apoptosis‐inducing effects of compounds 1 – 4 were evaluated against HCT116 cancer cells. Compound 2 exhibited the strongest activity in inducing apoptosis on HCT116 cells within the range of 10–30 μM . In addition, the caspase activation, the release of cytochrome c from mitochondria, and the downregulation of Bcl‐2 protein in HCT116 cells treated with compound 2 were detected.     

Molecular insights into the binding selectivity of a synthetic ligand DAAG to Fc fragment of IgG

Affinity chromatography with synthetic ligands has been focused as the potential alternative to protein A‐based chromatography for antibody capture because of its comparable selectivity and efficiency. Better understanding on the molecular interactions between synthetic ligand and antibody is crucial for improving and designing novel ligands. In this work, the molecular interaction mechanism between Fc fragment of IgG and a synthetic ligand (DAAG) was studied with molecular docking and dynamics simulation. The docking results on the consensus binding site (CBS) indicated that DAAG could bind to the CBS with the favorable orientation like a tripod for the top‐ranked binding complexes. The ligand‐Fc fragment complexes were then tested by molecular dynamics simulation at neutral condition (pH 7.0) for 10 ns. The results indicated that the binding of DAAG on the CBS of Fc fragment was achieved by the multimodal interactions, combining the hydrophobic interaction, electrostatic interaction, hydrogen bond, and so on. It was also found that multiple secondary interactions endowed DAAG with an excellent selectivity to Fc fragment. In addition, molecular dynamics simulation conducted at acidic condition (pH 3.0) showed that the departure of DAAG ligand from the surface of Fc fragment was the result of reduced interaction energies. The binding modes between DAAG and CBS not only shed light on the molecular mechanisms of DAAG for antibody purification but also provide useful information for the improvement of ligand design. Copyright © 2014 John Wiley & Sons, Ltd.     

Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH₂-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27^Kip1 protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27^Kip1 significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27^Kip1 degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.     

Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development

The mammalian urogenital sinus (UGS) develops in a sex specific manner, giving rise to the prostate in the male and the sinus vagina in the embryonic female. Androgens, produced by the embryonic testis, have been shown to be crucial to this process. In this study we show that retinoic acid signaling is required for the initial stages of bud development from the male UGS. Enzymes involved in retinoic acid synthesis are expressed in the UGS mesenchyme in a sex specific manner and addition of ligand to female tissue is able to induce prostate-like bud formation in the absence of androgens, albeit at reduced potency. Functional studies in mouse organ cultures that faithfully reproduce the initiation of prostate development indicate that one of the roles of retinoic acid signaling in the male is to inhibit the expression of Inhba, which encodes the βA subunit of Activin, in the UGS mesenchyme. Through in vivo genetic analysis and culture studies we show that inhibition of Activin signaling in the female UGS leads to a similar phenotype to that of retinoic acid treatment, namely bud formation in the absence of androgens. Our data also reveals that both androgens and retinoic acid have extra independent roles to that of repressing Activin signaling in the development of the prostate during fetal stages. This study identifies a novel role for retinoic acid as a mesenchymal factor that acts together with androgens to determine the position and initiation of bud development in the male UGS epithelia.     

小鼠心肌肥厚发展过程中心电图动态变化

目的：探讨小鼠心肌肥厚发展过程中心电图的动态变化。方法：复制小鼠压力超负荷性心肌肥厚模型,连续动态监测小鼠从心肌肥厚早期至心力衰竭发展过程中的不同阶段体表心电图改变。结果：①对照组和模型组术后2周内小鼠未见自发性心律失常,而模型组术后5周、9周和13周小鼠出现自发性心律失常,主要表现为频发的室性早搏以及阵发性室性心动过速,心律失常发生率分别为15％、28％和63％。②与同期对照相比,术后2周、5周、9周和13周组动物伽间期以及帆间期明显延长,分别延长20．4％、32．7％、49．7％、61．0％和27．1％、32．1％、43．9％、59．1％（P〈0．01）。③心肌肥厚小鼠心电图的另一个特征为J波变化。所有对照组动物心电图均为正向J波,而模型组动物从2周开始J波正向值下降,5周逐渐变平,到13周时完全翻转。④与同期对照相比,模型组的PR间期没有改变,但术后2周RR问期轻微缩短。结论：心肌肥厚小鼠自发性心律失常发生率逐渐增加,QT间期进行性延长,J波幅值逐渐降低,表明随着疾病的进展心室复极化异常逐渐加重。