zVAD-induced autophagic cell death requires c-Src-dependent ERK and JNK activation and reactive oxygen species generation

The treatment of L929 fibrosarcoma cells with zVAD has been shown to induce necroptosis. However, whether autophagy is involved or not in this event remains controversial. In this study, we re-examined the role of autophagy in zVAD-induced cell death in L929 cells and further elucidated the signaling pathways triggered by caspase inhibition and contributing to autophagic death. First, we found that zVAD can stimulate LC3-II formation, autophagosome and autolysosome formation, and ROS accumulation. Antioxidants, beclin 1 or Atg5 silencing, and class III PtdIns3K inhibitors all effectively blocked ROS production and cell death, suggesting ROS accumulation downstream of autophagy contributes to cell necrosis. zVAD also stimulated PARP activation, and the PARP inhibitor DPQ can reduce zVAD-induced cell death, but did not affect ROS production, suggesting the increased ROS leads to PARP activation and cell death. Notably, our data also indicated the involvement of Src-dependent JNK and ERK in zVAD-induced ROS production and autophagic death. We found caspase 8 is associated with c-Src at the resting state, and upon zVAD treatment this association was decreased and accompanied by c-Src activation. In conclusion, we confirm the autophagic death in zVAD-treated L929 cells, and define a new molecular pathway in which Src-dependent ERK and JNK activation can link a signal from caspase inhibition to autophagy, which in turn induce ROS production and PARP activation, eventually leading to necroptosis. Thus, in addition to initiating proteolytic activity for cell apoptosis, inactivated caspase 8 also functions as a signaling molecule for autophagic death.     

Biotic communities of freshwater marshes and mangroves in relation to saltwater incursions: implications for wetland regulation

An ecosystem-level study was conducted in the Guandu wetlands insubtropical coastal Taiwan to examine how salinity influences the abundance,diversity, and structure of biotic communities. We surveyed eight permanentstudy sites, spanning freshwater marshes, to the gate on the dyke, andmesohaline mangroves representing a gradient of the extent of saltwaterincursions. Analyses of abiotic variables showed that salinity was the primarydetermining factor for discriminating habitat types in the wetlands, butcommunities differed in their sensitivity to salinity. The composition of plantand insect communities was most affected by the salinity gradient, suggestingthe utility of these communities for ecological monitoring of saltwaterincursions. However, spatial changes in communities at higher trophic levels,including macrobenthos, mollusks, fish, and birds, could not be explained simplyby the salinity gradient. Instead, changes in these communities were morerelevant to the composition of other biotic communities. Our results show thatspecies richness and diversity of plant communities were higher in the marshesthan in the mangroves. Nevertheless, insect communities censused in themangroves had higher diversity, despite lower abundance and species richness.Macrobenthos surveyed in the mangroves showed higher biomass and number of taxa.Mollusks and fish were also more abundant at sites near the gate compared to themarsh sites. This suggests that maintaining a tidal flux by means of gateregulation is necessary for conserving the spatial heterogeneity andbiodiversity of coastal wetlands.     

On the biogenesis of cytokinins in roots of Phaseolus vulgaris

Roots of intact bean plants were supplied with [¹⁴C]adenine by pulse-chase experiments. The rate of incorporation of radioactivity into tRNA and oligonucleotides of roots as well as the content of radioactive labeled cytokinin nucleotides in these RNA fractions were determined. On the average, 1/70 of the radioactivity incorporated into tRNA was localized in N⁶(²isopentenyl)adenosine. The half life of tRNA was estimated to be 65–70 h. Shortly after the pulse period, oligonucleotides contained zeatin riboside at a ratio of 1:800, on the basis of radioactivity. The half life of these oligonucleotides was determined to be about 8 h. The main free radioactive cytokinin of roots and leaves was zeatin. Comparing the rate of degradation of ¹⁴C-labeled tRNA and the oligonucleotides of roots and the rate of appearance of radioactive cytokinins in roots and leaves, we found strong indications for their dependency. The results contradict the hypothesis of de novo synthesis of cytokinins in roots of intact bean plants.Abbreviations AMP adenosine monophosphate - IPA N⁶(²isopentenyl)adenosine - IPAde N⁶(²isopentenyl)adenosine - Z zeatin - ZR zeatinriboside - TLC thin-layer chromatography - HPLC high performance liquid chromatography Part of the doctoral thesis, Bonn 1980     

High-performance liquid chromatography with concanavalin A immobilized by metal interactions on the stationary phase

Concanavalin A (Con A) was immobilized via metal interactions on macroporous, microparticulate silica support having covalently bound iminodiacetic acid functions (IDA-silica) chelated with Cu(II) at the surface. The amount of copper and of Con A in the column could readily be controlled by the conditions used for chelating the metal by IDA-silica and for immobilization of the lectin. The retention behavior of columns packed with the stationary phase did not change under a wide range of elution conditions, indicating no loss of immobilized lectin. However, the Con A proper could readily be removed from the column at pH 3.0 or together with Cu(II) by perfusion with EDTA at neutral pH. Columns containing Con A immobilized by this technique exhibited dual retention behavior for proteins, glycoproteins, and carbohydrates according to the pertinent glycan-lectin or protein-metal interactions. The glycoproteins, peroxidase and alpha 1-acid glycoprotein, were retained by the Con A moiety and eluted with eluents containing competing sugars, whereas the proteins, beta-lactoglobulin, alpha-chymotrypsinogen A, and ribonuclease A and B were retained by the chelated copper and were eluted and separated with eluents containing sodium chloride or borate. Binding constants of glycosides on the immobilized Con A were evaluated chromatographically and found to be one-third to two-thirds those reported in the literature on the basis of experiments in free solution.     

Feasibility of protein spray coating using a fluid-bed Würster processor

This article documents a feasibility study on coating fine powders with protein solutions using a Würster spray coater (GPCG-1 from Glatt Air Techniques, Ramsey, NJ). Spray coating was based on a fluid-bed process where fluidized microcarriers were coated inside the Würster column and dried in the fluidization chamber. Recombinant human deoxyribonuclease (rhDNase) was used as the model protein. Lactose powders of two different size ranges, 53-125 and 125-250 mum, were used as the model microcarrier. The amount of protein applied was varied to obtain coatings of varying thickness. The extent of rhDNase loading determined experimentally was found to be consistent with the theoretical value and was also confirmed visually by scanning electron microscopy. The coating showed a strong integrity after being subjected to mechanical force. However, the protein suffered serious aggregation during coating, most likely due to the thermal stress of the process. Aggregation was significantly reduced when rhDNase was formulated with calcium ions, consistent with the observation that Ca(2+) thermally stabilized the protein (as determined by scanning microcalorimetry) in aqueous solution. Thus, our study demonstrates that spray coating, particularly when used in conjunction with rational stabilization strategies, is a feasible alternative to other methods of preparing dried pharmaceutical proteins. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 560-566, 1997.     

Eps8 protein facilitates phagocytosis by increasing TLR4-MyD88 protein interaction in lipopolysaccharide-stimulated macrophages

Toll-like receptors (TLRs) are crucial in macrophage phagocytosis, which is pivotal in host innate immune response. However, the detailed mechanism is not fully defined. Here, we demonstrated that the induction of Src and Eps8 in LPS-treated macrophages was TLR4- and MyD88-dependent, and their attenuation reduced LPS-promoted phagocytosis. Confocal microscopy indicated the colocalization of Eps8 and TLR4 in the cytosol and at the phagosome. Consistently, both Eps8 and TLR4 were present in the same immunocomplex regardless of LPS stimulation. Inhibition of this complex formation by eps8 siRNA or overexpression of pleckstrin homology domain-truncated Eps8 (i.e. 261-p97(Eps8)) decreased LPS-induced TLR4-MyD88 interaction and the following activation of Src, focal adhesion kinase, and p38 MAPK. Importantly, attenuation of Eps8 impaired the bacterium-killing ability of macrophages. Thus, Eps8 is a key regulator of the LPS-stimulated TLR4-MyD88 interaction and contributes to macrophage phagocytosis.