Alphaproteobacteria dominate active 2-methyl-4-chlorophenoxyacetic acid herbicide degraders in agricultural soil and drilosphere

2-Methyl-4-chlorophenoxyacetic acid (MCPA) is a widely used phenoxyalkanoic acid herbicide and subject to aerobic microbial degradation. Earthworms stimulate both growth and activity of MCPA-degrading bacteria in soil. Thus, active MCPA degraders in soil and drilosphere (i.e. burrow walls, gut content and cast) were assessed by 16S rRNA stable isotope probing in soil columns under experimental conditions designed to minimize laboratory incubation biases. Agriculturally relevant concentrations of [(13) C]MCPA (20 μg g(dw) (-1)) were degraded in soil within 23 and 27 days in the presence and absence of earthworms respectively. Total 16S rRNA analysis revealed 73 operational taxonomic units indicative of active Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Planctomycetes, Proteobacteria and Verrucomicrobia in soil and drilosphere derived material. Seven operational taxonomic units indicative of Alpha-, Beta-, Gammaproteobacteria and Firmicutes consumed MCPA-[(13) C]. Dominant consumers of MCPA-[(13) C] were Alphaproteobacteria (Sphingomonadaceae and Bradyrhizobiaceae) in soil and drilosphere. Beta- (Comamonadaceae) and Gammaproteobacteria (Xanthomonadaceae) were also important MCPA-[(13) C] consumers in burrow walls only, indicating that earthworms favour betaproteobacterial MCPA degraders. In oxic microcosms with bulk soil, burrow walls and cast, 20 and 300-400 μg g(dw) (-1) [(13) C]MCPA were consumed within 24 h and 20 days respectively. Gut contents did not facilitate the degradation of [(13) C]MCPA. Sphingomonadaceae dominated MCPA-[(13) C] consumers in bulk soil and burrow wall microcosms, while Beta- and Gammaproteobacteria (Burkholderiacea, Comamonadaceae, Oxalobacteraceae and Xanthomonadaceae) dominated MCPA-[(13) C] consumers in microcosms of cast, indicating that the latter taxa are prone to respond to MCPA in cast. The collective data indicated that Alphaproteobacteria are major MCPA degraders in soil and drilosphere.     

Evaluation of an in vitro coculture model for the blood-brain barrier: Comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources

Summary Cocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.     

Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha

Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.     

The flower and the butterfly constructed wetland system at Koh Phi Phi—System design and lessons learned during implementation and operation

In 2007, a constructed wetland system was implemented on the tourist island of Koh Phi Phi in Southern Thailand. This paper presents the process of planning, designing and implementing the system and discusses the performance and operational issues 3 years after implementation. The system is an international lighthouse project showing the potential for aesthetical integration of constructed wetland systems in the built environment. The system comprises a wastewater collection system for the main business and hotel area of the island, a pumping station and a pressure pipe system to the treatment facility, a multi-stage constructed wetland system, and a system for reuse of treated wastewater. The treatment chains consist of vertical-flow, horizontal subsurface flow and free water surface flow units. Because the treatment system is located at the centre of the island, surrounded by resorts, restaurants and shops, the systems are designed with terrains as scenic landscaping. The wastewater is treated to meet the Thai effluent standards for total suspended solids and nitrogen, but because of inadequate pre-treatment and removal of oil and grease prior to the system, the standards for oil and grease and BOD are not met. A number of challenges during construction and operation have caused problems with clogging of the vertical flow constructed wetlands and given rise to odour problems. Safeguards were prepared, but not effectively activated, mainly because no key-person or key-authority took responsibility for managing the system.     

Renal sulfatides: sphingoid base-dependent localization and region-specific compensation of CerS2-dysfunction

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer­amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound’s function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS² (in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS² analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.     

Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line

Intracellular Ca²⁺ mobilization events were assessed in mouse L cells, which contain native prostaglandin E₁ receptors and transfected human ₂ adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca²⁺]_i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca²⁺]_i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca²⁺ release. [Ca²⁺]₁ in these cells was also increased by prostaglandin E₁, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP₁, IP₂, and IP₃. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP₃ production which leads to an elevation in [Ca²⁺];. We propose that this cyclic AMP dependent activation of the IP₃ generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca²⁺]_i intracellular [Ca²⁺]_i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP₂ Phosphatidylinositol 4,5-bisphosphate - IP₂ Inositol 4 Phosphate - IP₂ Inositol 4,5 Bisphosphate - IP₃ Inositol Trisphosphate - PGE₁ Prostaglandin E₁ - PBS Phosphate Buffered Saline Solution     

The influence of biomass and structure of the crustacean plankton on the water transparency in the Saidenbach storage reservoir

Planktonic crustacean biomass as well as structure are important factors influencing water transparency. The significant dependence of the water quality (Secchi depth) on the concentration and the share of the Daphnia biovolume and not on the total Crustacea biovolume in the Saidenbach reservoir indicates that the density of the Crustacea is only a measure of the cleaning performance, if Daphnia dominates. Using the mean size, the influence of the crustacean structure on the Secchi depth can be recorded. If big size categories prevail (like Daphnia) the water transparency is high. The mainly occurrence of little species (Mesocyclops, Bosmina) results in lower Secchi depths. However, a well defined (significant) relationship is being prevented by the different feeding behaviour of the several species.     

Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth

The ER‐bound kinase/endoribonuclease (RNase), inositol‐requiring enzyme‐1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box‐binding protein‐1 (XBP1) or the RNA substrates of IRE1‐dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide‐derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross‐talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P₃) 5‐phosphatase‐2 (INPPL1) is a direct target of miR‐2137, which controls PI(3,4,5)P₃ levels in macrophages. The modulation of cellular PI(3,4,5)P₃/PIP₂ ratio and anabolic mTOR signaling by the IRE1‐induced miR‐2137 demonstrates how the ER can provide a critical input into cell growth decisions.     

AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin

The A kinase-anchoring protein AKAP79/150 is a postsynaptic scaffold molecule and a key regulator of signaling events. At the postsynapse it coordinates phosphorylation and dephosphorylation of receptors via anchoring kinases and phosphatases near their substrates. Interactions between AKAP79 and two Ca(2+) -binding proteins caldendrin and calmodulin have been investigated here. Calmodulin is a known interaction partner of AKAP79/150 that has been shown to regulate activity of the kinase PKC in a Ca(2+) -dependent manner. Pull-down experiments and surface plasmon resonance biosensor analyses have been used here to demonstrate that AKAP79 can also interact with caldendrin, a neuronal calcium-binding protein implicated in regulation of Ca(2+) -influx and release. We demonstrate that calmodulin and caldendrin compete for a partially overlapping binding site on AKAP79 and that their binding is differentially dependent on calcium. Therefore, this competition is regulated by calcium levels. Moreover, both proteins have different binding characteristics suggesting that the two proteins might play complementary roles. The postsynaptic enrichment, the complex binding mechanism, and the competition with calmodulin, makes caldendrin an interesting novel player in the signaling toolkit of the AKAP interactome.