Studying non-covalent enzyme carbohydrate interactions by STD NMR

Saturation transfer difference NMR spectroscopy is used to study non-covalent interactions between four different glycostructure transforming enzymes and selected substrates and products. Resulting binding patterns represent a molecular basis of specific binding between ligands and biocatalysts. Substrate and product binding to Aspergillus fumigatus glycosidase and to Candida tenuis xylose reductase are determined under binding-only conditions. Measurement of STD effects in substrates and products over the course of enzymatic conversion provides additional information about ligand binding during reaction. Influences of co-substrates and co-enzymes in substrate binding are determined for Schizophyllum commune trehalose phosphorylase and C. tenuis xylose reductase, respectively. Differences between ligand binding to wild type enzyme and a corresponding mutant enzyme are shown for Corynebacterium callunae starch phosphorylase and its His-334-->Gly mutant. The resulting binding patterns are discussed with respect to the possibility that ligands do not only bind in the productive mode.     

Inhibition of the mitochondrial respiratory chain function abrogates quartz induced DNA damage in lung epithelial cells

Respirable quartz dust has been classified as a human carcinogen by the International Agency for Research on Cancer. The aim of our study was to investigate the mechanisms of DNA damage by DQ12 quartz in RLE-6TN rat lung epithelial type II cells (RLE). Transmission electron microscopy and flow-cytometry analysis showed a rapid particle uptake (30 min to 4 h) of quartz by the RLE cells, but particles were not found within the cell nuclei. This suggests that DNA strand breakage and induction of 8-hydroxydeoxyguanosine - as also observed in these cells during these treatment intervals - did not result from direct physical interactions between particles and DNA, or from short-lived particle surface-derived reactive oxygen species. DNA damage by quartz was significantly reduced in the presence of the mitochondrial inhibitors rotenone and antimycin-A. In the absence of quartz, these inhibitors did not affect DNA damage, but they reduced cellular oxygen consumption. No signs of apoptosis were observed by quartz. Flow-cytometry analysis indicated that the reduced DNA damage by rotenone was not due to a possible mitochondria-mediated reduction of particle uptake by the RLE cells. Further proof of concept for the role of mitochondria was shown by the failure of quartz to elicit DNA damage in mitochondria-depleted 143B (rho-0) osteosarcoma cells, at concentrations where it elicited DNA damage in the parental 143B cell line. In conclusion, our data show that respirable quartz particles can elicit oxidative DNA damage in vitro without entering the nuclei of type II cells, which are considered to be important target cells in quartz carcinogenesis. Furthermore, our observations indicate that such indirect DNA damage involves the mitochondrial electron transport chain function, by an as-yet-to-be elucidated mechanism.     

Characterization of Dynamic Behaviour of MCF7 and MCF10A Cells in Ultrasonic Field Using Modal and Harmonic Analyses

Treatment options specifically targeting tumour cells are urgently needed in order to reduce the side effects accompanied by chemo- or radiotherapy. Differences in subcellular structure between tumour and normal cells determine their specific elasticity. These structural differences can be utilised by low-frequency ultrasound in order to specifically induce cytotoxicity of tumour cells. For further evaluation, we combined in silico FEM (finite element method) analyses and in vitro assays to bolster the significance of low-frequency ultrasound for tumour treatment. FEM simulations were able to calculate the first resonance frequency of MCF7 breast tumour cells at 21 kHz in contrast to 34 kHz for the MCF10A normal breast cells, which was due to the higher elasticity and larger size of MCF7 cells. For experimental validation of the in silico-determined resonance frequencies, equipment for ultrasonic irradiation with distinct frequencies was constructed. Differences for both cell lines in their response to low-frequent ultrasonic treatment were corroborated in 2D and in 3D cell culture assays. Treatment with ~ 24.5 kHz induced the death of MCF7 cells and MDA-MB-231 metastases cells possessing a similar elasticity; frequencies of > 29 kHz resulted in cytotoxicity of MCF10A. Fractionated treatments by ultrasonic irradiation of suspension myeloid HL60 cells resulted in a significant decrease of viable cells, mostly significant after threefold irradiation in intervals of 3 h. Most importantly in regard to a clinical application, combined ultrasonic treatment and chemotherapy with paclitaxel showed a significantly increased killing of MCF7 cells compared to both monotherapies. In summary, we were able to determine for the first time for different tumour cell lines a specific frequency of low-intensity ultrasound for induction of cell ablation. The cytotoxic effect of ultrasonic irradiation could be increased by either fractionated treatment or in combination with chemotherapy. Thus, our results will open new perspectives in tumour treatment.     

Cdx2 determines the fate of postnatal intestinal endoderm

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.     

Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.     

Extending the dynamic range of label-free mass spectrometric quantification of affinity purifications

Affinity purification (AP) of protein complexes combined with LC-MS/MS analysis is the current method of choice for identification of protein-protein interactions. Their interpretation with respect to significance, specificity, and selectivity requires quantification methods coping with enrichment factors of more than 1000-fold, variable amounts of total protein, and low abundant, unlabeled samples. We used standardized samples (0.1-1000 fmol) measured on high resolution hybrid linear ion trap instruments (LTQ-FT/Orbitrap) to characterize and improve linearity and dynamic range of label-free approaches. Quantification based on spectral counts was limited by saturation and ion suppression effects with samples exceeding 100 ng of protein, depending on the instrument setup. In contrast, signal intensities of peptides (peak volumes) selected by a novel correlation-based method (TopCorr-PV) were linear over at least 4 orders of magnitude and allowed for accurate relative quantification of standard proteins spiked into a complex protein background. Application of this procedure to APs of the voltage-gated potassium channel Kv1.1 as a model membrane protein complex unambiguously identified the whole set of known interaction partners together with novel candidates. In addition to discriminating these proteins from background, we could determine efficiency, cross-reactivities, and selection biases of the used purification antibodies. The enhanced dynamic range of the developed quantification procedure appears well suited for sensitive identification of specific protein-protein interactions, detection of antibody-related artifacts, and optimization of AP conditions.     

Land use intensification alters ecosystem multifunctionality via loss of biodiversity and changes to functional composition

Global change, especially land‐use intensification, affects human well‐being by impacting the delivery of multiple ecosystem services (multifunctionality). However, whether biodiversity loss is a major component of global change effects on multifunctionality in real‐world ecosystems, as in experimental ones, remains unclear. Therefore, we assessed biodiversity, functional composition and 14 ecosystem services on 150 agricultural grasslands differing in land‐use intensity. We also introduce five multifunctionality measures in which ecosystem services were weighted according to realistic land‐use objectives. We found that indirect land‐use effects, i.e. those mediated by biodiversity loss and by changes to functional composition, were as strong as direct effects on average. Their strength varied with land‐use objectives and regional context. Biodiversity loss explained indirect effects in a region of intermediate productivity and was most damaging when land‐use objectives favoured supporting and cultural services. In contrast, functional composition shifts, towards fast‐growing plant species, strongly increased provisioning services in more inherently unproductive grasslands.     

cAMP-dependent protein kinase and Ca2+ influx through L-type voltage-gated calcium channels mediate Raf-independent activation of extracellular regulated kinase in response to glucagon-like peptide-1 in pancreatic beta-cells

Glucagon like peptide-1 (GLP1) is a G(s)-coupled receptor agonist that exerts multiple effects on pancreatic beta-cells, including the stimulation of insulin gene expression and secretion. In this report, we show that treatment of the mouse pancreatic beta-cell line MIN6 with GLP1 leads to the glucose-dependent activation of Erk. These effects are mimicked by forskolin, a direct activator of adenylate cyclase, and blocked by H89, an inhibitor of cAMP-dependent protein kinase. Additionally, we provide evidence that GLP1-stimulated activation of Erk requires an influx of calcium through L-type voltage-gated calcium channels and the activation of calcium/calmodulin-dependent protein kinase II. GLP1-stimulated activation of Erk is blocked by inhibitors of MEK, but GLP1 does not induce the activation of A-Raf, B-Raf, C-Raf, or Ras. Additionally, dominant negative forms of Ras(N17) and Rap1(N17) fail to block GLP1-stimulated activation of Erk. In conclusion, our results indicate that, in the presence of stimulatory concentrations of glucose, GLP1 stimulates the activation of Erk through a mechanism dependent on MEK but independent of both Raf and Ras. This requires 1) the activation of cAMP-dependent protein kinase, 2) an influx of extracellular Ca(2+) through L-type voltage-gated calcium channels, and 3) the activation of CaM kinase II.     

Genome Reduction Is Associated with Bacterial Pathogenicity across Different Scales of Temporal and Ecological Divergence

Emerging bacterial pathogens threaten global health and food security, and so it is important to ask whether these transitions to pathogenicity have any common features. We present a systematic study of the claim that pathogenicity is associated with genome reduction and gene loss. We compare broad-scale patterns across all bacteria, with detailed analyses of Streptococcus suis, an emerging zoonotic pathogen of pigs, which has undergone multiple transitions between disease and carriage forms. We find that pathogenicity is consistently associated with reduced genome size across three scales of divergence (between species within genera, and between and within genetic clusters of S. suis). Although genome reduction is also found in mutualist and commensal bacterial endosymbionts, genome reduction in pathogens cannot be solely attributed to the features of their ecology that they share with these species, that is, host restriction or intracellularity. Moreover, other typical correlates of genome reduction in endosymbionts (reduced metabolic capacity, reduced GC content, and the transient expansion of nonfunctional elements) are not consistently observed in pathogens. Together, our results indicate that genome reduction is a consistent correlate of pathogenicity in bacteria.