Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: II. Investigation of the ManNAc kinase functionality

N-Acetylmannosamine (ManNAc) is the physiological precursors to all sialic acids that occur in nature. As variations in the sialic acid decoration of cell surfaces can profoundly affect cell-cell, pathogen-cell, or drug-cell interactions, the enzymes that convert ManNAc into sialic acid are attractive targets for the development of drugs that specifically interrupt sialic acid biosynthesis or lead to modified sialic acids on the surface of cells. The first step in the enzymatic conversion of ManNAc into sialic acid is phosphorylation, yielding N-acetylmannosamine-6-phosphate. The enzyme that catalyzes this conversion is the N-acetylmannosamine kinase (ManNAc kinase) as part of the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Here, we employed saturation transfer difference (STD) NMR experiments to study the binding of ManNAc and related ligands to the ManNAc kinase. It is shown that the configuration of C1 and C4 of ManNAc is crucial for binding to the enzyme, whereas the C2 position not only accepts variations in the attached N-acyl side chain but also tolerates inversion of configuration. Our experiments also show that ManNAc kinase maintains its functionality, even in the absence of Mg(2+). From the analysis of the STD NMR-derived binding epitopes, it is concluded that the binding mode of the N-acylmannosamines critically depends on the N-acyl side chain. In conjunction with the relative binding affinities of the ligands obtained from STD NMR titrations, it is possible to derive a structure-binding affinity relationship. This provides a cornerstone for the rational design of drugs for novel therapeutic applications by altering the sialic acid decorations of cell walls.     

Soil respiration under elevated CO2 and its partitioning into recently assimilated and older carbon sources

Plant and Soil - Efflux of soil CO2 (soil respiration) plays a crucial role in the global carbon cycle and may be strongly altered by global change. In this study, we measured soil respiration in...     

Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.     

Effect of phytosterols and phytostanols on the solubilization of cholesterol by dietary mixed micelles: an in vitro study

The effect of a plant sterol, beta-sitosterol (SI), and a plant stanol, sitostanol (SS), on the solubilization of cholesterol (CH) by model dietary mixed micelles was examined under in vitro conditions with the use of gas chromatography, isothermal titration calorimetry, NMR spectroscopy and cryogenic transmission electron microscopy techniques. Free SI and SS were shown to reduce the concentration of CH in dietary mixed micelles via a dynamic competition mechanism. CH, SI and SS affect the microstructure of lipid vesicles and influence the process of amphiphilic self-assembly of nutrients in the gut with the formation of dietary mixed micelles in a similar manner. Therefore, substitution of CH by phytosterols and phytostanols in the diet does not lead to the notable changes in the mechanism of dietary mixed micelle formation and does not affect the process of the intestinal transport of nutrients and drugs via the micellar diffusion mechanism. Our experimental findings demonstrate that the introduction of plant sterols and plant stanols into the diet is clearly beneficial for the reduction of the intestinal uptake of cholesterol. Due to the limited capacity of dietary mixed micelles to embody hydrophobic sterol/stanol molecules, the micellar concentration of cholesterol is reduced and hence, its transport towards the intestinal brush border membrane decreases.     

Mechanical regulation of HB-GAM expression in bone cells

Bone adaption upon mechanical stimulation is accompanied by changes in gene expression. In this context we investigated the influence of mechanical loading on heparin binding growth associated molecule (HB-GAM) expression, an extracellular matrix molecule which in cell culture has been shown to stimulate the differentiation of osteoblasts. We obtained information on the participating signal transduction pathways using a mitogenic loading regimen. Specific inhibitors of various signal transduction pathways were added to loaded cells and to unloaded controls. By semi-quantitative PCR studies we demonstrated a rapid decrease of HB-GAM expression in primary osteoblasts and SaOs-2 cells by 20-30% upon mechanical loading within 30min. We showed that the RGD-integrin interaction is involved in the regulation of HB-GAM expression. Furthermore, integrity of the cytoskeleton, stretch-activated, and voltage-sensitive Ca(2+) channels as well as gap junctional communication are necessary for the downregulation of HB-GAM expression by mechanical loading.     

Spinal Loads during Cycling on an Ergometer

Cycling on an ergometer is an effective exercise for improving fitness. However, people with back problems or previous spinal surgery are often not aware of whether cycling could be harmful for them. To date, little information exists about spinal loads during cycling. A telemeterized vertebral body replacement allows in vivo measurement of implant loads during the activities of daily living. Five patients with a severe compression fracture of a lumbar vertebral body received these implants. During one measurement session, four of the participants exercised on a bicycle ergometer at various power levels. As the power level increased, the maximum resultant force and the difference between the maximum and minimum force (force range) during each pedal revolution increased. The average maximum-force increases between the two power levels 25 and 85 W were 73, 84, 225 and 75 N for the four patients. The corresponding increases in the force range during a pedal revolution were 84, 98, 166 and 101 N. There were large variations in the measured forces between the patients and also within the same patient, especially for high power levels. In two patients, the maximum forces during high-power cycling were higher than the forces during walking measured on the same day. Therefore, the authors conclude that patients with back problems should not cycle at high power levels shortly after surgery as a precaution.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

Monthly to interannual variability of microbial eukaryote assemblages at four depths in the eastern North Pacific

The monthly, seasonal and interannual variability of microbial eukaryote assemblages were examined at 5 m, the deep chlorophyll maximum, 150 m and 500 m at the San Pedro Ocean Time-series station (eastern North Pacific). The depths spanned transitions in temperature, light, nutrients and oxygen, and included a persistently hypoxic environment at 500 m. Terminal restriction fragment length polymorphism was used for the analysis of 237 samples that were collected between September 2000 and December 2010. Spatiotemporal variability patterns of microeukaryote assemblages indicated the presence of distinct shallow and deep communities at the SPOT station, presumably reflecting taxa that were specifically adapted for the conditions in those environments. Community similarity values between assemblages collected 1 month apart at each depth ranged between ∼20% and ∼84% (averages were ∼50–59%). The assemblage at 5 m was temporally more dynamic than deeper assemblages and also displayed substantial interannual variability during the first ∼3 years of the study. Evidence of seasonality was detected for the microbial eukaryote assemblage at 5 m between January 2008 and December 2010 and at 150 m between September 2000 and December 2003. Seasonality was not detected for assemblages at the deep chlorophyll a maximum, which varied in depth seasonally, or at 500 m. Microbial eukaryote assemblages exhibited cyclical patterns in at least 1 year at each depth, implying an annual resetting of communities. Substantial interannual variability was detected for assemblages at all depths and represented the largest source of temporal variability in this temperate coastal ecosystem.     

Label‐free analysis of human cerebrospinal fluid addressing various normalization strategies and revealing protein groups affected by multiple sclerosis

The aims of the study were to: (i) identify differentially regulated proteins in cerebrospinal fluid (CSF) between multiple sclerosis (MS) patients and non‐MS controls; (ii) examine the effect of matching the CSF samples on either total protein amount or volume, and compare four protein normalization strategies for CSF protein quantification. CSF from MS patients (n = 37) and controls (n = 64), consisting of other noninflammatory neurological diseases (n = 50) and non neurological spinal anesthetic subjects (n = 14), were analyzed using label‐free proteomics, quantifying almost 800 proteins. In total, 122 proteins were significantly regulated (p < 0.05), where 77 proteins had p‐value <0.01 or AUC value >0.75. Hierarchical clustering indicated that there were two main groups of MS patients, those with increased levels of inflammatory response proteins and decreased levels of proteins involved in neuronal tissue development (n = 30), and those with normal protein levels for both of these protein groups (n = 7). The main subgroup of controls clustering with the MS patients showing increased inflammation and decreased neuronal tissue development were patients suffering from chronic fatigue. Our data indicate that the preferable way to quantify proteins in CSF is to first match the samples on total protein amount and then normalize the data based on the median intensities, preferably from the CNS‐enriched proteins.