Exposure to 16 Hz Pulsed Electromagnetic Fields Protect the Structural Integrity of Primary Cilia and Associated TGF-ß Signaling in Osteoprogenitor Cells Harmed by Cigarette Smoke.

Cigarette smoking (CS) is one of the main factors related to avoidable diseases and death across the world. Cigarette smoke consists of numerous toxic compounds that contribute to the development of osteoporosis and fracture nonunion. Exposure to pulsed electromagnetic fields (PEMF) was proven to be a safe and effective therapy to support bone fracture healing. The aims of this study were to investigate if extremely low frequency (ELF-) PEMFs may be beneficial to treat CS-related bone disease, and which effect the duration of the exposure has. In this study, immortalized human mesenchymal stem cells (SCP-1 cells) impaired by 5% cigarette smoke extract (CSE) were exposed to ELF-PEMFs (16 Hz) with daily exposure ranging from 7 min to 90 min. Cell viability, adhesion, and spreading were evaluated by Sulforhodamine B, Calcein-AM staining, and Phalloidin-TRITC/Hoechst 33342 staining. A migration assay kit was used to determine cell migration. Changes in TGF-ß signaling were evaluated with an adenoviral Smad2/3 reporter assay, RT-PCR, and Western blot. The structure and distribution of primary cilia were analyzed with immunofluorescent staining. Our data indicate that 30 min daily exposure to a specific ELF-PEMF most effectively promoted cell viability, enhanced cell adhesion and spreading, accelerated migration, and protected TGF-ß signaling from CSE-induced harm. In summary, the current results provide evidence that ELF-PEMF can be used to support early bone healing in patients who smoke.

N-glycosylations of human α1,3-fucosyltransferase IX are required for full enzyme activity.

Human α1,3-fucosyltransferase IX catalyzes the transfer of l-fucose from guanosine diphosphate-ß-L-fucose to N-acetyllactosamine, generating a Lewis X epitope, and is thereby involved in the synthesis of fucosylated cell surface glycoconjugates. It contains three putative N-glycosylation sites (Asn62, Asn101 and Asn153). The current study considers the functional role of these potential N-glycosylations within the enzyme. We produced truncated variants of human fucosyltransferase IX containing the soluble extracellular catalytic domain. To analyze the relevance of each N-glycosylation site, several genomic mutant DNAs encoding a glutamine (Gln/Q) instead of the asparagine residue were created prosperously using site-directed mutagenesis and subsequently expressed in Spodoptera frugiperda cells applying a baculovirus expression system. After production and purification of these variants of human FucT IX, the wild-type (wt) enzyme and the variants were characterized regarding their activity and kinetic properties. The variants showed lower activity than the wt FucT, whereas the individual N-glycosylation sites had different effects on the enzyme activity and kinetic parameters. While the single variant N62Q still showed ∼60% of wt activity and N101Q retained ∼30% activity, replacement of Asn153 by glutamine led to an almost complete loss of enzymatic activity. The same could be observed for variants missing two or more putative N-glycosylation sites, which indicated the importance of N-glycosylation for enzyme stability and activity.

Fluorescently labeled substrates for monitoring α1,3-fucosyltransferase IX activity.

Fucosylation is often the final process in glycan biosynthesis. The resulting glycans are involved in a variety of biological processes, such as cell adhesion, inflammation, or tumor metastasis. Fucosyltransferases catalyze the transfer of fucose residues from the activated donor molecule GDP-ß-L-fucose to various acceptor molecules. However, detailed information about the reaction processes is still lacking for most fucosyltransferases. In this work we have monitored α1,3-fucosyltransferase activity. For both donor and acceptor substrates, the introduction of a fluorescent ATTO dye was the last step in the synthesis. The subsequent conversion of these substrates into fluorescently labeled products by α1,3-fucosyltransferases was examined by high-performance thin-layer chromatography coupled with mass spectrometry as well as dual-color fluorescence cross-correlation spectroscopy, which revealed that both fluorescently labeled donor GDP-ß-L-fucose-ATTO 550 and acceptor N-acetyllactosamine-ATTO 647N were accepted by recombinant human fucosyltransferase IX and Helicobacter pylori α1,3-fucosyltransferase, respectively. Analysis by fluorescence cross-correlation spectroscopy allowed a quick and versatile estimation of the progress of the enzymatic reaction and therefore this method can be used as an alternative method for investigating fucosyltransferase reactions.

A Novel Method to Achieve Precision and Reproducibility in Exposure Parameters for Low-Frequency Pulsed Magnetic Fields in Human Cell Cultures.

The effects of extremely low-frequency electromagnetic field (ELF-MF) exposure on living systems have been widely studied at the fundamental level and also claimed as beneficial for the treatment of diseases for over 50 years. However, the underlying mechanisms and cellular targets of ELF-MF exposure remain poorly understood and the field has been plagued with controversy stemming from an endemic lack of reproducibility of published findings. To address this problem, we here demonstrate a technically simple and reproducible EMF exposure protocol to achieve a standardized experimental approach which can be readily adopted in any lab. As an assay system, we chose a commercially available inflammatory model human cell line; its response to magnetic fields involves changes in gene expression which can be monitored by a simple colorimetric reporter gene assay. The cells were seeded and cultured in microplates and inserted into a custom-built, semi-automated incubation and exposure system which accurately controls the incubation (temperature, humidity, CO2) and magnetic-field exposure conditions. A specific alternating magnetic field (<1.0% spatial variance) including far-field reduction provided defined exposure conditions at the position of each well of the microplate. To avoid artifacts, all environmental and magnetic-field exposure parameters were logged in real time throughout the duration of the experiment. Under these extensively controlled conditions, the effect of the magnetic field on the cell cultures as assayed by the standardized operating procedure was highly reproducible between experiments. As we could fully define the characteristics (frequency, intensity, duration) of the pulsed magnetic field signals at the position of the sample well, we were, for the first time, able to accurately determine the effect of changing single ELF-MF parameters such as signal shape, frequency, intensity and duty cycle on the biological response. One signal in particular (10 Hz, 50% duty cycle, rectangular, bipolar, 39.6µT) provided a significant reduction in cytokine reporter gene expression by 37% in our model cell culture line. In sum, the accuracy, environmental control and data-logging capacity of the semi-automated exposure system should greatly facilitate research into fundamental cellular response mechanisms and achieve the consistency necessary to bring ELF-MF/PEMF research results into the scientific mainstream.

Primary human osteoblasts with reduced alkaline phosphatase and matrix mineralization baseline capacity are responsive to extremely low frequency pulsed electromagnetic field exposure - Clinical implication possible.

For many years electromagnetic fields (EMFs) have been used clinically with various settings as an exogenous stimulation method to promote fracture healing. However, underlying mechanisms of action and EMF parameters responsible for certain effects remain unclear. Our aim was to investigate the influence of defined EMFs on human osteoblasts' and osteoclasts' viability and function. Primary human osteoblasts and osteoclasts were treated 3 times weekly for 21 days during their maturation process using the Somagen® device (Sachtleben GmbH, Hamburg, Germany), generating defined extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs). Certain ELF-PEMF treatment significantly increased the total protein content (up to 66%), mitochondrial activity (up to 91.1%) and alkaline phosphatase (AP) activity (up to 129.9%) of human osteoblasts during the entire differentiation process. Furthermore, ELF-PEMF treatment enhanced formation of mineralized matrix (up to 276%). Interestingly, ELF-PEMF dependent induction of AP activity and matrix mineralization was strongly donor dependent - only osteoblasts with a poor initial osteoblast function responded to the ELF-PEMF treatment. As a possible regulatory mechanism, activation of the ERK1/2 signaling pathway was identified. Maturation of osteoclasts from human monocytes was not affected by the ELF-PEMF treatment. In summary the results indicate that a specific ELF-PEMF treatment with the Somagen® device improves viability and maturation of osteoblasts, while osteoclast viability and maturation was not affected. Hence, ELF-PEMF might represent an interesting adjunct to conventional therapy supporting bone formation during fracture healing or even for the treatment of osteoporosis.

Comparison of expression systems for human fucosyltransferase IX.

Human fucosyltransferase IX (hFucT-IX) is a highly conserved alpha1,3 fucosyltransferase with a distinct acceptor and site specificity. hFucT-IX catalyses the transfer of activated fucose to a sugar acceptor, thereby forming the Lewis x epitope. This epitope is responsible for recognition phenomena throughout the body e.g. in tumour growth. Detailed characterisation of hFucT-IX structure-function relationships by kinetic and X-ray structure analysis is prerequisite to the development of enzyme inhibitors for clinical applications such as the suppression of tumour metastasis. For these analyses substantial amounts of hFucT-IX are desirable. Since hFucT-IX is not present in considerable amounts in common cells an overproduction of recombinant hFucT-IX is appropriate. To evaluate the best system for this overproduction we compared different strategies employing prokaryotes (Escherichia coli), mammalian cells and insect cells. Insect cells were tested using stable and baculoviral expression strategies. Current results favour the use of the baculoviral expression system for further experiments.

High nitrogen supply and carbohydrate content reduce fungal endophyte and alkaloid concentration in Lolium perenne.

The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent. Here, we infected two perennial ryegrass (Lolium perenne) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues. Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration. The results stress the need for wider quantification of fungal endophytes in the grassland-foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems.