Extremely low frequency electromagnetic fields and heat shock can increase microvesicle motility in astrocytes

The effect of extremely low frequency electromagnetic fields (EMF) on microvesicles was examined in rat astrocytes by video-enhanced microscopy in combination with a perfusable cell chamber. The EMF effect was compared with the effect of heat shock (HS) and with a combination of them both. The velocity of microvesicles was measured using image processing software (NIH Scion image 1.61). After exposure of astrocytes to EMF (50 Hz, 100microT, 1 h), the velocity of microvesicles in astrocytes increased from 0.32 +/- 0.03 microm/s (n = 120, 95% CI) in the untreated control group to 0.41 +/- 0.03 microm/s (n = 175, 95% CI). Fifteen minutes after HS (45 degrees C, 10 min) the microvesicles showed a velocity of 0.56 +/- 0.03 microm/s (n = 125, 95% CI). Combination of HS and EMF led to an increase in velocity up to 0.54 +/- 0.03 microm/s (n = 110, 95% CI). No significant difference between HS and HS+EMF was found. Compared to the untreated control group, the increased microvesicle velocity of the exposed cells might be a stress response of the cell. It is possibly a sign of intensified intracellular traffic required to adjust the metabolic needs.     

Conformational Ensembles of Antibodies Determine Their Hydrophobicity

A major challenge in the development of antibody biotherapeutics is their tendency to aggregate. One root cause for aggregation is exposure of hydrophobic surface regions to the solvent. Many current techniques predict the relative aggregation propensity of antibodies via precalculated scales for the hydrophobicity or aggregation propensity of single amino acids. However, those scales cannot describe the nonadditive effects of a residue’s surrounding on its hydrophobicity. Therefore, they are inherently limited in their ability to describe the impact of subtle differences in molecular structure on the overall hydrophobicity. Here, we introduce a physics-based approach to describe hydrophobicity in terms of the hydration free energy using grid inhomogeneous solvation theory (GIST). We apply this method to assess the effects of starting structures, conformational sampling, and protonation states on the hydrophobicity of antibodies. Our results reveal that high-quality starting structures, i.e., crystal structures, are crucial for the prediction of hydrophobicity and that conformational sampling can compensate errors introduced by the starting structure. On the other hand, sampling of protonation states only leads to good results when combined with high-quality structures, whereas it can even be detrimental otherwise. We conclude by pointing out that a single static homology model may not be adequate for predicting hydrophobicity.     

Silicon: The key element in early stages of biocalcification

Biocalcification is a widespread process of forming hard tissues like bone and teeth in vertebrates. It is also a topic connecting life sciences and earth sciences: calcified skeletons and shells deposited as sediments represent the earth's fossil record and are of paramount interest for biogeochemists trying to get an insight into the past of our planet. This study reports on the role of silicon in the early biocalcification steps, where silicon and calcium were detected on the surface of cyanobacteria (initial stage of lacustrine calcite precipitation) and in crustacean cuticles. By using innovative methodological approaches of correlative microscopy (AFM in combination with analytical TEM: EFTEM, EELS) the chemical form of silicon in biocalcifying matrices and organic-inorganic particles is determined. Previously, silicon was reported to be localized in active growth areas in the young bone of vertebrates. We have found evidence that biocalcification in evolutionarily distant organisms involves very similar initial phases with silicon as a key element at the organic-inorganic interface.     

The zymogen granule protein 2 (GP2) binds to scavenger receptor expressed on endothelial cells I (SREC-I)

The pancreatic zymogen granule membrane protein (GP2) is expressed by pancreatic acinar cells and M cells of the ileum. GP2 is the closest related homologue of the urine resident Tamm–Horsfall protein (THP). Recently, it was shown that THP is a ligand of various scavenger receptors (SRs). Therefore, we were interested, if GP2 has similar properties.cDNA of different SRs was stably transfected into a murine thymoma cell line. GP2 was recombinantly expressed, purified and biotinylated. Binding or uptake of GP2 by transfected cells or monocyte-derived dendritic cells (moDCs) was analyzed by flow-cytometry.GP2 is a binding partner of the scavenger receptor expressed on endothelial cells I (SREC-I) but not of SR-AI and SR-BI. The dissociation constant (K_d) of GP2 binding to SREC-I is 41.3 nM. SREC transfected cells are able to internalize GP2. moDCs express SREC-I and also bind and internalize GP2. Inhibition of SREC-I on moDCs with anti-SREC-I antibodies does not result in a decreased GP2 binding.Interaction of GP2 with SREC-I and uptake might have profound effects in antigen clearance and mediation of the immune response. In addition to SREC-I other presently unknown receptors for GP2 on DCs might be involved in this process.     

In Human Monocyte Derived Dendritic Cells SOCS1 Interacting with CYTIP Induces the Degradation of CYTIP by the Proteasome

CYTIP (cytohesin interacting protein) is an intracellular molecule induced in dendritic cells during maturation. CYTIP modulates the binding intensity of the adhesion molecule LFA-1. If dendritic cells are silenced for CYTIP they keep longer contacts with T-cells resulting in a lower T cell stimulation. We identified Suppressor of cytokine signaling-1 (SOCS-1) as a binding partner for CYTIP in human monocyte derived dendritic cells. In Western blot analyses we found that CYTIP expression is down regulated at later time points, starting at about 72 hours after induction of maturation. To investigate a possible role for SOCS-1 in taking CYTIP to the degradation machinery of the cell we measured endogenous CYTIP protein levels in mature dendritic cells transfected with SOCS-1 encoding plasmid in quantitative Western blot analyses. We observed lower amounts of endogenous CYTIP in mature dendritic cells transfected with SOCS-1 encoding plasmid compared with untransfected dendritic cells. Experiments with the proteasome-inhibitor Bortezomib/Velcade® show stable CYTIP expression levels in dendritic cells. In addition, we show that CYTIP in dendritic cells matured for 48 hours is ubiquitinated and thus ready for degradation. We here describe a newly identified binding partner of CYTIP, SOCS-1, and confirm its function in regulating the degradation of CYTIP by the proteasome.