Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.     

Innovative combination strategy to enhance effect and diminish adverse effects of glucocorticoids: another promise?

In a paper by Zimmermann and colleagues in this issue of Arthritis Research & Therapy, results of extended laboratory research with the drug combination of prednisolone and dipyridamole are reported. There seems to be a boost and extension of the glucocorticoid effect by the combination, without a clear increase of adverse effects, potentially allowing the application of lower dosages. However, laboratory models are not patients and the glucocorticoid mechanisms leading to effects and adverse effects are manifold. The next required step will be to demonstrate the improved therapeutic window in patients in adequate comparative clinical trials, assessing predefined beneficial effects and adverse effects in a standardized way.     

Triple benefits from spatial resource management

Standard fishery management models suggest that regulations designed to produce maximum rent should reduce effort – and thus employment – from its open-access level. Using a simple diffusion model, we show that the opposite can be true when the spatial distribution of effort, as well as the total amount of effort, can be controlled. Under certain ecological and economic circumstances that we describe, optimal spatial management can produce “triple benefits” compared to open access: an increase in rent, an increase in standing stock, and an increase in employment. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Green-to-Red Photoconversion of GCaMP

Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs—including GCaMP3, GCaMP5 and GCaMP6—can be converted from green to red following exposure to blue-green light (450–500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.     

Response to thyrotropin of normal thyroid follicular cell strain FRTL5 in hypergravity.

Thyroid hormones control every cell in the organisms and, as indicated by many hormonal changes in astronauts during and shortly after space missions, its complex regulation may be influenced by gravity. To test in vitro the effects of gravity environment on thyroid, we selected a unique cultured cell system: the FRTL5, a normal follicular thyroid cell strain in continuous culture, originally derived from adult rat thyroids. To establish if modifications of the gravitational environment may interfere with post-receptorial signal transduction mechanisms in normal mammalian cultured cells, following our previous microgravity experiments, we exposed thyrotropin-stimulated and unstimulated FRTL5 cells to hypergravity (5 g and 9 g) in a special low-speed centrifuge. At all thyrotropin doses tested, we found significant increases in terms of cyclic AMP production in FRTL5 thyroid cells. The data here reported correlate well with our previous microgravity data, showing that the FRTL5 cells functionally respond to the variable gravity force in a dose-dependent manner in terms of cAMP production following TSH-stimulation.     

Effects of branching spatial structure and life history on the asymptotic growth rate of a population

The dendritic structure of a river network creates directional dispersal and a hierarchical arrangement of habitats. These two features have important consequences for the ecological dynamics of species living within the network. We apply matrix population models to a stage-structured population in a network of habitat patches connected in a dendritic arrangement. By considering a range of life histories and dispersal patterns, both constant in time and seasonal, we illustrate how spatial structure, directional dispersal, survival, and reproduction interact to determine population growth rate and distribution. We investigate the sensitivity of the asymptotic growth rate to the demographic parameters of the model, the system size, and the connections between the patches. Although some general patterns emerge, we find that a species’ modes of reproduction and dispersal are quite important in its response to changes in its life history parameters or in the spatial structure. The framework we use here can be customized to incorporate a wide range of demographic and dispersal scenarios.     

The early marginal zone B cell-initiated T-independent type 2 response resists the proteasome inhibitor bortezomib

The proteasome inhibitor bortezomib is approved for the treatment of multiple myeloma and mantle cell lymphoma. We recently demonstrated that bortezomib eliminates autoreactive plasma cells in systemic lupus erythematosus mouse models, thereby representing a promising novel treatment for Ab-mediated diseases. In this study, we investigated the effects of bortezomib on the just developing and pre-existing T-dependent Ab response toward dinitrophenyl-keyhole limpet hemocyanin and the T-independent type 2 response toward (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP)-Ficoll in BALB/c mice. Bortezomib treatment strongly reduced T-dependent Ab titers mainly due to depletion of plasma cells. In contrast, the early T-independent type 2 response against i.v. administered NIP-Ficoll, which is predominantly dependent on marginal zone (MZ) B cells, resisted bortezomib. Upon bortezomib treatment, immunoproteasome subunits and the antiapoptotic unfolded protein response including NF-κB were induced in NIP-Ficoll-stimulated MZ B cells, but not in plasma cells and follicular B cells. In summary, bortezomib treatment decreases Ab titers arising from T-dependent immune responses predominantly by eliminating plasma cells. In contrast, the early T-independent type 2 response protecting the organism against blood-borne pathogens remains largely intact due to a remarkable resistance of MZ B cells against proteasome inhibition.     

Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.     

Only a Few Fungal Species Dominate Highly Diverse Mycofloras Associated with the Common Reed

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed Phragmites australis as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.