Lipidic pore formation by the concerted action of proapoptotic BAX and tBID

BCL-2 homology 3 (BH3)-only proteins of the BCL-2 family such as tBID and BIM(EL) assist BAX-type proteins to breach the permeability barrier of the outer mitochondrial membrane, thereby allowing cytoplasmic release of cytochrome c and other active inducers of cell death normally confined to the mitochondrial inter-membrane space. However, the exact mechanism by which tBID and BIM(EL) aid BAX and its close homologues in this mitochondrial protein release remains enigmatic. Here, using pure lipid vesicles, we provide evidence that tBID acts in concert with BAX to 1) form large membrane openings through both BH3-dependent and BH3-independent mechanisms, 2) cause lipid transbilayer movement concomitant with membrane permeabilization, and 3) disrupt the lipid bilayer structure of the membrane by promoting positive monolayer curvature stress. None of these effects were observed with BAX when BIM(EL) was substituted for tBID. Based on these data, we propose a novel model in which tBID assists BAX not only via protein-protein but also via protein-lipid interactions to form lipidic pore-type non-bilayer structures in the outer mitochondrial membrane through which intermembrane prodeath molecules exit mitochondria during apoptosis.     

Insulin-induced NADPH oxidase activation promotes proliferation and matrix metalloproteinase activation in monocytes/macrophages

Insulin stimulates superoxide (O₂^?) production in monocytes and macrophages. However, the mechanisms through which insulin induces O₂^? production are not completely understood. In this study, we (a) characterized the enzyme and the pathways involved in insulin-stimulated O₂^? production in human monocytes and murine macrophages, and (b) analyzed the consequences of insulin-stimulated O₂^? production on the cellular phenotype in these cells. We showed that insulin stimulated O₂^? production, and promoted p47^phox translocation to the plasma membrane. Insulin-induced O₂^? production and p47^phox translocation were prevented in the presence of specific inhibitors of PI3K and PKC. Insulin-mediated NADPH oxidase activation stimulated MMP-9 activation in monocytes and cell proliferation in macrophages. The effect of insulin on these phenotypic responses was mediated through NFκB, p38MAPK, and ERK 1/2 activation. Small-interfering RNA-specific gene silencing targeted specifically against Nox2 reduced the cognate protein expression, decreased insulin-induced O₂^? production, inhibited the turn on of NFκB, p38MAPK, and ERK 1/2, and reduced cell proliferation in macrophages. These findings suggest a pivotal role for NADPH oxidase in insulin-induced proliferation and proteolytic activation in monocytes and macrophages, respectively, and identify a pathway that may play a pathological role in hyperinsulinemic states.     

ADD CONTROL: advanced control solutions for waste water treatment

Instrumentation, control, and automation (ICA) in wastewater treatment enables the improvement of treatment plant performance without structural modifications of the plant. Even for wastewater treatment plants (WWTPs) meeting all criteria with respect to effluent concentrations and sludge disposal, ICA can be of interest as it can help to reduce energy consumption and operating costs of the plant. Simulations are a useful and cost-effective tool for designing and evaluating different control strategies. Simulation strategies developed with existing WWTP-specific simulation packages are based on ideal sensor and actuator behavior because signal noise and potential sensor and actuator failures are not considered. Real sensor and actuator behavior including failures, however, needs to be accounted for to ensure robust controller performance despite disturbances in sensor and actuator behavior. The ADD CONTROL project aims to design, implement, and validate a new simulation tool that allows for designing and testing “practical” control solutions. A multi-layer modeling architecture is proposed for the simulation tool to represent the hierarchical architecture for automation and control in full-scale WWTPs, and to separate mathematical modeling of components related to the treatment process from components describing instrumentation and actuation devices, and components related to automation and control. The developed simulation tool is implemented based on the TORNADO framework for modeling and virtual experimentation and the WEST^? product suite.     

Control of electron transfer by protein dynamics in photosynthetic reaction centers

Abstract

Trehalose and glycerol are low molecular mass sugars/polyols that have found widespread use in the protection of native protein states, in both short- and long-term storage of biological materials, and as a means of understanding protein dynamics. These myriad uses are often attributed to their ability to form an amorphous glassy matrix. In glycerol, the glass is formed only at cryogenic temperatures, while in trehalose, the glass is formed at room temperature, but only upon dehydration of the sample. While much work has been carried out to elucidate a mechanistic view of how each of these matrices interact with proteins to provide stability, rarely have the effects of these two independent systems been directly compared to each other. This review aims to compile decades of research on how different glassy matrices affect two types of photosynthetic proteins: (i) the Type II bacterial reaction center from Rhodobacter sphaeroides and (ii) the Type I Photosystem I reaction center from cyanobacteria. By comparing aggregate data on electron transfer, protein structure, and protein dynamics, it appears that the effects of these two distinct matrices are remarkably similar. Both seem to cause a “tightening” of the solvation shell when in a glassy state, resulting in severely restricted conformational mobility of the protein and associated water molecules. Thus, trehalose appears to be able to mimic, at room temperature, nearly all of the effects on protein dynamics observed in low temperature glycerol glasses.     

Conformational effects of histones H1 on DNA structure. Comparative study between H1-1, H1(0), H5 and sperm holothuria phi 0

Interactions of mammalian histones, H1-1 and H1(0), phi 0 from holothuria sperm and H5 with poly(dA-dT), poly(dG-dC) and poly(dG-me5dC) were measured by a nitrocellulose filter binding assay and circular dichroism. All of the proteins bound to every one of the polymers, but differed in the extent of binding, which depended on the polynucleotide/protein ratios and ionic strength. The order of retention of all polymers was phi 0 greater than H1-1 greater than H1(0). The binding of H1(0) to poly(dG-me5dC) was remarkably sensitive to ionic strength. The proteins caused changes in the spectral features of the polynucleotides, but differed in the type and extent of the change. Complexes prepared with H1-1 and H1(0) with all polymers showed a strongly negative psi spectrum. Complexes of poly(dA-dT) and phi 0, at a protein/polynucleotide ratio of 0.4, displayed a distinctive spectrum, giving the appearance of a Z-like DNA spectrum, at low ionic strength. At higher ionic strength the complexes showed a psi spectrum. Complexes of poly(dG-me5dC) in the Z or B conformation with phi 0 showed spectral features characteristic of a mixture of a Z-like and a psi spectrum. In contrast, H5 reduced the Z-DNA spectral features in the presence of Mg, and produced an inversion of the B spectrum up to a polynucleotide/protein ratio of 0.24. These findings demonstrate the ability of different proteins to produce changes in the conformation of DNA. This may reflect the ability of chromatin to undergo differential condensation, depending on both the base composition of DNA and the type of H1 histone bound to it.     

Selective removal of histone H1 from nucleosomes at low ionic strength

The method for removal of histone H 1 from chromatin by treatment with ion-exchange resin AG 50 WX 2 in the presence of 100 mM NaCl and 50 mM phosphate buffer (Thoma and Koller, 1977, Cell, 12, 101–107) results in production not only of H1-depleted chromatin but also free DNA. We have now modified this procedure so that the nucleosome is treated with the cation exchange resin in two steps, first in 50 mM sodium phosphate buffer and then in 50 mM sodium phosphate and 50 mM NaCl whereby histone H 1 is selectively removed without a release of free DNA at low resin concentrations.Abbreviations NaP Sodium phosphate buffer of molarities and pH as stated in the text - SDS Sodium dodecyl sulfate