Methyl-beta-cyclodextrin induces mitochondrial cholesterol depletion and alters the mitochondrial structure and bioenergetics

There is growing evidence of mitochondrial membrane raft-like microdomains that are involved in the apoptotic pathway. The aim of this study was to investigate the effect of methyl-beta-cyclodextrin (MβCD), being a well-known lipid microdomain disrupting agent and cholesterol chelator, on the structure and bioenergetics of rat liver mitochondria (RLM). We observed that MβCD decreases the function of RLM, induces changes in the mitochondrial configuration state and decreases the calcium chloride-induced swelling. These data suggest that disruption of mitochondrial raft-like microdomains by cholesterol efflux on one hand impairs mitochondrial bioenergetics, but on the other hand it protects the mitochondria from swelling.     

Substrate Specificity, Membrane Topology, and Activity Regulation of Human Alkaline Ceramidase 2 (ACER2)

Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Δypc1Δydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca²⁺ for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2ΔN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2ΔN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2ΔN13, but not ACER2ΔN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2ΔN13 or ACER2ΔN36 inhibited the glycosylation of integrin β1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca²⁺ for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.     

Extended Anti-inflammatory Action of a Degradation-resistant Mutant of Cell-penetrating Suppressor of Cytokine Signaling 3

Suppressor of cytokine signaling 3 (SOCS3) regulates the proinflammatory cytokine signaling mediated by the JAK/STAT signaling pathway. SOCS3 is rapidly induced and then targeted to the ubiquitin-proteasome pathway via a mechanism that requires the C-terminal SOCS box. Due to its rapid turnover, the intracellular stores of SOCS3 seem insufficient to control acute or protracted inflammatory diseases. Previously, we developed an intracellular protein therapy that uses a recombinant cell-penetrating form of SOCS3 (CP-SOCS3) to inhibit the JAK/STAT pathway and prevent cytokine-mediated lethal inflammation and apoptosis of the liver (Jo, D., Liu, D., Yao, S., Collins, R. D., and Hawiger, J. (2005) Nat. Med. 11, 892–898). The potent anti-inflammatory and cytoprotective activity of CP-SOCS3 prompted us to analyze its intracellular turnover, as compared with that of endogenous SOCS3 protein induced in macrophages by the proinflammatory agonists, interferon-γ and lipopolysaccharide. We found that the half-life (t½) of endogenous SOCS3 is 0.7 h in activated macrophages, compared with a t½ of 6.2 h for recombinant CP-SOCS3. Deletion of the SOCS box in CP-SOCS3 renders it more resistant to proteasomal degradation, extending its t½ to 29 h. Consequently, this SOCS box-deleted form of CP-SOCS3 displays persistent inhibitory activity for 24 h toward interferon-γ- and lipopolysaccharide-induced cytokine and chemokine production. Compared with the wild-type suppressor, this gain-of-function CP-SOCS3 mutant provides a longer acting inhibitor of cytokine signaling, a feature that offers a clear advantage for the intracellular delivery of proteins to treat acute or protracted inflammatory diseases.     

Combined vascular endothelial growth factor-A and fibroblast growth factor 4 gene transfer improves wound healing in diabetic mice

Background

Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice.

Methods

Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination.

Results

Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration.

Conclusion

Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.     

RNA FRABASE 2.0: an advanced web-accessible database with the capacity to search the three-dimensional fragments within RNA structures

Background  

Recent discoveries concerning novel functions of RNA, such as RNA interference, have contributed towards the growing importance of the field. In this respect, a deeper knowledge of complex three-dimensional RNA structures is essential to understand their new biological functions. A number of bioinformatic tools have been proposed to explore two major structural databases (PDB, NDB) in order to analyze various aspects of RNA tertiary structures. One of these tools is RNA FRABASE 1.0, the first web-accessible database with an engine for automatic search of 3D fragments within PDB-derived RNA structures. This search is based upon the user-defined RNA secondary structure pattern. In this paper, we present and discuss RNA FRABASE 2.0. This second version of the system represents a major extension of this tool in terms of providing new data and a wide spectrum of novel functionalities. An intuitionally operated web server platform enables very fast user-tailored search of three-dimensional RNA fragments, their multi-parameter conformational analysis and visualization.     

The genetic diversity and evolution of field pea (<Emphasis Type="Italic">Pisum</Emphasis>) studied by high throughput retrotransposon based insertion polymorphism (RBIP) marker analysis

Background  

The genetic diversity of crop species is the result of natural selection on the wild progenitor and human intervention by ancient and modern farmers and breeders. The genomes of modern cultivars, old cultivated landraces, ecotypes and wild relatives reflect the effects of these forces and provide insights into germplasm structural diversity, the geographical dimension to species diversity and the process of domestication of wild organisms. This issue is also of great practical importance for crop improvement because wild germplasm represents a rich potential source of useful under-exploited alleles or allele combinations. The aim of the present study was to analyse a major Pisum germplasm collection to gain a broad understanding of the diversity and evolution of Pisum and provide a new rational framework for designing germplasm core collections of the genus.     

Biodiversity in Oscypek, a traditional Polish cheese, determined by culture-dependent and -independent approaches

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures.     

Dysfunctional HDL: A novel important diagnostic and therapeutic target in cardiovascular disease?

High density lipoprotein (HDL) has many properties, which contribute to its atheroprotective role. However, some recent clinical trials have identified subjects with the progression of atherosclerosis despite normal levels of HDL cholesterol. This raises the question if all subfractions of HDL have the same properties. Moreover, recent investigations have shown that both acute and chronic inflammation may lead to structural and functional changes of HDL, which render the particles proinflammatory. Although therapeutic agents that increase HDL levels are now quite well established it is not clear whether they influence HDL quality. We review the current state of knowledge on the properties of HDL and factors/therapeutic agents which may restrain the transformation of normal HDL into dysfunctional HDL.     

Tree species effects on coupled cycles of carbon, nitrogen, and acidity in mineral soils at a common garden experiment

Forest biogeochemical cycles are shaped by effects of dominant tree species on soils, but the underlying mechanisms are not well understood. We investigated effects of temperate tree species on interactions among carbon (C), nitrogen (N), and acidity in mineral soils from an experiment with replicated monocultures of 14 tree species. To identify how trees affected these soil properties, we evaluated correlations among species-level characteristics (e.g. nutrient concentrations in leaf litter, wood, and roots), stand-level properties (e.g. nutrient fluxes through leaf litterfall, nutrient pools in stemwood), and components of soil C, N, and cation cycles. Total extractable acidity (acidity_tot) was correlated positively with mineral soil C stocks (R ²  = 0.72, P < 0.001), such that a nearly two-fold increase in acidity_tot was associated with a more than two-fold increase of organic C. We attribute this correlation to effects of tree species on soil acidification and subsequent mineral weathering reactions, which make hydrolyzing cations available for stabilization of soil organic matter. The effects of tree species on soil acidity were better understood by measuring multiple components of soil acidity, including pH, the abundance of hydrolyzing cations in soil solutions and on cation exchange sites, and acidity_tot. Soil pH and acidity_tot were correlated with proton-producing components of the soil N cycle (e.g. nitrification), which were positively correlated with species-level variability in fine root N concentrations. Soluble components of soil acidity, such as aluminum in saturated paste extracts, were more strongly related to plant traits associated with calcium cycling, including leaf and root calcium concentrations. Our results suggest conceptual models of plant impacts on soil biogeochemistry should be revised to account for underappreciated plant traits and biogeochemical processes.