Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector–DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses.     

Role of oxidative stress in geldanamycin-induced cytotoxicity and disruption of Hsp90 signaling complex

Heat shock protein 90 (Hsp90) is a chaperone protein regulating PC-12 cell survival by binding and stabilizing Akt, Raf-1, and Cdc37. Hsp90 inhibitor geldanamycin (GA) cytotoxicity has been attributed to the disruption of Hsp90 binding, and the contribution of oxidative stress generated by its quinone group has not been studied in this context. Reactive oxygen species (ROS) and cell survival were assessed in PC-12 cells exposed to GA or menadione (MEN), and Akt, Raf-1, and Cdc37 expression and binding to Hsp90 were determined. GA disrupted Hsp90 binding and increased ROS production starting at 1 h, and cell death occurred at 6 h, inhibited by N-acetylcysteine (NAC) without preventing dissociation of proteins. At 24 h, NAC prevented cytotoxicity and Hsp90 complex disruption. However, MnTBAP antioxidant treatment failed to inhibit GA cytotoxicity, suggesting that NAC acts by restoring glutathione. In contrast, 24 h MEN treatment induced cytotoxicity without disrupting Hsp90 binding. GA and MEN decreased Hsp90-binding protein expression, and proteasomal inhibition prevented MEN-, but not GA-induced degradation. In conclusion, whereas MEN cytotoxicity is mediated by ROS and proteasomal degradation, GA-induced cytotoxicity requires ROS but induces Hsp90 complex dissociation and proteasome-independent protein degradation. These differences between MEN- and GA-induced cytotoxicity may allow more specific targeting of cancer cells.     

Biodegradation kinetics of 1,4-benzoquinone in batch and continuous systems

Combining chemical and biological treatments is a potentially economic approach to remove high concentration of recalcitrant compounds from wastewaters. In the present study, the biodegradation of 1,4-benzoquinone, an intermediate compound formed during phenol oxidation by chlorine dioxide, was investigated using Pseudomonas putida (ATCC 17484) in batch and continuous bioreactors. Batch experiments were conducted to determine the effects of 1,4-benzoquinone concentration and temperature on the microbial activity and biodegradation kinetics. Using the generated data, the maximum specific growth rate and biodegradation rate were determined as 0.94 h⁻¹ and 6.71 mg of 1,4-benzoquinone l⁻¹ h⁻¹. Biodegradation in a continuous bioreactor indicated a linear relationship between substrate loading and biodegradation rates prior to wash out of the cells, with a maximum biodegradation rate of 246 mg l⁻¹ h⁻¹ observed at a loading rate of 275 mg l⁻¹ h⁻¹ (residence time: 1.82 h). Biokinetic parameters were also determined using the steady state substrate and biomass concentrations at various dilution rates and compared to those obtained in batch cultures.     

A mutation within the C-terminal domain of Sup35p that affects [PSI+] prion propagation

The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI+] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth. The involvement of Sup35p C-terminal domain into [PSI+] propagation is subject to debate. We previously showed that mutation of threonine 341 within Sup35p C-domain affects translation termination efficiency. Here, we demonstrate that mutating threonine 341 to aspartate or alanine results in synthetic lethality with [PSI+] and weakening of [PSI+] respectively. The corresponding Sup35D and Sup35A proteins assemble into wild-type like fibrils in vitro, but with a slower elongation rate. Moreover, cross-seeding between Sup35p and Sup35A is inefficient both in vivo and in vitro, suggesting that the point mutation alters the structural properties of Sup35p within the fibrils. Thus, Sup35p C-terminal domain modulates [PSI+] prion propagation, possibly through a functional interaction with the N and/or M domains of the protein. Our results clearly demonstrate that Sup35p C-terminal domain plays a critical role in prion propagation and provide new insights into the mechanism of prion conversion.     

580. FRITILLARIA KARELINII

Summary.  Fritillaria karelinii Fisch. ex D.Don ( Liliaceae ) is illustrated. Its relationships with other species and the position of subgenus Rhinopetalum are discussed. Instructions for its cultivation are given.     

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label‐free quantitative shotgun proteomic analysis was performed. A total of 2042 non‐redundant proteins were identified from the five temperature points. Fifty‐five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold‐responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 ( http://proteomecentral.proteomexchange.org/dataset/PXD000977 ).     

Aqueous Fraction of Beta vulgaris Ameliorates Hyperglycemia in Diabetic Mice due to Enhanced Glucose Stimulated Insulin Secretion,Mediated by Acetylcholine and GLP-1, and Elevated Glucose Uptake via Increased Membrane Bound GLUT4 Transporters

BackgroundThe study was designed to investigate the probable mechanisms of anti-hyperglycemic activity of B. Vulgaris.ConclusionFindings of the present study clearly prove the role of Acetylcholine and GLP-1 in the Insulin secreting activity of B. Vulgaris. Increased glucose uptake in the skeletal muscles and subsequent glycogen synthesis may also play a part in the anti-hyperglycemic activity of B. Vulgaris.     

Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

In relation to the growing recent interest in the establishment of sperm‐mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0–500 ng) of pcDNA/his/Lac‐Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live‐immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA‐incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X‐Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live‐immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live‐immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression. Mol. Reprod. Dev. 77:868–875, 2010. © 2010 Wiley‐Liss, Inc.     

Phylodynamics and human-mediated dispersal of a zoonotic virus

Understanding the role of humans in the dispersal of predominantly animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geopolitical boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.