Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Efficiently mining gene expression data via a novel parameterless clustering method

Clustering analysis has been an important research topic in the machine learning field due to the wide applications. In recent years, it has even become a valuable and useful tool for in-silico analysis of microarray or gene expression data. Although a number of clustering methods have been proposed, they are confronted with difficulties in meeting the requirements of automation, high quality, and high efficiency at the same time. In this paper, we propose a novel, parameterless and efficient clustering algorithm, namely, correlation search technique (CST), which fits for analysis of gene expression data. The unique feature of CST is it incorporates the validation techniques into the clustering process so that high quality clustering results can be produced on the fly. Through experimental evaluation, CST is shown to outperform other clustering methods greatly in terms of clustering quality, efficiency, and automation on both of synthetic and real data sets.     

Penetrating living cells using semiconductor nanowires

A recent publication by Kim et al. on penetrating both human embryonic kidney cells and mouse embryonic stem cells with Si nanowires highlights the increasing interest in using proven semiconductor materials not only to detect specific biomolecules in solutions but also to deliver genetic material or potentially screen for the presence of particular molecules at the cell level. Many semiconductors are biocompatible and this recent work has shown that penetrating cells with large diameters compared with those of the semiconductor nanowire is not fatal to the cell and that the cells remain functional for a few days.     

Risk of chronic myeloid and acute leukemia mortality after exposure to ionizing radiation among workers at four U.S. nuclear weapons facilities and a nuclear naval shipyard

A nested case-control study was conducted among workers at five U.S. nuclear facilities to evaluate leukemia mortality risk (excluding chronic lymphocytic) from ionizing radiation using worksite doses and adjusting for potential confounding. Conditional logistic regression was used to estimate the relative risk (RR) of exposed workers and the excess relative risk (ERR) per unit of radiation among 206 cases and 823 age-matched controls. Adjusting for sex and benzene, the RR of leukemia for workers receiving more than 10 mSv was higher compared to those receiving lower or no dose; however, the risk increase was attenuated in the highest dose group. The ERR per 10 mSv was 1.44% (95% CI: < -1.03%, 7.59%) but was higher for workers born after 1921 compared to workers born earlier or when excluding leukemias of uncertain type. Excluding the 7% who were high-dose workers (> 100 mSv), the sex- and benzene-adjusted ERR per 10 mSv was 6.82% (95% CI: -2.87%, 24.1%). The results suggest that risks among these nuclear workers are comparable to those observed in high-dose populations, although no evidence was observed of a positive quadratic dose-response term in this study. This large study is among the first to jointly evaluate benzene and ionizing radiation risk.     

Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system

BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.     

Development of poly(amino ester glycol urethane)/siRNA polyplexes for gene silencing

Nonviral vectors, with their low immunogenicity and lack of pathogenicity, offer significant promise for siRNA therapy with fewer safety concerns. Nonviral vectors were also preferred in most transient siRNA delivery due to their ease of preparation. Previously, we incorporated tertiary amines and polyethylene glycol (PEG) into poly(ester urethane) to synthesize a soluble poly(amino ester glycol urethane), PaE(G)U, as a novel DNA transfection reagent for transgene delivery. The aim of this study was to develop PaE(G)U/siRNA polyplexes for gene silencing. We characterized the properties of PaE(G)U/siRNA polyplexes and compared them with those of PaE(G)U/DNA polyplexes. Using the Alexa Fluor 488-labeled, nonsilencing control siRNA as the reporter, we visualized cellular uptake of PaE(G)U/siRNA polyplexes and optimized the mass ratio of PaE(G)U/siRNA for delivery at 80/1. At this ratio, the average diameter of polyplexes was 540 nm, which was significantly larger than the average diameter of PaE(G)U/DNA polyplexes at 155 nm for efficient DNA delivery. Using the optimized PaE(G)U/siRNA polyplexes, transient silencing of constitutive luciferase expression (up to 92%) was achieved in our recombinant human HT-1080 fibroblast model via anti-luciferase siRNA delivery. In conclusion, PaE(G)U/siRNA polyplexes were developed and optimized for cellular uptake to allow efficient gene silencing. Engineering of soluble biodegradable polymers to incorporate amino, ester, PEG, and urethane units in the backbone constitutes a useful approach for the future design of siRNA carriers.     

Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signalling

Disabled-2 (DAB2) is an adapter protein that plays a key role in cell proliferation and differentiation. We reported here that DAB2 is expressed in various regions of rat central nervous system and is most abundant in the olfactory bulb. The up-regulation of DAB2 upon 5,7-dihydroxytryptamine-induced spinal cord lesion implicates that DAB2 may participate in the regulation of neuronal plasticity. To investigate DAB2 function in the regulation of neurite outgrowth, the rat p59 and p82 form of DAB2 was individually and stably expressed in the PC12 cells. Both p59 and p82 inhibited nerve growth factor (NGF)-induced neurite outgrowth concomitantly with a decrease in the expression of neuron-specific cytoskeleton protein beta-tubulin III. To unveil the molecular mechanism of DAB2 in NGF signaling, we found that RhoA-GTPase activity was up-regulated in DAB2 stable lines whereas the Ras/MAPK and PI3-kinase/Akt signaling was not affected. The inhibitory effect of DAB2 on NGF-mediated neurite outgrowth was reversed by the pretreatment of Rho-kinase (ROCK) inhibitor Y27632, implicating that DAB2 modulates RhoA/ROCK signaling. Together, this study defines a role of DAB2 in the control of neuronal plasticity and demonstrates for the first time that DAB2 is a negative regulator in NGF-mediated neurite outgrowth.     

Degradation of polyethylene succinate (PES) by a new thermophilic <Emphasis Type="Italic">Microbispora</Emphasis> strain

Thermophilic actinomycetes were isolated from sediment of the Chingshuei hot spring in north Taiwan, and the strain HS 45-1 was selected from colonies which formed distinct clear zones on agar plate with emulsified polyethylene succinate (PES). The film of PES disappeared within 6 days in liquid cultures at 50°C. The strain HS 45-1 was also able to degrade poly (ε-carpolactone) (PCL) and poly (3-hydroxybutyrate) (PHB) films completely within 6 days in liquid cultures. Basing on the results of phynotypic characteristics, phylogenetic studies and DNA-DNA hybridization, strain HS 45-1 should be assigned to Micorbispora rosea subsp. taiwanensis.     

Identification of a hypothetical protein of plant pathogenic Xanthomonas campestris as a novel beta-galactosidase

Xc17L, a lactose-utilizing mutant of Xanthomonas campestris pv. campestris previously isolated by mutagenesis with nitrous acid, displays a level of beta-galactosidase 3.5-fold higher than that in the parental Xc17. In this study, the gene encoding the enzyme displaying a higher specific activity in Xc17L was inactivated by mini-Tn5 transposition.Sequencing revealed that the product (579 aa, 63.5 kDa) of this gene, designated galD, was previously annotated to encode a hypothetical protein on the genome. Mutation of the gene by marker exchange, complementation test and Western blot analysis together confirmed that galD is indeed the gene involved in beta-galactosidase elevation in Xc17L. With only the N-terminal region possessing similarity to the known beta-galactosidases and partially conserved consensus motif, GalD is recognized as a member of the glycosyl hydrolase family 35. Insertion with GmOmega, which causes polar effects, into the upstream genes followed by Western blotting showed that galD is cotranscribed with the upstream genes and expressed constitutively. Mutation in galD causes no significant changes including pathogenicity in the bacterium.