Hybrid vector designs to control the delivery, fate and expression of transgenes

One of the greatest challenges to gene therapy is the targetting of gene delivery selectively to the sites of disease and regulation of transgene expression without adverse effects. Ultimately, the successful realization of these goals is dependent upon improvements in vector design. Over the years, viral vector design has progressed from various types of replication-defective viral mutants to replication-conditioned viruses and, more recently, to 'gutted' and hybrid vectors, which have, respectively, eliminated expression of non-relevant or toxic viral genes and incorporated desired elements of different viruses so as to increase the efficacy of gene delivery in vivo. This review will focus on the different viral and cellular elements which have been incorporated into virus vectors to: improve transduction efficiencies; alter the entry specificity of virions; control the fate of transgenes in the host cells; and regulate transgene expression.     

Computational method for discovery of estrogen responsive genes

Estrogen has a profound impact on human physiology and affects numerous genes. The classical estrogen reaction is mediated by its receptors (ERs), which bind to the estrogen response elements (EREs) in target gene's promoter region. Due to tedious and expensive experiments, a limited number of human genes are functionally well characterized. It is still unclear how many and which human genes respond to estrogen treatment. We propose a simple, economic, yet effective computational method to predict a subclass of estrogen responsive genes. Our method relies on the similarity of ERE frames across different promoters in the human genome. Matching ERE frames of a test set of 60 known estrogen responsive genes to the collection of over 18,000 human promoters, we obtained 604 candidate genes. Evaluating our result by comparison with the published microarray data and literature, we found that more than half (53.6%, 324/604) of predicted candidate genes are responsive to estrogen. We believe this method can significantly reduce the number of testing potential estrogen target genes and provide functional clues for annotating part of genes that lack functional information.     

Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression

The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.     

Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers (bifilar enzyme-sensitive sites) in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined.     

Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells

We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.     

Biophysical characterization of Vpu from HIV-1 suggests a channel-pore dualism

Vpu from HIV-1 is an 81 amino acid type I integral membrane protein which consists of a cytoplasmic and a transmembrane (TM) domain. The TM domain is known to alter membrane permeability for ions and substrates when inserted into artificial membranes. Peptides corresponding to the TM domain of Vpu (Vpu(1-32)) and mutant peptides (Vpu(1-32)-W23L, Vpu(1-32)-R31V, Vpu(1-32)-S24L) have been synthesized and reconstituted into artificial lipid bilayers. All peptides show channel activity with a main conductance level of around 20 pS. Vpu(1-32)-W23L has a considerable flickering pattern in the recordings and longer open times than Vpu(1-32). Whilst recordings for Vpu(1-32)-R31V are almost indistinguishable from those of the WT peptide, recordings for Vpu(1-32)-S24L do not exhibit any noticeable channel activity. Recordings of WT peptide and Vpu(1-32)-W23L indicate Michaelis-Menten behavior when the salt concentration is increased. Both peptide channels follow the Eisenman series I, indicative for a weak ion channel with almost pore like characteristics.     

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and ¹H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide.

The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.     

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C₄ grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.