A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(-) hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins.

We created a "knockout" embryonic stem cell via targeted disruption of the phosphatidylinositol glycan class A (Pig-a) gene, resulting in loss of expression of cell surface glycosyl phosphatidylinositol-anchored proteins and reproducing the mutant phenotype of the human disease paroxysmal nocturnal hemoglobinuria. Morphogenesis of Pig-a- embryoid bodies (EB) in vitro was grossly aberrant and, unlike EB derived from normal embryonic stem cells, Pig-A EB produced no secondary hematopoietic colonies. Chimeric EB composed of control plus Pig-A- cells, however, appeared normal, and hematopoiesis from knock-out cells was reconstituted. Transfer in situ of glycosyl phosphatidylinositol-anchored proteins from normal to knock-out cells was demonstrated by two-color fluorescent analysis, suggesting a possible mechanism for these functional effects. Hematopoietic cells with mutated PIG-A genes in humans with paroxysmal nocturnal hemoglobinuria may be subject to comparable pathophysiologic processes and amenable to similar therapeutic protein transfer.

Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.

Intercellular transfer of a glycosylphosphatidylinositol (GPI)-linked protein: release and uptake of CD4-GPI from recombinant adeno-associated virus-transduced HeLa cells.

A diverse group of GPI-anchored protein structures are ubiquitously expressed on the external cell membranes of eukaryotes. Whereas the physiological role for these structures is usually defined by their protein component, the precise biological significance of the glycolipid anchors remains vague. In the course of producing a HeLa cell line (JM88) that contained a recombinant adeno-associated virus genome expressing a GPI-anchored CD4-GPI fusion protein on the surface of the cells, we noted the transfer of CD4-GPI to native HeLa cells. Transfer occurred after direct cell contact or exposure to JM88 cell supernatants. The magnitude of contact-mediated CD4-GPI transfer correlated with temperature. Supernatant CD4-GPI also attached to human red blood cells and could be cleaved with phosphatidylinositol-specific phospholipase C. The attached CD4-GPI remained biologically active after transfer and permitted the formation of syncytium when coated HeLa cells were incubated with glycoprotein 160 expressing H9 cells. JM88 cells provide a model for the production, release, and reattachment of CD4-GPI and may furnish insight into a physiologic role of naturally occurring GPI-anchored proteins. This approach may also allow the production of other recombinant GPI-anchored proteins for laboratory and clinical investigation.

Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers.

Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines.

Functional protection of dystrophic mouse (mdx) muscles after adenovirus-mediated transfer of a dystrophin minigene.

Fast skeletal muscles of mdx (X chromosome-linked muscular dystrophy) mice were injected after birth with a recombinant adenovirus containing a minidys- trophin gene, a 6.3-kbp cDNA coding for the N- and C-terminal ends of dystrophin. Adult muscles were challenged by forced lengthening during tetanic contractions. Stretch-induced mechanical and histological damages were much reduced in injected muscles, in direct proportion of the Miniber of fibers expressing minidystrophin. Damaged fibers were preferentially found among minidystrophin-negative regions. Minidystrostrophin confers an important functional and structural protection of limb muscles against high mechanical stress, even after a partial somatic gene transfer.

Interisland and interarchipelago transfer of stone tools in prehistoric Polynesia.

Tracing interisland and interarchipelago movements of people and artifacts in prehistoric Polynesia has posed a challenge to archaeologists due to the lack of pottery and obsidian, two materials most readily used in studies of prehistoric trade or exchange. Here we report the application of nondestructive energy-dispersive x-ray fluorescence (EDXRF) analysis to the sourcing of Polynesian artifacts made from basalt, one of the most ubiquitous materials in Polynesian archaeological sites. We have compared excavated and surface-collected basalt adzes and adze flakes from two sites in Samoa (site AS-13-1) and the Cook Islands (site MAN-44), with source basalts from known prehistoric quarries in these archipelagoes. In both cases, we are able to demonstrate the importing of basalt adzes from Tutuila Island, a distance of 100 km to Ofu Island, and of 1600 km to Mangaia Island. These findings are of considerable significance for Polynesian prehistory, as they demonstrate the movement of objects not only between islands in the same group (where communities were culturally and linguistically related) but also between distant island groups. Further applications of EDXRF analysis should greatly aid archaeologists in their efforts to reconstruct ancient trade and exchange networks, not only in Polynesia but also in other regions where basalt was a major material for artifact production.

Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain.

Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA.

Restoration of the impaired balance between pro- and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-1ra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-1ra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-1ra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-1ra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-1ra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.

Genetic transfer of a nonpeptide antagonist binding site to a previously unresponsive angiotensin receptor.

Mutational analysis based on the pharmacological differences between mammalian and amphibian angiotensin II receptors (AT receptors) previously identified 7 aa residues located in transmembrane domains (TMs) III (Val-108), IV (Ala-163), V (Pro-192, Thr-198), VI (Ser-252), and VII (Leu-300, Phe-301) of the rat AT receptor type 1b (rAT1b receptor) that significantly influenced binding of the nonpeptide antagonist Losartan. Further studies have shown that an additional 6 residues in the rAT1b receptor TMs II (Ala-73), III (Ser-109, Ala-114, Ser-115), VI (Phe-248), and VII (Asn-295) are important in Losartan binding. The 13 residues required for Losartan binding in the mammalian receptor were exchanged for the corresponding amino acids in the Xenopus AT receptor type a (xATa receptor) to generate a mutant amphibian receptor that bound Losartan with the same affinity as the rAT1b receptor (Losartan IC50 values: rAT1b, 2.2 +/- 0.2 nM: xATa, > 50 microM; mutant, 2.0 +/- 0.1 nM). To our knowledge, this is the first report of a gain-of-function mutant in which the residues crucial to formation of a ligand binding site in a mammalian peptide hormone receptor were transferred to a previously unresponsive receptor by site-directed mutagenesis. Ala substitutions and comparison of mammalian and amphibian combinatorial mutants indicated that TM III in the rAT1b receptor plays a key role in Losartan binding. Identification of residues involved in nonpeptide ligand binding will facilitate studies aimed at elucidating the chemical basis for ligand recognition in the AT receptor and peptide hormone receptors in general.

How can directories of open access repositories improve the reuse of learning objects in building engineering?

This paper studies the use of directories of open access repositories worldwide (DOARW) to search Spanish repositories containing learning objects in the field of building engineering (BE). Results show that DOARW are powerful tools, but deficiencies (indicated in this study) have to be solved in order to obtain more accurate searches, and to facilitate repository-finding for potential users who are seeking learning objects (LOs) for reuse. Aiming to contribute to the promotion of the reuse of Spanish LOs, this study exposes to the academic community all existing Spanish repositories with LOs, and in particular, the repositories that contain LOs in the field of BE. This paper also studies the critical mass of available content (LOs) in the field of BE in Spain. It has been found to be low.

Transfer of Pattern Recall Skills May Contribute to the Development of Sport Expertise

Superior recall of domain-specific patterns is well established as a defining attribute of expert performers. Recent studies on the developmental histories of expert team ball sport players (e.g. Baker, Côté, & Abernethy, 2003a) also suggest that experts characteristically receive exposure to a wide range of sports in their developing years and that this related sports experience may reduce the amount of sport-specific training needed to become an expert. This study examined whether the facilitation of expertise associated with other sport experience might arise from positive transfer of pattern recall skills from one sport to another. Expert netball, basketball and field hockey players and experienced non-experts performed a recall task for patterns of play derived from each of these sports. Experts from sports different to those shown in the presented pattern consistently outperformed non-experts in their recall of defensive player positions, suggesting some selective transfer of pattern recall skills may indeed be possible