Differential Role of PKC-Induced c-Jun in HTLV-1 LTR Activation by 12-O-Tetradecanoylphorbol-13-acetate in Different Human T-cell Lines

We have previously shown that TPA activates HTLV-1 LTR in Jurkat T-cells by inducing the binding of Sp1-p53 complex to the Sp1 site residing within the Ets responsive region 1 (ERR-1) of the LTR and that this activation is inhibited by PKCalpha and PKCepsilon. However, in H9 T-cells TPA has been noted to activate the LTR in two consecutive stages. The first stage is activation is mediated by PKCetta and requires the three 21 bp TRE repeats. The second activation mode resembles that of Jurkat cells, except that it is inhibited by PKCdelta. The present study revealed that the first LTR activation in H9 cells resulted from PKCetta-induced elevation of non-phosphorylated c-Jun which bound to the AP-1 site residing within each TRE. In contrast, this TRE-dependent activation did not occur in Jurkat cells, since there was no elevation of non-phosphorylated c-Jun in these cells. However, we found that PKCalpha and PKCepsilon, in Jurkat cells, and PKCetta and PKCdelta, in H9 cells, increased the level of phosphorylated c-Jun that interacted with the Sp1-p53 complex. This interaction prevented the Sp1-p53 binding to ERR-1 and blocked, thereby, the ERR-1-mediated LTR activation. Therefore, this PKC-inhibited LTR activation started in both cell types after depletion of the relevant PKCs by their downregulation. In view of these variable activating mechanisms we assume that there might be additional undiscovered yet modes of HTLV-1 LTR activation which vary in different cell types. Moreover, in line with this presumption we speculate that in HTLV-1 carriers the LTR of the latent provirus may also be reactivated by different mechanisms that vary between its different host T-lymphocyte subclones. Since this reactivation may initiate the ATL process, understanding of these mechanisms is essential for establishing strategies to block the possibility of reactivating the latent virus as preventive means for ATL development in carriers

Equipment specification for a space rated radar altimeter Final technical report

Equipment specifications for GEOS C mission altimeter

Water-Soluble Fullerene (C60) Derivatives as Nonviral Gene-Delivery Vectors

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch-Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both therapeutic and diagnostic agents

A Novel Mechanism Is Involved in Cationic Lipid-Mediated Functional siRNA Delivery

A key challenge for therapeutic application of RNA interference is to efficiently deliver synthetic small interfering RNAs (siRNAs) into target cells that will lead to the knockdown of the target transcript (functional siRNA delivery). To facilitate rational development of nonviral carriers, we have investigated by imaging, pharmacological and genetic approaches the mechanisms by which a cationic lipid carrier mediates siRNA delivery into mammalian cells. We show that 95% of siRNA lipoplexes enter the cells through endocytosis and persist in endolysosomes for a prolonged period of time. However, inhibition of clathrin-, caveolin-, or lipid-raft-mediated endocytosis or macropinocytosis fails to inhibit the knockdown of the target transcript. In contrast, depletion of cholesterol from the plasma membrane has little effect on the cellular uptake of siRNA lipoplexes, but it abolishes the target transcript knockdown. Furthermore, functional siRNA delivery occurs within a few hours and is gradually inhibited by lowering temperatures. These results demonstrate that although endocytosis is responsible for the majority of cellular uptake of siRNA lipoplexes, a minor pathway, probably mediated by fusion between siRNA lipoplexes and the plasma membrane, is responsible for the functional siRNA delivery. Our findings suggest possible directions for improving functional siRNA delivery by cationic lipids.National Institutes of Health (U.S.) (NIH Grant AI56267)National Institutes of Health (U.S.) (NIH Grant CA112967)National Institutes of Health (U.S.) (NIH Grant CA119349)Natural Sciences and Engineering Research Council of Canada (NSERC) (Post-doctoral fellowship

The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein

Leisure Behavior Pattern Stability During the Transition from Adolescence to Young Adulthood

Leisure is an important context in which human development occurs. Changes in leisure behavior patterns may indicate changing developmental needs or reflect contextual changes that impact leisure behavior. The transition from adolescence to young adulthood provides an excellent opportunity for the study of the stability of leisure behavior as individuals' contexts are changed with the adoption of adult roles and the potential for disruption of leisure patterns exists. Previous studies investigating leisure and the transition from adolescence to young adulthood have tended to be cross-sectional and focus on specific leisure behaviors rather than identifying patterns of leisure behavior. The present study involved a longitudinal investigation of leisure behavior patterns over a three-year period during the transition from adolescence to young adulthood, and determined the nature of leisure pattern stability and instability during this period. In general, leisure pattern stability was the most common pathway into young adulthood. The patterns of leisure behavior and the nature of the changes that occurred with the transition from adolescence to young adulthood differed to some degree for males and females, although similarities in patterns and transitions were also found.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45285/1/10964_2004_Article_411255.pd

Liposomes for use in gene delivery

Liposomes have a wide array of uses that have been continuously expanded and improved upon since first being observed to selfassemble into vesicular structures. These arrangements can be found in many shapes and sizes depending on lipid composition. Liposomes are often used to deliver a molecular cargo such as DNA for therapeutic benefit. The lipids used to form such lipoplexes can be cationic, anionic, neutral, or a mixture thereof. Herein physical packing parameters and specific lipids used for gene delivery will be discussed, with lipids classified according to overall charge

The Importance of and a Method for Including Transfection Efficiency Into Real-Time PCR Data Analyses

ABSTRACT: The polymerase chain reaction (PCR) is widely used to ascertain absolute or relative changes in the expression levels of specific genes as a function of cell type or in response to changes in environmental stimuli. Real-time PCR is an advance which allows for the analysis of gene expression over a wide range of initial cDNA concentrations, where the cDNA is the product of reverse transcriptase reactions applied to RNA samples. With the advent and advances in gene delivery technologies, it is now common for the cellular responses under scrutiny to be initiated via the expression of an exogenously delivered gene. When transfection (or transduction) is a part of the procedure used to prepare cell samples for real-time PCR, it is necessary to take the efficiency of gene delivery into account. Here a robust mathematical model for such analyses is derived, and validated with theoretical and experimental support. Comparison to existing analysis methods is presented to demonstrate the high significance of noting transfection, loading, and primer PCR efficiencies when processing PCR data