Adeno-associated virus-mediated gene transfer of a secreted form of TRAIL inhibits tumor growth and occurrence in an experimental tumor model

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno-associated virus (rAAV) vectors have a number of advantages including in vivo long-term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector. METHODS: A plasmid and rAAV2 vectors were constructed encoding sTRAIL composed of a leader sequence, the isoleucine zipper, and the active domain of TRAIL (aa 95-281). The functionality of sTRAIL was validated by cell viability, FACS analysis, caspase-3 activity, and TUNEL staining. rAAV-sTRAIL was injected intratumorally to nude mice bearing human A549 lung tumor cells. Nude mice received A549 tumor cells after intravenous delivery of rAAV-sTRAIL. The antitumor effect was then evaluated by measuring tumor regression and occurrence in the experimental animal. RESULTS: sTRAIL was released from cells transfected with the sTRAIL expression construct or transduced with rAAV-sTRAIL, and induced apoptosis in cancer cells, but spared normal fibroblast cells. Secreted sTRAIL formed oligomers including trimers with intersubunit disulfide. Purified sTRAIL exerted much lower cytotoxicity on primary human hepatocytes compared to recombinant TRAIL. Intratumoral delivery of rAAV-sTRAIL significantly inhibited growth of A549 tumors established in nude mice. A number of apoptotic tumor cells were detected by TUNEL staining in mice treated with rAAV-sTRAIL. Systemic pretreatment with rAAV-sTRAIL significantly inhibited tumor formation in nude mice. CONCLUSION: The results suggest that rAAV-sTRAIL may be useful for local or systemic cancer gene therapy for treating TRAIL-sensitive tumors.     

EP4 receptor regulates collagen type-I, MMP-1, and MMP-3 gene expression in human tendon fibroblasts in response to IL-1 beta treatment

Tendinopathy is accompanied by inflammation, tendon matrix degradation, or both. Inflammatory cytokine IL-1beta, which is a potent inflammatory mediator, is likely present within the tendon. The purpose of this study was to determine the biological impact of IL-1beta on tendon fibroblasts by assessing the expression of cPLA(2), COX-2, PGE(2) and its receptors (EPs), collagen type-I, and MMPs. We also studied the role of the p38 MAPK pathway in IL-1beta-induced catabolic effects. We found that IL-1beta increased the expression levels of cPLA(2) and COX-2, and also increased the secretion of PGE(2). Induction of MMPs, such as MMP-1 and MMP-3 at the mRNA level, was also observed after stimulation with IL-1beta. Furthermore, the presence of IL-1beta significantly decreased the level of collagen type-I mRNA in tendon fibroblasts. These effects were found to be mediated by selective upregulation of EP(4) receptor, which is a member of G-protein-coupled receptor that transduces the PGE(2) signal. Blocking EP(4) receptor by a specific chemical inhibitor abolished IL-1beta-induced catabolic effects. These results suggest that IL-1beta-induced catabolic action on tendon fibroblasts occurs via the upregulation of two key inflammatory mediators, cPLA(2) and COX-2, which are responsible for the synthesis of PGE(2). IL-1beta further stimulates the expression of EP(4) receptor, suggesting positive feedback regulation which may lead to accelerated catabolic processes in tendon fibroblasts. Studies using pathway-specific chemical inhibitors suggest that the p38 MAPK pathway is the key signaling cascade transducing IL-1beta-mediated catabolic effects. Collectively, our findings suggest that the EP(4) receptor mediates the IL-1beta-induced catabolic metabolism via the p38 MAPK pathway in human tendon fibroblasts and may play a major role in the tendon's degenerative changes often seen in the later stages of tendinopathy.     

Low level expression of prokaryotic tzs gene enhances growth performance of transgenic poplars

We modulated the level of a hormone gene expression in poplars using either 35S promoter (p35S) of cauliflower mosaic virus (CaMV) or aux promoter (pAUX) of A. rhizogenes. The transgenic poplars (Populus alba × P. tremula var. glandulosa), in which the bacterial trans-zeatin secretion (tzs) gene was attached either to the 35S promoter or to the aux promoter, were compared for their performance in tissue culture as well as in nursery. Northern blot analysis of total RNA probed with tzs coding region showed that the total tzs mRNA expression by p35S was approximately 200–300-fold higher than that driven by pAUX. In contrast, the cellular zeatin content of p35S-tzs transgenic poplars was merely 13-fold of those found in pAUX-tzs plants. Due to different levels of cellular zeatin levels, the two types of transgenic poplars showed different morphogenetic as well as growth responses. The p35S-tzs transgenic plants showed morphological characteristics typical of those treated with cytokinin in culture. These include multiple axillary shoot formation, thick stems, narrow leaves and absence of roots. In contrast, the pAUX-tzs plants had slightly higher cellular cytokinin levels than did control plants and showed a lower degree of cytokinin-related phenotypes, including a few axillary shoots in root-inducing media. Since p35S-tzs did not develop roots, only pAUX-tzs transgenic poplars could be transplanted to the nursery where they resumed a close-to-normal growth. Nevertheless, pAUX-tzs plants transferred to the nursery developed cytokinin-related phenotypes, including greater number of shoots, smaller leaves and slightly retarded growth in height, but with a high total biomass.     

Synthesis and phosphodiesterase 5 inhibitory activity of novel phenyl ring modified sildenafil analogues.

New sildenafil analogues containing an ether ring fused into the phenyl moiety, 6a--d and 7a--d, were efficiently synthesized from the readily available starting materials, 1a--d and 2, in five steps. Ab initio calculations indicated that introduction of a cyclic ether to the phenyl group might enhance the co-planarity of the molecule. The torsional angles were calculated to be 2--3 degrees for the 5-membered cyclic ether derivatives, 6a, 6c, 7a, and 7c, and 12--16 degrees for the 6-membered ones, 6b, 6d, 7b, and 7d. On the other hand, sildenafil showed the least co-planarity with the torsional angle of 23 degrees compared with the target compounds, 6a--d and 7a--d. In the enzyme assay, however, the in vitro PDE 5 inhibitory activity was found out to be inversely related to the degree of co-planarity. In other words, the least planar sildenafil showed the highest activity, and the most planar 5-membered cyclic ether derivatives were least active by 100--200-fold compared with sildenafil. Our study clearly demonstrated that the open chain 2'-alkoxy group of the phenyl ring, although less effective for inducing the co-planarity, seemed to act as a much better lipophilic requirement than the cyclic alkoxy moiety.     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

Theoretical analysis of the magnetocardiographic pattern for reentry wave propagation in a three-dimensional human heart model

We present a computational study of reentry wave propagation using electrophysiological models of human cardiac cells and the associated magnetic field map of a human heart. We examined the details of magnetic field variation and related physiological parameters for reentry waves in two-dimensional (2-D) human atrial tissue and a three-dimensional (3-D) human ventricle model. A 3-D mesh system representing the human ventricle was reconstructed from the surface geometry of a human heart. We used existing human cardiac cell models to simulate action potential (AP) propagation in atrial tissue and 3-D ventricular geometry, and a finite element method and the Galerkin approximation to discretize the 3-D domain spatially. The reentry wave was generated using an S1-S2 protocol. The calculations of the magnetic field pattern assumed a horizontally layered conductor for reentry wave propagation in the 3-D ventricle. We also compared the AP and magnetocardiograph (MCG) magnitudes during reentry wave propagation to those during normal wave propagation. The temporal changes in the reentry wave motion and magnetic field map patterns were also analyzed using two well-known MCG parameters: the current dipole direction and strength. The current vector in a reentry wave forms a rotating spiral. We delineated the magnetic field using the changes in the vector angle during a reentry wave, demonstrating that the MCG pattern can be helpful for theoretical analysis of reentry waves.     

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.     

Sphingosylphosphorylcholine generates reactive oxygen species through calcium-, protein kinase Cdelta- and phospholipase D-dependent pathways

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.