Nontoxic, Biodegradable Hyperbranched Poly(ß-amino ester)s for Efficient siRNA Delivery and Gene Silencing.

RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(ß-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

Exploration of photochemical reactions of N-trimethylsilylmethyl-substituted uracil, pyridone, and pyrrolidone derivatives.

Photochemical reactions of N-trimethylsilylmethyl-substituted uracil, pyridone and pyrrolidone derivatives were carried out to determine if silicone containing substituents have an impact on excited state reaction profiles. The results show that ultraviolet irradiation of N-trimethylsilylmethyl substituted uracils in the presence of substituted alkenes leads to efficient formation of both dimeric and cross [2+2]-cycloaddition products. Qualitatively similar observations were made in a study of the photochemistry of N-trimethylsilylmethyl-2-pyridone. The combined results demonstrate that [2+2]-photocycloaddition is a more efficient excited state reaction pathway for the uracil and pyridone substrates as compared to other processes, such as ylide-forming trimethylsilyl group C-to-O migration. Finally, photoreactions of N-trimethylsilylmethyl-2-pyrrolidone in solutions containing dipolarophiles, such as methyl acrylate, lead to the formation of the desilylation product, N-methyl-2-pyrrolidone by way of a simple, non-ylide generating, protodesilylation process. In addition, observations were made which show that orbital symmetry allowed photocycloreversion reactions of dimeric uracil derivatives, involving cyclobutane ring splitting, to take place. These processes, which lead to the formation of monomeric uracils, appear to be stimulated by the presence of electron donor groups on the cyclobutane ring, a likely result of a new SET promoted cyclobutane ring cleavage pathway. In the cases of N-trimethylsilylmethyl-substituted cyclobutane derivatives that possess phthalimide groups, highly efficient excited state cleavage of the cyclobutane moiety occurs to produce uracil derivatives and corresponding vinyl phthalimide.

Synthesis of novel cholesterol-based cationic lipids for gene delivery.

The new cholesterol-based cationic lipids B, C, and D with an ether linked spacer were synthesized by using aminopropyl chain extension with acrylonitrile. The cholesterol-based cationic lipid A with carbamoyl linkage were also synthesized in order to compare the difference in transfection efficiency of the two linkage types. To this end, GFP expression of these cationic lipids was confirmed respectively.

Sanguinarine induces apoptosis in A549 human lung cancer cells primarily via cellular glutathione depletion.

Sanguinarine is a plant-derived benzophenanthridine alkaloid and has been shown to possess anti-tumor activities against various cancer cells. In this study, we investigated whether sanguinarine induces apoptosis in A549 human lung cancer cells. Treatment of A549 cells with sanguinarine induced apoptosis in a dose- and time-dependent manner. Treatment with sanguinarine led to activation of caspases and MAPKs as well as increased MKP-1 expression. Importantly, pretreatment with z-VAD-fmk, a pan caspase inhibitor suppressed the sanguinarine-induced apoptosis in A549 cells. Moreover, pretreatment with NAC, a sulfhydryl group-containing reducing agent strongly suppressed the apoptotic response and caspase activation to sanguinarine. However, the sanguinarine-mediated cytotoxicity in A549 cells was not protected by pharmacological inhibition of MAPKs or MKP-1 siRNA-mediated knockdown of MKP-1. These results collectively suggest that sanguinarine induces apoptosis in A549 cells through cellular glutathione depletion and the subsequent caspase activation.

Cadmium specifically induces MKP-1 expression via the glutathione depletion-mediated p38 MAPK activation in C6 glioma cells.

Cadmium is a toxic heavy metal and an environmental pollutant. Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a negative regulator of the family of MAPK. In this study, we investigated the effect of heavy metals on MKP-1 expression in C6 rat glioma cells. Cadmium treatment induced MKP-1 at both protein and mRNA levels while cobalt or manganese treatment did not, suggesting the specificity. Cadmium treatment also depleted intracellular GSH and activated p38 MAPK, JNKs, and AKT. Profoundly, pretreatment with thiol-containing compounds NAC or GSH, but not vitamin E, blocked GSH depletion, 38 MAPK activation and MKP-1 expression by cadmium. Moreover, pharmacological inhibition of p38 MAPK by SB203580 suppressed the cadmium-induced MKP-1. Collectively, these results demonstrate that cadmium specifically induces MKP-1 by transcriptional up-regulation in C6 cells in a mechanism associated with the glutathione depletion-dependent p38 MAPK activation.

Prevention of CCl4-induced liver cirrhosis by ribbon antisense to transforming growth factor-beta1.

Transforming growth factor-beta1 (TGF-beta1) is an important mediator of tissue fibrosis, including liver cirrhosis. Ribbon-type antisense oligonucleotide to TGF-beta1 (TGF-beta1 RiAS) was designed and combined with cationic peptide derived from the nuclear localization signal of human immunodeficiency virus-1 Tat protein for enhanced cellular uptake. When Hepa1c1c7 cells were transfected with TGF-beta1 RiAS, the level of TGF-beta1 mRNA was reduced by >70%. TGF-beta1 RiAS, mismatched RiAS, and normal saline were each injected into mice via the tail vein, beginning the week after intraperitoneal CCl4 injection and continuing for 7 weeks, in order to determine whether TGF-beta1 RiAS prevents the fibrotic changes induced by the CCl4 injection. After 8 weeks of the experiment, all of the mice treated with TGF-beta1 RiAS survived, compared to 50% of the control group and 65% of the mismatched RiAS-treated group. Upon examining the biochemical effects on the liver, TGF-beta1 mRNA levels were reduced significantly only in the TGF-beta1 RiAS-treated group. Immunohistochemical studies showed a reduced accumulation of collagen and alpha-smooth muscle actin. Our experimental results suggest that ribbon antisense to TGF-beta1, with efficient uptake, effectively blocks the expression of TGF-beta1 and prevents fibrosis of the liver.

Bioactive glass-ionomer cement with potential therapeutic function to dentin capping mineralization.

We have developed a novel bioactive resin-modified glass-ionomer cement system with therapeutic function to dentin capping mineralization. In the system, the newly synthesized star-shape poly(acrylic acid) was formulated with water, Fuji II LC filler, and bioactive glass S53P4 to form resin-modified glass-ionomer cement. Compressive strength (CS) was used as a screening tool for evaluation. The commercial glass-ionomer cement Fuji II LC was used as a control. All the specimens were conditioned in simulated body fluid (SBF) at 37 degrees C prior to testing. The effect of aging in SBF on CS and microhardness of the cements was investigated. Scanning electron microscopy was used to examine the in vitro dentin surface changes caused by the incorporation of bioactive glass. The results show that the system not only provided strengths comparable to original commercial Fuji II LC cement but also allowed the cement to help mineralize the dentin in the presence of SBF. It appears that this bioactive glass-ionomer cement system has direct therapeutic impact on dental restorations that require root surface fillings.

Gene knockdown by large circular antisense for high-throughput functional genomics.

Single-stranded genomic DNA of recombinant M13 phages was tested as an antisense molecule and examined for its usefulness in high-throughput functional genomics. cDNA fragments of various genes (TNF-alpha, c-myc, c-myb, cdk2 and cdk4) were independently cloned into phagemid vectors. Using the life cycle of M13 bacteriophages, large circular (LC)-molecules, antisense to their respective genes, were prepared from the culture supernatant of bacterial transformants. LC-antisense molecules exhibited enhanced stability, target specificity and no need for target-site searches. High-throughput functional genomics was then attempted with an LC-antisense library, which was generated by using a phagemid vector that incorporated a unidirectional subtracted cDNA library derived from liver cancer tissue. We identified 56 genes involved in the growth of these cells. These results indicate that an antisense sequence as a part of single-stranded LC-genomic DNA of recombinant M13 phages exhibits effective antisense activity, and may have potential for high-throughput functional genomics.

Dexamethasone suppresses interleukin-1beta-induced human beta-defensin 2 mRNA expression: involvement of p38 MAPK, JNK, MKP-1, and NF-kappaB transcriptional factor in A549 cells.

Human beta-defensin (HBD)-2 is an inducible antimicrobial peptide that plays an important role in innate immunity. Glucocorticoids, on the other hand, exert immunosuppressive and anti-inflammatory actions. We have previously reported that interleukin (IL)-1beta induces HBD-2 mRNA expression through the activation of nuclear factor-kappaB (NF-kappaB) transcriptional factor, as well as p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), or phosphatidylinositol-3-kinase/AKT in A549 cells. In this study, we further investigated whether dexamethasone (Dex) controls IL-1beta-induced HBD-2 mRNA expression in A549 cells and the molecular mechanism associated with it. Dex suppressed IL-1beta-induced HBD-2 mRNA expression, which is mediated by a glucocorticoid receptor, at the transcriptional level. Interestingly, Dex attenuated IL-1beta-mediated activation of p38 MAPK and JNK, but not of AKT. Dex increased the expression of MAPK phosphatase (MKP)-1, which dephosphorylated p38 MAPK, but not JNK, by IL-1beta. However, although Dex did not inhibit the nuclear translocation of p65 NF-kappaB in response to IL-1beta, it profoundly inhibited NF-kappaB promoter- and HBD-2 promoter-driven luciferase activities. These results suggest that Dex acts to inhibit IL-1beta-induced HBD-2 mRNA expression through blockage of the nuclear transcriptional activation of p65 NF-kappaB as well as through inactivation of p38 MAPK and JNK. Specifically, Dex-induced MKP-1 expression is responsible for the inactivation of p38 MAPK, but not JNK, in response to IL-1beta in A549 cells.

Marked transfection enhancement by the DPL (DNA/peptide/lipid) complex.

A short peptide, corresponding to the nuclear localization signal of the human immunodeficiency virus-1 Tat protein, Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, was modified by adding a cysteine residue at the COOH terminus. The peptide was mixed with a reporter plasmid, and then with cationic lipids, to form a tripartite complex, DNA/peptide/lipid (DPL). Various cell lines were treated with the DPL complex and compared for transfection efficiency with those of the conventional DNA/lipid (DL) complex. With the simple inclusion of the peptide, the DPL complex showed much enhanced transfection. Meanwhile, the plasmid DNA mixed only with the peptide exhibited some improvement but with much lower transfection than the DPL complex. When the DPL complex was formed with various cationic lipids, the DOSPA/DOPE exhibited superior transfection efficiency than the other cationic lipids tested at the optimal ratio of 1:3:5 (w:w:w) in many cell types. At the optimal ratio of the DPL components, transfection efficiency was routinely shown to be approximately 10-fold higher for reporter gene expression than that of the conventional DL complex. Furthermore, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with antisense oligos, k-ras-RiAS, delivered as a DPL complex, tumor growth was markedly suppressed. This study shows that the DPL complex, which is easy to formulate by ordered mixing, can be employed for a much enhanced cellular uptake of a transgene both in vitro and in vivo.

Triptolide suppresses interleukin-1beta-induced human beta-defensin-2 mRNA expression through inhibition of transcriptional activation of NF-kappaB in A549 cells.

The immunosuppressive effect of triptolide has been associated with suppression of T-cell activation. However, the immunosuppressive effects of triptolide on innate immunity in the epithelial barrier remain to be elucidated. Human beta-defensin (HBD)-2 is an inducible antimicrobial peptide and plays an important role in the innate immunity. We have previously demonstrated that IL-1beta induced HBD-2 mRNA expression in A549 cells through activation of nuclear factor-kappaB (NF-kappaB) transcriptional factor as well as p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), or phosphatidylinositol-3-kinase (PI3K). In this study, we investigated effects of triptolide on IL-1beta-induced HBD-2 mRNA expression in A549 cells. Triptolide inhibited IL-1beta-induced HBD-2 mRNA expression in a dose-dependent manner. Addition of triptolide did not suppress activation of p38 MAPK, JNK, or PI3K in response to IL-1beta. Triptolide inhibited IL-1beta-induced MAPK phosphatase-1 expression at the transcriptional level and resulted in sustained phosphorylation of JNK or p38 MAPK, explaining the little effect of triptolide on IL-1beta-induced phosphorylation of these kinases. Although triptolide partially suppressed IL-1beta-mediated degradation of IkappaB-alpha and nuclear translocation of p65 NF-kappaB, triptolide potently inhibited NF-kappaB promoter-driven luciferase activity in A549 cells. These results collectively suggest that the inhibitory effect of triptolide on IL-1beta-induced HBD-2 mRNA expression in A549 cells seems to be at least in part mediated through nuclear inhibition of NF-kappaB transcriptional activity, but not inhibition of p38 MAPK, JNK, or PI3K. This inhibition may explain the ability of triptolide to diminish innate immune response.

Synthesis and preparation of novel 4-arm star-shape poly(carboxylic acid)s for improved light-cured glass-ionomer cements.

OBJECTIVE: The objective of this study was to synthesize and characterize novel 4-arm star-shape poly(acrylic acid)s (poly(AA)s) via atom-transfer radical polymerization (ATRP) technique, tether in situ light-curable methacrylate functionalities onto the poly(AA) backbone, use these star-shape poly(AA)s to formulate the light-cured glass-ionomer cements (LCGICs), and evaluate the mechanical strengths of the formed cements. MATERIALS AND METHODS: The 4-arm poly(AA)s were synthesized using ATRP and tethered with either 2-isocyanatoethyl methacrylate (IEM) or glycidyl methacrylate (GM). The polymers were formulated with 2-hydroxyethyl methacrylte (HEMA) or methacryloyl beta-alanine (MBA), water, initiators, and Fuji II LC filler. Compressive strength (CS) was used as a tool to evaluate the formed cements. The specimens were conditioned in distilled water at 37 degrees C for 24h prior to testing. RESULTS: The 4-arm poly(AA) showed a lower viscosity as compared to its linear counterpart. Both IEM-tethered and GM-tethered 4-arm poly(AA) constructed LCGICs showed significantly high mechanical strengths. Both types of co-monomer and grafting agent dramatically affected the mechanical strengths. The MBA-containing poly(AA) cements exhibited much higher CS than the HEMA-containing cements. The IEM-tethered poly(AA) cements showed much higher CS and DTS than the GM-tethered cements. CONCLUSIONS: This study developed a novel light-curable 4-arm star-shape poly(AA) system. The system was 13% in CS, 178% in DTS and 123% in FS, compared to Fuji II LC.

A novel comonomer-free light-cured glass-ionomer cement for reduced cytotoxicity and enhanced mechanical strength.

OBJECTIVE: The objective of this study was to develop a novel comonomer-free light-cured glass-ionomer system based on the 4-arm star-shape poly(acrylic acid). The mechanical strengths and in vitro cytotoxicity of the formed system were evaluated and compared with those of several representative commercial glass-ionomer cements. MATERIALS AND METHODS: The 4-arm poly(acrylic acid) was synthesized using ATRP and tethered with glycidyl methacrylate (GM). The GM-tethered polymer was formulated with water, photo-initiators, and Fuji II LC filler. Fuji II, Fuji II LC and Vitremer were used for comparison. Compressive strength (CS) and MTT assay were used as tools to evaluate the mechanical strengths and in vitro cytotoxicity of the cements, respectively. RESULTS: The experimental cement exhibited significantly high compressive, diametral tensile and flexural strengths as compared to commercial glass-ionomer cements, Fuji II, Fuji II LC and Vitremer. The effects of polymer/water (P/W) ratio, GM-grafting ratio, glass powder/polymer liquid (P/L) ratio and aging in water on strengths were significant. Similar to conventional glass-ionomer cement Fuji II, the eluates from the experimental cement showed little in vitro cytotoxicity to Balb/c mouse fibroblast cells, as compared to Fuji II LC and Vitremer that contain HEMA as a comonomer. CONCLUSIONS: It appears that this novel comonomer-free light-cured glass-ionomer cement will be a promising dental restorative because it demonstrated significantly improved mechanical strengths and almost no in vitro cytotoxicity as compared to current commercial light-cured glass-ionomer cements.

Novel injectable and in situ curable glycolide/lactide based biodegradable polymer resins and composites.

Novel in situ polymerizable liquid three-arm biodegradable oligomeric polyesters based upon glycolic acid (GA), L-lactic acid (LLA), and their copolymers are synthesized and characterized. Injectable and in situ curable polymer neat resins and their composites formulated with bioabsorbable beta-tricalcium phosphate are prepared at room temperature using photo- and redox-initiation systems, respectively. The cured neat resins show the initial compressive yield strength (YCS, MPa), modulus (M, MPa), ultimate compressive strength (UCS, MPa), and toughness (T, kN mm), ranging from 4.0 to 20.1, 201.5 to 730.2, 82.7 to 310.5, and 1.02 to 3.93. The cured composites show the initial YCS, M, UCS and T, ranging from 27.7 to 56.4, 1440 to 4870, 81.6 to 158.9, and 0.94 to 1.97. Increasing GA/LLA ratio increases all the initial compressive strengths of both neat resins and composites. Increasing filler content increases YCS and M but decreases UCS and T. A diametral tensile strength test shows the same trend as a compressive strength test. There seems to be an optimal flexural strength for the composite at the filler content around 43%. An increasing molar ratio increases curing time but decreases the degree of conversion (DC). An increasing filler content increases curing time but decreases exotherm and DC. During the course of degradation, all the materials show a burst degradation behavior within 24 h, followed by an increase in CS. The poly(glycolic acid) neat resin completely loses its strength at around Day 45. The composites completely lose their strengths at different time intervals, depending on their molar ratio and filler content. The degradation rate is found to be molar ratio and filler-content dependent.

Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR.

Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.

Leptomycin B, a metabolite of Streptomyces, inhibits the expression of inducible nitric oxide synthase in BV2 microglial cells.

Overexpression of inducible nitric oxide synthase (iNOS) and the resultant overproduction of NO has been implicated in neuronal inflammatory diseases. Leptomycin B (LMB), a metabolite of Streptomyces, has been identified as a specific inhibitor of CRM1 nuclear export receptor. In this study, we evaluated the effect of LMB on lipopolysaccharide (LPS)-induced iNOS expression in BV2 cells, a murine microglial cells and the associated mechanisms. LMB strongly inhibited LPS-induced iNOS protein and mRNA expressions in BV2 cells in which 10 ng/ml of LMB (18 nM) was sufficient to greatly down-regulate iNOS by LPS, suggesting the potency of LMB to inhibit iNOS. The data of iNOS promoter-driven luciferase assay further suggested that the LMB inhibitory effect was in part due to inhibition of iNOS transcription. However, LPS-induced activation of various intracellular signaling proteins, such as nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated kinases (ERKs), p38s, and c-Jun N-terminal kinases (JNKs), whose activations are known to be important for iNOS expression by LPS in BV2 cells, were not affected in the presence of LMB. Together, these results suggest that LMB inhibits iNOS expression in response to LPS in BV2 microglia, and the inhibition seems to be associated with blockage of CRM1-mediated iNOS mRNA nuclear export and also in part transcriptional down-regulation of iNOS, but not through modulation of NF-kappaB and the mitogen-activated protein kinase signaling pathways.

Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo.

Excessive proliferation of vascular smooth muscle cells (VSMCs) and neointimal formation are critical steps in the pathogenesis of atherosclerosis and restenosis after percutaneous transluminal angioplasty. In this study, we investigated the hypothesis that the activator protein-1 (AP-1) plays an important role in neointimal formation after vascular injury. A circular dumbbell AP-1 decoy oligodeoxynucleotide (CDODN) was developed as a novel therapeutic strategy for restenosis after angioplasty. This CDODN was more stable than the conventional phosphorothioate linear decoy ODN (PSODN) and maintained structural integrity on exposure to exonuclease III or serum. Transfection with AP-1 decoy ODNs strongly inhibited VSMC proliferation and migration, as well as glucose- and serum-induced expression of PCNA and cyclin A genes. Administration of AP-1 decoy ODNs in vivo using the hemagglutinating virus of Japan (HVJ)-liposome method virtually abolished neointimal formation after balloon injury to the rat carotid artery. Compared with PSODN, CDODN was more effective in inhibiting the proliferation of VSMCs in vitro and neointimal formation in vivo. Our results collectively indicate that AP-1 activation is crucial for the mediation of VSMC proliferation in response to vascular injury. Moreover, the use of stable CDODN specific for AP-1 activity in combination with the highly effective HVJ-liposome method provides a novel potential therapeutic strategy for the prevention of restenosis after angioplasty in humans.

Catalase induced expression of inflammatory mediators via activation of NF-kappaB, PI3K/AKT, p70S6K, and JNKs in BV2 microglia.

Catalase induces COX-2 or iNOS expression in some type of cells, but the mechanism remains unclear. Here we investigated the effect of catalase on COX-2 and iNOS expression in BV2 microglia and the inductive mechanism associated. Exposure of catalase to BV2 microglia induced expression of COX-2 and iNOS that was related with transcriptional up-regulation. Importantly, catalase-induced COX-2 and iNOS expression needed activations of NF-kappaB, PI3K/AKT, and JNKs, which were important for the transcriptional up-regulation of COX-2 and iNOS. Notably, rapamycin inhibition of p70S6K led to down-regulation of COX-2 and iNOS protein expression, but not steady-state mRNA expression and transcription, induced by catalase, suggesting that p70S6K is involved in increased COX-2 and iNOS mRNA translation by catalase. Interestingly, there was PI3K-dependent activation of AKT, p70S6K, JNKs, and NF-kappaB in response to catalase. These data collectively suggest catalase-induced COX-2 and iNOS expression in BV2 microglia is, in part at least, mediated through activation of multiple signaling proteins.

Novel amino acid-constructed polyalkenoates for dental glass-ionomer restoratives.

Several methacrylate or acrylate derivatives of natural amino acids were synthesized and characterized. Based upon these derivatives, novel amino acid-constructed polyalkenoic acids were prepared and used to formulate glass-ionomer cements (GICs) with Fuji II glass filler. The effects of type of derivatives, molar ratio, molecular weight, and powder/liquid ratio were investigated. The results show that amino acid-constructed polyalkenoic acids can be formed only from amino acid methacrylate derivatives or by copolymerization of methacrylate with acrylate derivatives. Strong hydrogen bond interactions failed the polymer formation from acrylate derivatives. The cement composed of poly(methacryloyl glutamic acid-co-acryloyl beta-alanine) with the molar ratio of 8:2 demonstrated the best mechanical strengths along with a workable viscosity. By using the powder/liquid ratio of 3.0/1, the experimental cement exhibited a significantly higher FS (27.7 MPa), and nearly the same CS (198.5 MPa) and DTS (11.8 MPa), as compared to Fuji II (18.9 for FS, 189.1 for CS, and 11.4 MPa for DTS). During aging, the cement showed a significant increase in strength over 24 h, followed by a slow increase over 6 months.

Inhibitory modulation of ATP-sensitive potassium channels by gallate-ester moiety of (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenolic substance found in green tea, is well recognized to be beneficial for human health. However, it is still controversial as to what dose of this compound is indeed good for human health. Though some recent studies have interestingly reported various beneficial effects of EGCG in cell culture system, however, plasma levels of EGCG attainable by oral regular intake in humans are normally in nanomolar range. However, potential side effects of EGCG when administered parenterally at higher concentration have not been thoroughly tested. Here, we evaluated the effect of EGCG on ATP-sensitive potassium (K(ATP)) channels expressed in Xenopus oocytes. EGCG inhibited the activity of the Kir6.2/SUR1 and Kir6.2DeltaC36 channels with IC(50) of 142+/-37 and 19.9+/-1.7microM, respectively. Inhibition of EGCG was also observed in Kir6.2/SUR2A or Kir6.2/SUR2B channels. Notably, (-)-epicatechin-3-gallate (ECG), another major polyphenolic substance in green tea, was found to reduce the channel activity with greater potency than EGCG. In contrast to EGCG and ECG, which have the gallic acid-ester moiety in their own structures, (-)-epigallocatechin and (-)-epicatechin exhibited very weak inhibition of the K(ATP) channel. Collectively, these results suggest that the gallate-ester moiety of epicatechins may be critical for inhibiting the K(ATP) channel activity via the pore-forming subunit Kir6.2 and this may be a possible mechanism by which green tea extracts or EGCG may cause unexpected side effects at micromolar plasma level.