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Biomedical subjects

Y Rojanasakul

Publications and source records attributed to Y Rojanasakul.

At least 19 recordsLinked to original sources
PubMed2001

Vanadium-induced nuclear factor of activated T cells activation through hydrogen peroxide.

The present study investigated the role of reactive oxygen species (ROS) in activation of nuclear factor of activated T cells (NFAT), a pivotal transcription factor responsible for regulation of cytokines, by vanadium in mouse embryo fibroblast PW cells or mouse epidermal Cl 41 cells. Exposure of cells to vanadium led to the transactivation of NFAT in a time- and dose-dependent manner. Scavenging of vanadium-induced H(2)O(2) with N-acety-L-cyteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor) or the chelation of vanadate with deferoxamine, resulted in inhibition of NFAT activation. In contrast, an increase in H(2)O(2) generation by the addition of superoxide dismutase or NADPH enhanced vanadium-induced NFAT activation. This vanadate-mediated H(2)O(2) generation was verified by both electron spin resonance and fluorescence staining assay. These results demonstrate that H(2)O(2) plays an important role in vanadium-induced NFAT transactivation in two different cell types. Furthermore, pretreatment of cells with nifedipine, a calcium channel blocker, inhibited vanadium-induced NFAT activation, whereas and ionomycin, two calcium ionophores, had synergistic effects with vanadium for NFAT induction. Incubation of cells with cyclosporin A (CsA), a pharmacological inhibitor of the phosphatase calcineurin, blocked vanadium-induced NFAT activation. All data show that vanadium induces NFAT activation not only through a calcium-dependent and CsA-sensitive pathway but also involved H(2)O(2) generation, suggesting that H(2)O(2) may be involved in activation of calcium-calcineurin pathways for NFAT activation caused by vanadium exposure.

Animals↗
PubMed2001

Immunoglobulin responses to experimental silicosis.

Silicosis is a crippling fibrotic lung disease induced by inhalation of crystalline silica. One feature of silicosis is systemic and pulmonary immune dysfunction characterized in part by elevations in serum and bronchoalveolar lavage (BAL) immunoglobulins. A major specific aim of the current report was to demonstrate that an experimental model of silicosis previously well characterized for the development of pulmonary inflammation and fibrosis would also exhibit increased levels of serum and BAL IgG and IgM similar to those of human silicosis. We also sought to document the anatomic compartments responsible for these immunoglobulin responses. To address these specific aims, we compared levels of IgG and IgM in serum and BAL from rats with experimental silicosis induced by inhalation of silica with levels of these immunoglobulins in titanium dioxide (TiO(2))- and sham (air)-exposed controls. The ability of mononuclear cell populations from lung, lung-associated lymph node, and spleen to produce IgG and IgM ex vivo were also compared. We found that experimental silicosis was associated with elevated IgG and IgM levels in blood and BAL relative to the control groups. Our findings also suggested that draining lung-associated lymph nodes (LALN) were the most important sites for increased IgG and IgM production in experimental silicosis, with lungs contributing to a lesser degree. Increased production in the LALN appeared related to marked expansion in total numbers, but not relative proportion, of B lymphocytes.

Administration, Inhalation↗
PubMed2001

Interleukin-10-mediated inhibition of free radical generation in macrophages.

Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines that are known to be transcriptionally regulated by nuclear factor-kappaB (NF-kappaB). Although still controversial, IL-10 has been shown to inhibit NF-kappaB activation through a process that involves proteolytic degradation of inhibitory subunit IkappaB-alpha. What is not known, however, is the mechanism by which IL-10 exerts its effect on IkappaB-alpha degradation. The present study investigates the possible role of reactive oxygen species (ROS) and their inhibition by IL-10 in NF-kappaB activation and IkappaB-alpha degradation in macrophages. Treatment of the cells with lipopolysaccharide (LPS) caused activation of NF-kappaB and rapid proteolysis of IkappaB-alpha as determined by the electrophoretic mobility shift assay, gene transfection, and Western blot. IL-10 pretreatment inhibited both NF-kappaB activation and IkappaB-alpha degradation. Both of these processes were also inhibited by ROS scavengers, catalase (H(2)O(2) scavenger), and sodium formate (.OH scavenger) but were minimally affected by superoxide dismutase (O scavenger). These results suggests that.OH radicals, formed by an H(2)O(2)-dependent, metal-catalyzed Fenton reaction, play a major role in this process. Electron spin resonance studies confirmed the formation of.OH radicals in LPS-treated cells. Addition of IL-10 inhibited both IkappaB-alpha degradation and generation of.OH radicals in response to LPS stimulation. These results demonstrate, for the first time, direct evidence for the role of IL-10 in ROS-dependent NF-kappaB activation.

Animals↗
PubMed2001

IgG subclass responses in experimental silicosis.

Silicosis is a crippling fibrotic lung disease induced by inhaling crystalline silica. In addition to fibrosis, silica inhalation by humans is associated with a number of immunological effects including increased levels of serum immunoglobulins (in particular IgG), increased prevalence of autoantibodies, and autoimmune disease. Recent studies using rodent models have shown that experimental silicosis is associated with a T-helper (TH)1 pattern of T-cell activation in the lungs and lung-associated lymph nodes after silica inhalation, which are also the sites of increased IgG production. We therefore hypothesized that the subclass distribution of IgG production occurring in experimental silicosis would suggest TH1 activation as the primary stimulus for IgG production. Using an ELISPOT assay, we found increased IgG-secreting spot-forming cells of all IgG subclasses in lung-associated lymph nodes taken from silica-exposed rats 3 to 4 months after aerosol exposure to silica. Neither TH1- nor TH2-dependent IgG subclass-secreting cells were selectively enhanced. Our findings suggest that TH1 activation alone does not account for increased production of IgG in experimental silicosis.

Administration, Inhalation↗
PubMed2001

Reactive oxygen species and molecular mechanism of silica-induced lung injury.

Silica particles are considered to be fibrogenic and carcinogenic agents, but the mechanisms of disease initiation and progression are not fully understood. This article summarizes the literature on the generation of reactive oxygen species (ROS) directly from interaction of silica with aqueous medium and from silica-stimulated cells. This article also discusses the role of ROS in silica-induced lung injury, with particular focus on the silica-induced NF-kappaB activation, including the molecular mechanisms of its regulation, its possible attenuation, and its relationship to silica-induced generation of cyclooxygenase II and TNF-alpha.

Air Pollutants, Occupational↗
PubMed2000

Novel non-endocytic delivery of antisense oligonucleotides.

Antisense oligonucleotides (ONs) have several properties that make them attractive as therapeutic agents. Hybridization of antisense ONs to their complementary nucleic acid sequences by Watson-Crick base pairing is a highly selective and efficient process. Design of therapeutic antisense agents can be made more rationally as compared to most traditional drugs, i.e., they can be designed on the basis of target RNA sequences and their secondary structures. Despite these advantages, the design and use of antisense ONs as therapeutic agents are still faced with several obstacles. One major obstacle is their inefficient cellular uptake and poor accessibility to target sites. In this article, we will discuss key barriers affecting ON delivery and approaches to overcome these barriers. Current methods of ON delivery will be reviewed with an emphasis on novel non-endocytic methods of delivery. ONs are taken up by cells via an endocytic process. The process of ON release from endosomes is a very inefficient process and, hence, ONs end up being degraded in the endosomes. Thus, ONs do not reach their intended site of action in the cytoplasm or nucleus. Delivery systems ensuring a cytoplasmic delivery of ONs have the potential to increase the amount of ON reaching the target. Here, we shall examine various ON delivery methods that bypass the endosomal pathway. The advantages and disadvantages of these methods compared to other existing methods of ON delivery will be discussed.

Animals↗
PubMed2000

Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis.

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H(2)O(2) by N-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H(2)O(2) generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53-/-) cells. These results demonstrate that vanadate induces p53 activation mainly through H(2)O(2) generation, and this activation is required for vanadate-induced apoptosis.

Acetylcysteine↗
PubMed2000

High-efficiency gene transfection of macrophages by lipoplexes.

Macrophage transfection studies are crucial for understanding gene regulation and expression. However, gene transfection in macrophages is difficult. We have shown here that macrophages are more resistant to gene transfection compared with other cell types. To further develop an efficient gene delivery system for macrophages, we evaluated various liposomal and non-liposomal agents including LipofectAMINE(R), Lipofectin(R), DOTAP, DEAE-dextran, and the DNA condensing agent protamine sulfate for their ability to promote gene transfection. CMV-luciferase was used as a reporter plasmid. Macrophage transfection was maximal at the DNA:LipofectAMINE:protamine ratio of 1:12:1 microg/ml. The LipofectAMINE formulation showed a 10-12-fold increase in transfection efficiency over DOTAP and a 4-5-fold increase over Lipofectin. This transfection method showed minimal toxicity at the concentrations tested and was at least 20-25-fold superior to the most frequently used DEAE-dextran method for macrophage transfection.

Animals↗
PubMed2000

Cellular delivery of functional peptides to block cytokine gene expression.

Advances in molecular and cellular biology have identified the cellular mediators that regulate many disease processes and have facilitated the development of new therapeutic agents that control these events. However, the size, complexity, and cellular inaccessibility of these therapeutic agents make their cellular delivery difficult. Here, we describe an efficient cellular delivery system that exploits the membrane-translocating ability of signal peptides to import functional peptides into cells. Molecular conjugates consisting of the signal import peptide (IP) and nuclear localization sequence (NLS) of the transcription factor NF-kappaB were synthesized. Electrophoretic mobility shift and enzyme-linked immunosorbent assays were used to assess the inhibitory effects of these synthetic peptides on agonist-induced NF-kappaB nuclear translocation and transcriptional activation. Our results indicated that the peptides were effective in inhibiting both the nuclear translocation and transcriptional activation of NF-kappaB. However, their effects required the presence of the IP moiety for efficient cellular entry of the NLS. Structural analysis of IP showed that the hydrophobic domain, and to a lesser extent the N-terminal domain, was responsible for the membrane translocating activity of IP.

Amino Acid Sequence↗
PubMed2000

Characterization of proteolytic activities of pulmonary alveolar epithelium.

Pulmonary alveolar type I epithelial cell and its progenitor, type II cell, present major transport and enzyme barriers for systemic delivery of pulmonary administered peptide drugs. The present study investigates the effect of cellular differentiation of type II to type I cells on their proteolytic activities, and evaluates the suitability of a continuous lung cell line, A549, for drug transport and degradation studies. High performance liquid chromatography was used to assess the degradation kinetics of two model peptide substrates, luteinizing hormone releasing hormone (LHRH) and [D-Ala(6)10-fold decrease in proteolytic activities for LHRH, as compared to type II cells. The continuous lung cell line A549 formed leaky monolayers and exhibited similar enzyme activities to the primary type II cells. The responsible enzymes for degradation of LHRH in type II and A549 cells were angiotensin converting enzyme (ACE), EP24.11, and EP24.15. In contrast, no EP24.15 or ACE activity was observed in type I-like pneumocytes and only a weak EP24.11 activity was detected. In all cell types, the degradation rate of [D-Ala(6)]-LHRH was about 3-8 times lower than that of LHRH. This peptide analog was resistant to degradation by EP24.15 and EP24.11, but was susceptible to ACE-mediated cleavage.

Animals↗
PubMed2000

Antioxidant properties of (-)-epicatechin-3-gallate and its inhibition of Cr(VI)-induced DNA damage and Cr(IV)- or TPA-stimulated NF-kappaB activation.

Electron spin resonance (ESR) spin trapping was utilized to investigate the scavenging effects on hydroxyl radicals (*OH) and superoxide radicals (O2*-) by (-)-epigallocatechin-3-gallate (EGCG), one of the major anticancer compounds in tea. The spin trap used was 5,5-dimethyl-pyrroline N-oxide (DMPO). The Fenton reaction (Fe2+ + H2O2-->Fe3+ + *OH + OH-) was used as a source of *OH radicals. EGCG efficiently scavenges *OH radicals with reaction rate of 4.62 x 10(11) M(-1)sec(-1), which is an order of magnitude higher than several well recognized antioxidants, such as ascorbate, glutathione and cysteine. It also scavenges O2*- radicals as demonstrated by using xanthine and xanthine oxidase system as a source of O2*- radicals. Through its antioxidant properties, EGCG exhibited a protective effect against DNA damage induced by Cr(VI). EGCG also inhibited activation of nuclear transcription factor NF-kappaB induced by Cr(IV) and 12-o-tetradecanoylphorbol-13-acetate (TPA). The present studies provide a mechanistic basis for the reported anticarcinogenic properties of EGCG and related tea products.

Antioxidants↗
PubMed2000

Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes.

PURPOSE: The objectives of this study are to investigate the toxicity associated with polycationic liposomes and to elucidate the underlying mechanism. We tested the hypothesis that the positive charge of liposomes is a key determinant of toxicity by testing differently charged liposomes in mice. METHODS: Differently charged liposomal systems including cationic liposomes, LipofectAMINE and DOTAP, and neutral and negative liposomes were evaluated for their toxicity after pulmonary administration in mice. LDH assay and differential cell counts were performed to measure toxicity and pulmonary inflammation, respectively. Reactive oxygen intermediates (ROI) were assessed by chemiluminescence. RESULTS: Instillation of cationic liposomes elicited dose-dependent toxicity and pulmonary inflammation. This effect was more pronounced with the multivalent cationic liposome LipofectAMINE as compared to the monovalent cationic DOTAP. Neutral and negative liposomes did not exhibit lung toxicity. Toxicity associated with cationic liposomes correlated with the oxidative burst induced by the liposomes. LipofectAMINE induced a dose-dependent increase in ROI generation. This effect was less pronounced with DOTAP and absent with neutral and negative liposomes. CONCLUSIONS: ROI play a key role in cationic lipid-mediated toxicity. Polyvalent cationic liposomes cause a release of ROI which are responsible for the pulmonary toxicity.

Animals↗
PubMed2000

Inhibition of endotoxin-induced lung inflammation by interleukin-10 gene transfer in mice.

Interleukin (IL)-10 is an anti-inflammatory cytokine that has great potential for use in the treatment of inflammatory and immune illnesses. In this study, gene transfer was used to induce IL-10 transgene expression in murine lungs for treatment of endotoxin-induced lung inflammation. Gene transfer was performed with a cytomegalovirus (CMV)-IL-10 plasmid with the aid of the liposomal agents LipofectAMINE and N-[1-(2,3-dioleoyl)propyl]-N,N, N-trimethylammonium methylsulfate (DOTAP). Administration of the endotoxin caused a marked increase in lung inflammation as indicated by increased tumor necrosis factor (TNF)-alpha release and neutrophil count. Pretreatment of the mice with IL-10 plasmid with and without LipofectAMINE had no inhibitory effect on lung inflammation and IL-10 transgene expression. LipofectAMINE by itself induced lung inflammation, an effect that was not observed with DOTAP. IL-10 plasmid when codelivered with DOTAP expressed biologically active IL-10 protein and caused a reduction in endotoxin-induced inflammation. Transgene expression was observed as early as 3 h after administration, peaked at 12 h, and declined thereafter. We conclude that IL-10 gene transfer is a feasible approach for the treatment of lung inflammation.

Animals↗
PubMed2000

Free radical-mediated transgene inactivation of macrophages by endotoxin.

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N-t-butyl-alpha-phenylnitrone, the H(2)O(2) scavenger catalase, and the.OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O(2)(-) scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that.OH formed by H(2)O(2)-dependent, metal-catalyzed Fenton reaction play a major role in this process.

Animals↗
PubMed2000

On the role of hydroxyl radical and the effect of tetrandrine on nuclear factor--kappaB activation by phorbol 12-myristate 13-acetate.

Nuclear factor kappaB (NF-kappaB) is considered to be an important target for therapeutic intervention because of its role in the regulation of proinflammatory and profibrotic mediators. The present study examined the role of hydroxyl (*OH) radical and the effect of tetrandrine, an alkaloid extracted from the Chinese medicinal herb Stephania tetrandra, on NF-kappaB activation by a tumor promoter, phorbol 12-myristate 13-acetate (PMA) in human lymphoid T cells (ie, Jurkat cells). Exogenous superoxide dismutase (SOD) enhanced the NF-kappaB activation by PMA, while catalase blocked it. Formate, a scavenger of *OH radical, also was inhibitory, as was deferoxamine, a metal chelator. These data suggest an important role of *OH radical in PMA-induced NF-kappaB activation. Incubation of the cells with tetrandrine prior to the stimulation of the cells was found to inhibit PMA-induced NF-kappaB activation. Tetrandrine activity was so potent that 50 microM of tetrandrine was sufficient to inhibit activation of NF-kappaB completely. Electron spin resonance (ESR) spin trapping was used to investigate the antioxidant action of tetrandrine using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Tetrandrine is an antioxidant for both *OH and superoxide (O2-)radicals. The reaction rate constant of tetrandrine with *OH is 1.4 x 10(10) M(-1)sec(-1), which is comparable with several well established antioxidants, such as ascorbate, glutathione, and cysteine. The Fenton reaction (Fe(II) + H2O2-->Fe(III) + *OH + OH-) and xanthine/xanthine oxidase were used as sources of *OH and O2- radicals. The free radical scavenging activity of tetrandrine is responsible for its inhibition of PMA-induced NF-kappaB activation.

Alkaloids↗
PubMed1999

Role of reactive oxygen species and p53 in chromium(VI)-induced apoptosis.

Apoptosis is a programmed cell death mechanism to control cell number in tissues and to eliminate individual cells that may lead to disease states. The present study investigates chromium(VI) (Cr(VI))-induced apoptosis and the role of reactive oxygen species (ROS) and p53 in this response. Treatment of human lung epithelial cells (A549) with Cr(VI) caused apoptosis as measured by DNA fragmentation, mitochondria damage, and cell morphology. Cr(VI)-induced apoptosis is contributed to ROS generation, resulting from cellular reduction of Cr(VI) as measured by flow cytometric analysis of the stained cells, oxygen consumption, and electron spin resonance spin trapping. Scavengers of ROS, such as catalase, aspirin, and N-acetyl-L-cysteine, decreased Cr(VI)-induced apoptosis, whereas NADPH and glutathione reductase, enhancers of Cr(VI)-induced ROS generation, increased it. p53 is activated by Cr(VI), mostly by ROS-mediated free radical reactions. Cr(VI)-induced ROS generation occurred within a few minutes after Cr(VI) treatment of the cells, whereas p53 induction took at least 5 h. The level of Cr(VI)-induced apoptosis was similar in both p53-positive cells and p53-negative cells independent of p53 status in the early stage (0-3 h) of Cr(VI) treatment. However, at the later stage (3-24 h), the level of the apoptosis is higher in p53-positive cells than in p53-negative cells. These results suggest that ROS generated through Cr(VI) reduction is responsible to the early stage of apoptosis, whereas p53 contributes to the late stage of apoptosis and is responsible for the enhancement of Cr(VI)-induced apoptosis at this stage.

Apoptosis↗