Effects of SOD/catalase mimetic platinum nanoparticles on radiation-induced apoptosis in human lymphoma U937 cells.

Since polyacrylic acid capped platinum nano-particles (nano-Pts) are known to have a unique ability to quench superoxide (O2(-)) and hydrogen peroxide (H2O2), the anti-oxidant activity of nano-Pts against apoptosis induced by x-irradiation in human lymphoma U937 cells was investigated. DNA fragmentation assay, Annexin V-FITC/PI by flow cytometry and Giemsa staining revealed a significant decrease in apoptosis induced by 10 Gy, when cells were pre-treated with nano-Pts in a dose-dependent manner. Pre-treatment with nano-Pts significantly decreased radiation-induced reactive oxygen species (ROS) production, Fas expression and loss of mitochondrial membrane potential as determined by flow-cytometry. Furthermore, western blot analysis also showed that the expression of cleaved caspase-3, Bid and cytosolic cytochrome-c were significantly reduced in nano-Pts pretreated cells. Due to the catalase mimetic activity of nano-Pts, these results indicate that pre-treatment of U937 cells with nano-Pts significantly protect radiation-induced apoptosis by inhibiting intracellular ROS (mainly H2O2), which plays a key role in the induction of apoptosis, because of no practical observation of intracellular O2(-) formation.

ARD1 binding to RIP1 mediates doxorubicin-induced NF-κB activation.

NF-κB is activated by several cellular stresses. Of these, the TNFα-induced activation pathway has been examined in detail. It was recently reported that receptor-interacting protein 1 (RIP1) is involved in DNA damage-induced NF-κB activation by forming a complex with the p53 interacting death domain protein (PIDD) and NF-κB essential modulator (NEMO) in the nucleus, although the underlying mechanism of this interaction has yet to be clarified. This study shows that siRNA knock-down of arrest-defective 1 protein (ARD1) abrogated doxorubicin- but not TNFα-induced activation. Conversely, the over-expression of ARD1 greatly enhanced NF-κB activation induced by doxorubicin. Immunoprecipitation experiments revealed that ARD1 interacted with RIP1 via the acetyltransferase domain. Furthermore, the over-expression of several domain-deleted ARD1 constructs demonstrated that the N-terminal and acetyltransferase domains of ARD1 were required for doxorubicin-induced NF-κB activation. Treatment of deacetylase inhibitor, trichostatin A, significantly increased doxorubicin-induced NF-κB activation in the presence of ARD1 but not acetyltransferase-defective ARD1 mutant. Moreover, N-terminal domain-deleted ARD1 could not be localized in the nucleus in response to doxorubicin treatment. These data indicate that the interaction between ARD1 and RIP1 plays an important role in the DNA damage-induced NF-κB activation, and that the acetyltransferase activity of ARD1 and its localization in to the nucleus are involved in such stress response.

The anti-inflammatory effects of platinum nanoparticles on the lipopolysaccharide-induced inflammatory response in RAW 264.7 macrophages.

OBJECTIVE: Platinum nanoparticles (nano-Pt) have been reported to possess anti-oxidant and anti-tumor activities. However, the biological activity and mechanism of action of nano-Pt in inflammation are still unknown. The present study was designed to determine the in-vitro anti-inflammatory effects of nano-Pt on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. METHODS: RAW 264.7 macrophages were used for the study. The LPS-induced production of reactive oxygen species (ROS) was determined by flow cytometry. The prostaglandin E(2) (PGE(2)) concentration was measured using a PGE(2) assay kit. The protein levels and mRNA expression of the pro-inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1ß and IL-6], along with cyclooxygenase (COX-2) and inducible nitric oxide synthase, were analyzed by Western blotting and reverse transcription-polymerase chain reaction analysis. The phosphorylation of extracellular signal regulated kinase (ERK1/2) and Akt, and the phosphorylation and degradation of inhibitory kappa B-alpha (IκB-α) was determined by Western blot analysis. RESULTS: Nano-Pt significantly reduced the LPS-induced production of intracellular ROS and inflammatory mediators. In addition, nano-Pt suppressed the phosphorylation of ERK1/2 and Akt, and significantly inhibited the phosphorylation/degradation of IκB-α as well as nuclear factor kappa-B (NFκB) transcriptional activity. CONCLUSION: These results suggest that the anti-inflammatory properties of nano-Pt may be attributed to their downregulation of the NFκB signaling pathway in macrophages, thus supporting the use of nano-Pt as an anti-inflammatory agent.

Neurological and pathological improvements of cerebral infarction in mice with platinum nanoparticles.

Ischemic stroke is a major, urgent neurologic disorder in which reactive oxygen species (ROS) are deeply involved in the detrimental effects. Platinum nanoparticle (nPt) species are a novel and strong scavenger of such ROS, so we examined the clinical and neuroprotective effects of nPts in mouse ischemic brain. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min. Upon reperfusion, nPt or vehicle was administered intravenously. At 48 hr after the tMCAO, motor function, infarct volume, immunohistochemistry of neurovascular components (endothelial NAGO, tight junctional occludin, and basal laminal collagen IV), and zymography for MMP-9 activity were examined. Superoxide anion generation at 2 hr after tMCAO was determined with oxidized hydroethidine. Compared with vehicle, treatment with nPts significantly improved the motor function and greatly reduced the infarct volume, especially in the cerebral cortex. Immunohistochemical analyses revealed that tMCAO resulted in a minimal decrease of NAGO and occludin but a great decrease of collagen IV and a remarkable increase of MMP-9. Treatment with nPts greatly reduced this decrease of collagen IV and activation of MMP-9 and, with large reductions of MMP-9 activation on zymography and superoxide production. The present study demonstrates that treatment with nPts ameliorates the neurological scores with a large reduction in infarct size as well as the preservation of outer components of the neurovascular unit (collagen IV) and inactivation of MMP-9. A strong reduction of superoxide anion production by nPts could account for such remarkable neurobehavioral and neuroprotective effects on ischemic stroke.

Expansion of nanotechnology for dentistry: effect of colloidal platinum nanoparticles on dentin adhesion mediated by 4-META/MMA-TBB.

PURPOSE: To investigate the effect of Colloidal Platinum Nanoparticles (CPN) on the bond strength between dentin and 4-META/MMA-TBB resin using different concentrations of CPN. MATERIALS AND METHODS: Twenty-five extracted human third molars were stored in 0.5% chloramine T. The occlusal dentin slices were prepared by grinding occlusal surfaces of each tooth and polishing with 600-grit silicon carbide paper under running water. One control and four experimental groups (2 specimens per group) were used as follows: a) dentin surfaces treated with 10-3 solution, followed by rinsing with water and subsequently an acrylic rod bonded with hand-mixed 4META/MMA-TBB resin (Super-Bond C&B, Sun Medical) (control); b) dentin surfaces treated with 10-3 etching solution, followed by rinsing with water and application of CPN (100% or 10%) as a primer solution for 60 s and rinsed with water for 20 s, then an acrylic rod bonded with Super-Bond C&B(Etch-CPN [100% or 10%]); c) dentin surfaces treated with CPN (100% or 10%) for 60 s, rinsed with water for 20 s, followed by application of 10-3 solution, then an acrylic rod bonded with Super-Bond C&B (CPN-Etch [100% or 10%]). After storage in 37°C water, specimens were sectioned into beams (cross-sectional area: 1 mm2) for microtensile bond strength testing at a crosshead speed of 1mm/min. The data were analyzed using the Games-Howell method (p < 0.05; n = 15). RESULTS: Etch-CPN (100), CPN-Etch(100) and CPN-Etch (10) showed significantly higher bond strengths compared to the control. When using 10% CPN, the highest bond strength was demonstrated. The bond strength of 4META/MMA-TBB resin was approximately doubled by CPN application. CONCLUSION: The results of this study showed that higher bond strengths are obtained when treating dentin with a lower concentration of CPN. Further evaluation to optimize conditions such as the application time and rinsing time are required.

Protective effects of platinum nanoparticles against UV-light-induced epidermal inflammation.

Intracellular reactive oxygen species (ROS) and apoptosis play important roles in the ultraviolet (UV)-induced inflammatory responses in the skin. Metal nanoparticles have been developed to increase the catalytic activity of metals, which is because of the large surface area of smaller particles. Platinum nanoparticles (nano-Pt) protected by poly acrylic acid were manufactured by reduction with ethanol. A marked increase in ROS production was observed in UV-treated HaCaT keratinocytes cell lines, while a decrease in ROS production was observed in nano-Pt-treated cells. Pretreatment of the cells with nano-Pt also caused a significant inhibition of UVB- and UVC-induced apoptosis. Furthermore, we found that mice treated with nano-Pt gel prior to UV irradiation showed significant inhibition of UVB-induced inflammation and UVA-induced photoallergy compared to UV-irradiated control mice. These results suggest that nano-Pt effectively protects against UV-induced inflammation by decreasing ROS production and inhibiting apoptosis in keratinocytes.

Platinum nanoparticle antioxidants inhibit pulmonary inflammation in mice exposed to cigarette smoke.

Recent evidence implicates increased oxidative stress as an important mechanism of the pulmonary inflammation that occurs in cigarette smokers. Since cigarette smoke (CS) contains and generates a large amount of reactive oxygen species (ROS) that elicit pulmonary inflammation, antioxidants may become effective therapeutic agents for CS-related inflammatory lung diseases, such as chronic obstructive pulmonary disease. Platinum nanoparticles stabilized with polyacrylate to form a stable colloid solution (PAA-Pt) are a new class of antioxidants that has been shown to efficiently quench ROS. In the present study we investigated the therapeutic effects of PAA-Pt on pulmonary inflammation in smoking mice. PAA-Pt or saline was administered intranasally to DBA/2 mice, which were then exposed to CS or control air daily for 3 days. Mice were sacrificed 4h after their final exposure to CS or control air. CS exposure caused depletion of antioxidant capacity, NFkappaB activation, and neutrophilic inflammation in the lungs of mice, and intranasal administration of PAA-Pt prior to CS exposure was found to inhibit these changes. Intranasal administration of PAA-Pt alone did not elicit pulmonary inflammation or toxicity. In in vitro experiments, treatment of alveolar-type-II-like A549 cells with PAA-Pt inhibited cell death after exposure to a CS extract. These results suggest that platinum nanoparticles act as antioxidants that inhibit pulmonary inflammation induced by acute cigarette smoking.

In vitro free radical scavenging activity of platinum nanoparticles.

A polyacrylic acid (PAA)-protected platinum nanoparticle species (PAA-Pt) was prepared by alcohol reduction of hexachloroplatinate. The PAA-Pt nanoparticles were well dispersed and homogeneous in size with an average diameter of 2.0 +/- 0.4 nm (n = 200). We used electron spin resonance to quantify the residual peroxyl radical ([Formula: see text]) generated from 2,2-azobis (2-aminopropane) dihydrochloride (AAPH) by thermal decomposition in the presence of O(2) and a spectrophotometric method to quantify the residual 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. PAA-Pt scavenged these two radicals in a dose-dependent manner. Platinum was the functional component. PAA-Pt reduced the rate of oxygen consumption required for linoleic acid peroxidation initiated by [Formula: see text] generated from AAPH, indicating inhibition of the propagation of linolate peroxidation. A thiobarbituric acid test also revealed dose-dependent inhibition of the linolate peroxidation by PAA-Pt. Fifty micromolar platinum, as PAA-Pt, completely quenched 250 microM DPPH radical for 5 min. Even when twice diluted in half, the PAA-Pt still quenched 100% of the 250 microM DPPH radical. The scavenging activity of PAA-Pt is durable. These observations suggest that PAA-Pt is an efficient scavenger of free radicals.

Improved bond performance of a dental adhesive system using nano-technology.

Since adhesive technology was introduced into dental field, metal-based restoration has been gradually replaced by metal-free restoration. Using the adhesive technology, minimum invasive technique has been possible in daily clinical practice as well as esthetic tooth-colored restorations have become very popular all over the world.One of the current issues of the dental adhesive is durability of bond between tooth structure and adhesive resin. Several approaches to overcome the issues have been carried out. Self-etching approach is believed to create durable bond because demineralization of superficial tooth surface is very shallow. Other approach is to utilize the inhibitor of enzymes which are suggested to catalyze the decomposition of resin composites and are always secreted within the oral environment.In the present study, Colloidal Platinum Nanoparticles (CPN) was applied before the application of 4-META/MMA-TBB resin cement as the third possibility to prolong the durability of bond. This implies that the use of the CPN solution would create higher conversion at the interface compared with conventional bonding procedures.

Effects of a potent antioxidant, platinum nanoparticle, on the lifespan of Caenorhabditis elegans.

We have shown that platinum nanoparticles (nano-Pt) are a superoxide dismutase (SOD)/catalase mimetic. Various data have shown extension of the Caenorhabditis elegans lifespan by antioxidant treatment. The present study was designed to elucidate the survival benefit conferred by nano-Pt, as compared to the well-known SOD/catalase mimetic EUK-8. At 0.5mM, nano-Pt significantly extended the lifespan of wild-type N2 nematodes and at 0.25 and 0.5mM, nano-Pt recovered the shortened lifespan of the mev-1(kn1) mutant, which is due to excessive oxidative stress. In both instances, EUK-8 at 0.05, 0.5, and 5mM did not extend nematode lifespan. Even when 0.4M paraquat was loaded exogenously, nano-Pt (0.1 and 0.5mM) and EUK-8 (0.5 and 5mM) were effective in rescuing worms. Moreover, 0.5mM nano-Pt significantly reduced the accumulation of lipofuscin and ROS induced by paraquat. We measured the in vitro dose-dependent quenching of O(2)(-) and H(2)O(2), indicating that nano-Pt is a more potent SOD/catalase mimetic than EUK-8. Nano-Pt prolonged the worm lifespan, regardless of thermotolerance or dietary restriction. Taken together, nano-Pt has interesting anti-ageing properties.

Apoptosis triggered by phagocytosis-related oxidative stress through FLIPS down-regulation and JNK activation.

Tumor necrosis factor-alpha (TNF-alpha)-activated neutrophils phagocytose and eliminate bacteria by using such oxidants as hydrogen peroxide (H(2)O(2)) and hypochlorous acid (HOCl), which is produced from H(2)O(2) by myeloperoxidase (MPO). Thereafter, neutrophils eventually undergo apoptosis to prevent excessive inflammation. However, it is unclear how this process is regulated. Here, we show that cotreatment of TNF-alpha-resistant neutrophilic HL-60 cells with taurine chloramine (TauCl), a detoxified form of HOCl, and TNF-alpha renders them susceptible to apoptosis, mostly by preventing nuclear factor-kappaB (NF-kappaB) activation. Of several NF-kappaB target genes tested, FLICE inhibitory protein short form (FLIP(S)) was specifically down-regulated by TauCl. TNF-alpha/TauCl cotreatment-induced apoptosis was largely blocked by stable expression of FLIP(S). Cotreatment with TNF-alpha and H(2)O(2) promoted apoptotic signaling via MPO activation and subsequent attenuation of FLIP(S) expression. TNF-alpha priming with H(2)O(2) or bacteria caused MPO-dependent apoptosis in human neutrophils. However, FLIP(S) knock-down by siRNA did not affect the viability of cells treated with TNF-alpha, implying that TauCl may affect another pathway in TNF-alpha-driven apoptosis. Indeed, oxidization of thioredoxin-1 (Trx-1) by TauCl induced the activation of apoptosis signal-regulating kinase 1 (ASK1) and cJun N-terminal kinase (JNK), thereby triggering TNF-alpha-mediated apoptosis. Taken together, these results indicate that the antiapoptotic signaling induced by TNF-alpha via NF-kappaB activation can be altered to promote apoptosis via H(2)O(2)-MPO-mediated FLIP(S) down-regulation and JNK activation.

Platinum nanoparticles have an activity similar to mitochondrial NADH:ubiquinone oxidoreductase.

This study was designed to examine if platinum nanoparticles have an activity similar to mitochondrial complex I, NADH:ubiquinone oxidoreductase. Platinum nanoparticles were prepared by a citrate reduction of H(2)PtCl(6) and protected by citrate itself and pectin (CP-Pt). Time- and dose-dependent decreases in NADH and a time-dependent increase in NAD(+) were observed in the presence of 50 microM CP-Pt; these observations were made using a spectrophotometric method in which the maximum absorption spectra at 340 and 260 nm were used for NADH and NAD(+), respectively. The required platinum concentration in CP-Pt to achieve a 50% oxidation of NADH for 3h was approximately 20 microM, and this NADH oxidation did not require oxygen as an electron acceptor. We also verified NAD(+) formation using an NAD(+)/NADH quantification kit. The absorption peak shift from 278 to 284 nm of 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (CoQ(1)) was observed by incubating CoQ(1) with CP-Pt in an aqueous buffer. A further analysis with HPLC revealed the reduction of CoQ(1) to CoQ(1)H(2) by CP-Pt. As a whole, platinum nanoparticles have an NADH:ubiquinone oxidoreductase-like activity. This suggests that platinum nanoparticles are a potential medicinal substance for oxidative stress diseases with suppressed mitochondrial complex I.

Platinum nanoparticle is a useful scavenger of superoxide anion and hydrogen peroxide.

Bimetallic nanoparticles consisting of gold and platinum were prepared by a citrate reduction method and complementarily stabilized with pectin (CP-Au/Pt). The percent mole ratio of platinum was varied from 0 to 100%. The CP-Au/Pt were alloy-structured. They were well dispersed in water. The average diameter of platinum nanoparticles (CP-Pt) was 4.7 +/- 1.5 nm. Hydrogen peroxide (H(2)O(2)) was quenched by CP-Au/Pt consisting of more than 50% platinum whereas superoxide anion radical (O(2)(-)) was quenched by any CP-Au/Pt. The CP-Au/Pt quenched these two reactive oxygen species in dose-dependent manners. The CP-Pt is the strongest quencher. The CP-Pt decomposed H(2)O(2) and consequently generated O(2) like catalase. The CP-Pt actually quenched O(2)(-) which was verified by a superoxide dismutase (SOD) assay kit. This quenching activity against O(2)(-) persisted like SOD. Taken together, CP-Pt may be a SOD/catalase mimetic which is useful for medical treatment of oxidative stress diseases.

Effect of platinum nanoparticles on cell death induced by ultrasound in human lymphoma U937 cells.

In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz ultrasound (US) at an intensity of 0.4 W/cm(2), 10% duty factor, 100 Hz PRF, for 2 min. Apoptosis induction was assessed by DNA fragmentation assay, cell cycle analysis and Annexin V-FITC/PI staining. Cell killing was confirmed by cell counting and microscopic examination. The mitochondrial and Ca(2+)-dependent pathways were investigated. Caspase-8 expression and autophagy-related proteins were detected by spectrophotometry and western blot analysis, respectively. Intracellular reactive oxygen species (ROS) elevation was detected by flow cytometry, while extracellular free radical formation was assessed by electron paramagnetic resonance spin trapping spectrometry. The results showed that Pt-NPs exerted differential effects depending on their internalization. Pt-NPs functioned as potent free radical scavengers when added immediately before sonication while pre-treatment with Pt-NPs suppressed the induction of apoptosis as well as autophagy (AP), and resulted in enhanced cell killing. Dead cells displayed the features of pyknosis. The exact mode of cell death is still unclear. In conclusion, the results indicate that US-induced AP may contribute to cell survival post sonication. To our knowledge this is the first study to discuss autophagy as a pro-survival pathway in the context of US. The combination of Pt-NPs and US might be effective in cancer eradication.

Nonrenewal of neurons in the cerebral neocortex of adult macaque monkeys.

The concept that, after developmental periods, neocortical neurons become numerically stable and are normally nonrenewable has been challenged by a report of continuous neurogenesis in the association areas of the cerebral cortex in the adult Macaque monkey. Therefore, we have reexamined this issue in two different Macaque species using the thymidine analog bromodeoxyuridine (BrdU) as an indicator of DNA replication during cell division. We found several BrdU+/NeuN+ (neuronal nuclei) double-labeled cells, but cortical neurons, distinguished readily by their size and cytological and immunohistochemical properties, were not BrdU positive. We examined in detail the frontal cortex, where it is claimed that the largest daily addition of neurons has been made, but did not see migratory streams or any sign of addition of new neurons. Thus, we concluded that, in the normal condition, cortical neurons of adult primates, similar to other mammalian species, are neither supplemented nor renewable.

Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus.

Neurogenesis in the dentate gyrus of the adult mammalian hippocampus has been proven in a series of studies, but the differentiation process toward newborn neurons is still unclear. In addition to the immunohistochemical study, electrophysiological membrane recordings of precursor cells could provide an alternative view to address this differentiation process. In this study, we performed green fluorescent protein (GFP)-guided selective recordings of nestin-positive progenitor cells in adult dentate gyrus by means of nestin-promoter GFP transgenic mice, because nestin is a typical marker for precursor cells in the adult dentate gyrus. The patch-clamp recordings clearly demonstrated the presence of two distinct subpopulations (type I and type II) of nestin-positive cells. Type I cells had a lower input resistance value of 77.1 M(Omega) (geometric mean), and their radial processes were stained with anti-glial fibrillary acidic protein antibody. On the other hand, type II nestin-positive cells had a higher input resistance value of 2110 MOmega and expressed voltage-dependent sodium current. In most cases, type II cells were stained with anti-polysialylated neural cell adhesion molecule. Taken together with a bromodeoxyuridine pulse-chase analysis, our results may reflect a rapid and dynamic cell conversion of nestin-positive progenitor, from type I to type II, at an early stage of adult neurogenesis in the dentate gyrus.

The natural phytochemical dehydroabietic acid is an anti-aging reagent that mediates the direct activation of SIRT1.

Dehydroabietic acid (DAA) is a naturally occurring diterpene resin acid of confers, such as pinus species (P. densiflora, P. sylvestris) and grand fir (Abies grandis), and it induces various biological actions including antimicrobial, antiulcer, and cardiovascular activities. The cellular targets that mediate these actions are largely unknown yet. In this report, we suggest that DAA is an anti-aging reagent. DAA has lifespan extension effects in Caenorhabditis elegans, prevents lipofuscin accumulation, and prevents collagen secretion in human dermal fibroblasts. We found that these anti-aging effects are primarily mediated by SIRT1 activation. Lifespan extension effects by DAA were ameliorated in sir-2.1 mutants and SIRT1 protein expression was increased, resulting in the deacetylation of SIRT1 target protein PGC-1α. Moreover, DAA binds directly to the SIRT1 protein independent of the SIRT1 substrate NAD(+) levels. Through a molecular docking study, we also propose a binding model for DAA-SIRT1. Taken together, our results demonstrate that the anti-aging effects are the first identified biological property of DAA and that the direct activation of SIRT1 enzymatic activity suggests the potential use of this natural diterpene, or related compounds, in age-related diseases or as a preventive reagent against the aging process.

Osmosensitive taurine transporter expression and activity in human corneal epithelial cells.

PURPOSE: To characterize in SV40-immortalized human corneal epithelial cells (tHCEC) osmosensitive taurine transporter gene and protein expression as well as its functional activity. To evaluate whether medium supplementation with taurine improves cell viability during a hypertonic challenge. METHODS: tHCEC were preincubated for up to 48 hours in hypertonic DMEM medium (i.e., up to 500 mosmol/kg). Taurine uptake was monitored through measurements of intracellular [3H]taurine accumulation. Gene and protein expression was detected by Northern and Western blot analyses, respectively. An amino acid analyzer measured intracellular cold taurine content. The live/dead assay evaluated with confocal microscopy determined cell viability. RESULTS: Na+-dependent taurine uptake occurred in an isotonic (310 mosmol/kg) medium. The apparent Michaelis-Menten constant, K(t), for taurine was 4.6 micro M, and uptake increased as a function of exposure time and rises in osmolality. Exposure for 12 hours to a 450 mosmol/kg medium increased uptake by 4.1-fold. However, after 48 hours of exposure to this medium, taurine uptake returned to its isotonic level. With time, biphasic changes occurred in taurine transporter gene and protein expression and taurine transport activity as well as elevating intracellular taurine content by 4.5-fold. Taurine medium supplementation for 48 hours improved cell viability. CONCLUSIONS: tHCEC express Na+-dependent osmosensitive taurine transport activity. The hypertonic-induced biphasic effects on gene and protein expression as well as transport activity suggest feedback regulation of these responses. Rises in intracellular taurine do not appear to be essential for osmoregulation, but instead enhance cell survival perhaps through a membrane stabilizer or an antioxidant effect.

Suppression of the TNFalpha-induced increase in IL-1alpha expression by hypochlorite in human corneal epithelial cells.

PURPOSE: In response to injury, activated neutrophils release tumor necrosis factor (TNF)-alpha and myeloperoxidase (MPO). TNFalpha in turn causes human corneal epithelial cells to secrete interleukin (IL)-1alpha, whereas MPO results in formation of HClO/OCl(-). The effect of HClO/OCl(-) on the expression of the IL-1alpha gene and protein is unknown. The current study was undertaken to examine in immortalized human corneal epithelial cells whether NaOCl alters TNFalpha-induced increases in expression of IL-1alpha gene and protein. METHODS: Semiquantitative RT-PCR and ELISA characterized IL-1alpha gene and protein expression, respectively. TNFalpha-induced nuclear transfer of nuclear factor (NF)-kappaB was measured by electrophoretic mobility shift assay (EMSA). The alpha isoform of inhibitory protein kappaB (IkappaBalpha) was identified by Western blot analysis. RESULTS: Exposure to NaOCl (0.75 mM) for 10 minutes caused suppression of TNFalpha-induced increases in IL-1alpha mRNA and protein, declines in NFkappaB nuclear transfer, and a modification of IkappaBalpha, based on a bandshift detected by Western blot analysis. Modified IkappaBalpha became resistant to TNFalpha-induced proteolysis. Methionine sulfoxide reductase A (MsrA, 10 micro M) eliminated the NaOCl-induced IkappaBalpha bandshift. CONCLUSIONS; NaOCl oxidizes IkappaBalpha at methionine residues and thereby suppresses dissociation of IkappaBalpha from NFkappaB. Decreased dissociation could in turn suppress TNFalpha-induced activation of NFkappaB, resulting in declines in expression of IL-1alpha gene and protein. These effects suggest that release of HClO/OCl(-) in vivo by activated neutrophils may counterbalance TNFalpha-induced NFkappaB-dependent secretion if IL-1alpha and suppress an excessive inflammatory reaction.

A glycine receptor antagonist, strychnine, blocked NMDA receptor activation in the neonatal mouse neocortex.

The NMDA receptor (NMDAR) is a Ca (2+)-permeable cation channel that plays a critical role in neural network formation during brain development. Since it is blocked in a voltage-dependent manner by extracellular Mg(2+), in order for the NMDA to be activated, the membrane must be strongly depolarized. Immature neurons in the developing neocortex can be depolarized by ligand-gated Cl(-) channels, such as the glycine receptor (GlyR) or GABA(A) receptor (GABA(A) R). We here assess the contribution of GlyRs to Ca(2+) influx via NMDARs in neonatal mouse cortical neurons. The GlyR antagonist, strychnine, was more effective in suppressing postsynaptic Ca(2+) influx than the GABA(A) R antagonist, picrotoxin, suggesting greater potentiation of NMDARs by GlyRs than by GABA(A) Rs. The GlyR, known to be endogenously activated at this stage, may play a critical role in neocortical development.