Our previous studies reported that methanol extract of Sanguisorbae radix from Sanguisorba officinalis L. (Rosaceae) prevented neuronal cell damage induced by Aβ (25-35) in vitro. The present study was carried out to investigate the effect of gallic acid isolated from Sanguisorbae radix on Aβ (25-35)-induced neurotoxicity using cultured rat cortical neurons. Gallic acid (0.1, 1 μM) showed a concentration-dependent inhibition on Aβ (25-35) (10 μM)-induced apoptotic neuronal death, as assessed by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Pretreatment of gallic acid inhibited 10 μM Aβ (25-35)-induced elevation of cytosolic Ca2+ concentration ([Ca2+]c) and generation of reactive oxygen species (ROS), which were measured by fluorescent dyes. Gallic acid also inhibited glutamate release into medium induced by 10 μM Aβ (25-35), which was measured by HPLC. These results suggest that gallic acid prevents Aβ (25-35)-induced apoptotic neuronal death by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release and generation of ROS, and that these effects of gallic acid may be partly associated with the neuroprotective effect of Sanguisorbae radix.
(Group B streptococcus, GBS) is an important pathogen affecting humans and animals as well as aquatic species. Between April 2003 and March 2006, bacteria were isolated from infected red tilapia sp. and Nile tilapia cultured in Thailand. They were identified as GBS by API 20 STREP, polymerase chain reaction (PCR) and multiplex PCR-based reverse line blot hybridization (mPCR/RLB). Genotyping indicated that they belonged to two genotypes. One of these genotypes has not been identified in any of 1792 GBS isolates genotyped by mPCR/RLB; it belongs to serotype Ia and contains genes encoding proteins Cα ( ) and Cβ ( ), three insertions sequences (IS , IS and IS ) and the group II intron GBSi1. The other has recently been identified among invasive human isolates in Hong Kong; it belongs to an uncommon subtype of serotype III (III-4), contains , three insertion sequences (IS , IS and IS ) and a tetracycline resistance gene ( M). Pathogenicity studies in red tilapia by experimental infection established that GBS is a pathogen of fish. Histopathological changes indicated that infection of tilapia with GBS produced disease with systemic involvement characterized by multiple necrotic foci in various tissues. The possible epidemiology and epizoology of these bacteria among human, mammal and aquatic species are discussed in this report.
Microglia are the main immunocompetent cells of the mammalian central nervous system (CNS). Activation of cultured microglial cells and subsequent release of nitric oxide and cytokines critically depends on intracellular calcium levels. Since microglia undergo dramatic morphological, biochemical and electrophysiological changes in response to pathological events in the CNS, we investigated temporal changes in expression levels of ion channels involved in cellular calcium homeostasis in mouse cortical microglial cells in culture. Specifically, we assessed the inward and delayed outward rectifier potassium currents ( I IRK and I DRK ), calcium (Ca 2+ ) release-activated Ca 2+ currents ( I CRAC ) and Ca 2+ -activated TRPM4-like currents ( I CAN ) in non-activated microglia and cells that were activated by exposure to lipopolysaccharide (LPS) between 3 and 48 h. Unstimulated microglial cells, subcultured from an astrocyte coculture, typically exhibited a ramified, rod-shaped morphology. During the first 3 days of culture cell size and shape were maintained, but the percentage of cells showing prominent I IRK went up and those expressing I DRK went down. Cells retaining I DRK exhibited smaller amplitudes, whereas those of I IRK and I CRAC were not affected. However, after 24 h of exposure to 1 Î¼g ml â1 LPS, most cells showed an amoeboid (âfried eggâ-shaped) morphology with a 62% increase in cell capacitance. At that point in time, only 14% of the cells revealed I IRK and 3% had I DRK exclusively, whereas the majority of cells expressed both currents. The amplitudes of I CRAC and I IRK progressively decreased after stimulation, whereas I DRK transiently reached a maximum after 6 h of LPS exposure and then returned to pre-stimulation expression levels. Cultured microglia also revealed TRPM4-like, Ca 2+ -activated non-selective currents ( I CAN ) with an EC 50 of 1.2 Î¼ m [Ca 2+ ] i . The expression levels of this current did not change significantly during and after 24 h of LPS exposure. We propose that LPS-induced down-regulation of I IRK and I CRAC will reduce the cell's capacity to produce significant calcium influx upon receptor activation and result in decreased sensitivity to exogenous stimulation. In this scenario, I CAN expression would remain constant, although its activity would automatically be reduced due to the diminished calcium influx capacity of the cell.
It has been suggested that accumulation of beta-amyloid (A beta) peptide triggers neurodegeneration, at least in part, via glutamate-mediated excitotoxicity in Alzheimer's disease (AD) brain. This is supported by observations that toxicity induced by A beta peptide in cultured neurons and in adult rat brain is known to be mediated by activation of glutamatergic N-methyl-d-aspartate (NMDA) receptors. Additionally, recent clinical studies have shown that memantine, a noncompetitive NMDA receptor antagonist, can significantly improve cognitive functions in some AD patients. However, very little is currently known about the potential role of memantine against A beta-induced toxicity. In the present study, we have shown that A beta(1-42)-induced toxicity in rat primary cortical cultured neurons is accompanied by increased extracellular and decreased intracellular glutamate levels. We subsequently demonstrated that A beta toxicity is induced by increased phosphorylation of tau protein and activation of tau kinases, i.e. glycogen synthase kinase-3 beta and extracellular signal-related kinase 1/2. Additionally, A beta treatment induced cleavage of caspase-3 and decreased phosphorylation of cyclic AMP response element binding protein, which are critical in determining survival of neurons. Memantine treatment significantly protected cultured neurons against A beta-induced toxicity by attenuating tau-phosphorylation and its associated signaling mechanisms. However, this drug did not alter either conformation or internalization of A beta(1-42) and it was unable to attenuate A beta-induced potentiation of extracellular glutamate levels. These results, taken together, provide new insights into the possible neuroprotective action of memantine in AD pathology.
Long ago, I set out to solve a problem, but something happened along the way: I was diverted by an unexpected observation. Thereafter, the direction of my research was guided at each stage by increasing familiarity with the experimental material and what could be done with it. The result was the birth of therapy with cultured keratinocytes. Subsequent developments soon led to the formation of the company Biosurface Technology (later taken over by the Genzyme Corporation), which provided autologous cultures for burn victims in many parts of the world. Further progress by others led to new therapeutic applications of cultured keratinocytes, such as treatment of an ocular disease and gene therapy. Unfortunately, there have developed serious regulatory problems that are a danger to future progress. As described in this brief history, the initial stages of development of cell therapy for the treatment of human disease were possible only because there was no restraint by committees or governmental regulations.
Abstract As materials are produced at smaller scales, the properties that make them especially useful for biological applications such as drug delivery, imaging or sensing applications also render them potentially harmful. There has been a reasonable amount of work addressing the interactions of biological fluids at material surfaces that demonstrates the high affinity of protein for particle surfaces and some looking at the role of particle surface chemistry in cellular associations, but mechanisms have been too little addressed outside the context of intended, specific interactions. Here, using cultured endothelium as a model for vascular transport, we demonstrate that the capacity of nanoparticle surfaces to adsorb protein is indicative of their tendency to associate with cells. Quantification of adsorbed protein shows that high binding nanoparticles are maximally coated in seconds to minutes, indicating that proteins on particle surfaces can mediate cell association over much longer time scales. We also remove many of the most abundant proteins from culture media which alters the profile of adsorbed proteins on nanoparticles but does not affect the level of cell association. We therefore conclude that cellular association is not dependent on the identity of adsorbed proteins and therefore unlikely to require specific binding to any particular cellular receptors.
Maturation of neuronal synapses is thought to involve mitochondria. Bcl-x L protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-x L is abundant. Here, we report that overexpression of Bcl-x L postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-x L , overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-x L or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-x L appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-x L coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-x L protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-x L positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation. Bcl-2 synaptic transmission mitochondria cell death ABT-737
As the applications of industrial nanoparticles are being developed, the concerns on the environmental health are increasing. Cytotoxicities of titanium dioxide nanoparticles of different concentrations (5, 10, 20 and 40 μg/ml) were evaluated in this study using a cultured human bronchial epithelial cell line, BEAS-2B. Exposure of the cultured cells to nanoparticles led to cell death, reactive oxygen species (ROS) increase, reduced glutathione (GSH) decrease, and the induction of oxidative stress-related genes such as heme oxygenase-1, thioredoxin reductase, glutathione- -transferase, catalase, and a hypoxia inducible gene. The ROS increase by titanium dioxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that titanium dioxide nanoparticles exert cytotoxicity by an apoptotic process. Furthermore, the expressions of inflammation-related genes such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), TNF-a, and C-X-C motif ligand 2 (CXCL2) were also elevated. The induction of IL-8 by titanium dioxide nanoparticles was inhibited by the pre-treatment with SB203580 and PD98059, which means that the IL-8 was induced through p38 mitogen-acitvated protein kinase (MAPK) pathway and/or extracellular signal (ERK) pathway. Uptake of the nanoparticles into the cultured cells was observed and titanium dioxide nanoparticles seemed to penetrate into the cytoplasm and locate in the peri-region of the nucleus as aggregated particles, which may induce direct interactions between the particles and cellular molecules, to cause adverse biological responses.
Melanin synthesis is an oxygen-dependent process that acts as a potential source of reactive oxygen species (ROS) inside pigment-forming cells. The synthesis of the lighter variant of melanin, pheomelanin, consumes cysteine and this may limit the capacity of the cellular antioxidative defense. We show that tyrosine-induced melanogenesis in cultured normal human melanocytes (NHM) is accompanied by increased production of ROS and decreased concentration of intracellular glutathione. Clinical atypical (dysplastic) nevi (DN) regularly contain more melanin than do normal melanocytes (MC). We also show that in these cultured DN cells three out of four exhibit elevated synthesis of pheomelanin and this is accompanied by their early senescence. By using various redox-sensitive molecular probes, we demonstrate that cultured DN cells produce significantly more ROS than do normal MC from the same donor. Our experiments employing single-cell gel electrophoresis (comet assay) usually reveal higher fragmentation of DNA in DN cells than in normal MC. Even if in some cases the normal alkaline comet assay shows no differences in DNA fragmentation between DN cells and normal MC, the use of the comet assay with formamidopyrimidine DNA glycosylase can disclose that the DNA of the cultured DN cells harbor more oxidative damage than the DNA of normal MC from the same person