Toxicological evaluation of S. involucrata culture: Acute, 90-day subchronic and genotoxicity studies.

Saussurea involucrata is an endangered plant that is used in traditional Chinese medicine. Through the use of plant cell culture techniques, preparations of Saussurea involucrata (S. involucrata) cell cultures have been developed and used to generate medicinal preparations. There have been few evidence-based analyses of the toxicological effects of S. involucrata culture conducted to date. Here, we conducted the experiments designed to assess the acute, subchronic, and genotoxic toxicological effects of S. involucrata culture. The genotoxic study was assessed through Ames, marrow micronucleus, and sperm malformation assays. The acute toxicity was assessed by orally administering in rats and mice at dose of 7500 mg/kg. Subchronic toxicity studies were then conducted by administering rats at doses of 500, 1000, or 1500 mg/kg for 90 days. No genotoxicity was observed at any tested dose levels, nor was any evidence of acute toxicity detected in treated mice or rats. Similarly, subchronic study of S. involucrata culture administration was not associated with any changes in rat food intake, weight, hematological parameters, organ weight, or organ histology. Then, we determined that the no observed adverse effect level of S. involucrata culture was greater than 1500 mg/kg in our 90-day toxicity study.

Measuring sunscreen protection against solar-simulated radiation-induced structural radical damage to skin using ESR/spin trapping: development of an ex vivo test method.

The in vitro star system used for sunscreen UVA-testing is not an absolute measure of skin protection being a ratio of the total integrated UVA/UVB absorption. The in vivo persistent-pigment-darkening method requires human volunteers. We investigated the use of the ESR-detectable DMPO protein radical-adduct in solar-simulator-irradiated skin substitutes for sunscreen testing. Sunscreens SPF rated 20+ with UVA protection, reduced this adduct by 40-65% when applied at 2 mg/cm(2). SPF 15 Organic UVA-UVB (BMDBM-OMC) and TiO(2)-UVB filters and a novel UVA-TiO(2) filter reduced it by 21, 31 and 70% respectively. Conventional broad-spectrum sunscreens do not fully protect against protein radical-damage in skin due to possible visible-light contributions to damage or UVA-filter degradation. Anisotropic spectra of DMPO-trapped oxygen-centred radicals, proposed intermediates of lipid-oxidation, were detected in irradiated sunscreen and DMPO. Sunscreen protection might be improved by the consideration of visible-light protection and the design of filters to minimise radical leakage and lipid-oxidation.

Effect of surface properties on nanoparticle-cell interactions.

The interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. These applications require a firm control over nanoparticle-cell interactions, which are mainly dictated by surface properties of nanoparticles. This critical Review presents an understanding of how synthetic and natural chemical moieties on the nanoparticle surface (in addition to nanoparticle shape and size) impact their interaction with lipid bilayers and cells. Challenges for undertaking a systematic study to elucidate nanoparticle-cell interactions are also discussed.

Fluorescent detection of lipid droplets and associated proteins.

Most eukaryotic cells can store excess lipid in cytosolic lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets with nile red and BODIPY 493/503 are included. The differences in the spectral properties of these two lipophilic dyes and advantages of each are discussed. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology. Immunofluorescent detection of adipophilin, a broadly expressed, lipid droplet-associated protein, widely used as a marker for lipid droplet accumulation, is presented as an example. Finally, a simple protocol for enhancing lipid droplet accumulation through supplementation with excess fatty acid is included.

Antioxidant effect of zinc in humans.

Oxidative stress is known to be an important contributing factor in many chronic diseases. We tested the hypothesis that in healthy normal volunteers zinc acts as an effective anti-inflammatory and antioxidant agent. Ten normal volunteers were administered daily oral zinc supplementation (45 mg zinc as gluconate) and 10 volunteers received placebo for 8 weeks. Plasma zinc, MDA, HAE, and 8-OHdG levels; LPS-induced TNF-alpha and IL-1beta mRNA; and ex vivo TNF-alpha-induced NF-kappaB activity in mononuclear cells (MNC) were determined before and after supplementation. In subjects receiving zinc, plasma levels of lipid peroxidation products and DNA adducts were decreased, whereas no change was observed in the placebo group. LPS-stimulated MNC isolated from zinc-supplemented subjects showed reduced mRNA for TNF-alpha and IL-1beta compared to placebo. Ex vivo, zinc protected MNC from TNF-alpha-induced NF-kappaB activation. In parallel studies using HL-60, a promyelocytic cell line, we observed that zinc enhances the upregulation of mRNA and DNA-specific binding for A20, a transactivating factor which inhibits the activation of NF-kappaB. Our results suggest that zinc supplementation may lead to downregulation of the inflammatory cytokines through upregulation of the negative feedback loop A20 to inhibit induced NF-kappaB activation. Zinc administration to human subjects with conditions associated with increased oxidative stress should be explored.

[Study on anti-tumor activity of extracts from cultured cells of Taxus chinensis].

OBJECTIVE: To analyze the cytotoxicity and inhibitory effect of extracts from cultured cells(from stems) of Taxus chinensis on cancer cell line SMMC-7721. METHODS: MTT assay for cell viability and flow cytometry for cell cycle analysis. RESULTS: MTT results showed that the extact by dichloromethane was more effective than that by methanol on SMMC-7721, IC50 were 0.0834 mg DCW.ml-1 and 0.8002 mg DCW.ml-1 respectively. SMMC-7721 cells in G2-M stage increased with higher concentration of dichloromethane extracts from Taxus chinensis cells and longer incubation. CONCLUSION: Extracts from cultured cells of Taxus chinensis could have cytotoxic effect on SMMC-7721 and could induce apoptosis of cancer cells.

Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus.

We investigated the mechanism of the immunomodulatory action of polysaccharide (ASP) isolated from a cell culture of Acanthopanax senticosus. ASP was found to directly increase the proliferation and differentiation of B cells, and the cytokine production of macrophage, but not the proliferation and cytokine production of T cells. Since ASP cannot penetrate the cell membrane due to its large molecular mass, such cellular activation may be caused by the surface binding of ASP to receptors expressed on B cells and macrophages. The possibility that TLRs, which are known to be involved in immune-related responses, may be the receptor(s) of ASP was investigated. The immunomodulating activities of ASP on the B cells and macrophages of C3H/HeJ mice, expressing a defective toll-like receptor (TLR)-4, were decreased versus the corresponding cells from C3H/HeN mice. In addition, the activities of ASP on B cells and macrophages were significantly reduced by treating the cells with antibodies to TLR4 and TLR2 prior to ASP, suggesting that both of them are the possible receptors of ASP. The ligation of TLRs induced by ASP was able to activate mitogen-activated protein kinases (MAPKs), such as Erk1/2, p38 and JNK, and the transcription factor NF-kappaB. Although ASP was shown to activate the TLR signaling cascades in the same manner as lipopolysaccharide (LPS), these two could be differentiated by the finding that polymyxin B (PMB), a specific inhibitor of LPS, did not significantly affect the activities of ASP on B cells and macrophages. Taken together, our results demonstrate that ASP, isolated from a cell culture of A. senticosus, activates B cells and macrophages by interacting with TLRs and leading to the subsequent activation of mitogen-activated protein kinases and NF-kappaB.

The rat myosin myr 5 is a GTPase-activating protein for Rho in vivo: essential role of arginine 1695.

myr 5 is an unconventional myosin (class IX) from rat that contains a Rho-family GTPase-activating protein (GAP) domain. Herein we addressed the specificity of the myr 5 GAP activity, the molecular mechanism by which GAPs activate GTP hydrolysis, the consequences of myr 5 overexpression in living cells, and its subcellular localization. The myr 5 GAP activity exhibits a high specificity for Rho. To achieve similar rates of GTPase activation for RhoA, Cdc42Hs, and Rac1, a 100-fold or 1000-fold higher concentration of recombinant myr 5 GAP domain was needed for Cdc42Hs or Rac1, respectively, as compared with RhoA. Cell lysates from Sf9 insect cells infected with recombinant baculovirus encoding myr 5 exhibited increased GAP activity for RhoA but not for Cdc42Hs or Rac1. Analysis of Rho-family GAP domain sequences for conserved arginine residues that might contribute to accelerate GTP hydrolysis revealed a single conserved arginine residue. Mutation of the corresponding arginine residue in the myr 5 GAP domain to a methionine (M1695) virtually abolished Rho-GAP activity. Expression of myr 5 in Sf9 insect cells induced the formation of numerous long thin processes containing occasional varicosities. Such morphological changes were dependent on the myr 5 Rho-GAP activity, because they were induced by expression the myr 5 tail or just the myr 5 Rho-GAP domain but not by expressing the myr 5 myosin domain. Expression of myr 5 in mammalian normal rat kidney (NRK) or HtTA-1 HeLa cells induced a loss of actin stress fibers and focal contacts with concomitant morphological changes and rounding up of the cells. Similar morphological changes were observed in HtTA-1 HeLa cells expressing just the myr 5 Rho-GAP domain but not in cells expressing myr 5 M1695. These morphological changes induced by myr 5 were inhibited by coexpression of RhoV14, which is defective in GTP hydrolysis, but not by RhoI117. myr 5 was localized in dynamic regions of the cell periphery, in the perinuclear region in the Golgi area, along stress fibers, and in the cytosol. These results demonstrate that myr 5 has in vitro and in vivo Rho-GAP activity. No evidence for a Rho effector function of the myr 5 myosin domain was obtained.

Negative regulation of two hyperproliferative keratinocyte differentiation markers by a retinoic acid receptor-specific retinoid: insight into the mechanism of retinoid action in psoriasis.

Retinoids down-regulate the expression of metalloproteinases, cytokines, and other genes involved in cell proliferation and inflammation. Tazarotene (AGN 190168), a retinoic acid receptor (RAR)-specific retinoid, is effective in the treatment of psoriasis, a hyperproliferative and inflammatory skin disease. Because negative regulation of genes appears to be important in the antiproliferative and antiinflammatory action of retinoids, we studied the down-regulation of genes in skin raft cultures by this antipsoriatic retinoid. By subtraction hybridization, we found that migration inhibitory factor-related protein (MRP-8) and skin-derived anti-leukoproteinase (SKALP) are down-regulated by AGN 190168. MRP-8 and SKALP are overexpressed in psoriatic lesions as compared to the normal epidermis, and they are markers of hyperproliferative keratinocyte differentiation. We also show that MRP-8 expression is retinoid inhibitable in cultured keratinocytes induced to differentiate with 10% serum or IFN-gamma, and that MRP-8 is inhibited by RAR but not by retinoid X receptor-specific retinoids in a dose-dependent manner. Finally, MRP-8, SKALP, and the previously characterized differentiation marker, transglutaminase I, are all down-regulated in vivo in psoriatic lesions after treatment with AGN 190168 in comparison to placebo. Taken together, these data suggest that these markers may be down-regulated by tazarotene in psoriasis through direct action on keratinocyte gene expression rather than by an overall tazarotene effect on lesional therapeutic status.

Characterization of densin-180, a new brain-specific synaptic protein of the O-sialoglycoprotein family.

We purified an abundant protein of apparent molecular mass 180 kDa from the postsynaptic density fraction of rat forebrain and obtained amino acid sequences of three tryptic peptides generated from the protein. The sequences were used to design a strategy for cloning the cDNA encoding the protein by polymerase chain reaction. The open reading frame of the cDNA encodes a novel protein of predicted molecular mass 167 kDa. We have named the protein densin-180. Antibodies raised against the predicted amino and carboxyl sequences of densin-180 recognize a 180 kDa band on immunoblots that is enriched in the postsynaptic density fraction. Immunocytochemical localization of densin-180 in dissociated hippocampal neuronal cultures shows that the protein is highly concentrated at synapses along dendrites. The message encoding densin-180 is brain specific and is more abundant in forebrain than in cerebellum. The sequence of densin-180 contains 17 leucine-rich repeats, a sialomucin domain, an apparent transmembrane domain, and a PDZ domain. This arrangement of domains is similar to that of several adhesion molecules, in particular GPIbalpha, which mediates binding of platelets to von Willebrand factor. We propose that densin-180 participates in specific adhesion between presynaptic and postsynaptic membranes at glutamatergic synapses.

Angiotensin II regulation of intracellular calcium in astroglia cultured from rat hypothalamus and brainstem.

This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca2+]i) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1-100 nM) increased [Ca2+]i in both polygonal and stellate astroglia when measured using fura-2 imaging fluorescence microscopy. Ang II increased [Ca2+]i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrode's solution (containing 2 mM CaCl2), the Ang II-stimulated increase in [Ca2+]i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady-state plateau of free Ca2+. In both cell types, the selective Ang II type 1 receptor subtype (AT1) antagonist losartan (1 microM) inhibited the Ang II-stimulated increase in [Ca2+]i. The selective AT2 antagonist PD 123319 (1 microM) did not inhibit the Ang II-stimulated increase in [Ca2+]i in either cell type. To define the sources of Ca2+ that participate in the Ang II-stimulated increase in [Ca2+]i in astroglia, experiments were performed in a nominally Ca(2+)-free Tyrode's solution. In either cell type, this resulted in only an initial transient increase of Ca2+ and no sustained plateau of Ca2+ when challenged with Ang II. Thapsigargin (5 microM), cyclopiazonic acid (10 microM), and ryanodine (10 microM), but not caffeine (1-10 mM), inhibited the initial rise in [Ca2+]i. The plateau increase of [Ca2+]i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 microM) and nifedipine (10 microM); in contrast, gadolinium (100 microM) had no effect on the plateau increase of [Ca2+]i. These results indicate that Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i of polygonal and stellate astroglia.

Similarity between rat brain nicotinic alpha-bungarotoxin receptors and stably expressed alpha-bungarotoxin binding sites.

The present results demonstrate stable expression of alpha-bungarotoxin (alpha-BGT) binding sites by cells of the GH4C1 rat pituitary clonal line. Wild-type GH4C1 cells do not express alpha-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor alpha2, alpha3, alpha4, alpha5, alpha7, beta2, or beta3 subunits. However, GH4C1 cells stably transfected with rat nicotinic receptor alpha7 cDNA (alpha7/GH4C1 cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of teh predicted size. The alpha7/GH4C1 cells also express saturable, high-affinity binding sites for 125I-labeled alpha-BGT, with a KD of 0.4 nM and Bmax of 3.2 fmol/10(6) intact cells. 125I-alpha-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or alpha7/GH4C1 cells. Furthermore, KD and Ki values for 125I-alpha-BGT binding sites on intact alpha7/GH4C1 cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the alpha-BGT binding sites expressed in alpha7/GH4C1 cells was similar to that of the native brain alpha-BGT receptor. Chronic exposure of alpha7/GH4C1 cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of alpha-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of alpha-BGT binding sites in transfected alpha7/GH4C1 cells resemble those for brain nicotinic alpha-BGT receptors. If the heterologously expressed alpha-BGT binding sites in the present study are composed solely of alpha7 subunits, the results could suggest that the rat brain alpha-BGT receptor has a similar homooligomeric structure. Alternatively, if alpha-BGT binding sites exist as heterooligomers of alpha7 plus some other previously identified or novel subunit(s), the data would indicate that the alpha7 subunits play a major role in determining properties of the alpha-BGT receptor.

Effects of high levels of fluoride on bone formation: an in vitro model system.

In order to develop an in vitro model for the study of the effects of different agents on biomineralization, a three-dimensional cell culture system was investigated at different levels of fluoride. Rat fetal osteoblasts were seeded onto collagen discs and maintained in a culture medium for 40 days. Results showed that, at 40 days, the cultured matrices had a Ca:P ratio, mineral content and Fourier transform infrared (FTIR) spectrum that were close to those seen for normal rat bone. Viable cells, observed by light microscopy, were present in the matrix at 40 days. The formation of a mineralized matrix in this experimental set-up provided a model for exploring in vitro the effects of high levels of fluoride on bone. The fluoride content of the mineral formed in the cultures showed a dose-dependent increase in fluoride content with time. Also, an increase in the crystallinity of the apatite in the presence of fluoride, was observed by FTIR. The Ca:P ratio and percentage mineral by weight showed no apparent differences among the groups. The three-dimensional model used for this study has the potential to be a powerful tool in the study of time-dependent effects of drugs and other factors on osteoblast cell functions and subsequently on matrix mineralization.

[A decrease in the damaging action of gamma irradiation on human lymphocytes by postradiation treatment with the preparation MDI]. / Snizhenie povrezhdaiushchego deistviia gamma-oblucheniia na limfotsity cheloveka pri postluchevoi obrabotke preparatom MDI.

The effect of MDI, the agent of plant origin, on the cell cycle and the number of micronuclei in human lymphocytes after gamma-irradiation has been studied. It has been found that the treatment of lymphocytes with MDI stimulates DNA synthesis and reduces the delay of irradiated cells in (G2 + M) phase. Moreover post-irradiation cell treatment with MDI reduces the number of damaged cells.

[A comparative analysis of the proteins from a cell culture and from field plants of the ecdysteroid producer Serratula coronata L]. / Sravnitel'nyi analiz belkov kletochnoi kul'tury i polevykh rastenii produtsenta ékdisteroidov Serratula coronata L.

Differences in the composition and amount of proteins synthesized in the cell culture and leaves of field plants Serratula coronata have been shown. They proceed from differences in intensity of synthesis of secondary metabolites, ecdysteroids, whose content in the cell culture is considerably lower.

[The correlation between the acidic composition of diacylglycerol and protein kinase C activation in cultures of rat cardiomyocytes]. / Correlazione tra composizione acidica del diacilglicerolo e attivazione della proteina cinasi C in colture di cardiomiociti di ratto.

We have studied the fatty acid composition of the diacylglycerol produced after different stimulation times with an alpha 1-agonist (phenylephrine) in cultures of beating neonatal rat cardiomyocytes, and we have related the acidic pattern to the time course of the translocation of protein kinase C from cytosol to the membrane, both in control cells and in cells grown in a medium supplemented with docosahexaenoic acid. Gas chromatography of the diacylglycerol produced after stimulation revealed significant differences between control cells and cells grown in the docosahexaenoic acid supplemented medium. In the control cells, in the early stimulation times, the higher protein kinase C activity was due to a higher relative molar content of arachidonic acid in the diacylglycerol; in the docosahexaenoic acid treated cells the lower but more persistent activation of the membrane-bound protein kinase C might be sustained by an enrichment of diacylglycerol with docosahexaenoic acid. The modification of the fatty acid composition of diacylglycerol can cause an alteration in the response of the cells to alpha 1-adrenoceptor stimulation.