Curcumin-Induced DNA Damage and Inhibited DNA Repair Genes Expressions in Mouse–Rat Hybrid Retina Ganglion Cells (N18)

Curcumin is reported to be a potent inhibitor of the initiation and promotion of many cancer cells. We investigated to examine whether or not curcumin induce DNA damage in mouse–rat hybrid retina ganglion cell line N18 cells. The Comet assay showed that incubation of N18 cells with 10, 25 and 30 μM of curcumin led to a longer DNA migration smear (Comet tail). The DNA gel electrophoresis showed that 20 μM of curcumin for 24 and 48 h treatment induced DNA damage and fragments in N18 cells. The real time PCR analysis showed that 20 μM of curcumin for 48 h treatment decreased ATM, ATR, BRCA1, 14-3-3σ, DNA-PK and MGMT mRNA, and ATM and MGMT mRNA expression were inhibited in a time-dependent manner. Our results indicate that curcumin caused DNA damage and inhibited DNA repair genes which may be the factors for curcumin-inhibited cell growth. H.-F. Lu and J.-S. Yang are contributed equally to this study.     

Production and characterization of soluble human lysosomal enzyme α-iduronidase with high activity from culture media of transgenic tobacco BY-2 cells

Lysosomal storage diseases (LSDs), that collectively represent over 50 disorders, are amenable to enzyme replacement therapies. However, the current methods used to commercially produce recombinant lysosomal enzymes for this purpose, most commonly Chinese Hamster Ovary cells and human fibroblasts, are prohibitively costly. Plant bioreactors hold great promise for economic production of functional human α-l-iduronidase (hIDUA; glycosaminoglycan α-l-iduronohydrolase; EC 3.2.1.76), the enzyme deficient in the human LSD, Mucopolysaccharidosis I. We have developed and tested an expression system using transgenic tobacco BY-2 cells to produce high amounts of active hIDUA. A plant signal peptide was essential for proper expression and secretion of the 78 kDa glycosylated hIDUA into the cultured media of transgenic BY-2 cells. The yield and activity of the secreted hIDUA from long-term cultures of transgenic BY-2 cell lines were as high as 10 μg/mL media and 53,000 pmol/min/mg proteins, respectively. Thus, this transgenic BY-2 cell line presents an attractive platform for economic production and easy downstream purification of hIDUA for enzyme replacement therapy. Furthermore, this system can be used for the production and purification of other human lysosomal enzymes or pharmaceuticals.     

Biological nitrification–denitrification with alternating oxic and anoxic operations using aerobic granules

Efficient nitrification and denitrification of wastewater containing 1,700 mgl⁻¹ of ammonium-nitrogen was achieved using aerobic granular sludge cultivated at medium-to-high organic loading rates. The cultivated granules were tested in a sequencing batch reactor (SBR) fed with 6.4 or 10.2 kg NH₄⁺-N m⁻³ day⁻¹, a loading significantly higher than that reported in literature. With alternating 2 h oxic and 2 h anoxic operation (OA) modes, removal rate was 45.5 mg NH₄⁺-N g⁻¹ volatile suspended solids⁻¹ h⁻¹ at 6.4 kg NH₄⁺-N m⁻³ day⁻¹ loading and 41.3 ± 2.0 at 10.2 kg NH₄⁺-N m⁻³ day⁻¹ loading. Following the 60 days SBR test, granules were intact. The fluorescence in situ hybridization and confocal laser scanning microscopy results indicate that the SBR-OA granules have a distribution with nitrifers outside and heterotrophs outside that can effectively expose functional strains to surrounding substrates at high concentrations with minimal mass transfer limit. This microbial alignment combined with the smooth granule surface achieved nitrification–denitrification of wastewaters containing high-strength ammonium using aerobic granules. Conversely, the SBR continuous aeration mode yielded a distribution with nitrifers outside and heterotrophs inside with an unsatisfactory denitrification rate and floating granules as gas likely accumulated deep in the granules.     

Expression of NAD<Superscript>+</Superscript>-dependent formate dehydrogenase in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> and its involvement in anaerobic metabolism and H<Subscript>2</Subscript> production

An expression system for NAD⁺-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H₂ yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H₂ production.     

Ca regulation of connexin 43 hemichannels in C6 glioma and glial cells

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca²⁺ ([Ca²⁺]_i) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca²⁺]_i was in the 500 nM range but the responses disappeared with larger [Ca²⁺]_i transients. Ca²⁺-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca²⁺-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca²⁺]_i triggers hemichannel opening, not by a direct action of Ca²⁺ on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca²⁺]_i and acting to restrain cellular ATP loss.     

Stratigraphy and palaeoenvironmental implications of Pleistocene and Holocene aeolian sediments in the Lhasa area,southern Tibet (China)

Along the middle and lower reaches of the Kyichu River and its tributaries (Lhasa area, southern Tibet), a multidisciplinary study was carried out in order to investigate the areal distribution, sedimentological properties, ages and palaeoenvironmental implications of aeolian deposits including intercalated palaeosols. This research was initiated to investigate to what extent southern Tibet is influenced by past human activity, as even recent evaluations perceive the present treeless desertic environment as natural. Fifteen profiles were recorded at an altitude of 3540–4580 m a.s.l. with subsequent sedimentological, geochronological (OSL, AMS ¹⁴C) and palaeobotanical (charcoal) analyses. Sediment properties of both loesses and aeolian sands reveal an origin from aeolian sorting of nearby fluvial deposits. The calculated ages are the oldest obtained thus far on aeolian sediments from southern and interior Tibet, revealing natural aeolian sedimentation before and around the Last Glacial Maximum (c. 20 ka). However, a distinct portion of Late Holocene sandy aeolian sediments also occurs. Both the evidence for the aeolian dynamics (widespread Pleistocene loess and aeolian sand deposition, local Late Holocene aeolian sand deposition, modern reactivation of widespread Pleistocene aeolian sands) and the palaeobotanical findings (Late Holocene vegetation change from a tree-bearing to a widely treeless landscape) provide evidence that the Lhasa area was strongly influenced by human activity since at least the Late Neolithic (c. 4200 cal yrs BP). Thus the present-day desertic environment might not primarily be a result of the semiarid climate or the high-altitude conditions, but rather of activities of the humans and their collateral effects. However, once established, this semi-natural ecosystem persisted, controlled by strong grazing, firewood extraction, erosion and harsh edaphic conditions, preventing the recovery of trees.     

Involvement of Matrix Metalloproteinases on the Inhibition of Cells Invasion and Migration by Emodin in Human Neuroblastoma SH-SY5Y Cells

Emodin (1,3,8-trihydroxy-6-methylanthaquinone), an active component present in the root and rhizome of Rheum palmatum L. (Polygonaceae) has anti-bacterial, anti-tumor, diuretic and vasorelaxant effects. However, its mechanism of action on the cell migration and invasion of human neuroblastoma cancer SH-SY5Y cells is not fully understood. In this study, firstly, the effects of emodin on the percentage of viable cells were examined by using MTT assay and it was found that emodin induced dose-and time-dependent inhibition in human neuroblastoma SH-SY5Y cells. Second, the effects of emodin on the migration and invasion of SH-SY5Y cells were examined by using wound assay and matrigel counting and the results showed that emodin suppressed the migration and invasion of SH-SY5Y cells. Third, we examined the effect of emodin on the levels of associated proteins by using Western blotting and the results indicated that emodin inhibited the levels of GRB2, RhoA, HIF-1α, VEGF, FAK, iNOS, COX2, p-p38, p-c-jun, MMP2, MMP9 and MMP7 but promoted the levels of PKC, PI3K, MEKK3 and NF-κB p65 that led to the inhibition of migration and invasion of SH-SY5Y cells in vitro.