2,3,7,8-tetrachlorobenzo-p-dioxin inhibits proliferation of SK-N-SH human neuronal cells through decreased production of reactive oxygen species

Oxidative stress has been known to be involved in the mechanism of toxic effects of various agents on many cellular systems. In this study we investigated the role of reactive oxygen species (ROS) in 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)-induced neuronal cell toxicity using SK-N-SH human neuroblastoma cells. TCDD inhibited proliferation of the cells in a dose-dependent manner, which was revealed by MTT staining, counting of cells stained with trypan blue and [ 3 H]thymidine uptake assay. TCDD also suppressed the basal generation of ROS in a time- and concentration-dependent manner assessed by 2',7'-dichlorofluorescein fluorescence. In addition, TCDD induced a dose-dependent inhibition of lipid peroxidation, a biomarker of oxidative stress, whereas it significantly increased the level of glutathione (GSH), an intracellular free radical scavenger in the cells. Moreover, TCDD altered the activities of major antioxidant enzymes; increase in superoxide dismutase (SOD) and catalase, but decrease in glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Red). Pretreatment with l -buthionine- S , R -sulfoximine (BSO, 50 μM), an inhibitor of GSH synthesis, significantly prevented the TCDD-induced reduction in lipid peroxidation and cell proliferation. Interestingly, exogenous application of an oxidant, H 2 O 2 (50 μM) markedly restored the inhibited cell proliferation induced by TCDD. Taken together, these results suggest that alteration of cellular redox balance may mediate the TCDD-induced inhibition of proliferation in human neuronal cells.     

Characterization of subpopulated articular chondrocytes separated by Percoll density gradient

The aim of this study was to develop a method for fractionation of articular chondrocytes from the entire thickness of the tissue. Isolated chondrocytes from rabbit articular cartilage fractionated by centrifugation in a discontinuous Percoll gradient resulted in four cell fractions with two differing properties. The lowest-density fraction consisted mainly of large cells with small nuclei proliferated actively, maintained the chondrocytic phenotype, and secreted larger amounts of proteoglycan. In contrast, the highest-density fraction consisted of small cells with large nuclei proliferated slowly, did not express the chondrocytic phenotype, and produced larger amounts of interleukin 1-induced nitric oxide. Comparing our results with other previous reports, we find that fraction 1 cells are likely originated from the deep layer of the articular cartilage, whereas fraction 4 cells are tentatively categorized as chondrocytes from the superficial layer of cartilage. Centrifugal fractionation of articular chondrocytes via Percoll density gradient permits clear separation of these heterogeneous cells into different phenotypic populations and allows distinguishing of cells from the different layers of articular cartilage. This simple novel method will provide ready separation of articular chondrocytes for the investigation of the pathogenesis of articular cartilage.     

Impact of electrode composition on electricity generation in a single-compartment fuel cell using Shewanella putrefaciens

The production of electricity by Shewanella putrefaciens in the absence of exogenous electron acceptors was examined in a single compartment fuel cell with different types of electrodes and varying physiological conditions. Electricity production was dependent on anode composition, electron donor type and cell concentration. A maximum current of 2.5 mA and a current density of 10.2 mW/m(2)electrode was obtained with a Mn(4+) graphite anode, 200 mM sodium lactate and a cell concentration of 3.9 g cell protein/ml. Current production by S. putrefaciens was enhanced 10-fold when an electron mediator (i.e., Mn(4+) or neutral red) was incorporated into the graphite anode.     

Unlocking the secrets of syndecans: transgenic organisms as a potential key

Heparan sulfate proteoglycans are known to modulate the activity of a large number of extracellular ligands thereby having the potential to regulate a great diversity of biological processes. The long-term studies in our laboratory have focused on the syndecans, one of the major cell surface heparan sulfate proteoglycan families. Most early work on syndecans involved biochemical studies that provided initial information on their structure and putative biological roles. In recent years, the development of transgenic organisms has allowed a more complete understanding of syndecan function. Studies with transgenic syndecan-1 and syndecan-3 mice have demonstrated an unforeseen role for syndecans in the regulation of feeding behavior. Syndecan-1 knockout mice display a reduced susceptibility to both Wnt-induced tumorigenesis and microbial pathogenesis. Experiments with Drosophila show that syndecan is first expressed upon cellularization in the early embryo, and may play a role in the early developmental stages of the fly. This review focuses on these diverse functions of the syndecans that have been elucidated by the use of transgenic mice and Drosophila as model systems. Published in 2003.     

Initial net CO2 uptake responses and root growth for a CAM community placed in a closed environment

To help understand carbon balance between shoots and developing roots, 41 bare-root crassulacean acid metabolism (CAM) plants native to the Sonoran Desert were studied in a glass-panelled sealable room at day/night air temperatures of 25/15 degrees C. Net CO(2) uptake by the community of Agave schottii, Carnegia gigantea, Cylindropuntia versicolor, Ferocactus wislizenii and Opuntia engelmannii occurred 3 weeks after watering. At 4 weeks, the net CO(2) uptake rate measured for south-east-facing younger parts of the shoots averaged 1.94 micro mol m(-2) s(-1) at night, considerably higher than the community-level nocturnal net CO(2) uptake averaged over the total shoot surface, primarily reflecting the influences of surface orientation on radiation interception (predicted net CO(2) uptake is twice as high for south-east-facing surfaces compared with all compass directions). Estimated growth plus maintenance respiration of the roots averaged 0.10 micro mol m(-2) s(-1) over the 13-week period, when the community had a net carbon gain from the atmosphere of 4 mol C while the structural C incorporated into the roots was 23 mol. Thus, these five CAM species diverted all net C uptake over the 13-week period plus some existing shoot C to newly developing roots. Only after sufficient roots develop to support shoot water and nutrient requirements will the plant community have net above-ground biomass gains.     

Crystal structures of unligated and CN-ligated Glycera dibranchiata monomer ferric hemoglobin components III and IV

Erythrocytes of the marine annelid, Glycera dibranchiata, contain a mixture of monomeric and polymeric hemoglobins. There are three major monomer hemoglobin components, II, III, IV (also called GMH2, 3, and 4), that have been highly purified and well characterized. We have now crystallized GMH3 and GMH4 and determined their structures to 1.4-1.8 A resolution. The structures were determined for these two monomer hemoglobins in the oxidized (Fe3+, ferric, or met-) forms in both the unligated and cyanide-ligated states. This work differs from two published, refined structures of a Glycera dibranchiata monomer hemoglobin, which has a sequence that is substantially different from any bona fide major monomer hemoglobins (GMH2, 3, or 4). The high-resolution crystal structures (presented here) and the previous NMR structure of CO-ligated GMH4, provide a basis for interpreting structure/function details of the monomer hemoglobins. These details include: (1) the strong correlation between temperature factor and NMR dynamics for respective protein forms; (2) the unique nature of the HisE7Leu primary sequence substitutions in GMH3 and GMH4 and their impact on cyanide ion binding kinetics; (3) the LeuB10Phe difference between GMH3 and GMH4 and its impact on ligand binding; and (4) elucidation of changes in the structural details of the distal and proximal heme pockets upon cyanide binding.     

Betaig-h3 supports keratinocyte adhesion,migration, and proliferation through alpha3beta1 integrin

betaig-h3 is an extracellular matrix protein and its expression is highly induced by TGF-beta and it has also been suggested to play important roles in skin wound healing. In this paper, we demonstrate that betaig-h3 is present in the papillary layer of dermis and synthesized in the basal keratinocytes in vivo and its expression is induced by TGF-beta in normal human keratinocytes (NHEK) and HaCaT cells. betaig-h3 mediates not only adhesion and spreading of keratinocytes but also supports migration and proliferation. These activities are mediated through interacting with alpha3beta1 integrin. Previously identified two alpha3beta1 integrin-interacting motifs of betaig-h3, EPDIM, and NKDIL, are responsible for these activities. The results suggest that betaig-h3 may regulate keratinocyte functions in normal skin and potentially during wound-healing process.     

GSH-dependent peroxidase activity of the rice (Oryza sativa) glutaredoxin,a thioltransferase

Glutaredoxin (Grx) is a 12-kDa thioltransferase that reduces disulfide bonds of other proteins and maintains the redox potential of cells. In addition to its oxidoreductase activity, we report here that a rice Grx (OsGrx) can also function as a GSH-dependent peroxidase. Because of this antioxidant activity, OsGrx protects glutamine synthetase from oxidative damage. Individually replacing the conserved Cys residues in OsGrx with Ser shows that Cys(23), but not Cys(26), is essential for the thioltransferase and GSH-dependent peroxidase activities. Kinetic characterization of OsGrx reveals that the maximal catalytic efficiency (V(max)/K(m)) is obtained with cumene hydroperoxide rather than H(2)O(2) or t-butyl hydroperoxide.     

A novel amylolytic enzyme from Thermotoga maritima,resembling cyclodextrinase and alpha-glucosidase,that liberates glucose from the reducing end of the substrates

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.