Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod, Achatina fulica

Heavy metal stress results in the production of O(2)(.-), H(2)O(2) and (.)OH, which affect various cellular processes, mostly the functioning of membrane systems. Cells are normally protected against free oxyradicals by the operation of intricate antioxidant systems. The aim of the present work is to examine the effect of CdCl(2) and ZnSO(4) on antioxidative enzyme activity in the gastropod, Achatina fulica. The concentrations of antioxidant enzymes--superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GPx)--and nonenzymatic antioxidants--glutathione and vitamin-C--were found to be decreased in both digestive gland and kidney of the gastropod, Achatina fulica treated with individual concentrations of 0.5 ppm and 1ppm of CdCl(2) and ZnSO(4), compared to that of control animals. Based on the above study, it is evident that Achatina fulica can be used as a bioindicator to monitor the environmental heavy metal pollution.     

Regional material property alterations in porcine femoral arteries with atheroma development

We have developed a novel methodology that permits assessment of regional vascular mechanical property alterations in the presence of atheroma in vivo employing a Yucatan miniswine model with induced lesions. Femoral arteries were imaged with intravascular ultrasound. Image data were segmented and, following three-dimensional reconstruction, underwent finite element and sensitivity analysis with optimization to identify regions with altered vascular mechanical properties. All regions were compared to histological analysis. In 12 animals with 8 weeks of endothelial cell denudation and high cholesterol diet (induced atherosclerosis), the elastic modulus initially decreased with early lesion development and then increased with increasing fibrosis-(elastic modulus-all values x10(4)Pa-mean+/-SEM) histologically normal (non-denuded control segment) elements 9.73+/-0.01, fatty elements 9.53+/-0.01, fibrofatty elements 9.41+/-0.03, and fibrous elements 9.68+/-0.02 (all p<0.001 vs. normal elements). Wall thickness, however, increased with atheroma formation. These data demonstrate decreasing vascular material properties with early lesions, followed by an increase as lesions progress. This methodology permits determination of areas with early atheroma development, follow atheroma progression, and potentially evaluate interventions aimed at decreasing atheroma load and normalizing vascular material properties.     

L166P mutant DJ-1, causative for recessive Parkinson's disease,is degraded through the ubiquitin-proteasome system

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.     

Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity

When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.     

Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies

Using X-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at liquid helium temperatures, the Cu(II) coordination geometry at the active site of bovine and human copper,zinc-superoxide dismutases (bSOD1 and hSOD1) treated with H(2)O(2) and bicarbonate (HCO(3)(-)) was examined. The time course EPR of wild type human SOD1 (WT hSOD1), W32F hSOD1 mutant (tryptophan 32 substituted with phenylalanine), and bSOD1 treated with H(2)O(2) and HCO(3)(-) shows an initial reduction of active site Cu(II) to Cu(I) followed by its oxidation back to Cu(II) in the presence of H(2)O(2). However, HCO(3)(-) induced a Trp-32-derived radical from WT hSOD1 but not from bSOD1. The mutation of Trp-32 by phenylalanine totally eliminated the Trp-32 radical signal generated from W32F hSOD1 treated with HCO(3)(-) and H(2)O(2). Further characterization of the free radical was performed by UV irradiation of WT hSOD1 and bSOD1 that generated tryptophanyl and tyrosyl radicals. Both proton ((1)H) and nitrogen ((14)N) ENDOR studies of bSOD1 and hSOD1 in the presence of H(2)O(2) revealed a change in the geometry of His-46 (or His-44) and His-48 (or His-46) coordinated to Cu(II) at the active site of WT hSOD1 and bSOD1, respectively. However, in the presence of HCO(3)(-) and H(2)O(2), both (1)H and (14)N ENDOR spectra were almost identical to those derived from native bSOD1. We conclude that HCO(3)(-)-derived oxidant does not alter significantly the Cu(II) active site geometry and histidine coordination to Cu(II) in SOD1 as does H(2)O(2) alone; however, the oxidant derived from HCO(3)(-) (i.e. carbonate anion radical) reacts with surface-associated Trp-32 in hSOD1 to form the corresponding radical.     

Numerical study on the effect of secondary flow in the human aorta on local shear stresses in abdominal aortic branches

Flow in the aortic arch is characterized primarily by the presence of a strong secondary flow superimposed over the axial flow, skewed axial velocity profiles and diastolic flow reversals. A significant amount of helical flow has also been observed in the descending aorta of humans and in models. In this study a computational model of the abdominal aorta complete with two sets of outflow arteries was adapted for three-dimensional steady flow simulations. The flow through the model was predicted using the Navier-Stokes equations to study the effect that a rotational component of flow has on the general flow dynamics in this vascular segment. The helical velocity profile introduced at the inlet was developed from magnetic resonance velocity mappings taken from a plane transaxial to the aortic arch. Results showed that flow division ratios increased in the first set of branches and decreased in the second set with the addition of rotational flow. Shear stress varied in magnitude with the addition of rotational flow, but the shear stress distribution did not change. No regions of flow separation were observed in the iliac arteries for either case. Helical flow may have a stabilizing effect on the flow patterns in branches in general, as evidenced by the decreased difference in shear stress between the inner and outer walls in the iliac arteries.     

Distinctive microbial ecology and biokinetics of autotrophic ammonia and nitrite oxidation in a partial nitrification bioreactor

Biological nitrogen removal (BNR) based on partial nitrification and denitrification via nitrite is a cost-effective alternate to conventional nitrification and denitrification (via nitrate). The goal of this study was to investigate the microbial ecology, biokinetics, and stability of partial nitrification. Stable long-term partial nitrification resulting in 82.1 +/- 17.2% ammonia oxidation, primarily to nitrite (77.3 +/- 19.5% of the ammonia oxidized) was achieved in a lab-scale bioreactor by operation at a pH, dissolved oxygen and solids retention time of 7.5 +/- 0.1, 1.54 +/- 0.87 mg O(2)/L, and 3.0 days, respectively. Bioreactor ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) populations were most closely related to Nitrosomonas europaea and Nitrobacter spp., respectively. The AOB population fraction varied in the range 61 +/- 45% and was much higher than the NOB fraction, 0.71 +/- 1.1%. Using direct measures of bacterial concentrations in conjunction with independent activity measures and mass balances, the maximum specific growth rate (micro(max)), specific decay (b) and observed biomass yield coefficients (Y(obs)) for AOB were 1.08 +/- 1.03 day(-1), 0.32 +/- 0.34 day(-1), and 0.15 +/- 0.06 mg biomass COD/mg N oxidized, respectively. Corresponding micro(max), b, and Y(obs) values for NOB were 2.6 +/- 2.05 day(-1), 1.7 +/- 1.9 day(-1), and 0.04 +/- 0.02 mg biomass COD/mg N oxidized, respectively. The results of this study demonstrate that the highly selective partial nitrification operating conditions enriched for a narrow diversity of rapidly growing AOB and NOB populations unlike conventional BNR reactors, which host a broader diversity of nitrifying bacteria. Further, direct measures of microbial abundance enabled not only elucidation of mixed community microbial ecology but also estimation of key engineering parameters describing bioreactor systems supporting these communities.     

Computer-aided design of biological circuits using TinkerCell

Synthetic biology is an engineering discipline that builds on modeling practices from systems biology and wet-lab techniques from genetic engineering. As synthetic biology advances, efficient procedures will be developed that will allow a synthetic biologist to design, analyze, and build biological networks. In this idealized pipeline, computer-aided design (CAD) is a necessary component. The role of a CAD application would be to allow efficient transition from a general design to a final product. TinkerCell is a design tool for serving this purpose in synthetic biology. In TinkerCell, users build biological networks using biological parts and modules. The network can be analyzed using one of several functions provided by TinkerCell or custom programs from third-party sources. Since best practices for modeling and constructing synthetic biology networks have not yet been established, TinkerCell is designed as a flexible and extensible application that can adjust itself to changes in the field.