Adaptive Mechanisms of Shewanella xiamenensis DCB 2-1 Metallophilicity.

The dose-dependent effects of single metals (Zn, Ni, and Cu) and their combinations at steady time-actions on the cell viability of the bacteria Shewanella xiamenensis DCB 2-1, isolated from a radionuclide-contaminated area, have been estimated. The accumulation of metals by Shewanella xiamenensis DCB 2-1 in single and multi-metal systems was assessed using the inductively coupled plasma atomic emission spectroscopy. To estimate the response of the bacteria's antioxidant defense system, doses of 20 and 50 mg/L of single studied metals and 20 mg/L of each metal in their combinations (non-toxic doses, determined by the colony-forming viability assay) were used. Emphasis was given to catalase and superoxide dismutase since they form the primary line of defense against heavy metal action and their regulatory circuit of activity is crucial. The effect of metal ions on total thiol content, an indicator of cellular redox homeostasis, in bacterial cells was evaluated. Genome sequencing of Shewanella xiamenensis DCB 2-1 reveals genes responsible for heavy metal tolerance and detoxification, thereby improving understanding of the potential of the bacterial strain for bioremediation.

Development of Multiplex PCR Coupled DNA Chip Technology for Assessment of Endogenous and Exogenous Allergens in GM Soybean.

Allergenicity assessment of transgenic plants and foods is important for food safety, labeling regulations, and health protection. The aim of this study was to develop an effective multi-allergen diagnostic approach for transgenic soybean assessment. For this purpose, multiplex polymerase chain reaction (PCR) coupled with DNA chip technology was employed. The study was focused on the herbicide-resistant Roundup Ready soya (RRS) using a set of certified reference materials consisting of 0, 0.1%, 0.5%, and 10% RRS. Technically, the procedure included design of PCR primers and probes; genomic DNA extraction; development of uniplex and multiplex PCR systems; DNA analysis by agarose gel electrophoresis; microarray development, hybridization, and scanning. The use of the asymmetric multiplex PCR method is shown to be very efficient for DNA hybridization with biochip probes. We demonstrate that newly developed fourplex PCR methods coupled with DNA-biochips enable simultaneous identification of three major endogenous allergens, namely, Gly m Bd 28K, Gly m Bd 30K, and lectin, as well as exogenous 5-enolppyruvyl shikimate-phosphate synthase (epsps) expressed in herbicide-resistant roundup ready GMOs. The approach developed in this study can be used for accurate, cheap, and fast testing of food allergens.

A comparison of DNA fragmentation methods - Applications for the biochip technology.

The efficiency of hybridization signal detection in a biochip is affected by the method used for test DNA preparation, such as fragmentation, amplification and fluorescent labelling. DNA fragmentation is the commonest methods used and it is recognised as a critical step in biochip analysis. Currently methods used for DNA fragmentation are based either on sonication or on the enzymatic digestion. In this study, we compared the effect of different types of enzymatic DNA fragmentations, using DNase I to generate ssDNA breaks, NEBNext dsDNA fragmentase and SaqAI restrictase, on DNA labelling. DNA from different Desulfovibrio species was used as a substrate for these enzymes. Of the methods used, DNA fragmented by NEBNext dsDNA Fragmentase digestion was subsequently labelled with the greatest efficiency. As a result of this, the use of this enzyme to fragment target DNA increases the sensitivity of biochip-based detection significantly, and this is an important consideration when determining the presence of targeted DNA in ecological and medical samples.

A novel cassette method for probe evaluation in the designed biochips.

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip.

Correlation between MMP-9 and extracellular cytokine HMGB1 in prediction of human ischemic stroke outcome.

Ischemic stroke (IS) outcome predictors include clinical features, biochemical parameters and some risk factors. The relations between two main players in the ischemic brain, MMPs and HMGB1, were estimated in the plasma of ischemic stroke patients stratified according to the Glasgow Outcome Scale and the Oxfordshire Community Stroke Project classification. IS patients exhibited higher plasma concentration of MMP-9 and the inflammatory cytokine HMGB1 compared with healthy controls. A full-blown correlation between MMP-9 activation and increased plasma MMP-9 concentration was observed in case of IS patients. A similar activity of MMP-2 and MMP-12 was characteristic of healthy volunteers and IS patients. In patients with ischemic stroke increased plasma levels of MMP-9 and HMGB1 are associated with a poor functional outcome and are significantly correlated with each other (P=0.0054). We suggest that diagnostic benefits will be obtained if plasma HMGB1 levels are measured for IS patients in addition to MMP-9.

Chromium (VI) can activate and impair antioxidant defense system.

The changes in glutathione-dependent cycle enzymes and catalase activities under Cr(VI)-induced oxidative stress were investigated in two distinct cell lines: L-41-human epithelial-like cells and HLF-fetal human diploid lung fibroblasts, which differ in tissue origin, proliferation, and antioxidant enzymes activities. The chromium concentrations from 1 to 5 µM cause nontoxic effects and activate antioxidant enzymes to overcome oxidative stress. In spite of some differences in the endogenous antioxidant activities, both cell lines reveal the same range of toxic concentrations (20-30 µM). The irreversible inhibition of glutathione-dependent antioxidant enzymes develops under toxic concentrations and serves as a marker of toxicity. The endogenous antioxidant activity influences time-dependent expression of Cr(VI) toxicity and the dynamics of antioxidant enzymes activity under nontoxic conditions. The cell antioxidant defense system is an important marker of the cell adaptive capacity under nontoxic and toxic conditions.

Response of antioxidant defense system to chromium (VI)-induced cytotoxicity in human diploid cells.

The aim of this study is to establish antioxidant indicators of chromium toxicity in fetal human lung fibroblasts (HLF). The results obtained corroborate and develop our earlier observation of low-dose and long-term action of Cr(VI) on human cells in culture. In the case of a nontoxic chromium dose, temporary oxidative stress is overcome by increased activity of the antioxidant system with correlation to cell cycle re-entry. The toxic concentrations misbalance the cell antioxidant defense systems and cause irreversible growth arrest and massive cell death by apoptosis. Sub-toxicity is defined as toxicity stretched in time. The activity of GPx (glutathione peroxidase) is proposed as a biomarker of oxidative stress caused by Cr(VI), and the GR (glutathione reductase) inhibition is considered as a marker of the toxicity developed under the complex Cr(VI) action. In HLF cells the glutathione dependent defense system is the first system destroyed in response to toxic chromium action. Only the balance between SOD (superoxide dismutase) and H(2)O(2) degrading enzymes (catalase and GPx), should play an important role in the fate of a cell, not individual enzymes.

Effect of chromium(VI) action on Arthrobacter oxydans.

Arthrobacter species is of interest because of its high potential for bioremediation. Bacteria can detoxify chromium, by either reduction or accumulation inside the bacteria and/or absorption of chromium(VI) (CrVI) on their surface, and efflux pump. The possible pathway of Cr(VI) reduction by Arthrobacter oxydans isolated from Columbia basalt rocks at a US DOE highly contaminated site (USA) has been considered in the present study. FTIR absorption spectroscopy showed that these bacteria reduce Cr(VI). In the present study the threshold Cr(VI) nontoxic concentration (35 microg/mL) for A. oxydans growing in liquid medium was estimated. Complete uptake of this concentration was achieved in about 10 days after chromium addition into the medium. At this concentration an increase in the protein isolated from the cell wall of A. oxydans was observed. This increased protein predominated independently of the growth phase at which Cr(VI) was added. Thermal analysis was used to identify any influence of Cr(VI) on the DNP complex of A. oxydans. According to the data obtained it can be supposed that Cr(VI) reduction predominantly occurs on the bacterial surface and that cell wall represents a permeable barrier for these bacteria at the non-toxic chromium action.

Estimation of the cellular antioxidant response to chromium action using ESR method.

In the present study, the antioxidant capacity of chromium-treated L-41 (human epithelial-like cells) was investigated by the ESR spin-trapping technique. The crude cell extracts of the cells grown in the presence of 2 microM (nontoxic) and 20 microM (toxic) chromium (VI) concentrations were tested in the model Fenton system with and without catalase-inhibitor sodium azide. The presented approach using the ESR technique along with inhibitors lets us discern cell extract defense capacity connected with the enzymatic activity in viable cells and the catabolic activity in dying cells.

Antioxidant capacity of cultured mammalian cells estimated by ESR method.

In the present study, the antioxidant capacity against hydrogen peroxide (H2O2), one of the stress-inducing agents, was investigated in two distinct cell lines: L-41 (human epithelial-like cells) and HLF (human diploid lung fibroblasts), which differ in tissue origin, life span in culture, proliferate activity, and special enzyme system activity. The cell antioxidant capacity against H2O2 was estimated by the electron spin resonance (ESR) spin-trapping technique in the Fenton reaction system via Fe+2 ion action with H2O2 resulting in hydroxyl radical generation. The effects of catalase inhibitors, such as sodium azide and 3-amino-1,2,4-triazole, on the antioxidant capacity of cells were tested. Based on our observation, it can be concluded that the defensive capacity of cells against H2O2 depends on the ratio between catalase/GPx/SOD and H2O2, especially at high-stress situations, and the intracellular balance of these enzymes are more important than the influence of the single component.

Effects of Cr(VI) long-term and low-dose action on mammalian antioxidant enzymes (an in vitro study).

In order to investigate the low-dose long-term Cr(VI) action on antioxidant enzymes in cultured mammalian cells we estimated the activity of glutathione dependent antioxidant enzymes, catalase and superoxide dismutase (SOD) under various chromium concentrations in human epithelial-like L-41 cells. The long-term action of 20 microM causes the toxicity that results in losing of the cell viability by activating the apoptotic process, as identified by morphological analysis, the activation of caspase-3, and DNA fragmentation. The toxic chromium concentration totally destroys glutathione antioxidant system, and diminishes the activity of catalase and cytosolic Cu, ZnSOD. The non-toxic concentration (2 microM) causes the activation of the antioxidant defense systems, and they neutralize the oxidative impact.