Disposable tyrosinase-peroxidase bi-enzyme sensor for amperometric detection of phenols

A new disposable amperometric bi-enzyme sensor system for detecting phenols has been developed. The phenol sensor developed uses horseradish peroxidase modified screen-printed carbon electrodes (HRP-SPCEs) coupled with immobilized tyrosinase prepared using poly(carbamoylsulfonate) (PCS) hydrogels or a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) matrix. Optimization of the experimental parameters has been performed with regard to buffer composition, pH, operating potential and storage stability. A co-operative reaction involving tyrosinase and HRP occurs at a potential of -50 mV versus Ag/AgCl without the requirement for addition of extraneous H(2)O(2), thus, resulting in a very simple and efficient system. Comparison of the electrode responses with the 4-aminoantipyrine standard method for phenol sample analysis indicated the feasibility of the disposable sensor system for sensitive "in-field" determination of phenols. The most sensitive system was the tyrosinase immobilized HRP-SPCE using PCS, which displayed detection limits for phenolic compounds in the lower nanomolar range e.g. 2.5 nM phenol, 10 nM catechol and 5 nM p-cresol.     

A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography

Here we describe a proteomic approach to detect fibrinolytic enzymes from the culture supernatant of Bacillus subtilis 168. Following isoelectric focusing without dithiothreitol, two gels, one for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the other for zymography, were run in parallel. After silver staining of SDS-PAGE and activity staining of zymography gel, the two gels were superimposed to detect protein spots that coincided with clear zones on the zymography gel. We identified four protein spots and characterized them with matrix-assisted laser desorption/ionization mass spectrometry. Database search revealed that four spots contained at least one of the extracellular serine proteases such as WprA and Vpr. This combined method of two-dimensional gel and zymography can be used as a powerful tool to detect proteases from various organisms.     

Diversity and varietal classification of Hibiscus syriacus L. with the heterogeneity within retrotransposon-like elements

Retrotransposons are present in multi-copy numbers that are integrated into plant genomes with considerable heterogeneous sequences within a single plant and between plant species, which allows the use of retrotransposons as additional sources of DNA polymorphism. A primer design for the sequence-tagged specific site and cleaved amplified polymorphic sequences (STS-CAPs) that are derived from retrotransposon-like sequences was developed for the molecular marker analysis in Hibiscus syriacus. This method was applied for the detection of sequence variations of intact retrotransposons that exist in plant genomes, which resulted in higher polymorphisms than in the amplified fragment length polymorphism (AFLP). Through STS-CAPs, specific fingerprinting data among H. syriacus varieties can be easily distinguished and generated with reproducible results. It could also be adapted to any species that possess multi-copy retrotransposons for varietal identification as well as the assessment of genetic relationships.     

Comparison of responses elicited by immunization with a Legionella species common lipoprotein delivered as naked DNA or recombinant protein

To evaluate the peptidoglycan-associated lipoprotein (PAL) antigen of Legionella pneumophila as a vaccine candidate, mice were immunized intramuscularly with pcDNA3-PAL and intraperitoneally with recombinant PAL (t-rPAL), which were compared for their ability to induce PAL-specific immune responses. The t-rPAL protein induced PAL-specific IgG antibody production significantly more than did pcDNA3-PAL. The IgG2a and IgG1 production was predominant after pcDNA3-PAL and t-rPAL administration, respectively. In particular, pcDNA3-PAL induced much higher PAL-specific cytotoxic T-lymphocyte responses than did t-rPAL. Furthermore, in vivo, CD19+ B-cell populations were dramatically increased by t-rPAL vaccination, suggesting a B-cell immunomodulatory activity of the lipoprotein. The PAL antigen was also conserved among Legionella species, as determined by PCR and immunoblot analyses. These results support a potential use of the t-rPAL protein and in particular DNA vaccines against Legionella infections.     

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.     

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.     

Enhanced detection and characterization of protocatechuate 3,4-dioxygenase in Acinetobacter lwoffii K24 by proteomics using a column separation

Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway.     

Structural and functional implications of C-terminal regions of alpha-synuclein

Aggregation of alpha-synuclein is thought to play a major role in the pathogenesis of Parkinson's disease (PD), which is characterized by the presence of intracytoplasmic Lewy bodies (LB) in the brain. alpha-Synuclein and its deletion mutants are largely unfolded proteins with random coil structures as revealed by CD spectra, fluorescence spectra, gel filtration chromatography, and ultracentrifugation. On the basis of its highly unfolded and flexible conformation, we have investigated the chaperone-like activity of alpha-synuclein in vitro. In our experiments, alpha-synuclein inhibited the aggregation of model substrates and protected the catalytic activity of alcohol dehydrogenase and rhodanese during heat stress. In addition, alpha-synuclein inhibited the initial aggregation of reduced/denatured lysozyme on the refolding pathway. Interestingly, deletion of the C-terminal regions led to the abolishment of chaperone activity, although largely unstructured conformations are maintained. Moreover, alpha-synuclein could inhibit the aggregation of various Escherichia coli cellular proteins during heat stress, and C-terminal deletion mutants could not provide any protection to these cellular proteins. Results with synthetic C-terminal peptides and C-terminal deletion mutants suggest that the second acidic repeat, (125)YEMPSEEGYQDYEPEA(140), is important for the chaperone activity of alpha-synuclein, and C-terminal deletion leads to the facilitated aggregation with the elimination of chaperone activity.