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Staphylococcus aureus is still one of the leading causes of both hospital- and community-acquired infections. Due to the very high percentage of drug-resistant strains, the participation of drug-tolerant biofilms in pathological changes, and thus the limited number of effective antibiotics, there is an urgent need to search for alternative methods of prevention or treatment for S. aureus infections. In the present study, biochemically characterized (HPLC/UPLC-QTOF-MS) acetonic, ethanolic, and water extracts from fruits and bark of Viburnum opulus L. were tested in vitro as diet additives that potentially prevent staphylococcal infections. The impacts of V. opulus extracts on sortase A (SrtA) activity (Fluorimetric Assay), staphylococcal protein A (SpA) expression (FITC-labelled specific antibodies), the lipid composition of bacterial cell membranes (LC-MS/MS, GC/MS), and biofilm formation (LIVE/DEAD BacLight) were assessed. The cytotoxicity of V. opulus extracts to the human fibroblast line HFF-1 was also tested (MTT reduction). V. opulus extracts strongly inhibited SrtA activity and SpA expression, caused modifications of S. aureus cell membrane, limited biofilm formation by staphylococci, and were non-cytotoxic. Therefore, they have pro-health potential. Nevertheless, their usefulness as diet supplements that are beneficial for the prevention of staphylococcal infections should be confirmed in animal models in the future.

Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1-4 (RXFP1-4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand-receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.

In order to achieve efficient extracellular expression of Sortase A (SrtA), various strategies in Pichia pastoris system were applied in this study. Among different constructed recombinant strains, the SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter was proved to be the best strain for the extracellular SrtA expression. After the optimization of fermentation conditions (induction 72 h at 28 °C, initial pH 6.0, supplemented with 1.5% methanol), the highest yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL at the shake-flask level, respectively. This is the first report on the efficient secretory expression of SrtA in P. pastoris and the yield of SrtA is the maximum compared with previous reports. In addition, the transpeptidation activity of extracellular SrtA was confirmed by the successful immobilization of enhanced green fluorescent protein (EGFP) onto Gly 3 -polystyrene beads., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Enterococcus faecalis (E. faecalis) is one of the major causes of biofilm infections. Berberine hydrochloride (BBH) has diverse pharmacological effects; however, the effects and mechanisms of BBH on E. faecalis biofilm formation and dispersion have not been reported. In this study, 99 clinical isolates from the urine samples of patients with urinary tract infections (UTIs) were collected and identified. Ten strains of E. faecalis with biofilm formation ability were studied. BBH inhibited E. faecalis biofilm formation and promoted the biofilm dispersion of E. faecalis. In addition, sortase A and esp expression levels were elevated during early E. faecalis biofilm development, whereas BBH significantly reduced their expression levels. The results of this study indicated that BBH effectively prevents biofilm formation and promotes biofilm dispersion in E. faecalis, most likely by inhibiting the expressions of sortase A and esp., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The immunoglobulin binding protein A (SpA) of Staphylococcus aureus is synthesized as a precursor with a C-terminal sorting signal. The sortase A enzyme mediates covalent attachment to peptidoglycan so that SpA is displayed on the surface of the bacterium. Protein A is also found in the extracellular medium, but the processes involved in its release are not fully understood. Here, we show that a portion of SpA is released into the supernatant with an intact sorting signal, indicating that it has not been processed by sortase A. Release of SpA was reduced when the native sorting signal of SpA was replaced with the corresponding region of another sortase-anchored protein (SdrE). Similarly, a reporter protein fused to the sorting signal of SpA was released to a greater extent than the same polypeptide fused to the SdrE sorting signal. Released SpA protected bacteria from killing in human blood, indicating that it contributes to immune evasion., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant.

Covalent attachment of surface proteins to the cell wall of Gram-positive bacteria requires a sortase-mediated transpeptidation reaction. In almost all Gram-positive bacteria, the housekeeping sortase, sortase A, recognizes the canonical recognition sequence LPXTG (X=any amino acid). The human pathogen Clostridium difficile carries a single putative sortase gene (cd2718) but neither transpeptidation activity nor specificity of CD2718 has been investigated. We produced recombinant CD2718 and examined its transpeptidation activity in vitro using synthetic peptides and MALDI-ToF(-ToF) MS analysis. We demonstrate that CD2718 has sortase activity with specificity for a (S/P)PXTG motif and can accommodate diaminopimelic acid as a substrate for transpeptidation., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Vancomycin-intermediate Staphylococcus aureus (VISA) strains present an increasingly difficult problem in terms of public health. However, the molecular mechanism for this resistance is not yet understood. In this study, we define the role of the msaABCR operon in vancomycin resistance in three clinical VISA strains, i.e., Mu50, HIP6297, and LIM2. Deletion of the msaABCR operon resulted in significant decreases in the vancomycin MIC (from 6.25 to 1.56 μg/ml) and significant reductions of cell wall thickness in strains Mu50 and HIP6297. Growth of the mutants in medium containing vancomycin at concentrations greater than 2 μg/ml resulted in decreases in the growth rate, compared with the wild-type strains. Mutation of the msaABCR operon also reduced the binding capacity for vancomycin. We conclude that the msaABCR operon contributes to resistance to vancomycin and cell wall synthesis in S. aureus., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The production of phytochelatins (PCs) provides an important means for plants to achieve tolerance to cadmium (Cd) toxicity. A reed gene encoding PC synthase (PaPCS) was isolated and its function tested through its heterologous expression in a strain of yeast sensitive to Cd. Subsequently, the Cd sensitive and high biomass accumulating species tall fescue was transformed either with PaPCS or PaGCS (a glutamyl cysteine synthetase gene of reed) on their own (single transformants), or with both genes together in the same transgene cassette (double transformant). The single and double transformants showed greater Cd tolerance and accumulated more Cd and PC than wild type plants, and their Cd leaf/root ratio content was higher. The ranking in terms of Cd and PC content for the various transgenic lines was double transformants>PaGCS single transformants>PaPCS single transformants>wild type. Thus PaGCS appears to exert a greater influence than PaPCS over PC synthesis and Cd tolerance/accumulation. The double transformant has interesting potential for phytoremediation.

Recent studies have identified rice (Oryza sativa) as a major dietary source of inorganic arsenic (As) and poses a significant human health risk. The predominant model for plant detoxification of heavy metals is complexation of heavy metals with phytochelatins (PCs), synthesized non-translationally by PC synthase (PCS) and compartmentalized in vacuoles. In this study, in order to restrict As in the rice roots as a detoxification mechanism, a transgenic approach has been followed through expression of phytochelatin synthase, CdPCS1, from Ceratophyllum demersum, an aquatic As-accumulator plant. CdPCS1 expressing rice transgenic lines showed marked increase in PCS activity and enhanced synthesis of PCs in comparison to non-transgenic plant. Transgenic lines showed enhanced accumulation of As in root and shoot. This enhanced metal accumulation potential of transgenic lines was positively correlated to the content of PCs, which also increased several-fold higher in transgenic lines. However, all the transgenic lines accumulated significantly lower As in grain and husk in comparison to non-transgenic plant. The higher level of PCs in transgenic plants relative to non-transgenic presumably allowed sequestering and detoxification of higher amounts of As in roots and shoots, thereby restricting its accumulation in grain.

Zinc-supplementation (20 μM) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

Rationale: Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component., Objective: To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation., Methods and Results: The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries., Conclusions: This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

To cost-efficiently produce biofuels, new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface-displayed multicellulase-containing complexes present in cellulolytic Clostridium and Ruminococcus species. In this study we created cellulolytic strains of Bacillus subtilis that display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by coexpressing them with the Bacillus anthracis sortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase from Clostridium thermocellum to the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface of B. subtilis via noncovalent interactions with a cell wall-attached cohesin module. We also demonstrate that it is possible to assemble multienzyme complexes on the cell surface. A three-enzyme-containing minicellulosome was displayed on the cell surface; it consisted of a cell wall-attached scaffoldin protein noncovalently bound to three cellulase-dockerin fusion proteins that were produced in Escherichia coli. B. subtilis has a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface of B. subtilis may yield cellulolytic bacteria with increased potency that can be used to degrade biomass.

The response of Populus tremula x tremuloides cv. Etrepole transgenic lines expressing the phytochelatin synthase TaPCS1 for Pb tolerance and accumulation was studied. In a hydroponic experiment, the concentrations of Pb in plants did not differ significantly between any of the transgenic lines assayed and the wild type (wt) plants, with any of the Pb solutions tested. However, total biomass and Pb accumulation were significantly higher in transgenic lines (PTa3, PTa5, PTa10) than in the control (wt) line when the plants were grown in solutions containing 0.75 and 1.5 mM Pb. The PTa3 and PTa5 lines accumulated 1.7 times more Pb than the wt plants. A concentration of 3.0 mM Pb was found to be toxic for both transgenic and wt plants. Biomass production was higher in transgenic lines PTa3 and PTaS than in the wt plants growing in M4 mining soil, accumulating more Pb and Zn than in the wt plants. When the plant material was grown in soil M15, none of the parameters differed significantly between the transgenic and wt plants. The different response in soils M4 and M15 indicated that the physicochemical properties of the soil play a determinant role in the phytoremediation potential.

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.

Phytochelatin synthase (PCS) is involved in the synthesis of phytochelatins (PCs), plays role in heavy metal detoxification. The present study describes for first time the functional expression and characterization of pcs gene of Anabaena sp. PCC 7120 in Escherichia coli in terms of offering protection against heat, salt, carbofuron (pesticide), cadmium, copper, and UV-B stress. The involvement of pcs gene in tolerance to above abiotic stresses was investigated by cloning of pcs gene in expression vector pGEX-5X-2 and its transformation in E. coli BL21 (DE3). The E. coli cells transformed with pGEX-5X-pcs showed better growth than control cells (pGEX-5X-2) under temperature (47 degrees C), NaCl (6% w/v), carbofuron (0.025 mg ml(-1)), CdCl2 (4 mM), CuCl2 (1mM), and UV-B (10 min) exposure. The enhanced expression of pcs gene revealed by RT-PCR analysis under above stresses at different time intervals further advocates its role in tolerance against above abiotic stresses.

The Staphylococcus aureus cell surface protein clumping factor A (ClfA) and the enzyme sortase A (SrtA), which attach surface proteins to the cell wall, have both been shown to be virulence factors in models of septic arthritis and sepsis. The mRNA levels of clfA, srtA and the putative housekeeping gene gyrase B (gyrB) in S. aureus were determined using real-time PCR during the course of sepsis/septic arthritis. Expression was measured in joints, being a target of localized infection, and in kidneys, representing a systemic compartment. In infected kidneys, the mRNA levels of clfA, srtA and gyrB were all decreasing over time, from day 3 of infection to day 14. The transcript numbers of clfA and srtA decreased faster in septic mice than in mice with a non-septic disease. The mRNA levels of clfA and gyrB in joints, though, were increasing during the course of infection. These differences suggest that the specific tissue environment is decisive for the differentiation of staphylococci. Also, there was a negative relationship between bacterial load in a tissue and the numbers of clfA, srtA and gyrB transcripts per colony-forming unit. Possibly enters the majority of bacteria a metabolically dormant steady state at high bacterial loads.

Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes.

We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent Kd for dimer formation of about 55 microM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.

To evaluate the role of phytochelatins and metallothioneins in heavy metal tolerance of black mangrove Avicennia germinans, 3-month-old seedlings were exposed to cadmium or copper for 30 h, under hydroponic conditions. Degenerate Mt2 and PCS primers were synthesized based on amino acid and nucleotide alignment sequences reported for Mt2 and PCS in other plant species found in GenBank. Total RNA was isolated from A. germinans leaves and two partial fragments of metallothionein and phytochelatin synthase genes were isolated. Gene expression was evaluated with reverse transcripatase-polymerase chain reaction (RT-PCR) amplification technique. Temporal analysis showed that low Cd2+ and Cu2+ concentrations caused a slight (but not significant) increase in AvMt2 expression after a 16 h exposure time, while AvPCS expression showed a significant increase under the same conditions but only after 4h. Results strongly suggest that the rapid increase in AvPCS expression may contribute to Cd2+ and Cu2+ detoxification. Moreover, we found that A. germinans has the capacity to over-express both genes (AvMt2 and AvPCS), which may constitute a coordinated detoxification response mechanism targeting non-essential metals. Nonetheless, our results confirm that AvPCS was the most active gene involved in the regulation of essential metals (e.g., Cu2+) in A. germinans leaves.

Glutaminyl cyclase (QC) catalyzes the N-terminal pyroglutamate formation of numerous hormones and peptides from their glutaminyl precursor. Pyroglutamate is a posttranslational or cotranslational modification important in many physiological and pathological processes. Here, we present the cloning of a QC cDNA from human bone marrow cDNA library. The protein was expressed in Escherichia coli system with the yields higher than approximately 10 mg/L bacterial culture, using a thioredoxin-tagged expression vector with several modifications. Based on high histidine content ( approximately 5%) of the protein, a convenient purification step by Ni-affinity chromatography was designed, leading to near homogeneity of the purified human QC. The identity of the recombinant human QC was confirmed by mass spectrometry and circular dichroism spectroscopy. The enzyme was active on both synthetic and physiological substrates, and the activity could be inhibited by several imidazole, triazole, and tetrazole derivatives. An atomic absorption analysis demonstrated that human QC contains one zinc ion per protein molecule. We also obtained the human QC crystals, which belong to cubic, tetragonal, and rhombohedral forms. Our works are useful to acquire new insights into human and animal QCs, particularly for future structural analysis and inhibitor designs.

Phytochelatins play an important role in heavy metal detoxification in plants as well as in other organisms. The Arabidopsis thaliana mutant cad1-3 does not produce detectable levels of phytochelatins in response to cadmium stress. The hypersensitivity of cad1-3 to cadmium stress is attributed to a mutation in the phytochelatin synthase 1 (AtPCS1) gene. However, A. thaliana also contains a functional phytochelatin synthase 2 (AtPCS2). In this study, we investigated why the cad1-3 mutant is hypersensitive to cadmium stress despite the presence of AtPCS2. Northern and Western blot analyses showed that expression of AtPCS2 is weak compared to AtPCS1 in both roots and shoots of transgenic Arabidopsis. The lower level of AtPCS2 expression was confirmed by RT-PCR analysis of wild type Arabidopsis. Moreover, no tissue-specific expression of AtPCS2 was observed. Even when AtPCS2 was under the control of the AtPCS1 promoter or of the cauliflower mosaic virus 35S promoter (CaMV 35S) it was not capable of fully complementing the cad1-3 mutant for cadmium resistance.

In Escherichia coli, heterologous production of Schizosaccharomyces pombe phytochelatin synthase (PCS) along with overproduction of E. coli serine acetyltransferase (SAT) and gamma-glutamylcysteine synthase (gammaECS) was achieved and resulted in the accumulation of phytochelatins in bacterial cells. Overproduction of either gammaECS alone or simultaneous production of all three proteins in bacterial cells were accompanied by reduced growth rate in liquid cultures. Interestingly, bacteria overproducing either gammaECS or both SAT and gammaECS (with elevated level of gamma-glutamylcysteine but not of phytochelatins) were able to accumulate more cadmium per dry weight than the control. However, the most efficient cadmium accumulation was observed in bacteria with elevated levels of all three proteins: SAT, gammaECS and PCS. Therefore, "pushing" the entire pathway might be the most promising approach in modification of bacteria for potential bioremediation purposes because the level of intermediates, cysteine and glutathione, can limit the rate of production of phytochelatins. However, in such bacteria other metabolic process might become limiting for efficient growth.

Crocin is a pharmacologically active component of Crocus sativus L. (saffron) that has been used in traditional Chinese medicine. In a previous study, we demonstrated that crocin inhibits apoptosis in PC-12 cells by affecting the function of tumor necrosis factor-alpha. In this study, we found that depriving cultured PC-12 cells of serum/glucose causes a rapid increase in cellular ceramide levels, followed by an increase in the phosphorylation of c-jun kinase (JNK). The accumulation of ceramide was found to depend on the activation of magnesium-dependent neutral sphingomyelinase (N-SMase), but not on de novo synthesis. The serum/glucose-deprived PC-12 cells also decreased the cellular levels of glutathione (GSH), which is the potent inhibitor of N-SMase. Treating the PC-12 cells with crocin prevented N-SMase activation, ceramide production, and JNK phosphorylation. We also found that the chemical can enhance the activities of GSH reductase and gamma-glutamylcysteinyl synthase (gamma-GCS), contributing to a stable GSH supply that blocks the activation of N-SMase. Thus our data suggest that crocin combats the serum/glucose deprivation-induced ceramide formation in PC-12 cells by increasing GSH levels and prevents the activation of JNK pathway, which is reported to have a role of the signaling cascade downstream ceramide for neuronal cell death.

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high alpha-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.

Free radicals are involved in the pathogenesis and/or progression of Parkinson's disease (PD). Several ergot derivative dopamine (DA) agonists have been reported to scavenge free radicals in vitro and to show a neuroprotective effect in vivo. We investigated the in vitro free radical scavenging and antioxidant activities of cabergoline, a long-acting ergot DA agonist, as well as its ability to activate glutathione (GSH), catalase (Cat) and superoxide dismutase (SOD) activating effects and its in vivo neuroprotective properties against 6-hydroxydopamine (6-OHDA) intracerebroventricularly (i.c.v.) in mice. The striatal DA turnover induced by i.c.v. injection of 6-OHDA was completely normalized by pretreatment with cabergoline. Moreover, cabergoline scavenged free radicals in vitro and significantly reduced lipid peroxidation in vitro and in vivo. Furthermore, daily administration of cabergoline to mice significantly increased striatal GSH levels by activation of RNA expressions of GSH-related enzymes, although striatal Cat and SOD activities did not change. In addition, our present results suggest that repeated administration of cabergoline attenuates both 6-OHDA-induced nigrostriatal DAergic dysfunction and DA neuronal cell death, since cabergoline also had a neuroprotective effect in the immunohistochemical experiment. In conclusion, our findings indicate that the multiple antioxidant mechanisms of cabergoline, such as activation of the GSH system and the direct free radical scavenging activity, may explain the neuroprotective effect of this ergot DA agonist.

The coffee components kahweol and cafestol (K/C) were reported to be protective against mutagenic damage by heterocylic amines and aflatoxin B1 in the rat, while in humans the consumption of coffee with a high K/C content was associated with a lower rate of colon tumors. An important mechanism of this antimutagenic effect appears to be the potential of K/C to induce glutathione-S-transferase (GST) and to enhance hepatic levels of glutathione (GSH), the co-factor of GST, which is independently involved in further protective mechanisms. In the present study, we investigated mechanisms and organ specificities (liver, kidney, lung, colon) of the K/C effect on GSH levels, and particularly the role of gamma-glutamylcysteine synthetase (GCS), the rate limiting enzyme of GSH synthesis. Chows containing one of four concentrations of either a 1:1 mixture of K/C (0.012-0.122%) or of cafestol alone (0.006-0.061%) were fed to male F344 rats for 10 days. In the K/C-treated livers, a dose-dependent increase of up to 2.4-fold in the activity of GCS was observed, being statistically significant even at the lowest dose, and associated with an increase in GSH of up to three-fold. Notably, the highest dose doubled the hepatic mRNAs of the heavy and light subunits of GCS, suggesting enhanced transcription. In the extrahepatic organs, GCS activity and GSH levels were increased as well, although more moderately than in the liver. Since enhancement of GCS had also been observed as a consequence of oxidative stress, the possibility of such an involvement in the actions of K/C was examined by determining hepatic thiobarbituric acid reactive substances and the ratio of oxidized and reduced GSH. However, no evidence of oxidative stress was detected. In summary, K/C increased GSH levels apparently through the induction of the rate limiting enzyme of GSH synthesis, which may be a key factor in the chemopreventive potential of coffee components.

The controlled step in de novo glutathione (GSH) synthesis is catalyzed by gamma-glutamylcysteine synthetase (gamma-GCS), a dimeric enzyme consisting of a heavy catalytic subunit (gamma-GCS-HS) and a light regulatory subunit (gamma-GCS-LS). We have previously reported that exposure to cadmium (Cd) induces pulmonary gamma-GCS-HS mRNA and protein, and that these alterations are accompanied by increases in GSH synthesis and its steady-state level. The current study was designed to test the hypothesis that Cd exposure also up-regulates the expression of the regulatory gamma-GCS subunit. By using northern blotting, we have demonstrated that a single Cd aerosol exposure of adult male Lewis rats results in time- and dose-dependent increases in pulmonary levels of gamma-GCS-LS mRNA. Transcripts of gamma-GCS-LS in rat lung are maximally elevated (8-fold) 2 h following Cd inhalation exposure and remain significantly higher than air controls at 24 h. This response is highly correlated with Cd dose, ranging from 0.9 to 5 mg Cd per m(3), and with lung Cd burden. We also observed Cd-induced up-regulation of gamma-GCS-LS mRNA expression in alveolar epithelial cells exposed to Cd in vitro, either acutely or after repeated passaging in Cd-containing medium. The magnitude of the gamma-GCS regulatory subunit induction observed in Cd-treated cells was approximately five times greater than the induction of the catalytic subunit. These modifications in the expression of gamma-GCS subunits may offer protection from Cd toxicity.

Previous studies indicate that the ability of cells to up-regulate levels of intracellular glutathione (GSH) synthesis may determine their sensitivity to MeHg exposure. The purpose of the current study is two-fold. First, we determined whether the vulnerability of the developing central nervous system (CNS) to MeHg lies in its intracellular GSH content. The intracellular GSH content and the activity of gamma-glutamyl cysteine synthetase (GCS) were determined with and without MeHg exposure in primary cultures of rat embryonic CNS cells. In addition, the effect of GSH modulation on MeHg-induced cytotoxicity was determined. Second, we characterized the mechanism of GCS regulation, initially by studying the GCS heavy chain subunit (GCS-HC). Primary embryonic limb bud cells were used as a reference cell type for comparing the response of CNS cells. The results indicate that constitutive intracellular GSH content, GCS activity, and GCS-HC mRNA and protein levels of CNS cells were approximately ten-, two-, five-, and ten-fold higher, respectively, than those in limb bud cells. A dose-dependent increase in GSH levels and GCS activity was observed in CNS and limb bud cells following 1 and 2 microM MeHg exposure for 20 hr. Further characterization of GCS up-regulation in CNS cells showed that the increase in GCS activity following MeHg exposure, unlike limb bud cells, was not accompanied by an elevation of GCS-HC mRNA and protein levels. Pretreatment with N-acetylcysteine led to a significant increase in intracellular GSH, while L-buthionine-(S,R)-sulfoximine (BSO) resulted in decreased GSH levels, however neither pretreatment had a significant impact on MeHg-induced cytotoxicity in either cell type. Our results suggest that although oxidative stress may mediate aspects of MeHg toxicity, disruption of GSH homeostasis alone is not responsible for the sensitivity of embryonic CNS cells to MeHg.

To define the essential amino acid residues of Yap1 in stress response, we generated yap1 mutations by in vitro mutagenesis, which cause defects in mediating resistance to the stress of H2O2, but not of CdCl2. Sequence analysis of the mutant yap1 genes revealed three point mutations and two truncation mutations near the carboxy-terminus. The truncation mutations resulted in hyperresistance to cadmium. Northern blot analysis of stress-induced levels of TRX2 and GSH1 mRNAs indicated that the ability of the mutant Yap1 protein to induce transcriptional activation of target genes correlates well with its ability to confer stress resistance. The carboxy-terminal domain of Yap1 appears to act negatively in cadmium resistance.