Tyrosol and its metabolites as antioxidative and anti-inflammatory molecules in human endothelial cells.

Tyrosol (Tyr) is a phenolic compound found in virgin olive oil. After ingestion, Tyr undergoes extensive first pass intestinal/hepatic metabolism. However, knowledge about the biological effects of Tyr metabolites is scarce. We chemically synthesized Tyr glucuronate (Tyr-GLU) and sulphate (Tyr-SUL) metabolites and explored their properties against oxidative stress and inflammation in TNF-α-treated human umbilical vein endothelial cells (hECs). Tyr and Tyr-SUL prevented the rise of reactive oxygen species, the depletion of glutathione, and the down-regulation of glutathione peroxidase 1, glutamate-cysteine ligase catalytic subunit, and heme oxygenase-1 genes. Tyr-SUL and to a lower extent Tyr and Tyr-GLU prevented the phosphorylation of NF-κB signaling proteins. Tyr-GLU and Tyr-SUL also prevented the over-expression of adhesion molecules at gene, protein, and secretory levels, and the adhesion (Tyr-SUL > Tyr-GLU) of human monocytes to hECs. In vivo, Tyr, and most notably Tyr-SUL in a dose-dependent manner, ameliorated plantar and ear edemas in mice models of acute and chronic inflammation. This study demonstrates the antioxidant and/or anti-inflammatory properties of Tyr metabolites, with Tyr-SUL being the most effective.

Effect of treatment with geraniol on ovalbumin-induced allergic asthma in mice.

BACKGROUND: Asthma, a complex highly prevalent airway disease, is a major public health problem for which current treatment options are inadequate. OBJECTIVE: To evaluate the antiasthma activity of geraniol and investigate its underlying molecular mechanisms. METHODS: In a standard experimental asthma model, Balb/c mice were sensitized with ovalbumin, treated with geraniol (100 or 200 mg/kg) or a vehicle control, during ovalbumin challenge. RESULTS: Treatment of ovalbumin-sensitized/challenged mice with geraniol significantly decreased airway hyperresponsiveness to inhaled methacholine. Geraniol treatment reduced eotaxin levels in bronchoalveolar lavage fluid and attenuated infiltration of eosinophils induced by ovalbumin. Geraniol treatment reduced TH2 cytokines (including interleukins 4, 5, and 13), increased TH1 cytokine interferon γ in bronchoalveolar lavage fluid, and reduced ovalbumin-specific IgE in serum. In addition, treatment of ovalbumin-sensitized/challenged mice with geraniol enhanced T-bet (TH1 response) messenger RNA expression and reduced GATA-3 (TH2 response) messenger RNA expression in lungs. Furthermore, treatment of ovalbumin -sensitized/challenged mice with geraniol further enhanced Nrf2 protein expression and activated Nrf2-directed antioxidant pathways, such as glutamate-cysteine ligase, superoxide dismutase, and glutathione S-transferase, and enhanced formation of reduced glutathione and reduced formation of malondialdehyde in lungs. CONCLUSION: Geraniol attenuated important features of allergic asthma in mice, possibly through the modulation of TH1/TH2 balance and activation the of Nrf2/antioxidant response element pathway.

Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor.

Glutathione and tryptophan metabolism are required for Arabidopsis immunity during the hypersensitive response to hemibiotrophs.

The hypersensitive response (HR) is a type of strong immune response found in plants that is accompanied by localized cell death. However, it is unclear how HR can block a broad range of pathogens with different infective modes. In this study, we report that γ-glutamylcysteine synthetase GSH1, which is critical for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to HR and block development of fungal pathogens with hemibiotrophic infective modes. We found that GSH1 is involved in the penetration2 (PEN2)-based entry control of the nonadapted hemibiotroph Colletotrichum gloeosporioides. However, Arabidopsis mutants specifically defective in entry control terminated further growth of the pathogen in the presence of HR cell death, whereas gsh1 mutants supported pathogen invasive growth in planta, demonstrating the requirement of GSH1 for postinvasive nonhost resistance. Remarkably, on the basis of the phenotypic and metabolic analysis of Arabidopsis mutants defective in Trp metabolism, we showed that biosynthesis of Trp-derived phytochemicals is also essential for resistance to C. gloeosporioides during postinvasive HR. By contrast, GSH1 and these metabolites are likely to be dispensable for the induction of cell death during postinvasive HR. Furthermore, the resistance to Ralstonia solanacearum 1/resistance to Pseudomonas syringae 4 dual Resistance gene-dependent immunity of Arabidopsis to the adapted hemibiotroph shared GSH1 and cytochromes P450 CYP79B2/CYP79B3 with postinvasive nonhost resistance, whereas resistance to P. syringae pv. maculicola 1 and resistance to P. syringae 2-based Resistance gene resistance against bacterial pathogens did not. These data suggest that the synthesis of glutathione and Trp-derived metabolites during HR play crucial roles in terminating the invasive growth of both nonadapted and adapted hemibiotrophs.

Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used.

Sulforaphane inhibits de novo synthesis of IL-8 and MCP-1 in human epithelial cells generated by cigarette smoke extract.

Chronic obstructive pulmonary disease (COPD) is currently the fifth leading cause of death worldwide. Exposure to cigarette smoke (CS) is the primary factor associated with the COPD development. CS activates epithelial cells to secrete chemokines such as interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) that recruit neutrophils and macrophages to the lung. These inflammatory cells then release additional chemokines and cytokines leading to chronic inflammation that initiates apoptosis in epithelial and endothelial cells and destruction of alveolar structure. Pulmonary epithelium responds to oxidative stress mediated by CS through activating NRF2-dependent pathways, leading to an increased expression of antioxidant and cytoprotective enzymes thereby providing a protective response against CS-induced lung injury. We hypothesized that activating NRF2-dependent cytoprotective gene expression with sulforaphane (SFN) affords protection against CS-induced lung damage by inhibiting chemokine production. Results indicate that in the human BEAS-2B epithelial cell line, 5 µM SFN activated NRF2-dependent gene expression by triggering the translocation of NRF2 to the nucleus and significantly increased the expression of NRF2-dependent genes such as NADPH quinone oxidoreductase-1, heme oxygenase-1, and glutamate cysteine ligase modulatory subunit. Cigarette smoke extract (CSE) exposure of BEAS-2B cells significantly increased production of both IL-8 and MCP-1. Production of both chemokines was significantly reduced with SFN given prior to CSE; SFN inhibited IL-8 and MCP-1 gene expression at the transcription level. Our results indicate that activating NRF2 pathways with SFN inhibits CSE-induced chemokine production in human epithelial cells. However, the mechanism by which the production of chemokines is inhibited through SFN still remains to be elucidated. SFN may enhance NRF2 transcriptional activity resulting in the inhibition of proinflammatory pathways such as NF-κB.

Vaccination with recombinant Leishmania donovani gamma-glutamylcysteine synthetase fusion protein protects against L. donovani infection.

Visceral leishmaniasis presents a serious health threat in many parts of the world. There is, therefore, an urgent need for an approved vaccine for clinical use to protect against infection. In this study, the ability of recombinant Leishmania donovani gamma-glutamyl cysteine synthetase protein (LdγGCS) alone or incorporated into a non-ionic surfactant vesicle (NIV) delivery system to protect against L. donovani infection was evaluated in a BALB/c mouse model. Immunization with LdγGCS alone or LdγGCS-NIV induced specific IgG1 and IgG2a antibodies compared to controls, with LdγGCS-NIV inducing significantly higher titers of both antibody classes (P < 0.05). Both formulations induced similar increases in splenocyte IFN-γ production following ex vivo antigen stimulation with LdγGCS compared with cells from control mice (P < 0.05). Similar levels of protection against infection were induced by LdγGCS alone and LdγGCS-NIV, based on their ability to suppress liver parasite burdens compared to control values (P < 0.01), indicating that using a carrier system did not enhance the protective responses induced by the recombinant protein. The results of this study indicate that LdγGCS may be a useful component in a vaccine against L. donovani.

Gamma-glutamylcysteine synthetase from Plasmodium berghei.

Gamma-glutamylcysteine synthetase (L-glutamate-L-cysteine ligase, gamma-GCS, EC 6.3.2.2.), the rate limiting enzyme in glutathione biosynthetic pathway has been analysed in the asexual erythrocytic stages of rodent malaria parasite, Plasmodium berghei and its host erythrocytes. Cell-free parasite isolated by saponin lysis contained about 2 and 8 times higher activity of gamma-GCS compared to P. berghei-infected and normal mice erythrocytes respectively. Subcellular fractionation revealed that the enzyme was mainly confined to the cytosolic part of the parasite. gamma-GCS from P. berghei was purified employing ammonium sulphate precipitation, Sephadex G-200 gel filtration and anionic exchange chromatography on DEAE-cellulose. There was 51.6 fold purification of enzyme and its specific activity was 39.5 U/mg. SDS-PAGE showed P. berghei gamma-GCS as a heterodimer dissociating into two non-identical sub-units of 66 kDa and 57 kDa. The enzyme was observed as white band of activity on native polyacrylamide gel stained for specific gamma-GCS activity. Km values for L-Cys, ATP and L-Glu were 0.53 mM, 0.92 mM and 0.75 mM, respectively. The inhibition of gamma-GCS activity by glutathione was found to be competitive with respect to glutamate (Ki=1.53 mM) and non competitive to ATP and cysteine. Antimalarial drugs did not show any significant effect on parasite gamma-GCS. Parasite enzyme induced humoral response in mice demonstrated by ELISA, IFA and immunoblotting and exhibited partial protection against P. berghei infection suggesting a significant role of P. berghei gamma-GCS in malaria control.

Nrf2 is a critical modulator of the innate immune response in a model of uveitis.

Uveitis is an inflammatory condition that can lead to blindness. It is therefore important to understand the pathophysiology against which to develop targeted therapy. Herein, we tested whether the oxidant-responsive transcription factor Nrf2 is involved in regulating the innate immune response and oxidative damage in the LPS uveitis model. As shown by dihydroethidium staining, intraperitoneally injected LPS increased reactive oxygen species in the retina and iris-ciliary body of Nrf2+/+ and Nrf2-/- mice. After LPS injection, ICAM-1, IL-6, TNF-alpha, COX-2, iNOS, and MCP-1 mRNAs were increased more in the retina and iris-ciliary body of Nrf2-/- than in those of Nrf2+/+ mice. NQO-1 and GCLM, two Nrf2-responsive antioxidant enzymes, had reduced expression in Nrf2+/+ retinas after LPS injection, but no change in expression in Nrf2-/- mice. The number of FITC-Con A-labeled leukocytes adherent to the retinal vascular endothelium increased after LPS treatment in both Nrf2+/+ and Nrf2-/- mice compared to control injections, with more adherent leukocytes in Nrf2-/- than in Nrf2+/+ mice. Pretreatment with the Nrf2 activator 1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)imidazole increased antioxidant gene expression in the retina, reduced inflammatory mediator expression, and reduced leukocyte adherence to retinal vasculature after LPS treatment in Nrf2+/+ mice, but had no effect on Nrf2-/- mice. Treatment targeting the Nrf2 pathway may be a new therapy for uveitis.

DNA vaccination against the parasite enzyme gamma-glutamylcysteine synthetase confers protection against Leishmania donovani infection.

In this study the potential of using Leishmania donovani gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, gamma-GCS) as a rational target for vaccine development was determined. Mice, immunised with plasmid containing the full gene sequence for gamma-GCS (pVAXgammaGCS) or plasmid alone (pVAX control), were challenged with a high dose of L. donovani amastigotes to give a stringent test of the ability of the vaccine to protect against infection. Vaccination with pVAXgammaGCS resulted in the production of specific IgG1 and IgG2a antibodies and resulted in significantly lower liver parasite burdens compared to controls. Protection was also associated with a significant increase in cell-mediated immunity, demonstrated as an increase in nitrite production by ConA stimulated splenocytes, an increase in the percentage of splenic CD3+CD4+ cells, and enhanced granuloma maturation, compared to control values.

Rapid isolation of monoclonal antibodies. Monitoring enzymes in the phytochelatin synthesis pathway.

Genomics projects have identified thousands of interesting new genes whose protein products need to be examined at the tissue, subcellular, and molecular levels. Furthermore, modern metabolic engineering requires accurate control of expression levels of multiple enzymes in complex pathways. The lack of specific immune reagents for characterization and monitoring of these numerous proteins limits all proteomic and metabolic engineering projects. We describe a rapid method of isolating monoclonal antibodies that required only sequence information from GenBank. We show that large synthetic peptides were highly immunogenic in mice and crude protein extracts were effective sources of antigen, thus eliminating the time-consuming step of purifying the target proteins for antibody production. A case study was made of the three-enzyme pathway for the synthesis of phytochelatins. Enzyme-linked immunosorbent assays and western blots with the recombinant proteins in crude extracts demonstrated that the monoclonal antibodies produced to synthetic peptides were highly specific for the different target proteins, gamma-glutamyl cysteine synthetase, glutathione synthetase, and phytochelatin synthase. Moreover, immunofluorescence localization studies with antibacterial gamma-glutamyl cysteine synthetase and antiglutathione synthetase antibodies demonstrated that these immune reagents reacted strongly with their respective target proteins in chemically fixed cells from transgenic plants. This approach enables research to progress rapidly from the genomic sequence of poorly characterized target genes, to protein-specific antibodies, to functional studies.

Gamma-glutamylcysteine synthetase from erythrocytes.

gamma-Glutamylcysteine synthetase was isolated by means of a three-step method in highly active (specific activity, about 1400 units/mg) and apparently homogeneous form from rat erythrocytes. The enzyme has a molecular weight of about 100,000, and is composed of two subunits (Mr approximately 75,000 and 25,000). The erythrocyte enzyme exhibits physicochemical, catalytic, and immunological properties that closely resemble those displayed by rat kidney gamma-glutamylcysteine synthetase. The isolation procedure described here, which was also successfully applied to isolation of the enzyme from sheep erythrocytes, may be useful in exploring the properties of mutant forms of the enzyme.