Evaluation of the mechanisms involved in leucine-induced oxidative damage in cerebral cortex of young rats

Maple syrup urine disease (MSUD) is a metabolic disorder caused by the deficiency of the activity of the mitochondrial enzyme complex branched-chain l-2-keto acid dehydrogenase. The metabolic block results in tissue and body fluid accumulation of the branched-chain amino acids leucine (Leu), isoleucine and valine, as well as of their respective α-keto acids. Neurological sequelae are usually present in MSUD, but the pathophysiologic mechanisms of neurotoxicity are still poorly known. It was previously demonstrated that Leu elicits oxidative stress in rat brain. In the present study we investigated the possible mechanisms involved in Leu-induced oxidative damage. We observed a significant attenuation of Leu-elicited increase of thiobarbituric acid-reactive substances (TBA-RS) measurement when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus trolox, dithiothreitol, glutathione, and superoxide dismutase, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of N^ω-nitro-l-arginine methyl ester or catalase (CAT) did not affect TBA-RS values. We also demonstrated an inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and gluthathione peroxidase, as well as a significant reduction in the membrane-protein thiol content from mitochondrial enriched preparations. Furthermore, dichlorofluorescein levels were increased although not significantly by Leu. Taken together, our present data indicate that an unbalance between free radical formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage.     

Elevated plasma homocysteine elicits an increase in antioxidant enzyme activity

Elevated plasma homocysteine is considered to be a risk factor for cardiovascular disease. The mechanisms for this effect are not fully understood but there is some evidence for a role for reactive oxygen species (ROS). This study was conducted to explore the effects of elevated plasma total homocysteine (tHcy) concentration on activity of antioxidant enzymes in the circulation. The study group consisted of 10 patients with inherited defects of homocysteine metabolism, from whom 41 blood samples were collected over a period of six months. Blood samples were also collected from 13 of their obligate heterozygous parents. For data analysis samples were classified as those with plasma tHcy < 20 μM or ≥ 20 μM. The activity of erythrocyte superoxide dismutase (SOD) and plasma glutathione peroxidase (GSHPx) was elevated in samples with plasma tHcy > 20 μM. Moreover, a significant correlation was demonstrated between plasma GSHPx activity, plasma glutathione peroxidase protein and plasma tHcy. In vitro studies confirmed that this observation was not due to a simple chemical enhancement of enzyme activity. Homocysteine protected GSHPx from loss of activity following incubation at 37°C. A similar effect was seen with another thiol-containing amino acid, cysteine. Results suggest that elevated plasma tHcy represents an oxidative stress, resulting in an adaptive increase in activity of antioxidant enzymes in the circulation.     

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat Sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 μM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.     

Enzymic Pathways Involved in Cell Response to H2O2

An influence of possible interaction of glutathione peroxidase and cyclooxygenase on the clonogenic survival of epithelial cells exposed in vitro to H₂O₂ was investigated. Indomethacin served as the inhibitor of cyclooxygenase, and the use of alkaline (7.5) or acidic (6.5) pH combined with controlled supply of glucose modified glutathione peroxidase activity. Indomethacin affected survival of cells exposed to H₂O₂ in a biphasic manner, enhancing cytotoxicity at lower hydrogen peroxide concentrations, and diminishing it at higher concentrations. The turning point moved gradually to higher concentrations of H₂O₂ corresponding to the augmented decomposition of hydrogen peroxide caused by increased activity of glutathione peroxidase. The data revealed that both enzymic pathways interact in the presence of H₂O₂, resulting in the overall cell survival different from that obtained after inhibition of either.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.     

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix

Background

Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood.

Methods

We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested.

Results

MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed.

Conclusions

Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins.

General significance

Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.     

Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.