Cytotoxicity induced by grape seed proanthocyanidins: Role of nitric oxide

Grape seed proanthocyanidin extract (GPSE) at high doses has been shown to exhibit cytotoxicity that is associated with increased apoptotic cell death. Nitric oxide (NO), being a regulator of apoptosis, can be increased in production by the administration of GSPE. In a chick cardiomyocyte study, we demonstrated that high-dose (500 μg/ml) GSPE produces a significantly high level of NO that contributes to increased apoptotic cell death detected by propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. It is also associated with the depletion of intracellular glutathione (GSH), probably due to increased consumption by NO with the formation of S-nitrosoglutathione. Co-treatment with L-NAME, a NO synthase inhibitor, results in reduction of NO and apoptotic cell death. The decline in reduced GSH/oxidized GSH (GSSG) ratio is also reversed. N-Acetylcysteine, a thiol compound that reacts directly with NO, can reduce the increased NO generation and reverse the decreased GSH/GSSG ratio, thereby attenuating the cytotoxicity induced by high-dose GSPE. Taken together, these results suggest that endogenous NO synthase (NOS) activation and excessive NO production play a key role in the pathogenesis of high-dose GSPE-induced cytotoxicity.     

Excretion of endogenous boldione in human urine: Influence of phytosterol consumption

Boldenone (17-hydroxy-androsta-1,4-diene-3-one, Bol) and boldione (androst-1,4-diene-3,17-dione, ADD), are currently listed as exogenous anabolic steroids by the World Anti-Doping Agency. However, it has been reported that these analytes can be produced endogenously. Interestingly, only for Bol a comment is included in the list on its potential endogenous origin. In this study, the endogenous origin of ADD in human urine was investigated, and the potential influence of phytosterol consumption was evaluated.We carried out a 5-week in vivo trial with both men (n = 6) and women (n = 6) and measured α-boldenone, β-boldenone, boldione, androstenedione, β-testosterone and α-testosterone in their urine using gas chromatography coupled to multiple mass spectrometry (GC–MS–MS). The results demonstrate that endogenous ADD is sporadically produced at concentrations ranging from 0.751 ng mL⁻¹ to 1.73 ng mL⁻¹, whereas endogenous Bol could not be proven. We also tested the effect of the daily consumption of a commercially available phytosterol-enriched yogurt drink on the presence of these analytes in human urine. Results from this study could not indicate a relation of ADD-excretion with the consumption of phytosterols at the recommended dose. The correlations between ADD and other steroids were consistently stronger for volunteers consuming phytosterols (test) than for those refraining from phytosterol consumption (control). Excretion of AED, bT and aT did not appear to be dependent on the consumption of phytosterols.This preliminary in vivo trial indicates the endogenous origin of boldione or ADD in human urine, independent on the presence of any structural related analytes such as phytosterols.     

Pacifastin-related peptides: structural and functional characteristics of a family of serine peptidase inhibitors

Members of the pacifastin family are serine peptidase inhibitors, found in arthropods and have many members within different insect orders. Based on their structural characteristics, inhibitors of this peptide family are divided into two groups (I and II). Members of both groups exhibit specificity towards different types of serine peptidases. In addition, group I inhibitors display species selectivity. The specificity and selectivity of these inhibitors depends on the nature of their P1 residue and on additional interaction sites at the inhibitor's surface. Functional analysis studies have shown that crustacean pacifastin plays a key role in the immune response, whereas insect pacifastin-like peptides have multiple regulatory functions in processes involved in immunity, reproduction, phase transition, etc.     

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them.     

An NADH:quinone oxidoreductase active during biodegradation by the brown-rot basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k(cat)/K(m) for the quinones is greater than 10(8) M(-1) s(-1). The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Synthesis and evaluation of caffeic acid amides as antioxidants

A series of amides of caffeic acid has been synthesised and their antioxidant properties evaluated as lipid peroxidation inhibitors. Anilides of caffeic acid were found to be very efficient antioxidants with IC50's of 0.3 microM.