Availability of non-carotenoid antioxidants affects the expression of a carotenoid-based sexual ornament

Carotenoids are responsible for much of the yellow, orange and red pigmentation in the animal kingdom, and the importance of such coloration as an honest signal of individual quality has received widespread attention. In particular, owing to the multiple roles of carotenoids as pigments, antioxidants and immunostimulants, carotenoid-based coloration has been suggested to advertise an individual's antioxidant or immune defence capacity. However, it has recently been argued that carotenoid-based signals may in fact be advertising the availability of different antioxidants, many of which (including various vitamins, antioxidant enzymes and minerals) are colourless and so would be uninformative as components of a visual signal, yet often have greater biological activity than carotenoids. We tested this hypothesis by feeding male sticklebacks (Gasterosteus aculeatus) a diet containing a fixed level of carotenoids and either low or high, but biologically realistic levels of the colourless antioxidant vitamins C and E. High-antioxidant diet males produced significantly more intensely coloured (but not larger) carotenoid-based regions of nuptial coloration and were preferred over size-matched males of the opposite diet treatment in mate-choice trials. Furthermore, there were positive correlations between an individual's somatic antioxidant activity and signal intensity. Our data suggest that carotenoid-based ornaments may honestly signal an individual's availability of non-carotenoid antioxidants, allowing females to make adaptive mate-choice decisions.     

The Formation of Free Lipid Radicals During the Electromanipulation of Protoplasts (Vicia faba)

Membrane lipid degradation of plant protoplasts of Vicia faba was characterized by the determination of ethane formation after electrofusion and electropermeabilization. Incubation of protoplasts with ascorbate or α-tocopherol before electromanipulation led to a decrease in ethane production. Fusion product yield could be increased by incubation with α-tocopherol. Using a simple model system, we investigated the active species involved in lipid peroxidation under conditions typically used for electromanipulation. The formation of lipid free radicals by electric pulses could be demonstrated.     

Interactions Of Vitamin E With Free Radicals And Membranes

-Tocopherol performs an antioxidant role in biological membranes by acting as a one-electron reductant. In micellar solutions it has been observed by pulse radiolysis that the micellar charge has a pronounced effect on the rate constant for repair of organic free radicals by -tocopherol. The interactions between -tocopherol and model bilayer lipid membranes have been studied by fluorescence spectroscopy. Quencing of -tocopherol fluorescence by acrylamide and some n-doxyl stearates shows the transverse distribution of -tocopherol in membranes to be affected by the physical state of the membrane lipids and by the salt concentration in the aqueous phase. Time-resolved fluorescence depolarization measurements, with a diphenylhexatriene-phospholipid conjugate as probe. demonstrate an increase in the bilayer order parameter on incorporation of -tocopherol into a membrane     

The Free Radical Scavenger, α-Lipoic Acid, Protects Against Cerebral Ischemia-Reperfusion Injury in Gerbils

-Lipoic acid (thioctic acid) was tested for its neuroprotective activity in a Mongolian gerbil model of forebrain ischemia/reperfusion. Adult gerbils were treated for 7 days with two intraperitoneal injections per day of -lipoic acid (20 mg/kg), vehicle or saline and on the 7th day the animals were subjected to 5 min of forebrain ischemia. Ischemic injury was assessed by monitoring the increases in locomotor activity and from the extent of damage to the CA1 hippocampal pyramidal cell layer after 5 days of recovery. By both criteria, -lipoic acid was neuroprotective against ischemia/reperfusion evoked cerebral injury.     

Validation of previous computer models and MD simulations of discoidal HDL by a recent crystal structure of apoA-I

HDL is a population of apoA-I-containing particles inversely correlated with heart disease. Because HDL is a soft form of matter deformable by thermal fluctuations, structure determination has been difficult. Here, we compare the recently published crystal structure of lipid-free (Δ185-243)apoA-I with apoA-I structure from models and molecular dynamics (MD) simulations of discoidal HDL. These analyses validate four of our previous structural findings for apoA-I: i) a baseline double belt diameter of 105 Å ii) central α helixes with an 11/3 pitch; iii) a “presentation tunnel” gap between pairwise helix 5 repeats hypothesized to move acyl chains and unesterified cholesterol from the lipid bilayer to the active sites of LCAT; and iv) interchain salt bridges hypothesized to stabilize the LL5/5 chain registry. These analyses are also consistent with our finding that multiple salt bridge-forming residues in the N-terminus of apoA-I render that conserved domain “sticky.” Additionally, our crystal MD comparisons led to two new hypotheses: i) the interchain leucine-zippers previously reported between the pair-wise helix 5 repeats drive lipid-free apoA-I registration; ii) lipidation induces rotations of helix 5 to allow formation of interchain salt bridges, creating the LCAT presentation tunnel and “zip-locking” apoA-I into its full LL5/5 registration.     

Rat lymphocytes express NMDA receptors that take part in regulation of cytokine production

Incubation of rat lymphocytes with homocysteine (HC) or homocysteic acid (HCA) was found to increase the stationary levels of free radicals in lymphocytes, the effect of both ligands being mediated by ionotropic receptors activated by N-methyl-D-aspactic acid (NMDA), the expression of which on rat lymphocyte membranes was earlier demonstrated. In agreement with these data, increase of free radicals in the lymphocyte cytoplasm is preceded by an increase in the intracellular calcium levels, activation of protein kinase C, nicotinamide adenine dinucleotide phosphate oxidase and/or nitric oxide synthase. Both HC and HCA increase the production of IFN-γ and TNF-α by lymphocytes and antagonist of NMDA receptors; MK-801 prevents this effect. The data presented show that rat lymphocyte membrane contains functionally active NMDA receptors, which regulate cytokine accumulation.     

The alpha-tocopherol content of leaves of pedunculate oak (Quercus robur L.)--variation over the growing season and along the vertical light gradient in the canopy

This study was performed in order to investigate whether the actual requirement for defence against photo-oxidative stress is reflected by the alpha-tocopherol (alpha-Toco) content in leaves of pedunculate oak (Quercus robur L.). Antioxidants and pigments were quantified in leaves that were collected on six days between May and September 2000 in a mixed pine/oak forest at canopy positions differing in light environment. Pools of hydrophilic antioxidants and photo-protective xanthophyll cycle pigments (V + A + Z) reflected the anti-oxidative demand, as these pools increased with the average light intensity to which the leaves were acclimated. The photo-protective demand was not the determinant of the alpha-Toco content of oak leaves, as (1) foliage of a young oak, exposed to low light levels in the understorey, contained higher amounts of this lipophilic antioxidant than leaves sampled from semimature oaks at canopy positions with a similar light environment, and (2) a strong increase in the alpha-Toco content over the growing season was detected at each investigated crown position, whereas the V + A + Z pool did not show a concomitant accumulation during leaf ageing. The rate of alpha-Toco accumulation differed distinctly between samples taken at different canopy positions.     

Protection of Heme Proteins by Vitamin E, Selenium, and β-Carotene Against Oxidative Damage in Rat Heart, Kidney, Lung and Spleen

Effects of the combination of vitamin E, selenium, and β-carotene on oxidative damage to rat heart, kidney, lung, and spleen were studied by measurement of the production of oxidized heme proteins (OHP) during spontaneous and prooxidant-induced oxidation. Male SD rats were fed with a vitamin E and selenium deficient diet or a diet supplemented with vitamin E, selenium, and β-carotene, Homogenates of heart, kidney, lung, and spleen were incubated at 37°C with and without the presence of bromotrichloromethane (CBrCl₃). The diet supplemented with antioxidants showed a strong protective effect against oxidative damage to heme proteins during the early stages of both spontaneous and CBrCl₃-induced oxidation in contrast to the antioxidant deficient diet. Synergism of multiple antioxygenic nutrients against oxidative damage to various animal tissues is discussed.     

Antioxidant Activities of Dihydrolipoic Acid and its Structural Homologues

The relationships between structure and antioxidant activity of dihydrolipoic acid (DHLA) were studied using homologues of DHLA: bisonor-DHLA (a derivative which lacks two carbons in the hydrophobic tail), tetranor-DHLA (which lacks four carbons) and a methyl ester derivative. It was observed that: i) DHLA homologues with shorter hydrocarbon tails (i.e., bisnor- and tetranor-DHLA) had greater ability to quench superoxide radicals (O^-₂); ii) no differences among homologues with different chain lengths were found for peroxyl radical (ROO) scavenging in aqueous solution, and iii) DHLA was the best membrane antioxidant in terms of ROO scavening and lipid peroxidation inhibition. Differences among the DHLA homologues in their antioxidant properties in polar and apolar environments generally agreed with differences in their partition coefficients. The methyl ester was the least effective antioxidant both in aqueous phase and in membranes. Tetranor-DHLA was found not only to be less effective in preventing ROO-induced lipid peroxidation, but also to induce lipid peroxidation in the presence of residual iron. Thus, the complexity of biological systems seems to complicate generalizations on the correlation of molecular structure with antioxidant activity of DHLA.     

Methyl vinyl ketone induces apoptosis in murine GT1-7 hypothalamic neurons through glutathione depletion and the generation of reactive oxygen species

,β-Unsaturated carbonyl compounds have been implicated in a number of environmentally-related diseases. Often, the presence of ,β-unsaturated carbonyl functionality as part of either an aliphatic or cyclic structure is considered a structural alert for cytotoxicity. We examined the cytotoxicity of methyl vinyl ketone (MVK), an aliphatic, straight-chain ,β-unsaturated carbonyl compound, in murine GT1-7 hypothalamic neurons. In addition to its widespread environmental occurrence, MVK was selected due to its extensive use in the chemical industry. Also, MVK is a close structural analog of hydroxymethylvinyl ketone that, in part, mediates the cytotoxic effects of 1,3-butadiene in vivo. It was found that MVK at low micromolar concentrations induced extensive cell death that retained key features of apoptosis such as chromatin condensation and DNA fragmentation. The MVK-induced apoptosis was associated with depletion of glutathione, disruption of mitochondrial transmembrane potential, and increased generation of reactive oxygen species (ROS). Supplementation of neuronal cells with Trolox offered partial, but significant, protection against the MVK-induced cytotoxicity, presumably due to scavenging of ROS in situ. The suggested sequence of events in the MVK-induced apoptosis in neuronal cells involves the depletion of cellular glutathione followed by an increased generation of ROS and finally the loss of mitochondrial function.     

Ubiquitin C-Terminal Hydrolase L1 Is Expressed in Mouse Pituitary Gonadotropes In Vivo and Gonadotrope Cell Lines In Vitro

The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating variousbiological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinatingenzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly andrestrictedly expressed in neural and reproductive tissues and plays significant roles inthese organs. Although the expression of UCH-L1 in the anterior pituitary gland has beenreported, the detailed localization and the role of UCH-L1 remain obscure. In the presentstudy, we detected UCH-L1 protein exclusively in hormone-producing cells, but notnon-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition,the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes andmammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed acomparative analysis using genetically UCH-L1-deficient gad mice.Significant decreases in the numbers of gonadotropes and mammotropes were observed ingad mice, suggesting a close involvement of UCH-L1 in these cells.Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Takentogether, this is the first report to definitely demonstrate the presence of UCH-L1 inmouse anterior pituitary gland, and our results might provide a novel insight for betterunderstanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in thereproduction.     

Free energy calculations on binding and catalysis by alpha-lytic protease: the role of substrate size in the P1 pocket

We present free energy calculations using molecular dynamics on different substrates of alpha-lytic protease in the gas phase, in solution, while forming a noncovalent Michaelis complex with the enzyme, and in a tetrahedral structure representing a transition state/intermediate for acylation by the enzyme. Various P1 substrates were studied, with P1 = Gly, Ala, Val, and Leu. In qualitative agreement with experiment, the enzyme was calculated to bind and catalyze most effectively substrates with P1 = Ala over those with P1 = Gly, Val or Leu. Also, the calculated relative solvation free energies of Gly----Ala and Ala----Val were in qualitative agreement with experimental values in corresponding model systems. However, the level of quantitative agreement with experiment achieved in our earlier study of relative binding and catalysis of native subtilisin and an Asn-155----Ala mutant was not achieved. We surmise that this is due to the greater difficulty in quantitatively simulating effects that are predominantly van der Waals and hydrophobic compared to those that are hydrogen bonding/electrostatic.     

A novel anti-peroxiredoxin autoantibody in patients with Kawasaki disease

Antibodies to the anti-oxidative peroxiredoxin (Prx) enzymes occur in both systemic autoimmune disease and vasculitis in adulthood. Because increased oxidative stress induces vasculitis in Kawasaki disease (KD), autoimmunity to Prxs in patients with KD was investigated. The presence of antibodies to Prx 1, 2 and 4 was analyzed by ELISA and Western blot. Of 30 patients with KD, 13 (43.3%) possessed antibodies to Prx 2, whereas these antibodies were present in only 1 of 10 patients (10.0%) with sepsis (4 with purulent meningitis and 6 with septicemia). In contrast, antibodies to Prx 1 and 4 were not detected in either group. There was no significant correlation among the titers of the three antibodies. Clinical parameters were compared between anti-Prx 2-positive and -negative patients. The presence of anti-Prx 2 antibodies correlated with a longer period of fever and poor response to high-dose γ-globulin therapy in patients with KD. Anti-Prx 2-positive patients had significantly greater excretion of urinary 8-isoprostaglandin than did anti-Prx 2-negative patients. These results provide the first evidence for an antibody to Prx 2 in patients with KD. They also suggest that this antibody might serve as a marker of disease severity and be involved in the pathophysiology of vasculitis in some patients with KD.     

Paradoxical enhancement of oxidative cell injury by overexpression of heme oxygenase-1 in an anchorage-dependent cell ECV304

There has been increasing evidence suggesting the potent anti-inflammatory roles of heme oxygenase-1 (HO-1) in protecting renal tubular epithelial cells, vascular endothelial cells, and circulating monocytes. Based on these findings, novel therapeutic interventions have been proposed to control the expression of endothelial HO-1 levels to ameliorate various vascular diseases. We evaluated the effect of HO-1 gene transfer into an anchorage-dependent cell, ECV304. Effect of HO-1 production on the cell injury induced by hydrogen peroxide was evaluated after hemin stimulation and after HO-1 gene transfection. Morphological changes and the induction of various anti-apoptotic proteins were examined at the same time. Levels of HO-1 expression were variable in different clones of HO-1-transfected ECV304 cells. Among these, the clones with moderate levels of HO-1 expression were significantly more resistant to oxidative stress. In contrast, those with the highest levels of HO-1 exhibited paradoxically enhanced susceptibility to oxidative injury. Interestingly, the cell survival after oxidative stress was in parallel with the levels of Bcl-2 expression and of fibronectin receptor, alpha5 integrin. It is suggested from these results, that excessive HO-1 not only leads to enhanced cell injury, but also prolongs the repair process of the injured endothelial tissue. However, HO-1 reduces the oxidative cell injury and protects the endothelial cells, if its expression is appropriately controlled. Copyright 2004 Wiley-Liss, Inc.     

Kinetic investigation of the effect of the amino acid side chains in the selective acidolysis of N-acyl-N,alpha,alpha-trialkyl glycine amides.

Accurate kinetic measurements of the rate constants for the acidolysis of five N-acetyl-N-(4-methoxybenzyl)-alpha,alpha-trialkyl glycine cyclohexyl amides in TFA were performed at 25 degrees C and the reactions monitored by HPLC. In all cases the results were consistent with a first order behaviour with respect to the substrate. No direct correlation was obtained with these data between the rate constant values and structure, but a good correlation coefficient was obtained when a multiple regression analysis was applied by taking advantage of a Taft equation using appropriate polar and steric substituent parameters. In a plot of the values observed for log k against those calculated by this equation all five points fell very close to the line of perfect correlation. The calculated sensitivity coefficients to polar and steric contributions were used to discuss the experimental results and showed that the acidolysis were comparatively less affected by steric effects than expected.