The role of the neurohormone melatonin as a buffer against macromolecular oxidative damage

This paper summarizes the recent findings which show that the neural hormone melatonin is a free radical scavenger and general antioxidant. When compared with other antioxidants melatonin seems to have greater efficacy in protecting against cellular oxidative stress. These findings illustrate that melatonin preserves macromolecules including DNA, protein and lipid from oxidative damage following the administration of the chemical carcinogen, safrole, after exposure to ionizing radiation, following glutathione depletion, and after administration of the free radical generating herbicide, paraquat. In vitro evidence shows that melatonin is a potent scavenger of the highly toxic hydroxyl radical and in vitro evidence suggests that melatonin is an important and powerful antioxidant. Considering its high lipophilicity and its non-toxic nature as well as its ability to readily cross the blood-brain barrier, the neurohormone melatonin may prove to be an effective and important molecule in the antioxidative defense system, especially in the central nervous system. Besides the ease with which melatonin enters the brain, neurons seem to accumulate readily this hormone.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.     

Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen

Pablos, Marta I., Russel J. Reiter, Jin-Ing Chuang, GenaroG. Ortiz, Juan M. Guerrero, Ewa Sewerynek, Maria T. Agapito, DanielaMelchiorri, Richard Lawrence, and Susan M. Deneke. Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen. J. Appl.Physiol. 83(2): 354-358, 1997.Hyperbaric oxygenexposure rapidly induces lipid peroxidation and cellular damage in avariety of organs. In this study, we demonstrate that the exposure ofrats to 4 atmospheres of 100% oxygen for 90 min is associated withincreased levels of lipid peroxidation products [malonaldehyde(MDA) and 4-hydroxyalkenals (4-HDA)] and withchanges in the activities of two antioxidative enzymes[glutathione peroxidase (GPX) and glutathione reductase (GR)], as well as in the glutathione status in the lungs and in the brain. Products of lipid peroxidation increased after hyperbaric hyperoxia, both GPX and GR activities were decreased, and levels oftotal glutathione (reduced+oxidized) and glutathione disulfide (oxidized glutathione) increased in both lung and brain areas (cerebralcortex, hippocampus, hypothalamus, striatum, and cerebellum) but not inliver. When animals were injected with melatonin (10 mg/kg) immediatelybefore the 90-min hyperbaric oxygen exposure, all measurements ofoxidative damage were prevented and were similar to those in untreatedcontrol animals. Melatonin's actions may be related to a variety ofmechanisms, some of which remain to be identified, including itsability to directly scavenge free radicals and its induction ofantioxidative enzymes via specific melatonin receptors.

     

Melatonin administration prevents lipopolysaccharide-induced oxidative damage in phenobarbital-treated animals

The protective effect of melatonin on lipopolysaccharide (LPS)-induced oxidative damage in phenobarbital-treated rats was measured using the following parameters: changes in total glutathione (tGSH) concentration, levels of oxidized glutathione (GSSG), the activity of the antioxidant enzyme glutathione peroxidase (GSH-PX) in both brain and liver, and the content of cytochrome P450 reductase in liver. Melatonin was injected intraperitoneally (ip, 4mg/kg BW) every hour for 4 h after LPS administration; control animals received 4 injections of diluent. LPS was given (ip, 4 mg/kg) 6 h before the animals were killed. Prior to the LPS injection, animals were pretreated with phenobarbital (PB), a stimulator of cytochrome P450 reductase, at a dose 80 mg/kg BW ip for 3 consecutive days. One group of animals received LPS together with N^w-nitro-L-arginine methyl ester (L-NAME), a blocker of nitric oxide synthase (NOS) (for 4 days given in drinking water at a concentration of 50 mM). In liver, PB, in all groups, increased significantly both the concentration of tGSH and the activity of GSH-PX. When the animals were injected with LPS the levels of tGSH and GSSG were significantly higher compared with other groups while melatonin and L-NAME significantly enhanced tGSH when compared with that in the LPS-treated rats. Melatonin alone reduced GSSG levels and enhanced the activity of GSH-PX in LPS-treated animals. Additionally, LPS diminished the content of cytochrome P450 reductase with this effect being largely prevented by L-NAME administration. Melatonin did not change the content of P450 either in PB- or LPS-treated animals. In brain, melatonin and L-NAME increased both tGSH levels and the activity of GSH-PX in LPS-treated animals. The results suggest that melatonin protects against LPS-induced oxidative toxicity in PB-treated animals in both liver and brain, and the findings are consistent with previously published observations related to the antioxidant activity of the pineal hormone.     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Transmethylation reactions regulate affinity and functional activity of chemotactic factor receptors on macrophages

Methylation mediated by S-adenosyl-l-methionine is required for the chemotaxis of mononuclear leukocytes. We investigated whether transmethylation reactions are required for normal functioning of chemotactic factor receptors. Three chemoattracrant-mediated functions in macrophages, chemotaxis, the stimulated release of arachidonic acid from membrane phospholipids and superoxide production, are markedly depressed by agents that inhibit cellular methylation reactions. Treatment of macrophages with methylation inhibitors decreased the affinity of the N-formylated chemoattractant receptor present on these cells by a factor of 4.5, but did not significantly alter the total receptor number. These results suggest that the N-formylated chemoattractant receptor on macrophages can exist in more than one affinity state and that an ongoing methylation reaction is required for the maintenance of the receptor in its higher affinity form. Inhibition of methylation lowers the affinity of the receptor and renders it no nfunctional or “uncoupled” in its ability to produce chemotaxis, superoxide and the release of arachidonic acid from leukocyte membranes.     

Red light and auxin effects on rubidium uptake by oat coleoptile and pea epicotyl segments

Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [⁸⁶Rb⁺] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10⁻⁷ molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light-enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [⁸⁶Rb⁺] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [⁸⁶Rb⁺] flux is not a general feature of phytochrome action.     

Phytochrome-controlled Hydrogen Ion Excretion by Avena Coleoptiles

A red light-induced, far red reversible stimulation of proton efflux from apical segments of etiolated Avena sativa L. cv. Victory coleoptiles was observed. The acidification responses to red light and also to auxin were not the consequence of respired CO(2). The response to red light was strongly inhibited by cycloheximide and carbonyl cyanide, m-chlorophenyl hydrazone, but mannitol had a stimulatory effect. Red light and auxin applied together yielded a greater than additive response, in comparison to the effects of the two stimuli applied separately.     

Fusion of neurohypophyseal membranes in vitro

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca²⁺ in micromolar concentrations. Mg²⁺ and Sr²⁺ were ineffective. Mg²⁺ inhibited Ca²⁺-induced fusion.In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca²⁺. Mg²⁺ and Sr²⁺ were ineffective in replacing Ca²⁺ as trigger for fusion or vasopressin release.Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosome were exposed to cold.     

Vitamin D receptor interaction with specific DNA. Association as a 1,25-dihydroxyvitamin D3-modulated heterodimer.

The vitamin D receptor (VDR) is a member of the steroid receptor gene family. In this report, we examine the nature of specific VDR DNA binding utilizing the vitamin D-responsive element derived from the human osteocalcin promoter. Association of the VDR with the human osteocalcin 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) responsive element (VDRE) in vitro was characterized on VDRE affinity columns by both weak and strong interactions. Weak interaction was a property of the VDR itself, monomeric in nature, and determined exclusively by the VDR's DNA-binding domain. Strong interaction, in contrast, was dependent upon an intact receptor molecule as well as a heterologous mammalian cell nuclear accessory factor (NAF). Heteromeric interaction between VDR and NAF was independent of the VDR DNA-binding domain, suggesting the presence of a functional dimerization domain separate from that for DNA binding. Direct association of NAF with immobilized VDR revealed that the interaction does not require the presence of DNA. Most importantly, while occupancy of the VDR by 1,25(OH)2D3 was not required for VDR interactions with either DNA or NAF, the presence of hormone increased the apparent relative affinity of the VDR for NAF approximately 10-fold. These studies suggest that high affinity association of the VDR with DNA requires both the DNA-binding domain as well as an additional independent structure located within the steroid-binding region. This protein subdomain interacts with NAF and is regulated by 1,25(OH)2D3.