Extension of life span and stress resistance of Drosophila melanogaster by long-term supplementation with melatonin

According to the free radical theory of aging, free radicals are involved in the production of changes in cellular metabolism that lead to a time-dependent functional decline in all living beings. Consequently, antioxidant and/or free radicals scavengers may retard the aging process. We explored the effect of melatonin on the life span of Drosophila melanogaster (Oregon wild strain). It was presumed that given the antioxidant and free radicals scavenger properties of melatonin, this hormone would prevent oxidative damage to the fly tissues and slow down the process of aging. Melatonin, added daily to the nutrition medium at a concentration of 100 microg/ml, increased significantly the life span of D. melanogaster. The maximum life span was 61.2 days in controls and 81.5 days in melatonin fed flies. Relative to the controls, the percentage increase in the melatonin fed flies was 33.2% in maximum life span, 19.3% in the onset of 90% mortality, and 13.5% in median life span. Furthermore, in a test of superoxide mediated toxicity it was shown that melatonin treatment increased the resistance of D. melanogaster to paraquat. Finally, the augmented resistance to an ambient temperature of 36 degrees C was also a demonstration of the antioxidative protection provided by the hormone.     

Mitochondrial DNA and life span changes in normal and dewinged Drosophila at different temperatures

At 11 and 30 degrees C there was no change in the amount or in the buoyant density of nuclear DNA of male Oregon R Drosophila melanogaster with aging. When normal flies were maintained at 11 degrees C (at which temperature they do not fly), mitochondrial DNA content declined gradually with aging and 39.4% of the original mitochondrial DNA was lost at the median survival time of 152 days of age. For normal flies maintained at 30 degrees C, 86.5% of the mitochondrial DNA was lost during aging at the median survival time of 25 days. In contrast only a 39.2% decrease in the mitochondrial DNA occurred in dewinged flies maintained at 30 degrees C. A slow phase of mitochondrial DNA loss appears to be related to aging and a fast phase to flight activity. Removing the wings of flies eliminates the fast phase of DNA loss but only slightly improves life span (by less than 10%). Lowering the environmental temperatures to 11 degrees C also eliminates fast phase DNA loss and decreases the rate of the slow phase DNA loss. We conclude that mitochondrial DNA loss is related to both physical activity and to the aging process itself.     

Temperature-dependent trade-offs between longevity and fertility in the Drosophila mutant, methuselah

Single gene, hypomorphic mutations which extend the life spans of cold-blooded animals, such as the methuselah (mth) mutation in the fruit fly, Drosophila melanogaster, may have additional, deleterious effects on overall fitness. The hypotheses tested here were: (i) that the extension of life span by mth might be temperature-dependent, and (ii) that it might be associated with depression of reproductive output, physical activity, or the rate of metabolism. The effect of mth on life span was smaller in magnitude than reported previously, and it was both sex-specific and temperature-dependent. Female longevity was increased only at 29 degrees C, whereas for male flies the extension of mean life span diminished progressively from 15-25% 25-29 degrees C to 2% at 18 degrees C, and the survival time at 4 degrees C was decreased by 22-39%. Conversely, the lifetime reproductive output of mth mutants was decreased at 29 degrees C, but increased at 18-22 degrees C. The walking speed of mth flies was significantly elevated, but mth had no effect on the rate of oxygen consumption at 25 degrees C. Collectively, the results demonstrate that where the life span is extended, there is an offsetting effect on reproductive output, suggesting that mth induces trade-off effects and is not a direct, mechanistic regulator of the aging process.     

Ascorbic acid in Drosophila and changes during aging.

The ascorbic acid content of Drosophila melanogaster was found to be high in the absence of a dietary source. The amount of ascorbic acid per fly declined with aging in both the Oregon R and Swedish C strains. The median life span at 25 degrees C was 45 days for Swedish C and 59 days for Oregon R. The amount of ascorbic acid in Swedish C flies (0.078 micrograms/fly) was higher than that for Oregon R (0.058 micrograms/fly) for newly emerged flies but the rate of decline with aging was greater for Swedish C than Oregon R. The decline in ascorbic acid content with aging was 70.4% for Swedish C versus 19.9% for Oregon R. A brief cold shock was found to significantly increase the amount of ascorbic acid in Oregon R flies. Feeding the precursor of ascorbic acid synthesis, L-gulonolactone, did not improve the life span. Life-time feeding of ascorbic acid did not improve the life span of either Swedish C or Oregon R flies.     

Aerobic and anaerobic contributions to sustained activity in Xenopus laevis

In conditions of declining water PO2, Xenopus obtains the majority of resting oxygen needs from lung breathing at 15 degrees and 25 degrees C. The critical oxygen tension was 120 +/- 9 mm Hg at 15 degrees C, and 90 +/- 10 mm Hg at 25 degrees C. During 30 min stimulation of activity to complete exhaustion at 15 degrees C, frogs exhibited an aerobic capacity of 1.7 microliter O2.g-1.h-1 and accumulated 2.22 mg lactate . g-1. Following activity these animals exhibited an oxygen debt of 49.2 microliter O2.g-1. At 25 degrees C, Xenopus had an aerobic capacity of 16.1 microliter O2.g-1 and accumulated 1.94 mg lactate . g-1. Following activity, 25 degrees C frogs exhibited an oxygen debt of 261.9 microliter O2.g-1. Thus at 15 degrees C, Xenopus acquires 85% of the ATP used during a bout of sustained exhaustive activity from anaerobic sources, and 98% at 25 degrees C. Recovery from exhaustive activity was complete after 5 h at 25 degrees C and 9 h at 15 degrees C. Comparison of these data with those of other amphibians stimulated to sustained activity suggests that aquatic amphibians can tolerate a larger lactate load than can terrestrial forms.     

Decreased life span and membrane damage of carbamylated erythrocytes in vitro.

Red blood cells exposed to cyanate (CNO) in vitro have a concentration-dependent decreased cell survival time associated with an inhibition of the ability of the cell membrane to synthesize lipids. The t1/2 of rabbit erythrocytes exposed to 30 mM or 50 mM cyanate for 1 hr at 37 degrees C is reduced from the normal 24 days to 15 and 9 days, respectively. The cyanate-induced defect in membrane lipid metabolism is irreversible. Carbamylation of membrane proteins and damage to metabolism are minimized by limiting exposure in vitro to 15 mM cyanate at 4 degrees C for 30 min. Cells carbamylated under these conditions do not have a shortened life span. Levels of globin carbamylation of 0.5 moles CNO/mole hemoglobin, shown to be clinically effective in prolonging the life span of sickle erythrocytes, are obtained under these conditions and reach maximal levels after only 30 min of incubation. Carbamylation of blood in CPD anticoagulant is inferior to either ACD or heparin. The findings indicate that adequate carbamylation of sickle erythrocytes with minimal red cell membrane damage can be achieved without significant modification of the standard plasmapheresis procedure utilized by the working blood bank.     

Effects of ambient temperature on free radical generation, antioxidant defenses and life span in the adult housefly, Musca domestica

Life spans of poikilotherms like the housefly are shortened by elevation of ambient temperature. The objective of this study was to examine the possible involvement of active oxygen species in temperature induced life-shortening of the adult male housefly. Effects of varied ambient temperature, 20 degrees C and 28 degrees C, on life span, cyanide-resistant respiration, H2O2 concentration, superoxide dismutase (SOD) and catalase activities and glutathione (GSH) concentration were examined. Average life span of flies raised at 28 degrees C was about 52% lower than those raised at 20 degrees C. Rate of cyanide-resistant respiration, an indicator of oxygen free radical generation, was higher in flies raised at 28 degrees C, whereas steady-state concentration of H2O2 was decreased at this temperature. Catalase activity and GSH concentration were lower at 28 degrees C while SOD activity was unaffected by the ambient temperature. Results of this study suggest that life-shortening effects of elevated ambient temperature may be due, in part, to increased oxidative stress.     

The relationship between oxygen radical generation and impairment of myocardial energy metabolism following post-ischemic reperfusion

Oxygen radical toxicity has been implicated in the pathogenesis of myocardial reperfusion injury. In the present study we sought to document the existence of a precise temporal relationship between the time course of free radical generation and the time course of alterations of myocardial energy metabolism during early reperfusion. Rabbit hearts perfused within the bore of a 31-Phosphorous NMR spectrometer were subjected to 30 min of total global ischemia at 37 degrees C. At reflow, 12 control hearts received a bolus of normal perfusate and 12 hearts recombinant human superoxide dismutase (h-SOD) as a 60,000 IU bolus followed by a 100 IU/ml infusion for 15 min. Ischemia resulted in similar depletion of tissue ATP and phosphocreatine (PCr) in the two groups. During the first minute of reflow, recovery of PCr was similar in both groups. However, PCr recovery arrested in control hearts after 2 min, at 63% of baseline, and averaged 64 +/- 4% after 45 min of reperfusion. In contrast, h-SOD treated hearts recovered 86.7% of baseline PCr content after 2 min, 102% after 10 min of reperfusion (P less than 0.001), and 93 +/- 6.4% at the end of the 45 min of reflow (P less than 0.01). The time course of free radical formation during reperfusion was assessed by EPR spectroscopy using both the frozen tissue and the spin trapping methodologies. In control hearts, peak generation of oxygen radicals was reached after 20 s of reflow. h-SOD treatment decreased concentrations of the oxygen-centered radicals in myocardial tissue and of the radical-adducts in the coronary effluent by approximately 80%. Thus, in reperfused hearts peak oxygen radical generation is followed by the occurrence of alterations in the recovery of high energy phosphate metabolism. Both events were largely prevented by administration of h-SOD at reflow. These results provide strong support for a link between oxygen free radical generation and post-ischemic reperfusion injury.     

The effect of temperature, stress, and cortisol on plasma IGF-I and IGFBPs in sunshine bass

The mechanisms through which stress and cortisol regulate insulin like growth factor-I (IGF-I) and insulin like growth factor binding proteins (IGFBPs) were studied in sunshine bass, by measuring plasma IGF-I and IGFBPs in fish maintained at 5, 10, 15, 20, 25, or 30 degrees C, fish subjected to an acute 15 min confinement stress at 25 and 30 degrees C, and fish fed 100 mg cortisol/kg feed. Plasma IGF-I concentrations were higher at 25 and 30 degrees C than at 20 degrees C and below. A 15 min confinement stress resulted in a decrease in IGF-I 2h post-confinement. Plasma concentrations of IGFBP with molecular weights of 24, 28, and 33 kDa were similar for fish acclimated to different temperatures, except for 5 degrees C where a 33-kDa IGFBP was significantly reduced. After a 15 min low-water stress at 25 degrees C, a 33-kDa IGFBP was reduced and IGFBPs with molecular weights of 24 and 28 kDa were increased at 2 and 6h, respectively. A 15 min low-water stress at 30 degrees C, resulted in no change in levels of a 33-kDa IGFBP over the 6-h recovery period. However, levels of a 24- and 28-kDa IGFBP were significantly increased at 2 and 6h, respectively. A single feeding with 100 mg cortisol/kg feed increased plasma cortisol but did affect plasma concentrations of IGF-I or any of the three IGFBPs. Acute stress appears to result in a decrease in IGF-I, but the mechanism of the decrease does not appear to be caused by cortisol released during the stress.     

Effect of temperature on naloxone treatment in canine hemorrhagic shock

The effects of temperature on naloxone treatment in canine hemorrhagic shock were examined in 24 dogs hemorrhaged to a mean arterial blood pressure of 35 mm Hg (ambient temperature, 21 degrees C). After two hours of hypotension, the blood reservoir was clamped with no return of shed blood. Dogs were divided into three groups: Control (n = 8) received normal saline (0.5 cc/kg/hr); naloxone-cold (n = 8) and -warm (n = 8) received naloxone (2 mg/kg bolus and 2 mg/kg/hr constant infusion). Body temperature was maintained in four dogs with a warming blanket, and four dogs received no external warming. Rectal temperature fell to 34.2 +/- 0.9 degrees C in naloxone-cold animals; naloxone-warm animals were maintained at 38.6 +/- 0.1 degrees C by external warming. Control dogs rapidly deteriorated after reservoir clamping (survival, 18.6 +/- 5 min). Naloxone infusion significantly increased survival regardless of body temperature (cold, 125 +/- 21 min; warm, 199 +/- 13 min). Naloxone transiently increased mean arterial pressure and dP/dt in the colder dogs, while coronary perfusion, myocardial oxygen metabolism, and plasma beta-endorphin levels were unchanged. In the warmer dogs, naloxone significantly improved hemodynamic function and myocardial perfusion as indicated by the increased mean arterial pressure, cardiac output, stroke volume, dP/dt, and coronary blood flow. Furthermore, naloxone reduced plasma beta-endorphin levels and corrected the metabolic derangements of shock in this group. Our data indicate hypothermia significantly diminished the beneficial effects of naloxone treatment in canine hemorrhagic shock.     

Conjugated dienes in ischemic and reperfused myocardium: an in vivo chemical signature of oxygen free radical mediated injury

Free radical reactions have been suggested to be important events in the mechanism of myocardial injury during ischemia and reperfusion. Most of the in vivo evidence implicating free radical mediated events in the etiology of myocardial injury has been based on intervention studies which document the efficacy of oxygen free radical scavengers in improving function or tissue salvage. We have assayed free fatty acid oxidation products (hydroxy conjugated dienes) derived from myocardial phospholipid as chemical markers of oxidative injury. Biopsies, harvested from canine myocardium subjected to cardiopulmonary bypass with no aortic crossclamp (control), from pre-ischemic, end ischemic (at the end of 45 min of global normothermic ischemia induced by aortic crossclamp) and at 5, 10, 15, 30 and 60 mins of reperfusion were analyzed for hydroxy conjugated dienes using HPLC with structural confirmation by GC-MS. All biopsies were taken from non-necrotic myocardium as indicated by gross tetrazolium staining of myocardial cross sections. Trace levels of hydroxy conjugated dienes could be detected in the preischemic biopsies or control biopsies harvested from hearts subjected to cardiopulmonary bypass with no ischemia. At the end of 45 min ischemia, however, a significant increase in 18:2 and 20:4 hydroxy isomers was detected and confirmed by GC-MS (P less than 0.05 vs. control). After 5 mins of reperfusion a further significant increase in hydroxy conjugated dienes was noted with 18:2, 20:4 and 22:6 isomers being identified (P less than 0.01 vs. end ischemia). By 30 min of reperfusion the concentration of phospholipid oxidation products had returned to pre-ischemic levels. This study presents the first chemically rigorous in vivo evidence for the formation of products of phospholipid oxidation (hydroxy conjugated dienes) during myocardial ischemia and reperfusion and supports the concept of oxygen free radical mediated lipid peroxidation. This study emphasizes the formation of phospholipid oxidation products during ischemia and particularly during the early phase of reperfusion and illustrates the transient nature of these products in myocardial phospholipid.     

Once daily therapy with isosorbide-5-mononitrate causes endothelial dysfunction in humans: evidence of a free-radical-mediated mechanism.

OBJECTIVES: The aim of the study was to determine if isosorbide-5-mononitrate (IS-5-MN) 120 mg, taken once daily for 7 days, is associated with evidence of endothelial dysfunction and whether this effect is determined by increased free radical production. BACKGROUND: Tolerance to nitroglycerin is associated with increased free radical production and abnormal endothelial function. To date, no data is available concerning the effect of IS-5-MN, administered in clinically employed dosages, on endothelial function in humans. METHODS: A total of 19 healthy volunteers were randomized in a double-blind fashion to therapy with IS-5-MN (120 mg once daily) or placebo. After 7 days of treatment, forearm blood flow responses to acetylcholine (Ach; 7.5, 15, and 30 microg/min) and N-monomethyl-L-arginine (L-NMMA; 1, 2, and 4 mumol/min) were measured. In a separate study, after 7 days of therapy with IS-5-MN 120 mg once daily, the responses to Ach were assessed during intra-arterial coinfusion of vitamin C (24 mg/min) or saline. RESULTS: As compared with placebo, IS-5-MN caused significant blunting of the responses to both Ach (peak responses: placebo 127 +/- 31%; IS-5-MN 52 +/- 24%) and L-NMMA (peak responses: placebo 41 +/- 5%; IS-5-MN 22 +/- 8%). Vitamin C completely restored the forearm blood flow responses to Ach (peak responses: vitamin C 180 +/- 33%; saline 107 +/- 17%). CONCLUSIONS: We document for the first time that IS-5-MN impairs endothelial function in humans in vivo. Suggesting a role of oxygen free radicals, nitrate-induced abnormalities in endothelium-dependent vasomotor responses were reversed by the antioxidant vitamin C.     

A Novel Second-Generation Polyolefin Container for Storage of Single-Donor Apheresis Platelets

A non-blow-molded LE-2 polyolefin (PO) container was developed to store single-donor apheresis platelet concentrates (PCs) processed by the Haemonetics Plasma Collecting System (PCS) at 22 degrees C for over 1 day. We molded PO containers with heat-sealing of light-weight polymer alloy films with a thickness of 0.25 mm. The film was made of polymer blends consisting of polypropylene, styrene ethylene butylene styrene-block copolymer, and ethylene ethylacrylate copolymer. The PO container with enough strength and flexibility in routine practice has 2 and 1.6 times higher oxygen and carbon dioxide gas transfer properties than standard polyvinyl chloride (PVC) plastic containers. PCs (1-1.9 x 10(11) platelets) processed from 450 ml platelet-rich plasma were stored in 0.6-liter PO containers at 22 degrees C with flatbed agitation for up to 6 days. The pH of PCs was well maintained at the mean values of 7.0 in PO containers after 6 days in the well-oxygenated condition. The energy metabolism of stored platelets was determined. Oxygen consumption rates of platelets stored in PO containers averaged 1.5 nmol/min/10(9) platelets. The rates of glucose consumption and lactate production were 0.4 and 0.8 nmol/min/10(11) platelets, respectively. The rates of adenosine triphosphate (ATP) generation of platelets, 9.7 nmol/min/10(9) platelets, in PO containers did not differ from those in the PVC containers. Aggregation responses to adenosine diphosphate and hypotonic shock response of platelets were better maintained in PO containers. The morphological changes into sphere forms with projections and the appearance of unclassified forms were more frequently observed in PO than in PVC containers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chlorpromazine inhibits loss of contractile function, compliance and ATP in ischemic rabbit heart

Using a modified Langendorff preparation, rabbit hearts were either continuously perfused at 37 degrees C for 150 min, in the presence of O2 and substrate, or after a 30 min equilibration period exposed to global ischemia followed by 30 min of reperfusion. Ischemia, for 90 min at 27 decrees C or for 60 min at 37 degrees C was compared. Perfusion pressure, heart rate, and ventricular volume were maintained constant. Contractile function and metabolic status were assessed. The effect of chlorpromazine, administered (30 mg/kg IP) 30 min prior to sacrifice, was compared to the untreated animal. (1) Chlorpromazine had little effect on the contractile function or metabolic status of hearts continuously perfused for 150 min in the presence of O2 and substrate. (2) The chloropromazine-treated hearts maintained contractile function and metabolic status at preischemic levels following exposure to 90 min of global ischemia at 27 degrees C and reperfusion at 37 degrees C. Untreated hearts exhibited a severe deterioration in both contractile and metabolic parameters under the same conditions. Both untreated and chlorpromazine-treated hearts exhibited loss of contractile function after 60 min ischemia at 37 degrees C; untreated hearts had undetectable ATP levels while chlorpromazine-treated hearts exhibited low levels of ATP. (3) Untreated hearts, exposed to 90 min of ischemia at 27 degrees C, exhibited a significant loss in compliance, while the compliance of chlorpromazine-treated hearts was unchanged from pre-ischemic levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic rate is not reduced by dietary-restriction or by lowered insulin/IGF-1 signalling and is not correlated with individual lifespan in Drosophila melanogaster

The link between resting metabolic rate and aging, measured as adult lifespan, was investigated in Drosophila melanogaster by (i) comparing lifespan and metabolic rate of individual flies, (ii) examining the effect of dietary-restriction on the metabolic rate of adult flies, and (iii) comparing the metabolic rate of wild-type and insulin/IGF-1 signalling mutant chico1 flies. The resting oxygen consumption of 65 individually housed and fully fed Drosophila was measured weekly throughout their lifetime. There was no significant difference in the mass-specific rate of oxygen consumption between cohorts that differed in lifespan. Nor was there any statistical correlation between mass-specific oxygen consumption and lifespan of individual Drosophila. The average mass-specific rate of oxygen consumption at 25 degrees C was 3.52+/-0.07 microl O2 mg(-1) h(-1). Variation in mass-specific metabolic rate explained only 4% of variation in individual life span in these flies. Contrary to predictions from the 'rate of living' theory of aging lifetime oxygen consumption was not constant and the lifespan of individual flies accounted for 91% of their lifetime oxygen consumption. An average Drosophila consumes about 3 ml O2 during its adult life. Dietary-restriction had no effect on mass-specific resting metabolic rate both when measured as oxygen consumption by respirometry and when measured as heat production by microcalorimetry. The mass-specific resting heat production of fully fed adult flies at 25 degrees C averaged 17.3+/-0.3 microW mg(-1). Similarly there was no difference in mass-specific metabolic rate of wild-type flies and longliving chico1 insulin/IGF-1 signalling mutant flies, either when measured as oxygen consumption or heat production. Thus, individual variation in lifespan in wild-type flies, and life extension by dietary-restriction and reduced insulin/IGF-1 signalling is not attributable to differences in metabolic rate.     

Antioxidants, metabolic rate and aging in Drosophila

In line with the (metabolic) rate-of-living theory of aging, previous work from this laboratory showed that the life-prolonging effect of the antioxidant thiazolidine carboxylic acid (TCA) in Drosophila was paralleled by a similar reduction of the oxygen consumption rate of the flies. To assess the generality of this phenomenon, several life-prolonging antioxidants were dietarily administered to the flies (in standard medium with 1% w/v of tocopherol-stripped corn oil) and their effects on metabolic rate and life span were determined. Respiration rate of groups of continuously agitated flies was measured in the Gilson respirometer. The studied antioxidants were as follows: (the numbers in parentheses are consecutively the antioxidant concentration in the medium in % wt/vol.; mean life span in days; and metabolic rate in microliter O2/mg fly per 24 h): vitamin E (0.4; 46.3; 58.5); 2,4-dinitrophenol (0.1; 45.7; 66.2); nordihydroguaiaretic acid (0.5; 45.6; 69.1); thiazolidine carboxylic acid (0.3; 53.1; 55.8); and control with no antioxidant added (0; 40.7; 73.3). All of these antioxidants at the tested concentrations reduced oxygen consumption rate and increased mean life span; there was a significant negative linear correlation (r = -0.87) between mean life span and metabolic rate. These data suggest that some antioxidants may inhibit respiration rate in addition to their protective effect against free radical-induced cellular damage.     

Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus

The effect of temperature on the ability of Aedes aegypti to transmit dengue (DEN) 2 virus to rhesus monkeys was assessed as a possible explanation for the seasonal variation in the incidence of dengue hemorrhagic fever in Bangkok, Thailand. In two laboratory experiments, a Bangkok strain of Ae. aegypti was allowed to feed upon viremic monkeys infected with DEN-2 virus. Blood-engorged mosquitoes were separated into two groups and retained at constant temperatures. Virus infection and transmission rates were determined for Ae. aegypti at intervals ranging from 4 to 7 days during a 25-day incubation period. Results of the first experiment for mosquitoes infected with a low dose of DEN-2 virus and maintained at 20, 24, 26, and 30 degrees C, indicated that the infection rate ranged from 25% to 75% depending on the incubation period. However, DEN-2 virus was transmitted to monkeys only by Ae. aegypti retained at 30 degrees C for 25 days. In the second experiment, the infection rate for Ae. aegypti that ingested a higher viral dose, and incubated at 26, 30, 32, and 35 degrees C ranged from 67% to 95%. DEN-2 virus was transmitted to monkeys only by mosquitoes maintained at greater than or equal to 30 degrees C. The extrinsic incubation period was 12 days for mosquitoes at 30 degrees C, and was reduced to 7 days for mosquitoes incubated at 32 degrees C and 35 degrees C. These results imply that temperature-induced variations in the vector efficiency of Ae. aegypti may be a significant determinant in the annual cyclic pattern of dengue hemorrhagic fever epidemics in Bangkok.     

Analysis of the stability of stored adenosine 5'-monophosphate used for bronchoprovocation

Adenosine 5'-monophosphate (AMP) bronchial challenge has been shown to be very useful tool in the diagnosis of asthma. Freshly test solutions are prepared just prior to each test in most of the studies. The objective of this study was to assess the stability of AMP solutions at different temperatures using a reversed-phase high-performance liquid chromatography assay. Sodium salt AMP solutions in concentrations of 0.03 mg/ml and 400 mg/ml were analyzed. One aliquot was kept at room temperature (20-25 degrees C) and the others were refrigerated at 4 degrees C. Room temperature stored samples were analyzed daily. Refrigerated stored samples were analyzed daily for first 15 days and then weekly. The duration of the study was 25 weeks. Samples were injected into the chromatograph column in quadruplicate and quantification was based on the arithmetic mean and standard deviation (+/-SD) of four measurements. Room temperature stored samples at concentrations of 0.03 mg/ml showed a mean percent variation greater than 10% at day 9 and greater than 75% at day 14. Samples at concentrations of 400 mg/ml maintained almost the initial concentration during the first 10 days, but decomposition occurred thereafter. In contrast, there was no significant degradation of refrigerated stored samples throughout the study period. We conclude the exposure to room temperature of AMP solutions results in a substantial loss of the initial concentration, but the shelf life of adequately prepared stock AMP solutions stored at 4 degrees C is at least 25 weeks.     

Lifelong alpha-tocopherol supplementation increases the median life span of C57BL/6 mice in the cold but has only minor effects on oxidative damage

The effects of dietary antioxidant supplementation on oxidative stress and life span are confused. We maintained C57BL/6 mice at 7 +/- 2 degrees C and supplemented their diet with alpha-tocopherol from 4 months of age. Supplementation significantly increased (p = 0.042) median life span by 15% (785 days, n = 44) relative to unsupplemented controls (682 days, n = 43) and also increased maximum life span (oldest 10%, p = 0.028). No sex or sex by treatment interaction effects were observed on life span, with treatment having no effect on resting or daily metabolic rate. Lymphocyte and hepatocyte oxidative DNA damage and hepatic lipid peroxidation were unaffected by supplementation, but hepatic oxidative DNA damage increased with age. Using a cDNA macroarray, genes associated with xenobiotic metabolism were significantly upregulated in the livers of female mice at 6 months of age (2 months supplementation). At 22 months of age (18 months supplementation) this response had largely abated, but various genes linked to the p21 signaling pathway were upregulated at this time. We suggest that alpha-tocopherol may initially be metabolized as a xenobiotic, potentially explaining why previous studies observe a life span extension generally when lifelong supplementation is initiated early in life. The absence of any significant effect on oxidative damage suggests that the life span extension observed was not mediated via any antioxidant properties of alpha-tocopherol. We propose that the life span extension observed following alpha-tocopherol supplementation may be mediated via upregulation of cytochrome p450 genes after 2 months of supplementation and/or upregulation of p21 signaling genes after 18 months of supplementation. However, these signaling pathways now require further investigation to establish their exact role in life span extension following alpha-tocopherol supplementation.     

Role of serum in stimulation of polymorphonuclear leukocyte, luminol-dependent chemiluminescence by influenza A

Granulocyte membrane perturbation activates oxidative metabolism with the release of highly reactive species (O2-, H2O2, OH., and 'O2) and emission of light (chemiluminescence (CL)). Using the CL response as a measure of oxidative metabolism, we assayed the effects of influenza A on the granulocyte respiratory burst. Human polymorphonuclear leukocytes (PMNs) were isolated by Ficoll-Hypaque cushioning and dextran sedimentation. The isolated PMNs were incubated with egg-grown influenza A (H3N2) virus, or a medium control, in the presence of 1 microM luminol and fresh autologous serum (10%). No light emission occurred during the incubation of PMNs with the medium control. Influenza A (33 to 50% egg-infective-doses (EID50):1 PMN) stimulated PMN light emission with a maximal response (48,386 +/- 10,764 cpm/10(6) PMN) occurring at 37 degrees CL was dependent on the virus dose with a diminished response (6,041 +/- 3,200 cpm/10(6) PMN) occurring at a lower infectivity of 10 EID50:1 PMN. Chemiluminescence responses were similar with infective and with noninfective virus particles (heat inactivated, 56 degrees C X 2 h). Fresh serum was necessary for the influenza virus to cause a CL response. A significant correlation (p less than 0.01) existed between the level of light emission and the hemagglutination-inhibiting (HI) antibody titer to influenza A of the autologous serum. Virus in the absence of detectable antibody did not stimulate CL. The virus-associated CL was completely inhibited if autologous serum was heated (56 degrees C X 30 min) or if the PMNs were pretreated with cytochalasin B (5 mcg/ml X 5 min). These findings suggest that influenza A-associated PMN CL requires antibody, complement, and phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)