Iron induces oxidative stress and may alter the rate of aging in the housefly, Musca domestica

Iron is known to play a catalytic role in the generation of oxygen free radicals in vitro. The present study was conducted in order to determine the in vivo effects of iron intake. Administration of 2 mM ferrous chloride to adult male houseflies in their drinking water significantly shortened their life span, increased the concentration of inorganic peroxides and chloroform-soluble fluorescent material, and stimulated the activity of catalase. Levels of superoxide dismutase activity, glutathione and oxygen utilization were unaffected. Overall, these results indicate that iron causes oxidative stress in vivo and may influence the rate of aging.     

Effect of age and ambient temperature on n-pentane production in adult housefly, Musca domestica

The objective of this study was to examine the relationship between lipid peroxidation and aging in the male housefly. Metabolic rate of flies is known to be higher and life span shorter at elevated ambient temperature. Evolution of n-pentane and level of thiobarbituric acid (TBA) reactive material were used as indicators of lipid peroxidation. n-Pentane accumulated by houseflies in vivo and by whole body homogenates of houseflies, in response to tert-butyl hydroperoxide (1 mM), increased with age. n-Pentane accumulation in vivo was markedly higher at higher ambient temperature. Furthermore, n-pentane generated by flies in vivo and by fly homogenates in vitro tended to be lower in flies raised at a lower ambient temperature. TBA-reactive material, elicited by tert-butyl hydroperoxide, was augmented in older flies, but no significant difference was found between flies aged at different ambient temperatures. Analysis of fatty acids in housefly homogenates indicated an age-associated increase in the ratio of polyunsaturated to saturated fatty acids.     

Effects of exogenous antioxidants on the levels of endogenous antioxidants, lipid-soluble fluorescent material and life span in the housefly, Musca domestica

Effects of exogenous antioxidant administration (0.5% and 2% ascorbate, beta-carotene and alpha-tocopherol in sucrose) on life-span, metabolic rate, activities of superoxide dismutase and catalase, levels of glutathione, inorganic peroxides and chloroform-soluble fluorescent material (lipofuscin) were examined in adult male houseflies. Administration of antioxidants at a level of 0.5% did not affect life-span, whereas, 2% ascorbate and alpha-tocopherol decreased average life-span. Metabolic rate of flies was unaffected, except by 2% ascorbate, which caused a decrease. Superoxide dismutase activity was depressed by 2% ascorbate at all ages, and by beta-carotene and alpha-tocopherol in older flies. Catalase activity was unaffected except by alpha-tocopherol at younger ages. Glutathione concentration was decreased by ascorbate and beta-carotene at both concentrations administered. Inorganic peroxides (H2O2) were increased by 2% beta-carotene and alpha-tocopherol. Only high concentrations of ascorbate and beta-carotene decreased the level of soluble fluorescent material. Results suggest that administration of exogenous antioxidants causes a compensatory depression of endogenous defenses.     

Effects of diethyldithiocarbamate on life span, metabolic rate, superoxide dismutase, catalase, inorganic peroxides and glutathione in the adult male housefly, Musca domestica

The effect of inactivation of superoxide dismutase, by diethyldithiocarbamate, on life span, metabolic rate, superoxide dismutase activity, catalase activity, inorganic peroxide concentration and glutathione levels in adult male houseflies was determined. The general objective was to examine the role of free radical defenses in the aging process. Houseflies were administered 0. 1 and 10 mM diethyldithiocarbamate in their drinking water. Administration of diethyldithiocarbamate caused an extension in the average life spans of the populations while the metabolic rates were depressed. Superoxide dismutase activity was significantly reduced. Inorganic peroxide concentration and catalase activity declined in rough correspondence to the decrease in the metabolic rate. The levels of reduced glutathione were significantly elevated whereas oxidized glutathione concentrations remained relatively unaffected. The results are interpreted to indicate that a decrease in superoxide dismutase activity in the housefly, by diethyldithiocarbamate administration, is compensated by an elevation in reduced glutathione levels and reduction of oxygen consumption, suggesting the existence of alternative free radical defenses in vivo.     

Correlates of longevity in two strains of the housefly, Musca domestica

The general objective of this study was to identify biochemical correlates of longevity in the housefly by comparing two strains of flies that have different longevities. The average and the maximum life spans of the longer-lived "Cambridge" strain flies were 46% and 23%, respectively, greater than the shorter-lived "Thuron" strain flies. The hypothesis that longer-lived organisms have relatively more efficient mechanisms to minimize oxidative stress and maintain a relatively more reduced redox potential was tested. All measurements were made on 8-day-old male flies maintained under identical conditions. Flies of the longer-lived strain had a lower metabolic rate and contained lesser amounts of H2O2 and thiobarbituric acid-reactants than the flies of the shorter-lived strain. Reduced glutathione concentration and activities of catalase, glutathione reductase and thioltransferase were higher in the longer-lived strain indicating that longer-lived flies manifest lower levels of oxidative stress and greater ability to maintain a relatively more reducing environment than the shorter-lived flies. Superoxide dismutase (SOD) activity was similar in the two strains, but the SOD/metabolic rate ratio was higher in the longer-lived strain. Total activity of glutathione S-transferases was comparable in the two strains suggesting that differences in detoxification ability are not correlated with longevity. Only S-glutamylcysteine synthetase activity was greater in the shorter-lived strain suggesting that variation in longevity is not due to reduction in the ability to synthesize GSH. Overall, the results support the view that parameters associated with oxidative stress play a role in the aging process of the houseflies.     

Relationship between life expectancy and endogenous DNA single-strand breakage, strand break induction and DNA repair capacity in the adult housefly, Musca domestica

The objective of this study was to investigate the relationship between genomic damage and the physiological rate of aging. Endogenous DNA single-strand breaks, susceptibility of DNA to exogenously induced strand breaks and the capacity to repair strand breakage were compared, using the alkaline elution technique, in flies of the same chronological age but with different life expectancy. Distinctions between physiological and chronological ages were made (1) by experimentally altering the life spans of houseflies by varying the level of physical activity, and (2) by phenotypic selection of short- and long-lived cohorts from the same population. The degree of endogenous DNA single-strand breaks was found to be unrelated to physiological age. However, flies selected for relatively shorter life expectancy exhibited a greater susceptibility to exogenously-induced (gamma-irradiation) single-strand breakage. Flies with a longer life expectancy exhibited a more efficient repair capacity to reverse single-strand breakage than those with a shorter life expectancy.     

Effect of gymnemic acid on single taste hairs of the housefly, Musca domestica.

The effect of a gymnemate salt extract (KGE) on the behavioral and neurophysiological taste responses of Musca domestica to sugar and salt were studied. After exposure of single taste hairs to KGE, the behavioral positive response to a just-suprathreshold sugar stimulus was abolished, and the neuronal firing pattern was modified in a form that ranged from a waxing and waning of impulse frequently to an abolishment of impulses. The neurophysiological response to a salt stimulus was similarly modified, although the waxing and waning of spike frequency was more rapid and firing returned to a near-control pattern sooner. Neurophysiological studies were replicated with two other fly genera. The results uniformly suggest a narcotic effect on the dendrites of taste neurons.     

Spontaneous and evoked quantal neurotransmitter release at the neuromuscular junction of the larval housefly,Musca domestica

The release of neurotransmitter was monitored at the neuromuscular junctions of larval housefly ventrolateral muscles 6a and 7a, using intracellular recording, and a loose patch clamp technique to isolate discrete release sites. Transmitter release occurred spontaneously and could also be evoked by neural stimuli. Spontaneous discharges consisted of events which were randomly distributed in time and of bursts of temporally ordered events. Evoked and spontaneous release occurred in a quantal manner. The quantal content of evoked excitatory postsynaptic currents (EPSCs) was dependent upon the extracellular calcium concentration, increasing with a 3.8 power dependency. The relationship between the quantal content of a response and extracellular calcium concentration was offset by the presence of magnesium in the bathing saline. The rates of decay of miniature EPSCs (mEPSCs) and EPSCs were also found to increase with extracellular calcium concentration, consistent with a non-diffusion limited block of the glutamate receptor-channel complex by calcium ions (K _B 2.5×10⁴ s^–1 M^–1,P<0.01). The frequency of random mEPSCs (0.26±0.32 Hz,n=24 cells) was independent of the extracellular calcium concentration. Random mEPSCs were not inhibited by 1 M tetrodotoxin which blocked mEPSC bursts and neurally evoked responses. EPSCs evoked during mEPSC bursts had a significantly lower quantal content than those EPSCs recorded from the same nerve terminal between bursting, indicating that both of these forms of release recruited quanta from a common pool of transmitter. Following a neurally evoked EPSC the mEPSC frequency was potentiated severalfold, this delayed release was influenced by EPSCs with large quantal contents evoked in saline containing elevated calcium concentrations. The alpha-cyano pyrethroid insecticide, cypermethrin, when applied at 10 nM concentration to this NMJ, caused a sustained 10-fold increase of transmitter quanta (240% of control), and blocked neurally evoked EPSCs after ca. 20 min (22°C). mEPSC frequency remained elevated for several hours after treatment with cypermethrin (ca. 3 Hz), before declining below control levels (0.26 Hz). A model based upon the frequency of mEPSC discharges, the quantal content of evoked responses, and the association constants of calcium to the four binding sites of calmodulin was used to investigate a possible role for this metalloprotein in both the spontaneous and evoked release of neurotransmitter. The probable lack of involvement of calmodulin in the mechanism of neurotransmitter release at the larval housefly neuromuscular junction is discussed in the light of this model, as are the implications of these findings for vesicular amino acid release in relation to pyrethroid insecticide action.Abbreviations used [Ca] ionic calcium concentration - CaM calmodulin - NMJ neuromuscular junction - EPSC excitatory postsynaptic current (prefix m denotes miniature EPSC) A preliminary presentation of some of these results have been presented at Neurotox '88, Neuropharmacology and Pesticide Action, Nottingham, UK     

The polyunsaturated fatty acids, EPA and DPA exert a protective effect in the hippocampus of the aged rat

Age is characterized by deficits in synaptic function identified by decreased performance of aged animals in spatial learning tasks and reduced ability of animals to sustain long term potentiation (LTP). Several cellular and molecular events are correlated with these deficits, many of which are indicative of age-related neuroinflammatory and oxidative cell stress. It is significant that agents which decrease microglial activation are commonly associated with restoration of function. We set out to examine whether the n-3 polyunsaturated fatty acid docosapentaenoic acid (DPA), which is a metabolite of eicosapentaenoic acid (EPA), could modulate the age-related increase in microglial activation and the associated increase in oxidative changes and therefore impact on synaptic function in aged rats. We demonstrate that docosapentaenoic acid possesses neurorestorative effects and is capable of downregulating microglial activation. The data show that it also decreases the coupled activation of sphingomyelinase and caspase 3, probably because of its ability to decrease age-related oxidative changes, and consequently attenuates the age-related decrease in spatial learning and long-term potentiation.     

Effect of age on endogenous DNA single-strand breakage,strand break induction and repair in the adult housefly,Musca domestica

It has been suggested that genomic alterations involving DNA damage and the ability to repair such damage play an important role in cellular sensecence. In this study, endogenous DNA single-strand breaks, the susceptibility of DNA to induced strand breakage and the capacity to repair these breaks were compared in postmitotic cells from young (3-day-old) and old (23-day-old) houseflies. DNA single-strand breaks did not accumulate during normal aging in the housefly. However, cells of the old flies exhibited a greater sensitivity to single-strand breakage induced by γ-radiation and UV light. The capacity to repair these exogenously induced single-strand breaks declined with age. Results fo not support the view that DNA single-strand breaks are casual factor in aging in the housefly. An age-related increase in the susceptibility to undergo single-strand breakage suggests alterations in chromatin during the aging process.     

Purification and Molecular Identification of an Antifungal Peptide from the Hemolymph of Musca domestica （housefly）

Antibacterial and antifungal peptides found in houseflies （Musca domestica） in large number are indispensable components of its immune defense mechanism. In this study the anterior tip of the larvae of housefly was cut off with a pair of fine scissors and hemolymph was collected and exuded in an ice-cold test tube. From the hemolymph an antifungal substance was isolated by solid-phase extraction combined with reverse phase-high performance liquid chromotography （RP-HPLC） and named as Musca domestica antifungal peptide-1 （MAF-1）. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE） showed its molecular weight was 17 kDa. UV absorption spectra revealed that this antifungal substance possessed the characteristics of protein peptides. Analysis by fingerprint-identification and tandem mass spectrometry suggested MAF-I was an unknown protein. Edman degradation identified the sequence of 30 amino acids of its N-terminal which matched no peptide in the MASCOT search database, indicating MAF-1 was a novel insect antifungal peptide. Mass spectrometry showed the precise molecular weight of MAF-1 was 17203.384 Da. Its isoelectric point was acidic.     

Regional differences in age-related lipofuscin accumulation in the female hamster brainstem

Lipofuscin accumulation is a characteristic feature of senescent postmitotic neuronal cells but estrogen may have protecting effects by inhibiting its formation. In the present ultrastructural study, lipofuscin accumulation was studied in 2 estrogen-α-receptive brainstem areas: nucleus pararetroambiguus (NPRA) and the commissural part of the solitary tract nucleus/A2 catecholaminergic group (NTScom/A2) and compared with the estrogen-insensitive medial tegmental field (mtf), in young (23 weeks) and aged (95 weeks) female hamsters. In the aged animals, extensive intracytoplasmic lipofuscin accumulation was observed. A total number of 6450 neurons were classified in 4 categories. Levels were significantly elevated in each of the brain areas studied. Lipofuscin accumulation was strongest in the mtf, less in NPRA, and remarkably less in the area of NTScom/A2. In conclusion, the observed differences in lipofuscin accumulation suggest: (1) considerable regional differences in the degree of neuronal vulnerability; and (2) a possible neuroprotective role for estrogen, because the degree of accumulation is inversely related to the density of the estrogen receptors, varying from nonreceptive (mtf) to NPRA and NTScom/A2 (most receptive).     

The potential interactions between polyunsaturated fatty acids and colonic inflammatory processes

n-3 Polyunsaturated fatty acids (PUFAs) are recognized as having an anti-inflammatory effect, which is initiated and propagated via a number of mechanisms involving the cells of the immune system. These include: eicosanoid profiles, membrane fluidity and lipid rafts, signal transduction, gene expression and antigen presentation. The wide-range of mechanisms of action of n-3 PUFAs offer a number of potential therapeutic tools with which to treat inflammatory diseases. In this review we discuss the molecular, animal model and clinical evidence for manipulation of the immune profile by n-3 PUFAs with respect to inflammatory bowel disease. In addition to providing a potential therapy for inflammatory bowel disease there is also recent evidence that abnormalities in fatty acid profiles, both in the plasma phospholipid membrane and in perinodal adipose tissue, may be a key component in the multi-factorial aetiology of inflammatory bowel disease. Such abnormalities are likely to be the result of a genetic susceptibility to the changing ratios of n-3 : n-6 fatty acids in the western diet. Evidence that the fatty acid components of perinodal adipose are fuelling the pro- or anti-inflammatory bias of the immune response is also reviewed.     

Relationship between selected bacteria and the growth of immature house flies, Musca domestica, in an axenic test system.

To investigate the relationship between immature (maggot) house flies, Musca domestica, and bacteria, we compared the development of sterile first-instar maggots in each of 10 pure blood agar cultures of bacteria with growth on sterile blood agar (negative control) and on standard house fly rearing medium (positive control). Nine species of bacteria representing gram-negative and gram-positive rods, coccoid, and micrococcoid cell types supported house fly growth on blood agar. One bacterium, a strain of Bacillus cereus, inhibited maggot growth. The percent pupation for maggots that developed in the presence of eight of nine bacteria (range, 41-69%), was significantly greater than in sterile blood agar (0-5%), and did not differ significantly from maggot growth in the rearing medium (50-90%). Average pupal weight for maggots that developed on blood agar with bacteria ranged from 19 to 21 mg, a reflection of favorable growth conditions. Average pupal weight in the presence of three bacteria (19.9, 19.4, and 19.4 mg) was significantly less than respective pupal weights in house fly rearing medium (24.0, 22.3, and 22.1 mg), but there was no difference in average pupal weight with six bacteria and the house fly rearing medium. These findings illustrate that bacteria or their metabolic products are essential as nutrients for house fly maggot growth in blood agar; a wide variety of adventitious bacteria can contribute to the suitability of an organic substrate for maggot growth; and a naturally occurring isolate of B. cereus limits house fly maggot growth in blood agar, a relationship that has not been reported previously.     

Morphology of the puparia of the housefly, Musca domestica (Diptera: Muscidae) and blowfly, Chrysomya megacephala (Diptera: Calliphoridae)

Examination of the puparia of the housefly, Musca domestica L. and blowfly Chrysomya megacephala (F.), through scanning electron microscopy (SEM), revealed many differences in the profile of their morphology. Special attention was focused on puparial characteristics used to differentiate between the two fly species studied, and between other forensically important flies. Results of this study indicate that the housefly puparia are almost evenly rounded at both ends and the anterior spiracle bears six papillae. A pair of pupal respiratory horns is found laterally before the posterior boundary of the first abdominal segment, bearing numerous papillae that have a longitudinal opening along the oval convex base. The peritreme of each posterior spiracle forms a crude forward or reverse D-shape, encircling three sinuous slits. The blowfly pupariums anterior spiracle contains 8–12 papillae. The pupal respiratory horns protrude slightly and in some specimens a group of ~38 globules on the bubble-like membrane may be observed. Each of the posterior spiracles is more or less an oval- shaped peritreme, encircling three straight spiracular slits. The anatomical features presented herein allow for the differentiation of puparia of the two fly species studied and could prove useful in future forensic entomological assessments.     

Characterisation of the interaction between WRN,the helicase/exonuclease defective in progeroid Werner's syndrome,and an essential replication factor,PCNA

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werner's syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.     

Relationship between dietary retinol and lipofuscin in the retinal pigment epithelium

A variety of evidence suggests that autoxidation of cellular components probably plays a significant role in the age-related accumulation of lipofuscin, or age-pigment, in the mammalian retinal pigment epithelium (RPE). Among the likely candidates for conversion into RPE lipofuscin fluorophores via autoxidative mechanisms are vitamin A compounds, which are present in the retina and RPE in high concentrations. Vitamin E, an important lipid antioxidant, is likely to inhibit vitamin A autoxidation. Experiments were conducted to evaluate the significance of vitamin A autoxidation in the deposition of lipofuscin in the RPE. Albino rats were fed diets either supplemented with or lacking vitamin E. Each of these two groups of animals was further subdivided into three groups which were fed different levels of vitamin A palmitate: none, 14.0 mumol/kg diet, and 80.5 mumol/kg diet. After 26 weeks, the animals were killed and the RPE lipofuscin contents were determined by both fluorescence measurements and quantitative ultrastructural morphometry. Vitamin A palmitate deficiency led to significant reductions in RPE lipofuscin deposition, relative to the amounts of this pigment present in the groups receiving vitamin A palmitate in their diets. The relative magnitude of the vitamin A effect was greater in the vitamin E-supplemented groups than in the groups fed the diets deficient in vitamin E. This finding suggests that vitamin E interacts with vitamin A ester metabolites in vivo in a more complex manner than simply acting as an antioxidant protectant. Rats fed the diets containing the higher level of vitamin A palmitate failed to display elevated RPE lipofuscin contents relative to those in the rats fed 14.0 mumol of vitamin A palmitate/kg diet. Failure of high vitamin A intake to enhance RPE lipofuscin deposition may have been due to the fact that intake of vitamin A above normal levels did not lead to an elevation in vitamin A content of the retinal tissue. Establishing an effect of vitamin A deficiency on RPE lipofuscin deposition and characterization of the interactions between vitamins E and A are important steps toward defining precisely the molecular and cellular mechanisms underlying age-pigment accumulation in the RPE.     

Adulticidal and larvicidal activity of Beauveria bassiana and Metarhizium anisopliae against housefly, Musca domestica (Diptera: Muscidae), in laboratory and simulated field bioassays

The susceptibility of the adult and larval stage of housefly, Musca domestica L. (Diptera: Muscidae), to two entomopathogenic fungi, Metarhizium anisopliae (Metsch.) Sor. and Beauveria bassiana (Bals.) Vuill., was evaluated under laboratory and simulated field bioassays. Bioassays on adult houseflies were carried out at different conidial concentrations ranging from 10³ to 10⁹ conidia/ml in petri plate and minichamber assays. Absolute mortality was observed within 4–5 days at all the concentrations tested. M. anisopliae was found to be more effective with LC₅₀ of 6.75 × 10⁷ conidia/ml compared with 1.21 × 10⁸ conidia/ml of B. bassiana in petri plate bioassay. Similar trend was observed in minichamber bioassay. Larvicidal activity evaluated through petri plate bioassay also indicated that M. anisopliae was more effective larvicide with LC₅₀ of 4.1 × 10⁸ conidia/ml as against 3.31 × 10⁹ conidia/ml of B. bassiana. Larvicidal activity was further evaluated in simulated field condition of decaying waste matrix using dry conidial formulations (10⁸ conidia/g) of both the fungi. Larval mortality obtained in this assay was 43% (B. bassiana) and 63% (M. anisopliae). Remarkably better performance of M. anisopliae as an adulticidal and larvicidal agent over B. bassiana in laboratory bioassays as well as simulated field conditions suggests that it may have good potential to become part of an integrated housefly control program.     

来源于n-3多不饱和脂肪酸的新型抗炎介质Resolvin,docosatriene及neuroprotectin

研究表明,n-3多不饱和脂肪酸能调节免疫,缓解急性和慢性炎症疾病.传统观点认为n-3多不饱和脂肪酸的抗炎机制是通过竞争环氧酶和脂氧酶从而减少来源于花生四烯酸(AA)的促炎性介质的生成,或通过影响酶与细胞因子的基因表达,抑制促炎症因子产生、调节黏附分子表达来调节免疫功能.但近期研究发现,二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)可在代谢过程中产生一类新型的脂质介质(Resolvin、docosatriene),其在炎症衰退阶段的渗出物中可被检测到,这些代谢中间物都具有潜在的抗炎及免疫调节活性,从而在新的方面揭示了n-3多不饱和脂肪酸发挥抗炎及免疫调节活性的分子机制.