Photolysis of cholesterol during biological experiments

1. When dilute aqueous emulsions of radioactive cholesterol are exposed to daylight, extensive photolysis may take place. This results chiefly in the formation of substances more polar than cholesterol, some of which are probably acidic. Substances less polar than cholesterol are formed to a smaller extent. Photolysis is greatest when the emulsions are strongly acidic or strongly alkaline. 2. Photolysis takes place rapidly if radioactive cholesterol stored in the dry state, either on glass or on filter paper, is exposed to daylight. 3. If precautions are taken to minimize exposure to daylight during analysis and storage of the samples, the amount of non-enzymic alteration of cholesterol in biological experiments may be negligible.     

Signaling or Not-Signaling: Variation in Vulnerability and Defense Tactics of Armored Ground Crickets (Acanthoplus Speiseri: Orthoptera, Tettigoniidae, Hetrodinae)

Male Orthoptera singing from exposed perches are at risk from acoustically- and visually-hunting predators. The defensive reactions of armored ground crickets (Acanthoplus speiseri) include falling silent, dropping from their perch, alarm stridulation and autohaemorrhaging. Male and female ground crickets show different reactivity (i.e. the number or intensity of defense tactics used) to predation, depending on level of exposure: calling males were more reactive when approached during daylight, compared with in the dark. During daylight, calling males were more reactive than silent, cryptic, males and females. The level of response presumably reflected the riskiness of the individual’s behavior and situation at that time. Plasticity of response to predation allows individuals to balance risky behavior (i.e. acoustic signaling from exposed perches) by being more reactive to potential threats.     

Lack of protection by carotenes against gamma-radiation damage in Phycomyces

Carotenes could protect cells from radiation damage by chemically quenching the free radicals and the activated chemical species originated by the exposure. We tested this hypothesis with strains of the zygomycete Phycomyces blakesleeanus that contained different carotenes (phytoene, lycopene, β-carotene) or different concentrations of β-carotene. Pairs of strains were cultured together, exposed to a maximum of 73 Gy γ-radiation from a ⁶⁰Co source, and allowed to recover and grow further together on limited resources. Irradiation did not affect the relative abundance of each strain in the resulting spore crop. Thus, carotenes did not protect the fungal cells against γ-radiation and did not influence their recovery from damage caused by the exposure. Received: 12 October 1995 / Accepted in revised form: 18 March 1996     

Prior temperature exposure affects subsequent chilling sensitivity

The chilling sensitivity of small discs or segments of tissue excised from chillingsensitive species was significantly altered by prior temperature exposure subsequent to holding the tissue at chilling temperatures as measured by a number of physiological processes sensitive to chilling. This temperature conditioning was reversible by an additional temperature exposure before chilling, and mature-green and red-ripe tomato tissue exhibit similar chilling sensitivities. Exposing pericarp discs excised from tomato fruit (Lycopersicon esculentum Mill. cv. Castelmart), a chilling-sensitive species, to temperatures from 0 to 37°C for 6 h before chilling the discs at 2.5°C for 4 days significantly altered the rate of ion leakage from the discs, but had no effect on the rate of ion leakage before chilling and only a minimal effect on discs held at a non-chilling temperature of 12°C. Exposing chillingsensitive tissue to temperatures below that required to induce heat-shock proteins but above 20°C significantly increased chilling sensitivity as compared to tissue exposed to temperatures between 10 and 20°C. Rates of ion leakage after 4 days of chilling at 2.5°C were higher from fruit and vegetative tissue of chilling-sensitive species (Cucumis sativus L. cv. Poinsett 76, and Cucurbita pepo L. cv. Young Beauty) that were previously exposed for 6 h to 32°C than from similar tissue exposed to 12°C. Exposure to 32 and 12°C had no effect on the rate of ion leakage from fruit tissue of chilling tolerant species (Malus domestica Borkh. cv. Golden Delicious, Pyrus communis L. cv. Bartlett). Ethylene and CO₂ production were higher and lycopene synthesis was lower in chilled tomato pericarp discs that were previously exposed for 6 h to 32°C than the values from tissue exposed to 12°C for 6 h before chilling. Increased chilling sensitivity induced by a 6 h exposure to 32°C could be reversed by subsequent exposure to 12°C for 6 h.     

Aggregate Size Distribution of Ammonia-Oxidizing Bacteria and Archaea at Different Landscape Positions

To quantify the spatial distribution of ammonia-oxidizing bacteria (AOB) and archaea (AOA) and to determine nitrification activity in soil aggregates along a landscape, soil samples were collected from three landscape positions (shoulder, backslope, and toeslope) at two pasture sites with contrasting climatic conditions. The abundance of AOB and AOA was estimated by quantifying their respective bacterial and archaeal amoA gene copies using real-time polymerase chain reaction. Soil organic C (SOC), total N (TN), and the potential nitrification rate (PNR) were measured in aggregate size ranges (4–1, 1–0.25, and 0.25–0.05 mm). At site 1, a decreasing trend in PNR was observed as the size of aggregates decreased. Both bacterial and archaeal amoA genes were higher in the macroaggregates (4–1 and 1–0.25 mm) than in the microaggregates (0.25–0.05 mm) along the landscape. At site 2, PNR was higher in the smallest size of aggregates. In the 0.25–0.05-mm fraction, the abundance of bacterial and archaeal amoA genes was equal to, or greater than, those found in larger aggregate sizes. The relative abundance of archaeal amoA gene and the PNR correlated with relative SOC and TN contents along the landscapes. The positive relationship between relative archaeal amoA gene abundance and PNR suggests that nitrification in the studied pastures is probably driven by ammonia-oxidizing Thaumarchaeota.     

The antioxidant and anti-inflammatory properties of lycopene in mice lungs exposed to cigarette smoke

Lycopene is a carotenoid with known antioxidant and anti-inflammatory properties. We aimed to evaluate the in vitro and in vivo effects of lycopene on reducing the redox imbalance and inflammation induced by cigarette smoke (CS). For the in vitro study, J774A.1 (macrophages) cells were incubated in the presence of 0.5, 1.0, 2.0, 4.0, 8.0, 10.0 and 25 μM of lycopene for 3, 6 and 24 h or in the presence of 0.1%, 0.25%, 0.5%, 0.625%, 1.25%, 2.25%, 5% and 10% cigarette smoke extract (CSE) for 3, 6 and 24 h to assess cell viability and measurement of intracellular reactive oxygen species (ROS). For the in vivo study, 40 mice were divided into 5 groups: a control exposed to ambient air (CG), a vehicle-control group that received 200 μl of sunflower oil by orogastric gavage, a group exposed to CS and two groups administered lycopene (diluted in sunflower oil) at doses of either 25 or 50 mg/kg/day prior to exposure to CS (LY25+CS and LY50+CS). The total treatment time lasted 5 days. A cell viability decrease was observed at 10- and 25-μM concentrations of lycopene in 3, 6 and 24 h compared with CG. There was an increase of ROS production in 24 h in CS compared with CG. Lycopene concentrations of 1 μM and 2 μM were able to reduce the production of ROS in 24 h compared with CS. In the bronchoalveolar lavage fluid, the total number of leukocytes increased in the CS group compared with the control groups (CG). Administration with lycopene at the highest dose suppressed this CS-induced increase in leukocytes. Lipid peroxidation and DNA damage increased in the CS group compared with that in the controls, and this increase was suppressed by lycopene at the highest dose. In contrast, superoxide dismutase activity decreased in the CS group compared with that in the controls. Catalase activity also increased in the CS group compared with that in both control groups, and this increase was suppressed in LY25+CS and LY50+CS. There was an increase in the levels of tumor necrosis factor-α, interferon-γ and interleukin-10 after exposure to CS, and these effects were suppressed by both doses of lycopene. These data elucidate the role of lycopene as an antioxidant and anti-inflammatory agent in these two models of short-term exposure to CS.     

Serum from rats fed red or yellow tomatoes induces Connexin43 expression independently from lycopene in a prostate cancer cell line

Epidemiologic studies suggested a protective effect of tomatoes against prostate cancer brought by lycopene, a carotenoid conferring the red colour of tomatoes. However, intervention studies on patients have shown that the preventive effect of tomato was more potent than that of lycopene. The aim of this study was to compare the effects of red tomato, yellow tomato (devoid of lycopene) and lycopene on Connexin43 (Cx43) expression, a protein regulating cell growth, on a prostate cancer cell line expressing the androgen receptor. Cells were incubated with serum from rats fed a control diet (CS) or control diet supplemented with red tomato (RTS), yellow tomato (YTS) or lycopene beadlets (LBS). After exposure of the cells to RTS or YTS for 48 h, the expression of Cx43 was significantly increased compared to cells exposed to CS. Whereas LBS effect was not significantly different. The cells incubated with RTS and LBS had similar levels of lycopene, while those incubated with YTS contained no lycopene. These data first show that serum nutritionally enriched with red and yellow tomatoes could up-regulate Cx43 turn-over in PC3AR cells independently from lycopene level. Within the physiological approach used in the present study, it can be concluded that compounds other than lycopene contribute to the preventive effect of tomatoes.     

Dietary lycopene attenuates cigarette smoke-promoted nonalcoholic steatohepatitis by preventing suppression of antioxidant enzymes in ferrets

Cigarette smoke (CS) is an independent risk factor in development of nonalcoholic steatohepatitis (NASH) and fibrosis. Lycopene, a carotenoid naturally occurring in tomatoes, has been shown to be a protective agent against tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced NASH. In the present study using a ferret model we investigated whether CS promotes NASH and whether dietary lycopene can inhibit CS-promoted NASH development, and if so, what potential mechanisms were involved. Ferrets were divided into 4 groups (n=12−16/group): control, NNK/CS exposed, NNK/CS plus low-dose lycopene (2.2 mg/kg BW/day), and NNK/CS plus high-dose lycopene (6.6 mg/kg BW/day) groups, for 26 weeks. Results showed that hepatic steatosis, infiltrates of inflammatory cells, and the number and size of inflammatory foci in liver, together with key genes involved in hepatic fibrogenesis were higher in the NNK/CS group compared to the control group; a lycopene diet reversed these changes to the levels of the control group. Interestingly, a major lycopene cleavage enzyme, beta-carotene 9’,10’-oxygenase (BCO2), which recently has been recognized to play metabolic roles beyond cleavage function, was down-regulated by NNK/CS exposure, but this decrease was prevented by lycopene feeding. NNK/CS exposure also downregulated liver expression of antioxidant enzymes and upregulated oxidative stress marker, which were all prevented by lycopene. In conclusion, our results suggest that CS can promote development of NASH and liver fibrosis in ferrets, which is associated with downregulation of BCO2 and impairment of antioxidant system in liver; dietary lycopene may inhibit CS-promoted NASH by preventing suppression of BCO2 and decline in antioxidant network.     

Lycopene Protects against Hypoxia/Reoxygenation-Induced Apoptosis by Preventing Mitochondrial Dysfunction in Primary Neonatal Mouse Cardiomyocytes

Background

Hypoxia/reoxygenation(H/R)-induced apoptosis of cardiomyocytes plays an important role in myocardial injury. Lycopene is a potent antioxidant carotenoid that has been shown to have protective properties on cardiovascular system. The aim of the present study is to investigate the potential for lycopene to protect the cardiomyocytes exposed to H/R. Moreover, the effect on mitochondrial function upon lycopene exposure was assessed.

Methods and Findings

Primary cardiomyocytes were isolated from neonatal mouse and established an in vitro model of H/R which resembles ischemia/reperfusion in vivo. The pretreatment of cardiomyocytes with 5 µM lycopene significantly reduced the extent of apoptosis detected by TUNEL assays. To further study the mechanism underlying the benefits of lycopene, interactions between lycopene and the process of mitochondria-mediated apoptosis were examined. Lycopene pretreatment of cardiomyocytes suppressed the activation of the mitochondrial permeability transition pore (mPTP) by reducing the intracellular reactive oxygen species (ROS) levels and inhibiting the increase of malondialdehyde (MDA) levels caused by H/R. Moreover, the loss of mitochondrial membrane potential, a decline in cellular ATP levels, a reduction in the amount of cytochrome c translocated to the cytoplasm and caspase-3 activation were observed in lycopene-treated cultures.

Conclusion

The present results suggested that lycopene possesses great pharmacological potential in protecting against H/R-induced apoptosis. Importantly, the protective effects of lycopene may be attributed to its roles in improving mitochondrial function in H/R-treated cardiomyocytes.     

Activity patterns at the Arctic Circle: nocturnal eagle owls and interspecific interactions during continuous midsummer daylight

Circadian rhythms result from adaptations to biotic and abiotic environmental conditions that cycle through the day, such as light, temperature, or temporal overlap between interacting species. At high latitudes, close to or beyond the polar circles, uninterrupted midsummer daylight may pose a challenge to the circadian rhythms of otherwise nocturnal species, such as eagle owls Bubo bubo. By non‐invasive field methods, we studied eagle owl activity in light of their interactions with their main prey the water vole Arvicola amphibius, and their competitor the white‐tailed eagle Haliaeetus albicilla during continuous midsummer daylight on open, treeless islands in coastal northern Norway. We evaluated circadian rhythms, temporal overlap, exposure, and spatial distribution. The owls maintained a nocturnal activity pattern, possibly because slightly dimmer light around midnight offered favourable hunting conditions. The eagles were active throughout the 24‐h period as opposed to the strictly diurnal rhythm reported elsewhere, thus increasing temporal overlap and the potential for interference competition between the two avian predators. This may indicate an asymmetry, with the owls facing the highest cost of interference competition. The presence of eagles combined with constant daylight in this open landscape may make the owls vulnerable to interspecific aggression, and contrary to the available literature, eagle owls rarely exposed themselves visually during territorial calls, possibly to avoid detection by eagles. We found indications of spatial segregation between owls and eagles reflecting differences in main prey, possibly in combination with habitat‐mediated avoidance. Eagle owl vocal activity peaked in the evening before a nocturnal peak in visual observations, when owls were active hunting, consistent with the hypothesis of a dusk chorus in nocturnal bird species. The owls may have had to trade‐off between calling and foraging during the few hours around midnight when slightly dimmer light reduced the detection risk while also providing better hunting conditions     

Somatic embryogenesis and plant regeneration in olive (Olea europaea L.)

Leaf discs from olive (Olea europaea L.) grown in vitro and immature zygotic embryos collected at 50, 75, 90 and 105 days after full bloom were tested for their somatic embryogenic capacity. The embryos were grown in half-strength MS medium and half-strength OM medium with BAP combinated with either 2,4-D or NAA. Incubation was either in an initial dark period followed by 16h daylight or in 16h daylight throughout. Somatic embryogenesis, approx. 40%, mostly directly from the embryos, was observed only in 75-day-old embryos in medium containing low cytokinin and auxin concentrations. Differentiation was inhibited by 2,4-D whereas NAA did not. In leaf discs and younger and older zygotic embryos, only callus and root formation was observed. Somatic embryos were germinated and then potted-up to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphtaleneacetic acid     

Efficiency of subaquatic light traps

Subaquatic light traps are often used for sampling of the aquatic insects; however, their efficiency has not been tested yet. Here, we describe the use of such traps, illustrate the design of a simple trap allowing standardised sampling, and present results of assessment of the light trap efficiency according to the different turbidity levels, as well as model insects’ escape rates. The effect of turbidity on the capture efficiency was not significant for Sigara lateralis (Leach, 1817), Notonecta glauca Linnaeus, 1758 and Corixa punctata (Illiger, 1807) and was marginally significant for Chaoborus sp. Escape rates were affected by the entrance size and daylight exposure time. Although subaquatic light traps are helpful for standardised sampling, their entrance should be adequate for the target species, and the traps should be removed soon after the dawn or the resulting specimen counts should be corrected for the daylight exposure time before the trap removal.     

Role of ascorbate in lung cellular toxicity mediated by light-exposed parenteral nutrition solution

Abstract

Neonatal lung injury has been induced experimentally by infusion of multivitamin-containing light-exposed parenteral nutrition (PN) solutions. The objective was to explore the role of ascorbate in toxic effects of light-exposed PN on primary cultured foetal rat lung epithelial cells. Hydroperoxides were measured in 3% amino acid solutions at baseline, immediately after addition of either multivitamins or ascorbate alone (400 μg/mL) and again after a 24-h period of exposure to (or protection from) ambient light. Cellular toxicity was assessed by [C¹⁴]adenine release. Multivitamins or ascorbate alone increased hydroperoxides in PN, which was attenuated by light protection. Light-exposed PN containing multivitamins was more toxic to cells than baseline or light-protected PN. Exposure to ascorbate at concentrations both lower (< 5 μg/mL) and higher (> 1000 μg/mL) than normally contained in PN-induced oxidant-mediated cell death, as indicated by protective effects of hydroperoxide and hydroxyl radical scavengers. This study concludes that ascorbate generates toxic amounts of peroxide in PN solutions. The types and physiological importance of hydroperoxides induced by pro-oxidant effects of ascorbate require further evaluation in vivo.     

Immunohistochemical detection of phosphorylated rhodopsin in light-exposed retina of living mouse with in vivo cryotechnique.

The purpose of this study is to analyze the time-dependent molecular states of rhodopsin (Rho) phosphorylation in the specimens originating from eyeballs cryoimmobilized in situ in living animals. Whole eyeballs of living mice under various dark- and light-exposure conditions were quickly frozen using the in vivo cryotechnique with isopentane-propane cryogen cooled down in liquid nitrogen (-196C). The frozen whole-mount eyeballs were freeze substituted in acetone containing paraformaldehyde and embedded in paraffin wax. Deparaffinized sections were immunostained with anti-phosphorylated (334)Ser Rho (P-Rho334) antibody. Immunoreactivity of P-Rho334 was specifically recognized in the outer segments of mouse retinas exposed to daylight. In the 12-h dark-adapted retinas, P-Rho334 immunoreactivity was completely eliminated. Moreover, in other retinas dark adapted for 12 or 36 hr and then exposed under the safety red light for 2 min, it was still barely recognized. Even in the eyeballs exposed to strong visible light for 10 sec, it was not detected. However, after 30, 60, and 180 sec of visible light exposure, P-Rho334 immunoreactivity was definitely recovered, similar to that under daylight condition. This is a new immunohistochemical approach to visualize the time-dependent Rho phosphorylation of living mice using the in vivo cryotechnique, in which changes could be detected within seconds following exposure to light.     

Isomerization of dietary lycopene during assimilation and transport in plasma

Diets of individuals were supplemented with tomatoes, either cooked or as tomato purée in order to compare uptake of lycopene from intact and homogenized fruit tissue matrices. Following a diet containing cooked tomatoes over three consecutive 7-day periods, little change in the carotenoid levels in plasma lipoproteins occurred. In contrast, a diet supplemented with concentrated tomato purée, over a 2 week period, caused a significant (p < 0.05) increase in lycopene levels in plasma, showing that the lycopene within intact cells is less bioavailable than that from processed tissue. The isomeric composition of plasma lycopene was significantly different to that of the ingested purée. A number of cis-isomers (predominantly 5-cis, 13-cis and 9-cis-) were detected in plasma, that are not present in the lycopene from purée. The significance of the increase in lycopene following dietary supplementation with respect to bioavailability and the causes of isomerization are discussed.     

Pyrethroid susceptibility and behavioral avoidance in Anopheles epiroticus,a malaria vector in Thailand

The physiological susceptibility to insecticides and the behavioral responses of four wild‐caught populations of female Anopheles epiroticus to synthetic pyrethroids (deltamethrin, permethrin, and alpha‐cypermethrin) were assessed. Test populations were collected from different localities along the eastern coast, Trat (TR), Songkhla (SK), and Surat Thani (ST) and one population from the western coast, Phang Nga (PN). Results showed that all four populations of An. epiroticus were susceptible to all three synthetic pyrethroids tested. Behavioral responses to test compounds were characterized for all four populations using an excito‐repellency test system. TR displayed the strongest contact excitation (‘irritancy’) escape response (76.8% exposed to deltamethrin, 74.1% permethrin, and 78.4% alpha‐cypermethrin), followed by the PN population (24.4% deltamethrin, 35% permethrin, and 34.4% for alpha‐cypermethrin) by rapidly escaping test chambers after direct contact with surfaces treated with each active ingredient compared with match‐paired untreated controls. Moderate non‐contact repellency responses to all three compounds were observed in the TR population but were comparatively weaker than paired contact tests. Few mosquitoes from the SK and ST populations escaped from test chambers, regardless of insecticide tested or type of trial. We conclude that contact excitation was a major behavioral response in two populations of An. epiroticus, whereas two other populations showed virtually no escape response following exposure to the three pyrethroids. The explanation for these large unexpected differences in avoidance responses between pyrethroid‐susceptible populations of the same species is unclear and warrants further investigation.     

Effect on lycopene, β-carotene,ascorbic acid and phenolic content in tomato fruits infected by Alternaria alternata and its toxins (TeA,AOH and AME)

Tomato is considered as one of the most important sources of nutrients such as lycopene, β-carotene, flavonoids, ascorbic acid (vitamin C) and hydroxyl-cinnamic acid derivatives. The quality and quantity of nutrients in tomato fruits were decreased during the severe infection of Alternaria alternata. The present study deals with the estimation of lycopene, β-carotene, phenolic and ascorbic acid content in tomato fruits which were infected with A. alternata and its toxins such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). The lycopene, β-carotene, ascorbic acid and phenolic content were found lowest in pathogen-infected fruits i.e. (0.66 ± 0.03 mg/g), (0.14 ± 0.01 mg/g), (1.89 ± 0.2 mg/g) and (0.58 ± 0.05 mg/g), respectively, followed by toxins-treated samples as compared to the control. The results concluded that A. alternata mostly affects the nutritional values of tomato fruits due to the combined effect of the toxins.     

Experimental exposure of the blue mussel (Mytilus edulis, L.) to the toxic dinoflagellate Alexandrium fundyense: Histopathology, immune responses, and recovery

Mussels (Mytilus edulis) were exposed to cultures of the toxic dinoflagellate Alexandrium fundyense or the non-toxic alga Rhodomonas sp. to evaluate the effects of the harmful alga on the mussels and to study recovery after discontinuation of the A. fundyense exposure. Mussels were exposed for 9 days to the different algae and then all were fed Rhodomonas sp. for 6 more days. Samples of hemolymph for hemocyte analyses and tissues for histology were collected before the exposure and periodically during exposure and recovery periods.Mussels filtered and ingested both microalgal cultures, producing fecal pellets containing degraded, partially degraded, and intact cells of both algae. Mussels exposed to A. fundyense had an inflammatory response consisting of degranulation and diapedesis of hemocytes into the alimentary canal and, as the exposure continued, hemocyte migration into the connective tissue between the gonadal follicles. Evidence of lipid peroxidation, similar to the detoxification pathway described for various xenobiotics, was found; insoluble lipofuchsin granules formed (ceroidosis), and hemocytes carried the granules to the alimentary canal, thus eliminating putative dinoflagellate toxins in feces. As the number of circulating hemocytes in A. fundyense-exposed mussels became depleted, mussels were immunocompromised, and pathological changes followed, i.e., increased prevalences of ceroidosis and trematodes after 9 days of exposure. Moreover, the total number of pathological changes increased from the beginning of the exposure until the last day (day 9). After 6 days of the exposure, mussels in one of the three tanks exposed to A. fundyense mass spawned; these mussels showed more severe effects of the toxic algae than non-spawning mussels exposed to A. fundyense.No significant differences were found between the two treatments during the recovery period, indicating rapid homeostatic processes in tissues and circulating hemocytes.