Characteristics associated with rapid decline in forced expiratory volume

Though the mechanism of tissue damage induced by colonic inflammation in ulcerative colitis is unknown, it has been established that the inflammatory mediator and potent neutrophil (PMN) chemotaxin, leukotriene B4(LTB4), is present in elevated amounts in the inflamed mucosa. The unique role of 5-lipoxygenase in the production of leukotrienes has made it a target for inhibition. This study used a rat model of acute colonic inflammation induced by a single IP injection of Mitomycin-C to test the efficacy of a specific and potent 5-lipoxygenase inhibitor zileuton in the treatment of colonic inflammation. We hypothesized that after inducing colitis in rats with mitomycin-C, the administration of oral zileuton would inhibit leukotriene production, thus preventing PMN infiltration and subsequent tissue damage. Zileuton decreased colonic tissue damage as measured by Histological score. However, zileuton did not significantly decrease neutrophil infiltration measured by mucosal PMN or myeloperoxidase (MPO) levels. Although zileuton was successful in significantly decreasing the frequency of severe colitis in our model, the fact that the decrease in PMN count and MPO level was not statistically significant suggests that another mechanism may be involved in its anti-inflammatory effect.     

Eicosanoid synthesis by warm- and cold-acclimated American bullfrog (Rana catesbeiana) brain

Previous studies in bullfrogs have demonstrated the presence of leukotriene (LT)C4 binding sites in the brain. However, synthesis of eicosanoids by brain tissue has not been examined. Because prostaglandin (PG) synthesis differs in warm- and cold-acclimated bullfrog lung tissue, this study compared the synthesis of prostaglandins and leukotrienes in brains from warm-(22 degrees C) and cold-acclimated (5 degrees C) animals. Initial experiments determined that leukotriene and prostaglandin production rates were greatest during the initial 30 min time period. Therefore, tissues were incubated in Munsick's solution and gassed with 95% O2, 5% CO2 for 30 min. Media were analyzed by radioimmunoassay for LTC4, LTB4, PGE2, PGF2 alpha, TXB2, and 6-keto PGF1 alpha. In warm-acclimated bullfrog brains, production was as follows: LTC4 > PGE2 > 6-keto PGF1 alpha, thromboxane (TX)B2, LTB4, and PGF2 alpha. Brain tissues from cold-acclimated animals incubated at 22 degrees C produced significantly greater quantities of PGE2 and 6-keto PGF1 alpha than did brains from warm-acclimated animals. Stimulation of TXB2 levels was observed when the animal was stunned with a blow to the head prior to decapitation. Indomethacin, a cyclooxygenase inhibitor, decreased prostaglandin but not leukotriene synthesis. Epinephrine (4 x 10(-8) M), the amphibian sympathetic postganglionic neurotransmitter, stimulated leukotriene synthesis by brains from warm-acclimated bullfrogs, and the effect was blocked with the 5-lipoxygenase inhibitor MK-886 (5 x 10(-5) M). These results clearly indicate that the bullfrog brain synthesized both leukotrienes and prostaglandins. Further studies are necessary to determine their function in the amphibian central nervous system.     

Evaluation of the antiinflammatory activity of a dual cyclooxygenase-2 selective/5-lipoxygenase inhibitor, RWJ 63556, in a canine model of inflammation

Sterile perforated polyethylene spheres (wiffle golf balls) were implanted s.c. in beagle dogs. A local inflammatory reaction was elicited within the spheres by injecting carrageenan. Changes in leukocyte count, prostaglandin E2, thromboxane B2 and leukotriene B4 levels were monitored in fluid samples collected over a 24-hr period. Blood samples were also collected at various time points and analyzed for prostaglandin E2 and leukotriene B4 production after ex vivo calcium ionophore treatment. Effects of standard antiinflammatory agents (aspirin, indomethacin, dexamethasone, tenidap and zileuton) and newer cyclooxygenase-2 (COX-2) selective agents (nimesulide, nabumetone and SC-58125) were determined after oral administration. Ex vivo inhibition of cyclooxygenase product synthesis (prostaglandin E2, thromboxane B2) in whole blood was used as an indicator of activity for the constitutive COX-1 isoform, although inhibition of the synthesis of these mediators in the chamber exudate during an inflammatory process is believed to represent COX-2 inhibition. Treatment effects on leukotriene B4 production were also determined both ex vivo in whole blood and in the fluid. All of the compounds tested, except aspirin, inhibited leukocyte infiltration into the fluid exudate. Inhibitors that exert their effects on both isozymes of cyclooxygenase attenuate production of cyclooxygenase metabolites in both the inflammatory exudate and in peripheral blood ex vivo, although COX-2 selective inhibitors only demonstrated activity in the exudate. A 5-lipoxygenase inhibitor (zileuton), a corticosteroid (dexamethasone) and a dual COX-2 selective/5-lipoxygenase inhibitor (RWJ 63556) had similar profiles in that they all inhibited cell infiltration and eicosanoid production in the fluid and also attenuated leukotriene B4 production in both the fluid and blood.     

Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.     

5-Lipoxygenase reaction products modulate alveolar macrophage phagocytosis of Klebsiella pneumoniae

The leukotrienes are potent lipid mediators of inflammation formed by the 5-lipoxygenase-catalyzed oxidation of arachidonic acid. Although the effects of leukotrienes on neutrophil chemotaxis and activation have been established, their role in modulating innate host defense mechanisms is poorly understood. In a previous study (M. Bailie, T. Standiford, L. Laichalk, M. Coffey, R. Strieter, and M. Peters-Golden, J. Immunol. 157:5221-5224, 1996), we used 5-lipoxygenase knockout mice to establish a critical role for endogenous leukotrienes in pulmonary clearance and alveolar macrophage phagocytosis of Klebsiella pneumoniae. In the present study, we investigated the role of specific endogenous leukotrienes in phagocytosis of K. pneumoniae and explored the possibility that exogenous leukotrienes could restore phagocytosis in alveolar macrophages with endogenous leukotriene synthesis inhibition and enhance this process in leukotriene-competent cells. Rat alveolar macrophages produced leukotriene B4 (LTB4), LTC4, and 5-hydoxyeicosatetraenoic acid (5-HETE) during the process of phagocytosis, and the inhibition of endogenous leukotriene synthesis with zileuton and MK-886 dramatically attenuated phagocytosis. We also observed a reduction in phagocytosis when we treated alveolar macrophages with antagonists to the plasma membrane receptors for either LTB4, cysteinyl-leukotrienes, or both. In leukotriene-competent cells, LTC4 augmented phagocytosis to the greatest extent, followed by 5-HETE and LTB4. These 5-lipoxygenase reaction products demonstrated similar relative abilities to reconstitute phagocytosis in zileuton-treated rat alveolar macrophages and in alveolar macrophages from 5-lipoxygenase knockout mice. We conclude that endogenous synthesis of all major 5-lipoxygenase reaction products plays an essential role in phagocytosis. The restorative and pharmacologic effects of LTC4, LTB4, and 5-HETE may provide a basis for their exogenous administration as an adjunctive treatment for patients with gram-negative bacterial pneumonia.     

Toxoplasma gondii alters eicosanoid release by human mononuclear phagocytes: role of leukotrienes in interferon gamma-induced antitoxoplasma activity

Toxoplasma gondii tachyzoites markedly alter the profile of eicosanoids released by human mononuclear phagocytes. Freshly isolated, 2-h adherent human monocytes release both cyclooxygenase (e.g., thromboxane [TX] B2, prostaglandin [PG] E2) and 5-lipoxygenase (e.g., leukotriene [LT] B4, LTC4) products of arachidonic acid metabolism after stimulation by the calcium ionophore A23187 or ingestion of opsonized zymosan particles or heat-killed T. gondii. However, after incubation with viable T. gondii, normal and chronic granulomatous disease monocytes release only the cyclooxygenase products TXB2 and PGE2 and fail to form LTB4, LTC4, or other 5-lipoxygenase products. Monocytes maintained in culture for 5 d lose this capacity to release TXB2 and PGE2 after incubation with T. gondii. T. gondii significantly inhibit calcium ionophore A23187-induced LTB4 release by monocyte-derived macrophages; heat-killed organisms do not affect this calcium ionophore A23187-induced release of LTB4. T. gondii-induced inhibition of LTB4 release by calcium ionophore A23187-stimulated monocyte-derived macrophage is reversed by interferon (IFN)-gamma treatment of the monolayers. LTB4 induced extensive damage to the cellular membranes and cytoplasmic contents of the organisms as observed by transmission electron microscopy. Exogenous LTB4 (10(-6) M) induced intracellular killing of ingested T. gondii by non-IFN-gamma-treated monocyte-derived macrophages. IFN-gamma-induced antitoxoplasma activity in monocyte-derived macrophages was inhibited by the selective 5-lipoxygenase inhibitor zileuton but not by the cyclooxygenase inhibitor indomethacin. These findings suggest a novel role for 5-lipoxygenase arachidonic acid products in human macrophage IFN-gamma-induced antitoxoplasma activity.     

Does the repeated gambles procedure measure impulsivity in social drinkers

Ethology of Polyrhachis laboriosa, an ant species from equatorial Africa, is little known. No field observation of a nuptial flight of these ants was ever made. We describe two nuptial flights observed in a laboratory colony of P. laboriosa at a 3 days interval. They both occurred in the morning while the nest was kept in near darkness (less than 2 lux of daylight). Flying activity of the alates was suppressed within 1 h by their exposure to daylight of about 140 lux, and within several minutes by their exposure to a lamp emitting white light of 3,000 lux and acting as a source of heat. On the day following the first flight the alates and the workers showed exceptionally high level of mutual grooming. The alates, in particular the males, were transported by workers to the brood chambers whenever they strayed outside and after the nuptial flights.     

Effects of 1-chloro-2,4,6-trinitrobenzene on 5-lipoxygenase activity and cellular leukotriene synthesis

5-Lipoxygenase (EC 1.13.11.34) is the key enzyme in the regulation of leukotriene synthesis. Here, the effects of various substituted nitrobenzene compounds on 5-lipoxygenase activity and the formation of leukotriene B4 (LTB4) were studied in polymorphonuclear leukocytes (PMNL), B lymphocytes, and human whole blood. 1-Chloro-2,4,6-trinitrobenzene (TNCB) was found to inhibit calcium ionophore A23187-induced leukotriene synthesis in PMNL in a biphasic manner. Thus, 1.0 microM TNCB caused 50% inhibition of LTB4 formation, but only 16% inhibition was found at 10 times higher concentration. In contrast, this higher concentration of TNCB activated the synthesis of LTB4 when PMNL were stimulated with arachidonic acid alone, demonstrating that TNCB can exert both stimulatory and inhibitory effects on leukotriene synthesis depending on the experimental conditions. The inhibitory effect of 1.0 microM TNCB on ionophore A23187-induced leukotriene synthesis could be circumvented by addition of exogenous arachidonic acid. At high concentrations of TNCB (25-100 microM), the drug blocked ionophore A23187-induced leukotriene synthesis. TNCB also inhibited LTB4 formation in B lymphocytes, as well as in human whole blood. The activity of recombinant 5-lipoxygenase was inhibited by TNCB, and reduced glutathione or beta-mercaptoethanol counteracted this inhibition. This suggests that TNCB might inhibit 5-lipoxygenase by alkylating thiol groups. TNCB possessed a high specificity for 5-lipoxygenase with only modest inhibitory effects on 12-lipoxygenase (EC 1.13.11.31), 15-lipoxygenase (EC 1.13.11.12), and phospholipase A2 (EC 3.1.1.4) activities. Taken together, these results show that TNCB can both specifically inhibit and stimulate leukotriene formation and might be useful in further studies on the regulation of 5-lipoxygenase.     

Leukotrienes D4 and E4 produced in myocardium impair coronary flow and ventricular function after two hours of global ischaemia in rat heart

OBJECTIVE: Leukotrienes D4 and E4 are potent coronary vasoconstrictors and myocardial depressants. The aim was to investigate the contribution of myocardial leukotrienes to impairment of coronary flow and recovery of contractile function in rat hearts subjected to 2 h of global ischaemia. METHODS: Rat hearts were mounted on a working Langendorff apparatus and perfused with oxygenated Krebs-Henseleit solution at 37 degrees C for 30 min. Hearts were then arrested with either standard potassium crystalloid cardioplegic solution (n = 6), or with cardioplegic solution containing the leukotriene D4, E4 receptor antagonist Ly171883 (n = 6). Arrested hearts were maintained at 15 degrees C for 2 h, then rewarmed to 37 degrees C during 30 min working reperfusion. Coronary effluent was analysed by radioimmunoassay for leukotriene C4, D4, E4, and F4 levels. Immediately prior to cardiac arrest, and again after 30 min reperfusion, coronary flow, and aortic outflow and pressure were measured. RESULTS: Postischaemic leukotriene levels were increased compared to preischaemic levels in both groups [pooled measurements: 133.3 (SD 136.4) v 20.7(17.8) pg.0.1 ml-1, p < 0.05]. Postischaemic coronary vascular resistance was increased by 80% in controls (p < 0.001) compared to 19% (p = NS) in treated hearts. In addition, functional recovery was significantly greater in treated hearts compared to controls [82(3)% v 53(3)% for coronary flow; 79(3)% v 50(2)% for cardiac output; 82(4)% v 54(3)% for stroke work]. CONCLUSIONS: Leukotrienes are endogenously produced by the heart, and this production is significantly increased after global ischaemia and reperfusion. Reversal of significantly increased coronary vascular resistance coupled with improved functional recovery in hearts treated with LY171883 demonstrates an important contribution of endogenously produced leukotrienes to coronary vascular impairment and functional stunning of the globally ischaemic, reperfused heart.     

Effects of continuous light and darkness on the eyes of the troglobitic salamander Typhlotriton spelaeus

Larval Typhlotriton spelaeus collected from five caves in Pulaski Co., Missouri, were kept as larvae or induced to transform in darkness or continuous fluorescent illumination. Larvae maintained in darkness for 215 and 279 days had smaller eyes, smaller rod inner and outer segments, and fewer metaphase figures in the germinative zone of the neural retina than comparable larvae maintained in light (258 lux). Except for visual cell size, differences were small and for each characteristic exceptions were observed. One larva kept in light showed early retinal degeneration comparable to that in transformed adults to T. spelaeus. All larvae exhibited optomotor behavior both before and after the experiment. Among animals induced to transform by L-thyroxin and maintained in darkness 111 to 366 days, visual cell and pigment epithelium degeneration was more extensive and more frequent than in animals kept for the same length of time in light (237-298 lux). In darkness the frequency of animals with retinal degeneration increased between 111 and 366 days. In light some animals exhibited pigment epithelium reduction with normal visual cells, and others had free, pigmented cells in the subretinal space. These effects were not comparable to degeneration in darkness. Eyelids covered the eyes of only a few animals in both light and dark treatments. The extent of eyelid encroachment over the eye greater in darkness than in light. Most animals exhibited optomotor responses after experiments, but responses of animals kept in darkness were impaired in comparison to those of animals kept in light.     

Light-induced apoptosis: differential timing in the retina and pigment epithelium

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.     

What makes a mother? Interviews with women involved in egg donation and surrogacy

The fish pineal organ contains typical and, in some species, modified photoreceptor cells involved in the photoperiodic control of melatonin production. In the majority of species studied, the rhythm in melatonin production is driven by an intra-pineal circadian oscillator synchronized by the light:dark cycle. In the present study, it is shown that the endogenous rhythm in melatonin release of superfused pike pineals maintained under constant darkness is expressed at temperatures of 19 degrees C, 20 degrees C, 25 degrees C, and 30 degrees C (period > 24 hr), but not at temperatures of 10 degrees C and 15 degrees C. Under constant darkness, pineal fractions containing either typical photoreceptors, modified photoreceptors, or both behaved like total organs. Dissociated pike pineal cells, cultured statically at 20 degrees C, expressed a high amplitude rhythm in melatonin secretion under a light:dark cycle. Under constant darkness, circadian oscillations, which appeared better sustained than in organ culture, were also observed. This study provides the first evidence that the rhythmic production of melatonin, by a fish pineal, is driven by a population of circadian oscillators or clocks. It is hypothesized that each typical and modified photoreceptor might be the locus of a circadian clock. Damping of the overall rhythm under constant darkness might reflect the desynchronization (uncoupling) between these clocks and/or damping of individual oscillators.     

Comprehensive asthma management: guidelines for clinicians

Asthma is a chronic inflammatory disease characterized by reversible airway obstruction and nonspecific airway hyperreactivity. Asthma is managed in steps according to disease symptoms and severity. Treatment goals are to decrease symptoms, improve pulmonary function, and reduce overall morbidity and the associated cost of medical care. Antiasthma drugs are a key component of asthma management that are classified as either long-term-control medications that control symptoms and prevent disease exacerbations, or quick-relief medications that rapidly relieve airway obstruction and acute asthma symptoms. Several new leukotriene (LT) modulators have been developed that promise to improve asthma control, including LT receptor antagonists montelukast and zafirlukast and the 5-lipoxygenase inhibitor zileuton. Each decreases symptoms and the use of rescue medication, and improves pulmonary function in patients with mild intermittent to moderate persistent asthma.     

Mechanisms of platelet-activating factor-induced lipid body formation: requisite roles for 5-lipoxygenase and de novo protein synthesis in the compartmentalization of neutrophil lipids

Lipid bodies, lipid rich cytoplasmic inclusions, are characteristically abundant in vivo in leukocytes associated with inflammation. Because lipid bodies are potential reservoirs of esterified arachidonate and sites at which eicosanoid-forming enzymes may localize, we evaluated mechanisms of lipid body formation in neutrophils (PMN). Among receptor-mediated agonists, platelet activating factor (PAF), but not C5a, formyl-methyl-phenylalanine, interleukin 8, or leukotriene (LT) B4, induced the rapid formation of lipid bodies in PMN. This action of PAF was receptor mediated, as it was dose dependently inhibited by the PAF receptor antagonist WEB 2086 and blocked by pertussis toxin. Lipid body induction by PAF required 5-lipoxygenase (LO) activity and was inhibited by the 5-lipoxygenase-activating protein antagonist MK 886 and the 5-LO inhibitor zileuton, but not by cyclooxygenase inhibitors. Corroborating the dependency of PAF-induced lipid body formation on 5-LO, PMN and macrophages from wild-type mice, but not from 5-LO genetically deficient mice, formed lipid bodies on exposure to PAF both in vitro and in vivo within the pleural cavity. The 5-LO product inducing lipid body formation was not LTB4 but was 5(S)-hydroxyeicosatetraenoic acid [5(S)-HETE], which was active at 10-fold lower concentrations than PAF and was also inhibited by pertussis toxin but not by zileuton or WEB 2086. Furthermore, 5-HETE was equally effective in inducing lipid body formation in both wild-type and 5-LO genetically deficient mice. Both PAF- and 5(S)-HETE-induced lipid body formation were inhibited by protein kinase C (PKC) inhibitors staurosporine and chelerythrine, the phospholipase C (PLC) inhibitors D609 and U-73122, and by actinomycin D and cycloheximide. Prior stimulation of human PMN with PAF to form lipid bodies enhanced eicosanoid production in response to submaximal stimulation with the calcium ionophore A23187; and the levels of both prostaglandin (PG) E2 and LTB4 correlated with the number of lipid bodies. Furthermore, pretreatment of cells with actinomycin D or cycloheximide inhibited not only the induction of lipid body formation by PAF, but also the PAF-induced "priming" for enhanced PGE2 and LTB4 in PMN. Thus, the compartmentalization of lipids to form lipid bodies in PMN is dependent on specific cellular responses that can be PAF receptor mediated, involves signaling through 5-LO to form 5-HETE and then through PKC and PLC, and requires new protein synthesis. Since increases in lipid body numbers correlated with priming for enhanced PGE2 and LTB4 production in PMN, the induction of lipid bodies may have a role in the formation of eicosanoid mediators by leukocytes involved in inflammation.     

Extraocular light exposure does not suppress plasma melatonin in humans

Light affects the circadian axis in at least two ways. It can cause the acute suppression of pineal melatonin synthesis, and/or a phase-shift of the circadian oscillator. As recent evidence has suggested that extraocular light exposure may cause phase-shifts of the circadian clock, we have investigated whether suppression of melatonin can be induced by the same type of light exposure. In the first study subjects' eyes were exposed to white light (2250 lux for 30 mins) via a fibre optic cable. As expected, suppression of nighttime plasma melatonin levels (61 +/- 6%) was observed. In the second study, light of the same quality but higher intensity (14,000 or 67,500 lux for 180 mins) was delivered in the same manner to the popliteal region behind the subjects' knees, whilst shielding their eyes. No suppression of plasma melatonin levels (4 +/- 7%) was detected in any of the subjects. Thus, extraocular photoreception, if it exists in mammals, does not affect the suprachiasmatic nucleipineal pathway.     

Nicotine inhibits cytokine synthesis by mouse colonic mucosa

We examined the in vivo effect of nicotine on the synthesis of (pro)inflammatory mediators by mouse colonic mucosa. The synthesis of lipid mediators such as the prostanoids prostaglandin E2, 6-keto-prostaglandin F1 alpha and thromboxane B2, the 5-lipoxygenase products leukotriene B4 and leukotriene C4 and the platelet activating factor was not affected, whereas the synthesis of the pro-inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha was completely abolished. The beneficial effects of smoking and nicotine in ulcerative colitis could be attributed to this inhibition.     

Influence of photosensitizers and light on the life span of Drosophila

The life span of adult Drosophila melanogaster fruit flies changed when they were fed two different photosensitizers. Methylene blue decreased the median life span by 49% when present in the food at a concentration of 0.001 M. Another photosensitizer, riboflavin, produced no changes in life span under the same conditions of a 12:12 h light/dark cycle at a daytime light intensity of 1000 lux. Flies exposed to constant darkness lived 43.2% longer than those exposed to constant light at a light intensity of 2000 lux. Under these conditions, riboflavin increased the life span of the flies exposed to constant light by as much as 25%. We conclude that riboflavin confers some degree of protection against the effects of constant light exposure. The completely different results obtained with riboflavin and methylene blue suggest a possible mechanism for photoageing involving photodynamic action mediated through the production of singlet oxygen.     

Mechanism of pyocyanin- and 1-hydroxyphenazine-induced lung neutrophilia in sheep airways

Pyocyanin (Pyo) and 1-hydroxyphenazine (1-HP) are extracellular products of Pseudomonas aeruginosa. To test whether these products were capable of producing an inflammatory response in the airways, combinations of Pyo and 1-HP at concentrations of 10(-4) and 10(-5) M were instilled into sheep airways, and indexes of inflammation were assessed by bronchoalveolar lavage (BAL) 24 h later. Challenge with the phenazines caused a significant dose-dependent increase in the number of cells and neutrophils recovered by BAL. Control challenges produced no such changes. The lung neutrophilia was accompanied by an increased concentration of albumin in BAL. The increases in BAL neutrophils and albumin could be blocked by treating the sheep with the 5-lipoxygenase inhibitor zileuton. Neither 1-HP nor Pyo was chemotactic to neutrophils when tested in vitro, but when alveolar macrophages (AM) were cultured in vitro in the presence of both Pyo and 1-HP (1 microM), the supernatants caused neutrophil chemotaxis. Analysis of AM culture supernatants incubated with the combination of pigments showed significant increases in leukotriene B4 and interleukin-8, and blocking these mediators separately or together reduced AM supernatant-induced neutrophil chemotaxis. We conclude that local instillation of Pyo and 1-HP can initiate an inflammatory response in the airways of sheep in vivo. This effect can be explained, in part, by the release of chemotactic factors produced by AM.     

Roles of suprachiasmatic nuclei and intergeniculate leaflets in mediating the phase-shifting effects of a serotonergic agonist and their photic modulation during subjective day

Serotonin (5-HT) has been implicated in the phase adjustment of the circadian system during the subjective day in response to nonphotic stimuli. Two components of the circadian system, the suprachiasmatic nucleus (SCN) (site of the circadian clock) and the intergeniculate leaflet (IGL), receive serotonergic projections from the median raphe nucleus and the dorsal raphe nucleus, respectively. Experiment 1, performed in golden hamsters housed in constant darkness, compared the effects of bilateral microinjections of the 5-HT1A/7 receptor agonist, 8-hydroxydipropylaminotetralin (8-OH-DPAT; 0.5 microgram in 0.2 microliter saline per side), into the IGL or the SCN during the mid-subjective day. Bilateral 8-OH-DPAT injections into either the SCN or the IGL led to significant phase advances of the circadian rhythm of wheel-running activity (p < .001). The phase advances following 8-OH-DPAT injections in the IGL were dose department (p < .001). Because a light pulse administered during the middle of the subjective day can attenuate the phase-resetting effect of a systemic injection of 8-OH-DPAT, Experiment 2 was designed to determine whether light could modulate 5-HT agonist activity at the level of the SCN and/or the IGL. Serotonergic receptor activation within the SCN, followed by a pulse of light (300 lux of white light lasting 30 min), still induced phase advances. In contrast, the effect of serotonergic stimulation within the IGL was blocked by a light pulse. These results indicate that the respective 5-HT projections to the SCN and IGL subserve different functions in the circadian responses to photic and nonphotic stimuli.     

Hypothalamic and suprachiasmatic uptake of serotonin in vitro: twenty-four-hour changes in male and proestrous female rats

Twenty-four-hour changes in the in vitro serotonin (5-HT) uptake capacity of hypothalamic homogenates and of "Vibratome" slices of the suprachiasmatic nuclear region (SNR) of the hypothalamus were studied in 60-90-day-old Holtzman (albino) rats. Animals acclimated to artificially illuminated (fluorescent, LD 12:12) and temperature controlled (22 +/- 2 C) rooms were killed 6% each of 8 time points. Synaptosomal fractions from homogenates of whole hypothalamus, and slices of the SNR were incubated for 20 min in Krebs-Henseleit buffer with [3H]5-HT. Males showed a single daily peak in SNR uptake at the start of darkness, and a minimum near the onset of light, while a more complex pattern containing 3 peaks and minima occurred in uptake by hypothalamic homogenates. Proestrous females showed a single high amplitude peak SNR uptake during the critical period, just prior to the plasma LH peak determined in the same animals by radioimmunoassay. It is suggested that this short-term and 4-fold increase in SNR uptake of 5-HT may serve to limit free 5-HT and its inhibitory or other effects on the gonadotropin release hormone system and thereby on LH release and ovulation.