Effects of anisotonicity on pentose-phosphate pathway,oxidized glutathione release and t-butylhydroperoxide-induced oxidative stress in the perfused liver of air-breathing catfish,<Emphasis Type="Italic">Clarias batrachus</Emphasis>

Both hypotonic exposure (185 mOsmol/l) and infusion of glutamine plus glycine (2 mmol/l each) along with the isotonic medium caused a significant increase of¹⁴CO₂ production from [1-¹⁴C]glucose by 110 and 70%, respectively, from the basal level of 18.4 ± 1.2 nmol/g liver/min from the perfused liver ofClarias batrachus. Conversely, hypertonic exposure (345 mOsmol/l) caused significant decrease of¹⁴CO₂ production from [1-¹⁴C]glucose by 34%.¹⁴CO₂ production from [6-¹⁴C]glucose was largely unaffected by anisotonicity. The steady-state release of oxidized glutathione (GSSG) into bile was 1.18 ±0.09 nmol/g liver/min, which was reduced significantly by 36% and 34%, respectively, during hypotonic exposure and amino acid-induced cell swelling, and increased by 34% during hypertonic exposure. The effects of anisotonicity on¹⁴CO₂ production from [1-¹⁴C]glucose and biliary GSSG release were also observed in the presence of t-butylhydroperoxide (50 (Amol/1). The oxidative stress-induced cell injury, caused due to infusion of t-butylhydroperoxide, was measured as the amount of lactate dehydrogenase (LDH) leakage into the effluent from the perfused liver; this was found to be affected by anisotonicity. Hypotonic exposure caused significant decrease of LDH release and hypertonic exposure caused significant increase of LDH release from the perfused liver. The data suggest that hypotonically-induced as well as amino acid-induced cell swelling stimulates flux through the pentose-phosphate pathway and decreases loss of GSSG under condition of mild oxidative stress; hypotonically swollen cells are less prone to hydroperoxide-induced LDH release than hypertonically shrunken cells, thus suggesting that cell swelling may exert beneficial effects during early stages of oxidative cell injury probably due to swelling-induced alterations in hepatic metabolism.     

β2-Adrenoceptor Agonist-Induced Down-Regulation After Short-Term Exposure

Abstract

We examined the effect of duration of β₂-adrenergic receptor (β₂AR) occupancy by isoproterenol on specific binding of ¹²⁵l-lodocyanopindolol (¹²⁵I-ICYP) in membranes from rat L6 myoblasts. Ten minute exposure caused a time- and concentration-dependent maximal decrease in ¹²⁵-?YP binding 24 hours after exposure equal to that following continuous exposure (p < 0.05). Low temperature, concanavalin A, H89 and ICI 118,551 blocked the decline in ¹²⁵I-ICYP binding during the first hour following exposure probably representing receptor sequestration to a compartment or change to a form incapable of ligand binding. Compared to controls, receptor binding 4 and 24 hours following exposure was reduced 56 ± 8.7% and 72 ± 8.8%, respectively (p < 0.05), and was blocked by ICI 118,551 but not CGP12177. Isoproterenol-induced, but not forskolinstimulated, cAMP accumulation was reduced 35% 24 hours following exposure (p < 0.05). ¹²⁵I-ICYP binding in intact L6 cells 4 and 24 hours after exposure were respectively 56 ± 8.9 and 61 ± 13% of controls (p < 0.05). Following agonist exposure, CHO cell membranes expressing human β₂ARs exhibited ¹²⁵I-ICYP binding 85 ± 2.0% and 6 ± 2.8% of control values 4 and 24 hours, respectively (p < 0.05). A model predicting that full occupation of the β₂AR activates receptor degradation explains our results that agonist-induced down-regulation of β₂AR does not require continuous presence of the agonist.     

Evaluation of increased proportion of cells with unusually high sister chromatid exchange counts as a cytogenetic biomarker for lead exposure

Sister chromatid exchange (SCE) frequency and high-frequency cells (HFCs) were analyzed in 50 storage battery plant workers with mean blood lead level (BLL) of 40.14±9.99 μg/dL. The mean BLL in the control group (n=30) was 9.77±1.67 μg/dL. This difference in mean BLLs between control and exposed group was statistically significant (p<0.05) and reflects clearly the lead exposure in the workers. Urinary aminolevulinic acid (U-ALA) was also determined in both control (3.37±0.89 mg ALA/g creatinine) and exposed groups (12.39±6.18 mg ALA/g creatinine) and U-ALA excretion was statistically higher (p<0.05) in lead-exposed workers. The relationship between biomarkers of lead exposure/effect and HFC percentage was higher than the relationship between biomarkers of lead exposure/effect and SCE frequency. Accordingly, HFC analysis seemed to be more sensitive than the SCE analysis as a cytogenetic biomarker for lead exposure. Additionally, the statistically significant correlation (r ²=0.880, p<0.01) between U-ALA excretion and HFC percentage in lead-exposed workers supported the probability of ALA mediated indirect mechanism for lead genotoxicity.     

Comparison Between 5-Aminosalicylic Acid (5-ASA) and Para-Aminosalicylic Acid (4-PAS) as Potential Protectors Against Mn-Induced Neurotoxicity

Manganese (Mn) is an essential metal for biological systems; however, occupational or clinical exposure to high levels of Mn can produce a neurological disorder called manganism. Oxidative stress and neuroinflammation play major roles in the Mn-induced neurodegeneration leading to dysfunction of the basal ganglia. We investigated the toxic effects of MnCl₂ in an immortalized rat brain endothelial cell line (RBE4) and the protective effects of the radical scavenging aminosalicylic acids, 5-aminosalicylic acid (5-ASA) and 4-aminosalicylic acid (4-PAS). Mn cytotoxicity was determined with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) activity. A significant decrease in MTT reduction concomitant with increased LDH release was noted in RBE4 cells exposed for 24 h to MnCl₂ (600 and 800 μM; p?<?0.0001). Our results establish that compared to 4-PAS, 5-ASA has greater efficacy in protecting RBE4 cells from Mn-induced neurotoxicity after preexposure to MnCl₂ 800 μM (p?<?0.0001).     

Nitrite and Nitrate Levels in Individual Molluscan Neurons: Single-Cell Capillary Electrophoresis Analysis

Abstract: Cell and tissue concentrations of NO₂^? and NO₃^? are important indicators of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide release. We adapted the capillary electrophoresis technique to quantify NO₂^? and NO₃^? levels in single identified buccal neurons and ganglia in the opisthobranch mollusc Pleurobranchaea californica, a model system for the study of the chemistry of neuron function. Neurons were injected into a 75-µm separation capillary and the NO₂^? and NO₃^? were separated electrophoretically from other anions and detected by direct ultraviolet absorbance. The limits of detection for NO₂^? and NO₃^? were <200 fmol (<4 µM in the neurons under study). The NO₂^? and NO₃^? levels in individual neurons varied from 2 mM (NO₂^?) and 12 mM (NO₃^?) in neurons histochemically positive for NADPH-diaphorase activity down to undetectable levels in many NADPH-diaphorase-negative cells. These results affirm the correspondence of histochemical NADPH-diaphorase activity and nitric oxide synthase in molluscan neurons. NO₂^? was not detected in whole ganglion homogenates or in hemolymph, whereas hemolymph NO₃^? averaged 1.8 ± 0.2 × 10^?3M. Hemolymph NO₃^? in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnalis (3.2 ± 0.2 × 10^?5M) and for another opisthobranch, Aplysia californica (3.6 ± 0.7 × 10^?4M). Capillary electrophoresis methods provide utility and convenience for monitoring NO₂^?/NO₃^? levels in single cells and small amounts of tissue.     

Response of the mouse lung transcriptome to welding fume: effects of stainless and mild steel fumes on lung gene expression in A/J and C57BL/6J mice

Background

Nitrogen dioxide (NO₂) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO₂ is also produced endogenously in the lung during acute inflammatory responses. NO₂ can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c⁺ antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c⁺ cells in NO₂-promoted allergic sensitization.

Methods

We systemically depleted CD11c⁺ cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO₂ followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c⁺ cells from wildtype mice were studied after exposure to NO₂ and ovalbumin for cellular phenotype by flow cytometry and in vitro cytokine production.

Results

Transient depletion of CD11c⁺ cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c⁺ cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO₂ exposure. By 48 hours, CD11c⁺MHCII⁺ DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c⁺CD11b^- and CD11c⁺CD11b⁺ pulmonary cells exposed to NO₂ in vivo increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647⁺ CD11c⁺MHCII⁺ DCs present in MLN from NO₂-exposed mice by 48 hours. Co-cultures of ova-specific CD4⁺ T cells from naïve mice and CD11c⁺ pulmonary cells from NO₂-exposed mice produced IL-1, IL-12p70, and IL-6 in vitro and augmented antigen-induced IL-5 production.

Conclusions

CD11c⁺ cells are critical for NO₂-promoted allergic sensitization. NO₂ exposure causes pulmonary CD11c⁺ cells to acquire a phenotype capable of increased antigen uptake, migration to the draining lymph node, expression of MHCII and co-stimulatory molecules required to activate naïve T cells, and secretion of polarizing cytokines to shape a Th2/Th17 response.     

Cytogenetic analysis of buccal cells from shoeworkers and pathology and anatomy laboratory workers exposed to n-hexane,toluene, methyl ethyl ketone and formaldehyde

People employed in the shoe manufacture and repair industry are at an increased risk for cancer, the strongest evidence being for nasal cancer and leukaemia. A possible causal role for formaldehyde is likely for cancer of the buccal cavity and nasopharynx. Exfoliated buccal cells are good source of tissue for monitoring human exposure to inhaled and ingested occupational and environmental genotoxicants. To assess the cytogenetic damage related to occupational exposure to airborne chemicals during shoe-making and the processes in pathology and anatomy laboratories, the micronuclei (MN) count per 3000 cells was measured in buccal smears from shoe-workers (group I, n = 22) exposed to mainly n-hexane, toluene and methyl ethyl ketone (MEK) and from anatomy and pathology staff (group II, n = 28) exposed to formaldehyde (FA). Eighteen male university staff were used as controls. The mean time-weighted average (TWA) concentrations of n-hexane, toluene and MEK in 10 small shoe workshops were 58.07 p.p.m., 26.62 p.p.m. and 11.39 p.p.m., respectively. The measured air concentrations of FA in the breathing zone of the anatomy and pathology laboratory workers were between 2 and 4 p.p.m. Levels of 2,5-hexadione (2,5-HD) and hippuric acid (HA), metabolic markers of n-hexane and toluene exposure, respectively, were significantly higher in the urine of workers in group I than in control subjects (p < 0.001 and p < 0.01, respectively). The mean (±SD) MN frequencies in buccal mucosa cells from workers in group I, group II and controls were 0.62±0.45%, 0.71±0.56% and 0.33±0.30%, respectively (p < 0.05 and p < 0.05 compared with controls for group I and group II, respectively). The effects of smoking, age and duration of exposure on the frequency of micronucleated buccal cells from workers in all three groups studied were also evaluated. Overall, the results suggest that occupational exposure to organic solvents, mainly n-hexane, toluene, MEK and FA, may cause cytogenetic damage in buccal cells and that use of exfoliated buccal cells seems to be appropriate to measure exposure to organic solvents.     

Pelagic nitrification and denitrification rates in an Arctic fjord during early spring

This study addresses factors governing nitrification and denitrification rates, along with the abundance of the bacterial groups likely involved in these activities, in Kongsfjorden, an Arctic fjord at Ny-Ålesund, Svalbard. The fjord was sampled three times during the month of March 2008 as day length and direct solar radiation increased. Although initially well mixed, cooler and more saline, the fjord became stratified, warmer and less saline during late March. The concentrations of NH₄ ⁺ (4.4?±?1.6 to 6?±?1.6 μM) and NO₂ ^? (1?±?0.3 to 1.2?±?0.4 μM) increased progressively with the decrease in NO₃ ^? (6.1?±?1.3 to 3.8?±?1.5 μM), reflecting the onset of primary productivity. Nitrification rates and the culturable population of nitrifiers decreased significantly from 1.6?±?0.9 to 0.4?±?0.1 ng at NH₄ ⁺-N l^?1 h^?1 and 5.1?±?0.3?×?10² to 29?±?14 cells l^?1, respectively. In contrast, denitrification rates increased (2.4?±?0.5 to 4.6?±?1.3 ng-at NO₃ ^?-N l^?1 h^?1), although the abundance of culturable denitrifiers did not vary significantly. A significant correlation of nitrifiers with NO₃ ^? during early March (p?<?0.01, r?=?0.51) indicated that nitrifiers may play an important role in regulating the NO₃ ^? pool and thereby in controlling the abundance of denitrifiers. However, the contribution of nitrification to the total NO₃ ^? pool decreased with time. Experimental simulations were also set up to understand the impact of change in duration of light and progressive increase in temperature on these processes. The application of 24 h light inhibited nitrification, suggesting that during peak Arctic summer the contribution of nitrification to the nitrate pool is minimal. It was also observed that a brief exposure to light (≤6 h) was enough to hamper nitrification rates. Experimental simulations suggested that a gradual increase in temperature in the fjord may enhance the magnitude of nitrification and denitrification in the fjord.     

Decreased superoxide anion radical production by rat alveolar macrophages following inhalation of ozone or nitrogen dioxide

$\text{In vivo}$ exposure of rats to ozone or nitrogen dioxide results in a dose-dependent decrease in superoxide anion radical production (O₂^?·) by alveolar macrophages isolated from the exposed animals. When alveolar macrophages from ozone-exposed animals were stimulated with phorbol myristate acetate (PMA, a non-phagocytic stimulus of O₂^?· production) the decrease in O₂^?· production ranged from 85.9% of control at 3.2 ppm-hrs ozone to 7% of control at 10.5 ppm-hrs. In a similar fashion, O₂^?· production by PMA-stimulated macrophages from NO₂-exposed rates ranged from 78% of control at 18.3 ppm-hrs NO₂ down to 14.5% of control at 51 ppm-hrs. Since the viability of the alveolar macrophages obtained from ozone or nitrogen dioxide-exposed animals was 88% or better in all cases as judged by both Trypan blue exclusion and lactate dehydrogenase release, the decreased ability of these cells to produce superoxide anion radical cannot be attributed to a pollutant effect on cell viability. This diminution in superoxide anion radical production by alveolar macrophages from the pollutant-exposed animals might account, in part, for the ability of these 2 air pollutants to potentiate bacterial infections in laboratory animals.     

Alterations of the glutathione redox cycle status in fumonisin B1-treated pig kidney cells

Fumonisin B₁ (FB₁) causes equine leukoencephalomalacia, porcine pulmonary edema, and liver tumors and chronic nephritis in rats. To investigate mechanisms by which FB₁ induces toxicity, effects of FB₁ on cellular glutathione (GSH) redox status and GSH depletion on FB₁ toxicity in pig kidney (LLC-PK₁) cells were studied. Treatment of LLC-PK₁ cells with 50 μM FB₁ for 24 hours significantly decreased cellular GSH contents from 56 ± 3.2 to 42.7 ± 4.4 nmol/mg protein (p < 0.05) and increased the activities of glutathione reductase (GR) from 25.7 ± 2.4 to 35.7 ± 5.0 μmol NADPH/mg protein (p < 0.05). The activities of glutathione peroxidase (GSHpx), catalase, and Cu,Zn-superoxide dismutase (SOD) were not changed by this treatment. Treatment of LLC-PK₁ cells for 12 hours with 0.1. mM buthionine sulfoximine (BSO), a selective inhibitor of the enzyme γ-glutamylcysteine synthetase that catalyzes the rate-limiting reaction in de novo GSH synthesis, decreased cellular GSH levels to about 20% of that found in the control cells. The cells pretreated with 0.1 mM BSO for 12 hours were significantly sensitized to the FB₁ cytotoxicity as determined by a long-term survival assay (p < 0.05). The results demonstrate that FB₁ changes GSH redox cycle status in LLC-PK₁ cells, and GSH may play a role in cytoprotection against FB₁ toxicity. © 1997 John Wiley & Sons, Inc.     

Changes in glucose transporter expression and nitric oxide production are associated with liver injury in diabetes

In diabetes mellitus (DM), both hyperglycaemia and hyperlipidaemia can initiate accumulation of fat in the liver, which might be further mediated by inducible nitric oxide synthase. We have studied changes in GLUT1, nitric oxide (NO^·) concentration and liver damage in two rat DM models. STZ model was induced by strepozotocin 50 mg/kg. HS model was induced by high‐fat diet and 30 mg/kg streptozotocin. GLUT1 expression was studied by means of real‐time RT‐PCR and immunohistochemistry. Production of NO^· was monitored by means of erythrocyte sedimentation rate spectroscopy of Fe‐DETC‐NO complex. Liver damage was assessed using histological activity index (HAI). NO^· concentration was increased in the liver of STZ rats, but it did not change in HS rats (control 36.8 ± 10.3; STZ 142.1 ± 31.1; HS 35.4 ± 9.8 ng/g). Liver HAI was higher in STZ group, 8.6 ± 0.17 versus HS 4.7 ± 0.31, p < 0.05. GLUT1 protein expression was elevated only in STZ group, 16 ± 3 cells/mm² versus Control 5 ± 2 cells/mm², p = 0.007. Hyperglycaemia sooner causes severe liver damage in rat models of DM, compared with hyperlipidaemia, and is associated with increased NO^· production. GLUT1 transporter expression might be involved in toxic effects of glucose in the liver. We have obtained novel data about association of GLUT1 expression and NO^· metabolism in the pathogenesis of liver injury in DM. Increased GLUT1 expression was observed together with overproduction of NO^· and pronounced liver injury in severely hyperglycaemic rats. On the contrary, moderately hyperglycaemic hyperlipidaemic rats developed only moderate liver steatosis and no increase in GLUT1 and NO^·. GLUT1 overexpression might be implicated in the toxic effects of glucose in the liver. Glycotoxicity is associated with oxidative stress and NO^· hyperproduction. GLUT1 and NO^· metabolism might become novel therapeutic targets in liver steatosis. Copyright © 2015 John Wiley & Sons, Ltd.     

THERMOREGULATORY EFFECT IN HUMANS OF SUPPRESSED ENDOGENOUS MELATONIN BY PRE-SLEEP BRIGHT-LIGHT EXPOSURE IN A COLD ENVIRONMENT

This study investigated the physiological function of suppressed melatonin through thermoregulation in a cold environment. Interactions between thermoregulation directly affected by exposure to a cold environment and indirectly affected by endogenous melatonin suppression by bright-light exposure were examined. Ten male subjects were exposed to two different illumination intensities (30 and 5000 lux) for 4.5?h, and two different ambient temperatures (15 and 27°C) for 2?h before sleep under dark and thermoneutral conditions. Salivary melatonin level was suppressed by bright light (p?<?0.001), although the ambient temperature condition had no significant effect on melatonin. During sleep, significant effects of pre-sleep exposure to a cold ambient temperature (p?<?0.001) and bright light (p?<?0.01) on rectal temperature (T_re) were observed. Pre-sleep, bright-light exposure led to an attenuated fall in T_re during sleep. Moreover, T_re dropped more precipitously after cold exposure than thermoneutral conditions (cold: ?0.54?±?0.07°C/h; thermoneutral: ?0.16?±?0.03°C/h; p?<?0.001). Pre-sleep, bright-light exposure delayed the nadir time of T_re under thermoneutral conditions (p?<?0.05), while cold exposure masked the circadian rhythm with a precipitous decrease in T_re. A significant correlation between the T_re nadir and melatonin level (r?=??0.774, p?<?0.05) indicated that inter-individual differences with higher melatonin levels lead to a reduction in T_re after cold exposure. These results suggest that suppressed endogenous melatonin inhibits the downregulation of the body temperature set-point during sleep. (Author correspondence: ishibasi@design.kyushu-u.ac.jp)     

Effects of intraperitoneally‐administered vitamin E and selenium on the blood biochemical and haematological parameters in rats

The aim of this work was to determine the role of intraperitoneally‐administered vitamin E and selenium on the biochemical and haematological parameters in the blood of rats. Thirty‐two adult male Wistar rats were used in this study. All rats were randomly divided into four groups. The first group was used as the control. The second group was intraperitoneally administered with vitamin E (±‐α‐tocopheroryl acetate, 10 mg day⁻¹), the third group with Se (Na₂SeO₃ 0·2 mg over a day), and the fourth group with vitamin E and Se (vitamin E 10 mg + Na₂SeO₃ 0·2 mg over a day). This administration was done for 5 weeks. Blood samples were taken from animals at the end of the dosage period and biochemical parameters in serum samples and haematological parameters in total blood were determined. The levels of total cholesterol (p<0·01) and number of white blood cells (p<0·001) in blood were significantly higher in the vitamin E group than in the control group. The levels of ALP, total cholesterol (p<0·01) and number of white blood cells (p<0·01) in blood were significantly higher in the selenium group than in the controls. The levels of glucose (p<0·05), ALP (p<0·01), total cholesterol (p<0·001) and number of white blood cells (p<0·01) were higher in the vitamin E and selenium combined group than in the controls. Other parameters considered within this trial (ALT, LDH, creatinine, albumin, total protein, amylase, creatine kinase, HDL, triglycerides, total lipid, sodium, chloride, uric acids, red blood cell, haemoglobin, packed cell volume, MCV, MCH, MCHC) did not show statistically significant differences between the control and injected groups. The results indicated that blood glucose and total cholesterol levels, ALP activity and white blood cell counts were significantly increased by intraperitoneal administration of vitamin E and selenium in rats. Copyright © 1999 John Wiley & Sons, Ltd.     

Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells

We recently reported that nitrogen dioxide (NO₂), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO₂ resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO₂ exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO₂ for 1–24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p < 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO₂-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar¹ Ile⁸] Ang II. Exposure of 5 ppm NO₂ for 1–24 hr provoked rapid increases in [³H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO₂ increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.     

Daily and Seasonal Variation in the Spectral Composition of Light Exposure in Humans

Light is considered the most potent synchronizer of the human circadian system and exerts many other non-image-forming effects, including those that affect brain function. These effects are mediated in part by intrinsically photosensitive retinal ganglion cells that express the photopigment melanopsin. The spectral sensitivity of melanopsin is greatest for blue light at approximately 480 nm. At present, there is little information on how the spectral composition of light to which people are exposed varies over the 24 h period and across seasons. Twenty-two subjects, aged 22±4 yrs (mean±SD) participated during the winter months (November–February), and 12 subjects aged 25±3 yrs participated during the summer months (April–August). Subjects wore Actiwatch-RGB monitors, as well as Actiwatch-L monitors, for seven consecutive days while living in England. These monitors measured activity and light exposure in the red, green, and blue spectral regions, in addition to broad-spectrum white light, with a 2 min resolution. Light exposure during the day was analyzed for the interval between 09:00 and 21:00 h. The time course of white-light exposure differed significantly between seasons (p?=?0.0022), with light exposure increasing in the morning hours and declining in the afternoon hours, and with a more prominent decline in the winter. Overall light exposure was significantly higher in summer than winter (p?=?0.0002). Seasonal differences in the relative contribution of blue-light exposure to overall light exposure were also observed (p?=?0.0006), in particular during the evening hours. During the summer evenings (17:00–21:00 h), the relative contribution of blue light was significantly higher (p?<?0.0001) (40.2±1.1%) than during winter evenings (26.6±0.9%). The present data show that in addition to overall light exposure, the spectral composition of light exposure varies over the day and with season.     

Kinetics of K-Cl Cotransport in Frog Erythrocyte Membrane: Effect of External Sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO₃) has been shown to mediate a large fraction of the total K⁺ transport. In the present study, Cl⁻-dependent and Cl⁻-independent K⁺ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K⁺ ([K⁺] _e and [K⁺] _i ) concentration. The dependence of ouabain-resistant Cl⁻-dependent K⁺ (⁸⁶Rb) influx on [K⁺] _e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K _m ) of 8.2 ± 1.3 mm and maximal velocity (V _max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl⁻-dependent K⁺ influx increased both K _m (12.8 ± 1.7 mm, P < 0.05) and V _max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K⁺] _e above 20 mm in isotonic media significantly reduced the Cl⁻-dependent K⁺ influx due to a reciprocal decrease of the external Na⁺ ([Na⁺] _e ) concentration below 50 mm. Replacing [Na⁺] _e by NMDG⁺ markedly decreased V _max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K _m (15.7 ± 2.1 mm, P < 0.03) of Cl⁻-dependent K⁺ influx. Moreover, NMDG⁺ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb⁺ uptake and K⁺ loss from red cells. Cell swelling did not affect the Na⁺-dependent changes in Rb⁺ uptake and K⁺ loss. In a nominally K⁺(Rb⁺)-free medium, net K⁺ loss was reduced after lowering [Na⁺] _e below 50 mm. These results indicate that over 50 mm [Na⁺] _e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K⁺, Cl⁻-dependent K⁺ loss in K⁺-free media was a linear function of [K⁺] _i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr⁻¹ (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K⁺ ions. The residual, ouabain-resistant K⁺ fluxes in NO₃ were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K⁺ effluxes in isotonic (∼0.014 hr⁻¹) and hypotonic (∼0.022 hr⁻¹) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999     

Physiological effects of a long term exposure to low concentrations of NH3, NO2 and SO2 on Douglas fir (Pseudotsuga menziesii)

The above-ground parts of two years old seedlings of Douglas fir (Pseudotsuga menziesii) were exposed to filtered air, NH₃, NO₂₊, SO₂ (66, 96 and 95 μg m^?3, respectively), to a mixture of NO₂+NH₃ (55 + 82 μg m^?3) or SO₂+NO₂ (128 + 129 μg m^?3), for 8 months in fumigation chambers. Both chlorophyll fluorescence and gas exchange measurements were carried out on shoots which had sprouted at the beginning of the exposure period. The chlorophyll fluorescence measurements were performed after 3 and 5 months of exposure (average shoot age 70 and 140 days, respectively). Light response curves of electron transport rate (J) were determined, in which J was deduced from chlorophyll fluorescence. In addition, light response curves of net CO₂ assimilation were determined after 5 months of exposure. After 3 months of exposure (average shoot age 70 days) all exposure treatments showed a lower maximum electron transport rate (J_max) as compared to the control shoots (filtered air). A large reduction (45%) was observed for shoots exposed to SO₂+NO₂. During the exposure period between 3 and 5 months (average shoot age 70 and 140 days, respectively) a decrease of J_max was observed for all treatments. J_max had further declined some time after termination of the exposure, when average shoot age was 310 days. Shoots exposed to SO₂ and SO₂+NO₂ also showed a reduction in maximum net CO₂ assimilation (P_max) as compared to the control shoots. However, shoots exposed to NO₂ showed no reduction and even a higher P_max was observed for shoots exposed to NH₃ or NO₂+NH₃. Needles of these treatments also showed a higher chlorophyll content which might explain the contradictory results obtained for these treatments: the increased amount of photosynthetic units counteracts the reduction in J_max and consequently no reduction in P_max is measured. Shoots exposed to SO₂ and SO₂+NO₂ also showed a reduction in maximum stomatal conductance (g_s). However, the stomatal opening was larger than could be expected on basis of their (maximum) CO₂ assimilation rate. Consequently, water use efficiency of these shoots was lower than that of the control shoots. Also shoots exposed to NO₂ had a lower water use efficiency due to a significantly higher maximum g_s. Shoots exposed to NH₃ showed a high transpiration rate in the dark, indicating imperfect stomatal closure.     

Effects of elevated nitrate concentration on early thyroid morphology in Atlantic salmon (Salmo salar Linnaeus, 1758)

A study was conducted to determine if exposure to elevated nitrate (NO₃^‐)(3.76 ± 0.07 and 93.15 ± 2.79) during embryonic development influences the morphology of thyroid tissue in newly hatched Atlantic salmon, Salmo salar. Digital three‐dimensional imaging of thyroid follicles in S. salar showed that the thyroid tissue in newly hatched alevin consisted mainly of spherical follicles dispersed throughout the lower jaw in close proximity to the ventral aorta and other blood vessels. With these digital models, the number of follicles, average volume per follicle, and total luminal volume were calculated for alevin exposed to well water or 93.15 mg L^?1 NO₃‐N during embryogenesis. There were no statistically significant differences in any of these parameters, suggesting that the normal thyroid development of S. salar could be relatively insensitive to excess environmental NO₃^‐. These results are promising for the culture of Atlantic salmon embryos in recirculating aquaculture systems that naturally accumulate NO₃^‐ as a product of nitrification.     

Differential growth response of <Emphasis Type="Italic">Ulva lactuca</Emphasis> to ammonium and nitrate assimilation

Controlled cultivation of marine macroalgal biomass such as Ulva species, notably Ulva lactuca, is currently studied for production of biofuels or functional food ingredients. In a eutrophic environment, this macrophyte is exposed to varying types of nutrient supply, including different and fluctuating levels of nitrogen sources. Our understanding of the influences of this varying condition on the uptake and growth responses of U. lactuca is limited. In this present work, we examined the growth response of U. lactuca exposed to different sources of nitrogen (NH₄⁺; NO₃⁻; and the combination NH₄NO₃) by using photo-scanning technology for monitoring the growth kinetics of U. lactuca. The images revealed differential increases of the surface area of U. lactuca disks with time in response to different N-nutrient enrichments. The results showed a favorable growth response to ammonium as the nitrogen source. The NH₄Cl and NaNO₃ rich media (50 μM of N) accelerated U. lactuca growth to a maximum specific growth rate of 16.4 ± 0.18% day⁻¹ and 9.4 ± 0.72% day⁻¹, respectively. The highest biomass production rate obtained was 22.5 ± 0.24 mg DW m⁻²·day⁻¹. The presence of ammonium apparently discriminated the nitrate uptake by U. lactuca when exposed to NH₄NO₃. Apart from showing the significant differential growth response of U. lactuca to different nitrogen sources, the work exhibits the applicability of a photo-scanning approach for acquiring precise quantitative growth data for U. lactuca as exemplified by assessment of the growth response to two different N-sources.     

Hydrogen peroxide modulates energy metabolism and oxidative stress in cultures of permanent human Müller cells MIO‐M1

In this study the influence of hydrogen peroxide (H₂O₂) on the redox state, NADH protein binding, and mitochondrial membrane potential in Müller cells is investigated. Cultures of permanent human Müller cells MIO‐M1 were exposed to H₂O₂ in 75 µM and 150 µM concentration for two hours. Fluorescence emission spectra and lifetimes were measured by two‐photon microscopy (excitation wavelength: 740 nm) at the mitochondria which were identified in the microscopic images by their fluorescence properties (spectra and intensity). Two hours of H₂O₂ exposure did not impair viability of MIO‐M1 cells in culture. Whereas the ratio of flavine‐ to NADH fluorescence intensity did not change under either H₂O₂ concentration, the mean lifetime was significantly different between controls, not exposed to H₂O₂, and the 150 µM H₂O₂ exposure (972 ± 63 ps vs. 1152 ± 64 ps, p = 0.014). One hour after cessation of the H₂O₂ exposure, the value retuned to that of the control (983 ± 36 ps). A hyperpolarization of the mitochondrial membrane under 150 µM H₂O₂ was found. These findings suggest a shift form free to protein‐bound NADH in mitochondria as well as a hyperpolarization of their inner membrane which could be related to an impairment of Müller cell function despite their preserved viability.

Exposure of human Müller cells to hydrogen peroxide for two hours results in a reversible change of protein binding of mitochondrial NADH upon unchanged redox ratio. The mitochondrial membrane potential is increased during exposure.