Reactive oxygen species mediate a cellular 'memory' of high glucose stress signalling.

AIMS/HYPOTHESIS: A long-term 'memory' of hyperglycaemic stress, even when glycaemia is normalised, has been previously reported in endothelial cells. In this report we sought to duplicate and extend this finding. MATERIALS AND METHODS: HUVECs and ARPE-19 retinal cells were incubated in 5 or in 30 mmol/l glucose for 3 weeks or subjected to 1 week of normal glucose after being exposed for 2 weeks to continuous high glucose. HUVECs were also treated in this last condition with several antioxidants. Similarly, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus alpha-lipoic acid in the last week. RESULTS: In human endothelial cells and ARPE-19 retinal cells in culture, as well as in the retina of diabetic rats, levels of the following markers of high glucose stress remained induced for 1 week after levels of glucose had normalised: protein kinase C-beta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, fibronectin, poly(ADP-ribose) Blockade of reactive species using different approaches, i.e. the mitochondrial antioxidant alpha-lipoic acid, overexpression of uncoupling protein 2, oxypurinol, apocynin and the poly(ADP-ribose) polymerase inhibitor PJ34, interrupted the induction both of high glucose stress markers and of the fluorescent reactive oxygen species (ROS) probe CM-H(2)DCFDA in human endothelial cells. Similar results were obtained in the retina of diabetic rats with alpha-lipoic acid added to the last week of normalised glucose. CONCLUSIONS/INTERPRETATION: These results provide proof-of-principle of a ROS-mediated cellular persistence of vascular stress after glucose normalisation.

Hypothesis: the 'metabolic memory', the new challenge of diabetes.

Large randomized studies have established that early intensive glycaemic control reduces the risk of diabetic complications, both micro- and macrovascular. However, epidemiological and prospective data support a long-term influence of early metabolic control on clinical outcomes. This phenomenon has recently been defined as 'metabolic memory.' Potential mechanisms for propagating this 'memory' are the non-enzymatic glycation of cellular proteins and lipids, and an excess of cellular reactive oxygen and nitrogen species, in particular originated at the level of glycated-mitochondrial proteins, perhaps acting in concert with one another to maintain stress signalling. Furthermore, the emergence of this 'metabolic memory' suggests the need for very early aggressive treatment aiming to 'normalize' glycaemic control and the addition of agents which reduce cellular reactive species and glycation in order to minimize long-term diabetic complications.

Melatonin rhythms in Atlantic salmon (Salmo salar) maintained under natural and out-of-phase photoperiods.

Diel changes in circulating melatonin were measured in juvenile Atlantic salmon, Salmo salar, maintained under natural and out-of-phase seasonal photocycles. Under natural daylengths of autumn, winter, spring, and summer circulating melatonin levels were inversely related to light intensity, with levels low during the day and high at night. The duration of the nocturnal increase in circulating melatonin was related to the duration of darkness, i.e., longer in winter than in summer. Under simulated seasonal photocycles circulating melatonin concentrations measured in August, October, and December were also elevated for the duration of darkness, irrespective of whether the photoperiods were synchronized or 6 months out-of-phase with the natural light and temperature cycles. Circulating melatonin also provided an accurate representation of the prevailing photoperiod in fish initially maintained on simulated natural photocycles, either synchronized or 6 months out-of-phase with the natural light cycle, and then held for 3 months on daylengths approximating the summer and winter solstices. Well-defined melatonin rhythms were always present, irrespective of time of year, photoperiod, and temperature. The amplitude of the nocturnal increase in circulating melatonin was similar in groups of fish maintained under simulated seasonal photoperiods 6 months out-of-phase with each other, but otherwise identical conditions, indicating that daylength per se did not influence the amplitude of the melatonin rhythm. The amplitude of the melatonin rhythm was slightly higher during the summer months, suggesting that temperature may modify circulating melatonin levels. These results demonstrate that circulating melatonin profiles always reflect the prevailing daylength and hence have the potential to provide the Atlantic salmon with accurate information on daily and calendar time, which could be utilised to time daily and seasonal events.

Effect of oral antacid administration on the pharmacokinetics of oral fluconazole.

Absorption and elimination of fluconazole after oral administration of a 100-mg capsule were unaffected by concomitant administration of an antacid containing aluminum and magnesium hydroxides.

Fluconazole levels in plasma and vaginal secretions of patients after a 150-milligram single oral dose and rate of eradication of infection in vaginal candidiasis.

Mean peak concentrations of fluconazole in plasma and vaginal secretions of females after a 150-mg single oral dose were shown to be 2.82 micrograms/ml and 2.43 micrograms/g, respectively. Our results indicate that clinically efficacious concentrations of fluconazole in vaginal secretions are easily achieved after this single oral dose.

Early social status and the development of life-history strategies in Atlantic salmon.

Atlantic salmon have a variable life cycle. In good growing conditions, underyearling fish may metamorphose into the migratory smolt phase during their second spring, or delay at least a further year. The strategy adopted by particular fish appears to become fixed during their first summer. This paper examines whether either feeding efficiency or dominance in mid-summer correlates with the life-history strategy adopted. Eighty fish were individually marked and their feeding efficiency (= mean handling time for food items) and dominance rank measured under laboratory conditions in mid-July. Growth rates of the fish were then monitored over the next three months, until developmental strategies became apparent. Discriminant and logistic regression analyses revealed that both dominance rank and size attained by July were independent, significant predictors of future developmental pattern (the age at metamorphosis being correctly predicted on the basis of rank and size in 84% of cases) whereas feeding efficiency had no effect. Thus fish that were dominant or larger two months after first feeding or both had a greater probability of migrating after only one year in freshwater than those more subordinate or smaller or both.

The effect of temperature on myogenesis in embryonic development of the Atlantic salmon (Salmo salar L.).

From fertilisation to hatching one group of salmon embryos was reared at ambient temperatures (fluctuating around 1.6 degrees C) and another at 10 degrees C. At Gorodilov stages 28, 30 and 33 transverse sections of whole embryos were obtained for light and electron microscopy. Total cross-sectional areas, fibre numbers, fibre diameters and myofibrillar areas of the white muscle of m. lateralis were measured. At hatching (stage 33, which occurred much earlier at the higher temperature), the higher temperature embryos had significantly larger (P less than 0.01) but fewer (P less than 0.05) muscle fibres. These larger fibres contained significantly more myofibrillar material (P less than 0.05) than the smaller fibres of the lower temperature embryos. Lesser differences were found at pre-hatching stages. Higher temperatures caused myofibre hypertrophy to increase at a greater rate than hyperplasia. Hence, the cellularity of the tissue produced under the different temperature regimes was quite different.

Transepithelial ion exchange in smolting atlantic salmon (Salmo Salar L.).

Whole-body (but predominantly gill) Na(+) exchange, gill Na(+)/K(+)/ATPase activity and seawater tolerance were examined in juvenile Atlantic salmon during the smolting period. Transepithelial net Na(+) gain decreased steadily from late February showing a net loss in April and early May, returning to approximate equilibrium in mid-May. This seasonal net loss of Na(+) to the environment occurred slightly after maximal gill epithelial Na(+)/K(+)/ATPase activity and preceded maximal seawater tolerance. The results are discussed in relation to changes in gill permeability and salt intake via the diet.

Bronchoalveolar lavage for diagnosing acute bacterial pneumonia.

The utility of gram stain and semiquantitative culture of the fluid retrieved by bronchoalveolar lavage (BAL) in identifying the causative agent in acute bacterial pneumonia was initially assessed in 92 patients. Fifteen of these patients presented with clinically active bacterial pneumonia; the remaining patients underwent bronchoscopy to evaluate other processes in the lung. Thirteen of the 15 patients with clinically active bacterial pneumonia had a BAL culture greater than or equal to 10(5) colony-forming units per milliliter of BAL fluid, whereas none of the other groups had a positive culture (chi 2 = 70.7, P less than .001). Gram stain of cytocentrifuged BAL fluid was positive (one or more organisms seen per 1,000 X field) only in those patients with an active bacterial pneumonia. Applying this technique, we studied 59 immunocompromised patients presenting with pulmonary infiltrates. Eight (21%) of the 39 patients presenting with microbial-related infiltrates proved to have acute bacterial pneumonia by BAL culture; the pneumonia resolved with appropriate antimicrobial therapy.

Use of the protected specimen brush in patients with endotracheal or tracheostomy tubes.

Twenty-one patients on mechanical ventilators for greater than 48 hours who had new localized infiltrates were evaluated using a quantitative culture technique of the involved lung compared to the non-involved lung. Based on the clinical course, response to antibiotics, or subsequent analysis of pathologic specimens, eight patients were felt to have acute bacterial pneumonia, while the remaining 13 were felt to have an alternative cause of their infiltrate. Cultures of the protected brush specimen of the involved lung in all eight cases of bacterial pneumonia had one or more organisms grown at a greater than 100 colony forming units (cfu) per ml while only one of the 13 cases of non-pneumonia had a culture from the involved area having greater than 100 cfu per ml (p less than 0.001). The non-involved area always grew fewer organisms than the involved area, and in 16 cases, there was no growth from the specimen obtained from the non-involved area.

Diel and seasonal changes in resting plasma cortisol levels in juvenile Atlantic salmon, Salmo salar L.

Plasma cortisol levels in juvenile Atlantic salmon, Salmo salar L., were higher at night than during the day from September to May, and were higher in the morning than at night from June to August. Mean nocturnal levels increased slightly from August (less than 1.4 ng/ml) to January (18.2 ng/ml), sharply to a peak in March (46.0 ng/ml), and then declined sharply again reaching minimal values in July (less than 1.4 ng/ml). With the exception of a February peak (7.7 ng/ml) mean diurnal levels also increased slightly from a September minimum of (less than 1.4 ng/ml) to April (3.3 ng/ml), then more sharply to plateau from May to July (8.2-11.0 ng/ml), and declined again to September. The data support the hypothesis that rising cortisol levels in spring represent a generalized stress response to behavioral and physiological maladaptation at smolting.

Bronchial reactivity increases soon after the immediate response in dual-responding asthmatic subjects.

To determine whether bronchial reactivity is augmented soon after an allergen-induced immediate asthmatic response, we compared reactivity to histamine before and immediately upon resolution of the immediate asthmatic response in seven subjects with mild asthma who were known to develop dual asthmatic responses after inhalation of Kentucky bluegrass allergen. Using a body plethysmograph and quiet breathing technique, specific airway resistance (SRaw) and reactivity to aerosol histamine were assessed on two days prior to allergenic challenge. The dose of histamine that doubled SRaw (PC200His) was determined by interpolation from histamine dose-response curves. On the day of allergenic challenge, each subject inhaled a concentration that was sufficient to induce a dual asthmatic response. Upon resolution of the immediate asthmatic response (45 to 105 minutes) after allergen, the PC200His in all cases had decreased more than 50 percent of its original value (PC200His for the group was 0.29 +/- 1.42 mg/ml [mean +/- SE], compared to 0.84 +/- 1.23 mg/ml initially). Seven or more days after the allergen, each subject had a PC200His comparable to original values. Our data indicate that airway reactivity in dual-responding asthmatic subjects markedly increases soon after the immediate asthmatic response and much before the late asthmatic response manifests clinically. Whether this early increase in bronchial reactivity is a putative requirement for, or shares common characteristics with the late asthmatic response requires further study.

Effects of leukotriene D4 and antagonists of arachidonic acid metabolism and calcium entry on guinea pig tracheal muscle responsiveness.

We studied the effects of antagonists of arachidonic acid metabolism and calcium entry on the responsiveness of airway smooth muscle to acetylcholine (ACh), potassium and norepinephrine. Responsiveness in vitro was assessed by measuring isometric contraction of guinea pig trachealis muscle upon chemical stimulation. We found that indomethacin potentiated the response of airway muscle to ACh and KCl, but not to norepinephrine. The indomethacin-induced potentiation observed was inhibited by both BW 755C and nifedipine. As occurred with indomethacin pretreatment, we also found that a noncontractile concentration of leukotriene D4 (LTD4) potentiated the responsiveness of tracheal muscle to both ACh and KCl. Our data suggest that indomethacin- and LTD4-potentiation of guinea pig airway muscle responsiveness to certain bronchoconstrictors may be mediated, at least in part, by enhanced extracellular Ca++ entry.

Leukotriene B4 potentiates airway muscle responsiveness in vivo and in vitro.

We studied the effects of leukotriene B4 (LTB4) on guinea pig airway muscle responsiveness in vivo and in vitro. Responsiveness in vivo was assessed by measuring specific airway resistance (SRaw) upon intravenous acetylcholine infusion in 5 unanesthetized, spontaneously breathing guinea pigs. We found that aerosolized LTB4, in a concentration that itself had no effect on baseline SRaw, caused a substantial increase in bronchial reactivity to i.v. ACh within 8 min of its administration. Responsiveness in vitro was assessed by measuring isometric contraction of the guinea pig trachealis upon stimulation by either chemical or electrical field stimuli. These studies in vitro showed that a concentration of LTB4 that itself did not cause contraction, potentiated airway muscle contraction to ACh and KCl, but not to norepinephrine. This effect of LTB4 was substantially reduced by nifedipine. Our data suggests that amounts of LTB4 that are themselves non-contractile in vivo or in vitro, may directly potentiate the responsiveness of airway smooth muscle to other bronchoconstrictors.