Phase-dependent effect of room light exposure in a 5-h advance of the sleep-wake cycle: implications for jet lag

The acute disruption in sleep quality, vigilance levels, and cognitive and athletic performance observed after transmeridian flights is presumed to be the result of a transient misalignment between the endogenous circadian pacemaker and the shifted sleep schedule. Several laboratory and field experiments have demonstrated that exposure to bright artificial light can accelerate circadian entrainment to a shifted sleep-wake schedule. In the present study, the authors investigated whether the schedule of exposure to indoor room light, to which urban dwellers are typically exposed, can substantially affect circadian adaptation to a simulated eastward voyage. We enrolled 15 healthy young men in a laboratory simulation of a Montreal-to-London voyage. Subjects were exposed to 6 h of room light (mean +/- SD: 379+/-10) prior to bedtime (n = 7) or when on a progressively advancing schedule (n = 8) early in the day. The remaining 10 hours of wakefulness were spent in dim light (4+/-1 lux). Circadian assessments, performed via the constant routine procedure, evaluated the phase of the endogenous circadian rhythms of core body temperature and plasma melatonin before and after 1 week on the shifted schedule. At the end of the study, only subjects exposed to room light on the advancing schedule expressed oscillations of the endogenous circadian pacemaker in phase with the new sleep-wake cycle. In this group, a mean advance shift of the nadir of core body temperature of +5:22+/-0:15 h was observed, with parallel shifts in plasma melatonin concentration and subjective alertness. The circadian rhythms of subjects exposed to room light later in the day remained much more adjusted to the departure than to the destination time zone. These results demonstrate that the schedule of exposure to room light can substantially affect circadian adaptation to a shifted sleep-wake schedule.     

Resistance to Fas-induced apoptosis in hepatocytes: role of GSH depletion by cell isolation and culture

The involvement of reduction/oxidation (redox) state in cell sensitivity to apoptosis has been suggested by several studies in which induction of apoptosis was shown to require oxidative stress or GSH extrusion. On the other hand, biochemical studies of caspases revealed that their activation necessitates a reduced cysteine in their active site. This is ensured by maintaining intact intracellular glutathione status during apoptotic induction as reported by in vivo studies. Therefore, we investigated the relationship between intracellular glutathione levels and the sensitivity of mouse hepatocytes in culture to Fas-induced apoptosis as well as potential mechanisms responsible for this sensitivity. We found that total and reduced glutathione levels are decreased by one-half after cell isolation procedure and further decline by 25% during cell culture for 2 h in normal Williams' E medium. Cell culture in medium supplemented with cysteine and methionine maintains glutathione at a level similar to that measured just after cell isolation. Results show that the capacity of Fas to activate caspase-8 and to induce apoptosis requires important intracellular glutathione levels and high GSH/total glutathione ratio. In conclusion, the present study shows that intracellular glutathione plays an important role in maintaining the apoptotic machinery functional and is thus capable of transmitting the apoptotic signal.     

Phospholipase D in Phytophthora infestans and its role in zoospore encystment

We show that differentiation of zoospores of the late blight pathogen Phytophthora infestans into cysts, a process called encystment, was triggered by both phosphatidic acid (PA) and the G-protein activator mastoparan. Mastoparan induced the accumulation of PA, indicating that encystment by mastoparan most likely acts through PA. Likewise, mechanical agitation of zoospores, which often is used to induce synchronized encystment, resulted in increased levels of PA. The levels of diacylglycerolpyrophosphate (DGPP), the phosphorylation product of PA, increased simultaneously. Also in cysts, sporangiospores, and mycelium, mastoparan induced increases in the levels of PA and DGPP. Using an in vivo assay for phospholipase D (PLD) activity, it was shown that the mastoparan-induced increase in PA was due to a stimulation of the activity of this enzyme. Phospholipase C in combination with diacylglycerol (DAG) kinase activity also can generate PA, but activation of these enzymes by mastoparan was not detected under conditions selected to highlight 32P-PA production via DAG kinase. Primary and secondary butanol, which, like mastoparan, have been reported to activate G-proteins, also stimulated PLD activity, whereas the inactive tertiary isomer did not. Similarly, encystment was induced by n- and sec-butanol but not by tert-butanol. Together, these results show that Phytophthora infestans contains a mastoparan- and butanol-inducible PLD pathway and strongly indicate that PLD is involved in zoospore encystment. The role of G-proteins in this process is discussed.     

Production of transglutaminase from Bacillus circulans on solid-state and submerged cultivations

A Bacillus circulans strain, isolated in the Amazon basin, produced a transglutaminase (EC 2.3 x 2.13) in both submerged and solid-state cultivation. Enzyme activity in the former case reached 0.69 U ml(-1) after 160 h cultivation on starch based medium, and in the latter 0.61 U ml(-1) after 60 h cultivation on soybean industrial fibrous residue, a non-expensive agro-industrial residue.     

Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.     

Peptidomic and proteomic analyses of the systemic immune response of Drosophila

Insects have developed an efficient host defense against microorganisms, which involves humoral and cellular mechanisms. Numerous data highlight similarities between defense responses of insects and innate immunity of mammals. The fruit fly, Drosophila melanogaster, is a favorable model system for the analysis of the first line defense against microorganisms. Taking advantages of improvements in mass spectrometry (MS), two-dimensional (2D) gel electrophoresis and bioinformatics, differential analyses of blood content (hemolymph) from immune-challenged versus control Drosophila were performed. Two strategies were developed: (i) peptidomic analyses through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and high performance liquid chromatography for molecules below 15 kDa, and (ii) proteomic studies based on 2D gel electrophoresis, MALDI-TOF fingerprinting and database searches, for compounds of greater molecular masses. The peptidomic strategy led to the detection of a large number of peptides induced in the hemolymph of challenged flies as compared to controls. Of these, 28 were characterized, amongst which were antimicrobial peptides. The 2D gel electrophoresis strategy led to the detection of 70 spots differentially regulated by at least fivefold after microbial infection. This approach yielded the identity of a series of proteins that were related to the Drosophila immune response, such as proteases, protease inhibitors, prophenoloxydase-activating enzymes, serpins and a Gram-negative binding protein-like protein. This strategy also brought to light new candidates with a potential function in the immune response (odorant-binding protein, peptidylglycine alpha-hydroxylating monooxygenase and transferrin). Interestingly, several molecules resulting from the cleavage of proteins were detected after a fungal infection. Together, peptidomic and proteomic analyses represent new tools to characterize molecules involved in the innate immune reactions of Drosophila.     

Involvement of the S100B in cAMP-Induced Cytoskeleton Remodeling in Astrocytes: A Study Using TRTK-12 in Digitonin-Permeabilized Cells

1. Stellation of astrocytes in culture involves a complex rearrangement of microfilaments, intermediate filaments, and microtubules, which reflects in part the plasticity of these cells observed during development or after injury.2. An astrocytic calcium-binding protein, S100B, has been implicated in the regulation of plasticity due to its ability to interact with cytoskeletal proteins.3. We used digitonin-permeabilized astrocytes to introduce TRTK-12, a peptide that binds to the C-terminal of S100B and blocks its interaction with cytoskeletal proteins.4. TRTK-12 was able to block cAMP-induced astrocyte stellation and this effect was dependent on the concentration of the peptide. These results support the idea that S100B has a modulatory role on astrocyte morphology.     

Modulation of a "CD59-like" protein in Naegleria fowleri amebae by bacteria

Found in soil and freshwater habitats, Naegleria fowleri are free-living amebae that cause a fatal disease in humans called Primary Amebic Meningoencephalitis. In the natural environment, amebae feed on bacteria. In the infected host, the amebae lyse and ingest nerve tissue. Recently, we have established that N. fowleri expresses a "CD59-like" surface protein, but the function of this protein in the ameba has not been elucidated. In mammalian cells, CD59 is a complement-regulatory protein that inhibits complement-mediated lysis of cells expressing this protein. In the present study, expression of the "CD59-like" protein in response to bacteria and bacterial toxins was investigated by Western immunoblot analysis. Co-culture of N. fowleri with log phase Escherichia coli or Pseudomonas aeruginosa resulted in differential expression of the "CD59-like" protein. Co-cultures of amebae and bacteria were examined by electron microscopy. The results of our study implicate a possible protective role of the "CD59-like" protein in response to bacterial predators and bacterial toxins, because amebae remained intact after co-culture with bacteria.     

Descriptive morphology of the reared eggs,larvae, and juveniles of the marine atherinid fish <Emphasis Type="Italic">Atherinomorus duodecimalis</Emphasis>

Embryonic, larval, and juvenile development of the marine atherinid Atherinomorus duodecimalis are described from laboratory-reared specimens. The eggs, measuring 1.15–1.24mm in diameter, were demersal and almost spherical in shape with numerous chorionic filaments. Hatching occurred 7 or 8 days after spawning, the newly hatched larvae measuring 4.5–5.1mm in body length (BL) and having 5+34=39 myomeres. Notochord flexion started at 6.9mm BL and finished at 8.3mm BL. Aggregate numbers of all fin rays were completed by 14mm BL. Eggs of this species could be distinguished from other known atherinid eggs on the basis of egg diameter and the arrangement, length, and number of chorionic filaments. Larvae were also distinctive in myomere counts, pigmentation, and locations of the anus and second dorsal fin origin.