Cellular utilization of cytosolic NADPH in kidney and liver cells from rats fed a normal or a vitamin D-deficient diet

The amount of reducing equivalents from NADPH generated by glucose 6-phosphate dehydrogenase activity (G6PD) used in mixed function oxidation (pathway I) or in reductive biosynthesis (pathway II) has been determined by cytochemical methods and microdensitometry in cells from the pars recta (PR) and distal convoluted tubule (DCT) of the kidney and from centrilobular (CL) and periportal (PP) hepatocytes from rats fed a normal or a vitamin D-deficient diet. In the kidney, pathway I activity was similar to that of pathway II in PR, whereas in DCT pathway II was markedly predominant. Feeding a vitamin D-deficient diet resulted in an increase in the total amount of reducing equivalents in PR and DCT. This increase was due to a rise in pathway I activity in the PR, whereas in the DCT the increase resulted from a stimulation of pathway II activity. Pathway I activity in PR was inversely correlated with plasma calcium, and was significantly decreased when calcium (1 mM) was added in vitro. In the liver the total amount of reducing equivalents generated by G6PD and both hydrogen pathways, was higher in CL than in PP hepatocytes. In CL cells, a vitamin D-deficient diet induced a significant increase in both NADPH pathways. Furthermore, in these cells pathway I activity was inversely related to plasma calcium and was significantly lowered when 1 mM calcium was added in vitro. It is concluded that vitamin D status and calcium influence the production and utilization of cytosolic reducing equivalents both in kidney and liver.     

In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdensitometry

A quantitative microdensitometric study has been designed to characterize in situ intestinal brush border-bound alkaline phosphatase of rat duodenal villosities. Intestinal slices were incubated with beta-glycerophosphate as substrate. Free phosphate liberated was precipitated in presence of a lead reagent as lead sulfide. The precipitate was quantified in situ by scanning and integrating microdensitometry. Kinetic parameters of the reaction were determined at 37 degrees C, pH 8.8, in the middle part of the villosities. Apparent Michaelis constant (Km) for beta-glycerophosphate was found to be 8.16 +/- 0.56 mM (mean +/- S.E.). Maximal enzyme activation was obtained at pH 8.5. Maximal inhibition of enzyme activity was observed in the presence of L-phenylalanine (30 mM) or theophylline (5 mM). Along the villosity axis, enzyme activity rose from the crypt up to the midportion of the villosity and finally decreased at the tip region. In phosphate-depleted rats, enzyme activity was increased in all portions of the villosity, with conservation of the same activity gradient. In this situation, kinetic analysis showed a marked decrease of Km, i.e. 4.56 +/- 0.39 mM (mean +/- S.E.) as compared to normal rats.     

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.     

Do leaf-litter decomposers control biofilm primary production and benthic algal community structure in forest streams? Insights from an outdoor mesocosm experiment

• 1. Forested headwater streams are generally considered to be light-limited ecosystems where primary production is reduced, and the main source of energy and nutrients is composed of allochthonous detritus. We hypothesised that in these ecosystems, the development of primary producers might also be limited by (1) competition for nutrients with leaf-litter decomposers (e.g. bacteria and fungi), and (2) leaf-litter leachates or allelopathic compounds produced by aquatic fungi.
• 2. To test these hypotheses, a 48-day mesocosm experiment was performed in 12 artificial streams containing stream water inoculated with epilithic biofilm suspensions collected from a forested headwater stream. Three different treatments were applied: control without leaf litter (C), microbially conditioned leaf litter added at the beginning of the experiment and left to decompose throughout the experiment (L), or leaf litter renewed three times during the experiment (RL).
• 3. We predicted that (1) the presence of litter, through microbial nutrient immobilisation and allelopathy, would reduce primary production and that (2) this effect would be amplified by litter renewal. We also predicted that nutrient competition would mean that (3) leaf-litter decomposers will alter primary producer community composition and physiology. These predictions were tested by analysing biofilm development, physiology, stoichiometry, and benthic algal community structure. To distinguish between the effects of nutrient immobilisation and allelopathy, the biofilm responses to leaf-litter leachates collected after different microbial conditioning durations were also measured in a parallel laboratory experiment.
• 4. Contrary to our expectations, by day 28, primary producer growth was higher in the mesocosms containing leaf litter (L and RL) despite the rapid decrease in dissolved nutrients when leaf litter was present. After 48 days, the lowest phototrophic biofilm development was observed when leaf litter was renewed (RL), whereas phototrophic biofilm development was similar in the C and L treatments. Biofilm stoichiometry indicated that this effect was most probably related to greater nitrogen limitation in the RL treatment. The presence of leaf litter also affected primary producers' photophysiology, which could be attributed to changes in taxonomic composition and to physiological adjustments of primary producers.
• 5. Laboratory measurements showed that despite a strong inhibition of primary producer growth by unconditioned leaf-litter leachates, microbially conditioned leaf litter had either low or no effects on the development of primary producers.
• 6. These results reveal that leaf-litter decomposers can have both positive and negative effects on primary producers underlining the need to consider microbial interactions when investigating the functioning of forested headwater streams.

     

Temperature and nutrient effects on the relative importance of brown and green pathways for stream ecosystem functioning: A mesocosm approach

1. In addition to global warming, aquatic ecosystems are currently facing multiple global changes among which include changes in nitrogen (N) loads. While several studies have investigated both temperature and N impacts on aquatic ecosystems independently, knowledge on their interactive effects remains scarce.
2. In forested headwater streams, decomposition of leaf litter represents the main process ensuring the transfer of nutrients and energy to higher trophic levels, followed by autochthonous primary production, mainly ensured by phototrophic biofilms. The main aim of this study was to disentangle the independent and combined effects of temperature increase and nutrient availability on the relative importance of brown and green processes involved in stream functioning. We hypothesised that water temperature and nutrients would lead to a general increase in leaf-litter decomposition and primary production, but that the intensity of these effects would be largely modulated by competitive interactions arising between microorganisms as well as by the top-down control of microorganisms by macro-invertebrates. Macro-invertebrates would, in turn, be bottom-up controlled by microbial resources quality.
3. To test these hypotheses, we conducted a 56-day experiment in artificial streams containing leaf litter, microbial decomposers and biofilm inoculum, and an assemblage of macro-invertebrates. Two water inorganic N:phosphorus (P) ratios (33 and 100, molar ratios) and two temperatures (ambient, +2°C) were manipulated, each treatment being replicated three times. Fungal and biofilm growth as well as leaf-litter decomposition and primary production were quantified. Top-down impacts of invertebrate primary consumers on brown and green compartments were evaluated using exclosures while bottom-up control was evaluated through the measurement of resource stoichiometry and fatty acid profiles, as well as quantification of macro-invertebrate growth and survival.
4. Contrary to expectations, microbial decomposition was not significantly stimulated by nutrient or temperature manipulations, while primary production was only improved under ambient temperature. In the + 2°C treatment with high N:P, greater biofilm biomass was associated with lower fungal development, which indicates competition for nutrients in these conditions. Temperature increased macro-invertebrate growth and leaf-litter consumption, but this effect was independent of any improvement of basal resource quality, suggesting that temperature mediated changes in consumer metabolism and activity was the main mechanism involved.
5. Most of our hypotheses that were based on simplified laboratory observations have been rejected in our semi-controlled mesocosms. Our study suggests that the complexity of biological communities might greatly affect the response of ecosystems to multiple stressors, and that interactions between organisms must be explicitly taken into account when investigating the impacts of global change on ecosystem functioning.