Mixotrophic growth of Phaeodactylum tricornutum on glycerol: growth rate and fatty acid profile

Mixotrophic growth of the eicosapentaenoic acid (EPA)producing diatom Phaeodactylum tricornutum UTEX640 was carried out in 1-L batch cultures under anexternal irradiance of 165 mol photons m^-2s^-1 by supplementing the inorganic culture mediumwith glycerol. The effect on the growth and the fattyacid profile was studied for different initialglycerol concentrations (0–0.1 M). The optimalglycerol concentration was 0.1 M.A lag phase was observed at high glycerolconcentrations. The present study also shows thatsuccessive additions of glycerol at 0.1M concentrationand using ammonium chloride as a nitrogen sourceremarkably increased the maximum biomass concentration(16.2 g L^-1) and maximum biomass productivity(61.5 mg L^-1 h^-1). These values wererespectively 9 and 8-fold higher than in thephotoautotrophically grown control. The level ofsaponifiable lipids in mixotrophically cultured cellswas significantly higher than in photoautotrophicallycultured cells and increased with the glycerolconcentration in the medium. The concentration ofstorage lipids, saturated and monounsaturated fattyacids, were enhanced but the EPA content did notchange significantly. The EPA content was around 2.2%of biomass dry weight. The maximum EPA yield was33.5 mg L^-1 d^-1 and was obtained in aculture containing 0.1 M glycerol, supplementedperiodically by ammonium chloride. This productivitywas 10-fold higher than the EPA productivity obtainedunder mixotrophic conditions.     

Detection of microsporidia in surface water: a one-year follow-up study

In order to estimate the rate and seasonal variation of Enterocytozoon bieneusi contamination of surface water, sequential samples of water from the River Seine in France were collected during a 1-year period. Each sample (300-600 l) was submitted to sequential filtrations, and the filters were then examined for microsporidia using light microscopy and nested polymerase chain reaction (PCR) for E. bieneusi. Amplified products were hybridized with a E. bieneusi-specific probe. Twenty-five samples of water were analyzed during 1 year. Microscopic examination of stained filters proved unreliable for the identification of spores. Using nested PCR, 16 of 25 specimens were positive (64%). Unexpectedly, E. bieneusi was identified in only one sample by specific hybridization underlining the lack of specificity of ours primers. Nevertheless, using DNA sequence analysis, unknown microsporidia species were identified in eight cases, which had highest scores of homology with Vittaforma corneae or Pleistophora sp. This study shows a low rate of water contamination by E. bieneusi suggesting that the risk of waterborne transmission to humans is limited.     

Unfolding and refolding in vitro of a tetrameric, alpha-helical membrane protein: the prokaryotic potassium channel KcsA

2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly alpha-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771-10777]. Here, we report that the effects on the protein structure of increasing concentrations of TFE in detergent solution include two successive protein concentration-dependent, cooperative transitions. In the first of such transitions, occurring at lower TFE concentrations, the tetrameric KcsA simultaneously increases the exposure of tryptophan residues to the solvent, partly loses its secondary structure, and dissociates into its constituent subunits. Under these conditions, simple dilution of the TFE permits a highly efficient refolding and tetramerization of the protein in the detergent solution. Moreover, following reconstitution into asolectin giant liposomes, the refolded protein exhibits nativelike potassium channel activity, as assessed by patch-clamp methods. Conversely, the second cooperative transition occurring at higher TFE concentrations results in the irreversible denaturation of the protein. These results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits, in which folding and oligomerization (or unfolding and dissociation in the other direction of the equilibrium process) are seemingly coupled processes. At higher TFE concentrations this is followed by the irreversible conversion of the unfolded monomers into a denatured protein form.     

Determination of the tissue distribution and excretion by accelerator mass spectrometry of the nonadecapeptide 14C-Moli1901 in beagle dogs after intratracheal instillation

Moli1901 is a 19 residue polycyclic peptide antibiotic which increases chloride transport and water mobilization in airway epithelium. These properties suggest that it may be a useful treatment for cystic fibrosis (CF). In this study, we used accelerator mass spectrometry (AMS) to quantify Moli1901 following administration of only 0.045 microCi of 14C-Moli1901 per dog. Limits of quantitation of AMS were 0.03 (urine) to 0.3 (feces) ng equiv. Moli1901/g. Administration of 14C-Moli1901 by intratracheal instillation (approximately 100 microg) into the left cranial lobe of the lung of beagle dogs resulted in retention of 64% of the dose in the left cranial lobe for up to 28 days. Whole blood and plasma concentrations of 14C were <5 ng/ml at all times after the dose. Concentrations of 14C in whole blood and plasma declined over the first day after the dose and rose thereafter, with the rise in plasma concentrations lagging behind those in whole blood. During the first 3 days after the dose, plasma accounted for the majority of 14C in whole blood, but after that time, plasma accounted for only 25-30% of the 14C in whole blood. Tissue (left and right caudal lung lobe, liver, kidney, spleen, brain) and bile concentrations were low, always less than 0.25% the concentrations found in the left cranial lung lobe. Approximately 13% of the dose was eliminated in urine and feces in 28 days, with fecal elimination accounting for about 10% of the dose. The data presented here are consistent with that obtained in other species. Moli1901 is slowly absorbed and excreted from the lung, and it does not accumulate in other tissues. Moli1901 is currently in the clinic and has proven to be safe in single dose studies in human volunteers and cystic fibrosis patients by the inhalation route. No information on the disposition of the compound in humans is available. This study in dogs demonstrates the feasibility of obtaining that information using 14C-Moli1901 and AMS.     

Genetic variation in the widespread lichenicolous fungus Marchandiomyces corallinus

The lichenicolous basidiomycete Marchandiomyces corallinus is widely distributed in North America and Europe, where it commonly is found on a variety of lichens. Theoretically either of these characteristics, a wide geographic range or generalized host ecology, could provide opportunities for genetic differentiation within this species. To determine how genetic variation is partitioned in M. corallinus, 12 fungal isolates were obtained from locations in North America and Europe; at two locations, in Washington County, Maine, and on the Isle of Mull in Scotland, fungi also were isolated from different lichen hosts. Vegetative mycelial compatibility tests were used to determine compatibility groupings from among the isolates; in addition, several PCR amplification products (RAPD, nuITS rDNA) were obtained for each isolate. A number of distinct compatibility groups were recognizable based on geography, not host ecology. In addition compatible isolates always were restricted to either North America or Europe. However RAPD markers indicated that compatible isolates are not always genetically identical. The presence of sequence heterozygosity at specific positions indicated that the isolates are heterokaryotic and a number of distinct haplotypes could be identified based on ITS variation at three separate locations. This type of genetic variation in these fungi suggests that sexual recombination is possible and that genetic differentiation has taken place recently as a result of geographic isolation, not host switching.     

Ependymal Cell Differentiation and GLUT1 Expression is a Synchronous Process in the Ventricular Wall

Ependymal cells appear to be totally differentiated during the first 3 weeks in the mouse brain. Early during postnatal development ependymal cells differentiate and undergo metabolic activation, which is accompanied by increased glucose uptake. We propose that ependymal cells induce an overexpression of the glucose transporter, GLUT1, during the first 2 weeks after delivery in order to maintain the early metabolic activation. During the first postnatal day, GLUT1 is strongly induced in the upper region of the third ventricle and in the ventral area of the rostral cerebral aqueduct. During the next 4 days, GLUT1 is expressed in all differentiated ependymal cells of the third ventricle and in hypothalamic tanycytes. At the end of the first week, ependymal cell differentiation and GLUT1 overexpression is concentrated in the latero-ventral area of the aqueduct. We propose that ependymal cell differentiation and GLUT1 overexpression is a synchronous process in the ventricular wall.     

Activity of antioxidant enzymes in response to cadmium in Crotalaria juncea

The aromatic amine, -phenethylamine, was identified in various field-grown leguminous plants by analyses with HPLC, GC, GC-MS and ¹H-NMR. High concentration of -phenethylamine was generally detected only in mature root nodules, but not in other plant organs such as root, stem, leaf, pod and grain. Occurrence was specific to the root nodules formed by Bradyrhizobium infection. Ten of eleven legume crops including soybean [Glycine max (L.) Merr.], pigeon pea [Cajanus cajan (L.) Millsp.], adzuki bean (Vigna angularis), mung bean [V. radiata (L.) Wilczek] and cowpea (V. unguiculata) contained this aromatic amine, but groundnut (Arachis hypogaea L.) also nodulated by Bradyrhizobium sp. did not. Root nodules collected from garden pea (Pisum sativum L.), broad bean (Vicia fava L.), kidney bean (Phaseolus vulgaris L.) and various other herbaceous legumes nodulated by Rhizobium sp., Mesorhizobium sp., Sinorhizobium sp. or Azorhizobium caulinodans, and root-nodulated, woody non-legumes, nodulated by Frankia spp., contained little -phenethylamine.The amount of -phenethylamine in Bradyrhizobium-infected nodules varied with the legume species and their cultivars, and most significantly, with nodule age. In field-grown soybean plants, nodule -phenethylamine attained maximum concentration at the flowering stage and far exceeded that of the major polyamines of soybean nodules, putrescine and spermidine.