Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus

Diarrheagenic Escherichia coli (ETEC) bearing CFA/I or CFA/II adhesive factors specifically adhere onto the brush border of the polarized epithelial human intestinal Caco-2 cells in culture. Heat-killed Lactobacillus acidophilus strain LB, that adheres onto Caco-2 cells, inhibits diarrheagenic Escherichia coli adhesion in a concentration-dependent manner. Since the L. acidophilus does not express ETEC-CFA adhesive factors, it can be postulated that the heat-killed L. acidophilus LB cells inhibit diarrheagenic E. coli attachment by steric hindrance of the human enterocytic ETEC receptors.     

Staphylococcus aureus FepA and FepB Proteins Drive Heme Iron Utilization in Escherichia coli

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Mineral and Bacterial Diversities of Desert Sand Grains from South-East Morocco

Mineralogy and microbiology of sand from Merzouga (Morocco) were simultaneously characterized, with the purpose of contributing to a better understanding of the geomicrobiology of deserts. In spite of very low measured bacterial biomass, bacterial diversity on each of the five defined mineralogical classes, was found high. An original grain by grain cultivation method enabled to obtain bacterial isolates with an unusually high recovery rate. The results of this study show that the genus Arthrobacter is well adapted to this environment with a preference for grains other than the dominant mineral quartz, and that the genera Chelatococcus and Saccharotrix are strongly attached to the grains.     

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.     

Demographic response of tundra small mammals to a snow fencing experiment

Snow cover is a key environmental component for tundra wildlife that will be affected by climate change. Change to the snow cover may affect the population dynamics of high‐latitude small mammals, which are active throughout the winter and reproduce under the snow. We experimentally tested the hypotheses that a deeper snow cover would enhance the densities and winter reproductive rates of small mammals, but that predation by mustelids could be higher in areas of increased small mammal density. We enhanced snow cover by setting out snow fences at three sites in the Canadian Arctic (Bylot Island, Nunavut, and Herschel Island and Komakuk Beach, Yukon) over periods ranging from one to four years. Densities of winter nests were higher where snow depth was increased but spring lemming densities did not increase on the experimental areas. Lemmings probably moved from areas of deep snow, their preferred winter habitat, to summer habitat during snow melt once the advantages associated with deep snow were gone. Our treatment had no effect on signs of reproduction in winter nests, proportion of lactating females in spring, or the proportion of juveniles caught in spring, which suggests that deep snow did not enhance reproduction. Results on predation were inconsistent across sites as predation by weasels was higher on the experimental area at one site but lower at two others and was not higher in areas of winter nest aggregations. Although this experiment provided us with several new insights about the impact of snow cover on the population dynamics of tundra small mammals, it also illustrates the challenges and difficulties associated with large‐scale experiments aimed at manipulating a critical climatic factor.     

Wastewater use in algae production for generation of renewable resources: a review and preliminary results

Microalgae feedstock production can be integrated with wastewater and industrial sources of carbon dioxide. This study reviews the literature on algae grown on wastewater and includes a preliminary analysis of algal production based on anaerobic digestion sludge centrate from the Howard F. Curren Advanced Wastewater Treatment Plant (HFC AWTP) in Tampa, Florida and secondary effluent from the City of Lakeland wastewater treatment facilities in Lakeland, Florida. It was demonstrated that a mixed culture of wild algae species could successfully be grown on wastewater nutrients and potentially scaled to commercial production. Algae have demonstrated the ability to naturally colonize low-nutrient effluent water in a wetland treatment system utilized by the City of Lakeland. The results from these experiments show that the algae grown in high strength wastewater from the HFC AWTP are light-limited when cultivated indoor since more than 50% of the outdoor illumination is attenuated in the greenhouse. An analysis was performed to determine the mass of algae that can be supported by the wastewater nutrients (mainly nitrogen and phosphorous) available from the two Florida cities. The study was guided by the growth and productivity data obtained for algal growth in the photobioreactors in operation at the University of South Florida. In the analysis, nutrients and light are assumed to be limited, while CO₂ is abundantly available. There is some limitation on land, especially since the HFC AWTP is located at the Port of Tampa. The temperature range in Tampa is assumed to be suitable for algal growth year round. Assuming that the numerous technical challenges to achieving commercial-scale algal production can be met, the results presented suggest that an excess of 71 metric tons per hectare per year of algal biomass can be produced. Two energy production options were considered; liquid biofuels from feedstock with high lipid content, and biogas generation from anaerobic digestion of algae biomass. The total potential oil volume was determined to be approximately 337,500 gallons per year, which may result in the annual production of 270,000 gallons of biodiesel when 80% conversion efficiency is assumed. This production level would be able to sustain approximately 450 cars per year on average. Potential biogas production was estimated to be above 415,000 kg/yr, the equivalent of powering close to 500 homes for a year.     

Heating and microwave assisted SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield

Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides.     

Cholesterol favors the anchorage of human dystrophin repeats 16 to 21 in membrane at physiological surface pressure

Dystrophin (DYS) is a filamentous protein that connects the cytoskeleton and the extracellular matrix via the sarcolemma, conferring resistance to muscular cells. In this study, interactions between the DYS R16–21 fragment and lipids were examined using Langmuir films made of anionic and zwitterionic lipids. The film fluidity was modified by the addition of 15% cholesterol. Whatever the lipid mixture examined, at low surface pressure (20 mN/m) few differences appeared on the protein insertion and the presence of cholesterol did not affect the protein/lipid interactions. At high surface pressure (30 mN/m), the protein insertion was very low and occurred only in zwitterionic films in the liquid-expanded phase. In anionic films, electrostatic interactions prevented the protein insertion outright, and caused accumulation of the protein on the hydrophilic part of the monolayer. Addition of cholesterol to both lipid mixtures drastically modified the protein–lipid interactions: the DYS R16–21 insertion increased and its organization in the monolayer appeared to be more homogeneous. The presence of accessible cholesterol recognition amino-acid consensus sequences in this fragment may enhance the protein/membrane binding at physiological lateral pressure. These results suggest that the anchorage of dystrophin to the membrane in vivo may be stabilized by cholesterol-rich nano-domains in the inner leaflet of sarcolemma.