Denitrifying Bacteria Active in Woodchip Bioreactors at Low-Temperature Conditions.

Woodchip bioreactor technology removes nitrate from agricultural subsurface drainage by using denitrifying microorganisms. Although woodchip bioreactors have demonstrated success in many field locations, low water temperature can significantly limit bioreactor efficiency and performance. To improve bioreactor performance, it is important to identify the microbes responsible for nitrate removal at low temperature conditions. Therefore, in this study, we identified and characterized denitrifiers active at low-temperature conditions by using culture-independent and -dependent approaches. By comparative 16S rRNA (gene) analysis and culture isolation technique, Pseudomonas spp., Polaromonas spp., and Cellulomonas spp. were identified as being important bacteria responsible for denitrification in woodchip bioreactor microcosms at relatively low temperature conditions (15°C). Genome analysis of Cellulomonas sp. strain WB94 confirmed the presence of nitrite reductase gene nirK. Transcription levels of this nirK were significantly higher in the denitrifying microcosms than in the non-denitrifying microcosms. Strain WB94 was also capable of degrading cellulose and other complex polysaccharides. Taken together, our results suggest that Cellulomonas sp. denitrifiers could degrade woodchips to provide carbon source and electron donors to themselves and other denitrifiers in woodchip bioreactors at low-temperature conditions. By inoculating these denitrifiers (i.e., bioaugmentation), it might be possible to increase the nitrate removal rate of woodchip bioreactors at low-temperature conditions.

Minimum current requirements for epidural stimulation test confirmation of epidural and intrathecal catheter placement.

BACKGROUND AND OBJECTIVES: The typical blind insertion of a catheter into the epidural space risks catheter misplacement into the intrathecal space. The epidural stimulation test is designed to confirm the correct epidural location of a catheter but may also detect unintended intrathecal catheter placement by evaluating the minimum electrical current required for appropriate motor stimulation. Using this test, we observed the minimum current requirements for appropriate motor stimulation of catheters placed in the epidural and intrathecal spaces. METHODS: In this prospective observational study, patients scheduled for epidural catheter placement and intrathecal catheter placement were evaluated by the epidural stimulation test. The epidural space was localized by using a loss-of-resistance technique with normal saline, and the intrathecal space was identified by advancing a Tuohy needle until a continuous flow of clear cerebrospinal fluid was obtained. Afterward, a catheter was placed in the appropriate space, and a nerve stimulator delivered progressively, increasing electrical current until an appropriate muscle contraction was palpated. The minimum milliamperage required for this muscle contraction was our primary outcome measure. RESULTS: Of 37 catheters intentionally placed in the epidural space, the mean current required to produce an appropriate palpable motor contraction was 7.8 +/- 3.3 mA with a range of 2 to 14 mA. Of the 11 catheters intentionally placed in the intrathecal space, the mean current required to produce an appropriate palpable motor contraction was 1.3 +/- 0.8 mA with a range of 0.05 to 2.4 mA. CONCLUSIONS: We conclude that the epidural stimulation test minimum electrical current requirement seems to be lower for intrathecal compared with epidural catheter placement.

Evidence for the interaction of the hereditary haemochromatosis protein, HFE, with the transferrin receptor in endocytic compartments.

HFE, the protein mutated in hereditary haemochromatosis type 1, is known to interact with the transferrin receptor (TfR) on the cell surface and during endocytosis [Gross, Irrinki, Feder and Enns (1998) J. Biol. Chem. 273, 22068-22074; Roy, Penny, Feder and Enns (1999) J. Biol. Chem. 274, 9022-9028]. However, whether they are capable of interacting with each other once inside the cell is not known. In the present study we present several lines of evidence that they do interact in endosome compartments. Cells expressing a chimaera of HFE protein with the cytoplasmic domain of lysosomal-associated membrane protein 1 (LAMP1) in place of its own (HFE-LAMP) show a decrease in the half-life of the TfR. This implies that the interaction between HFE and TfR in endosomes targets the TfR to lysosomal compartments. The interaction between TfR and HFE-LAMP was confirmed by immunoprecipitation, in addition to immunofluorescence studies. Addition of transferrin (Tf) to HFE-LAMP-expressing cells competes with HFE for binding to the TfR, thereby increasing the half-life of TfR and confirming that the HFE-LAMP-TfR complex reaches the cell surface prior to entering the endosomal vesicles and trafficking to the lysosome. These results raise the possibility that interaction of HFE and TfR in intracellular vesicles may play an important role in determining the function of HFE in iron homoeostasis, which is still unknown. Analysis of endosomal pH and the iron content of internalized Tf indicated that HFE does not appear to alter the unloading of iron from Tf in the endosome.

Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells.

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.