Upregulation of the expression of endogenous Mdr1 P-glycoprotein enhances lipid translocation in MDCK cells transfected with human MRP2

Various ABC transporters can translocate lipid molecules from the cytoplasmic into the exoplasmic leaflet of the plasma membrane bilayer. Two of these, MDR1 P-glycoprotein (Pgp) and MRP1, are multidrug transporters responsible for the resistance of various cancers against chemotherapy. We wanted to study whether MRP2, an ABC transporter of the bile canalicular membrane with a substrate specificity very similar to that of MRP1, is capable of translocating lipids. The translocation of short-chain lipids across the apical membrane of MDCK cells transfected with MRP2 was significantly higher than that in untransfected controls. However, the characteristics of the lipid translocation were similar to substrate transport by MDR1 and not MRP2: transport was strongly inhibited by classic MDR1 Pgp inhibitors, was independent of cellular glutathione, and was insensitive to a drug known to inhibit MRP2 activity. When tested by immunoblot, the MRP2-transfected cells expressed high levels of MRP2 but also of endogenous Mdr1. The expression of Mdr1 was unstable during maintenance of the cell line and correlated with the rate of lipid translocation across the apical membrane. We conclude that the observed increase in lipid transport in the MDCK cells transfected with MRP2 is the consequence of the upregulation of the expression of endogenous Mdr1 and that careful characterization of endogenous Mdr1 expression is needed in studies aimed to identify substrates of plasma membrane transporters.     

Effects of Grazing by the Free-Living Soil Amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis on Various Bacteria

Cultures of 10 different bacteria were used to serve as food sources for axenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis. The nonpigmented enterobacteriaceae Escherichia coli K-12 and Klebsiella aerogenes appeared to be excellent feed to all three amoebae. Hardly any growth or ammonium production was observed in tests with Chromatium vinosum and Serratia marcescens, which share the presence of pigmented compounds. Distinct differences in net ammonium production were detected and were correlated to the amoebal growth yield. In general, growth of amoebae and ammonium production increased in the order A. polyphaga, A. castellanii, and H. vermiformis.     

Methanobacterium thermoautotrophicum (strain ΔH) contains a membrane-bound cyclic 2,3-diphosphoglycerate hydrolase

Cyclic 2,3-diphosphoglycerate (cDPG) hydrolase activity was demonstrated in cofactor-free extract of Methanobacterium thermoautotrophicum (strain H), but not in crude extract. Only after ultrafiltration or dialysis of crude extract cDPG hydrolase activity could be shown. cCPG hydrolysis was optimal at pH 6.0 and 60°C. Hydrolysis of cDPG occurred under nitrogen or hydrogen atmosphere and was completely inhibited by oxygen. Phosphate and potassium chloride were also strong inhibitors: 50% inhibition occurred at 0.6–0.7 mM phosphate or 0.2 M KCl. The enzyme was localized in the membrane fraction and could be solubilized for approximately 60% by treatment with 25 mM of the detergent CHAPS. The K _m and the V _max for cDPG were determined at 60°C and were 59 mM and 216 mU/mg, respectively. Furthermore, cDPG hydrolase was dependent on the presence of Co²⁺. The role of cDPG and cDPG hydrolase is discussed.Abbreviations cDPG cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-diphosphoglycerate - 2-PG 2-phosphoglycerate - 3-PG 3-phosphoglycerate - PG phosphoglycerate - PEP phosphoenolpyruvate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - TRIS tris(hydroxymethyl)-aminomethane - DTT dithiothreitol - CHAPS 3-([3-cholamidopropyl]-dimethylammonio)-1-propanesulfonate - MOPS 3-(N-morpholino) propanesulfonic acid     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Purification and characterization of an extracellular β-glucosidase from the anaerobic fungus Piromyces sp. strain E2

An extracellular -glucosidase (EC 3.2.2.21) from the anaerobic fungus Piromyces sp. strain E2 was purified. The enzyme is a monomer with a molecular mass of 45 kDa and a pI of 4.15. The enzyme readily hydrolyzes p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, cellobiose, cellotriose, cellotetraose and cellopentaose but is not active towards Avicel, carboxymethylcellulose, xylan, p-nitrophenyl--d-galactoside and p-nitrophenyl--d-xyloside. To cleave p-nitrophenyl--d-glucoside the maximum activity is reached at pH 6.0 and 55°C, and the enzyme is stable up to 72 h at 40°C. Activity is inhibited by d-glucurono--lactone, cellobiose, sodium dodecyl sulfate, Hg²⁺ and Cu²⁺ cations. With p-nitrophenyl--d-glycoside, p-nitrophenyl--d-fucoside, and. cellobiose as enzyme substrates, the K _m and V _max balues are 1.5 mM and 25.5 IU·mg^-1, 1.1. mM and 133 IU·mg^-1, and 0.05 mM and 55.6 IU·mg^-1, respectively.     

Biomass and Biological Activity during the Production of Compost Used as a Substrate in Mushroom Cultivation

The production of a suitable substrate for the cultivation of the common white button mushroom, Agaricus bisporus, is referred to as composting. High microbiological activity causes temperatures of the composting material to rise as high as 80°C. At stacking, an optimal oxygen consumption rate of 140 μmol of O₂ h⁻¹ g (dry weight)⁻¹ was found in the compost at 50°C, whereas the oxygen consumption rate of the end product was lower at all temperatures tested. No significant differences were observed between biomass content and mineralization rate of ¹⁴C-labeled glutamate of the two composts. Biomass content was shown to be a major function of both temperature and the sampling site position in the stack. On the basis of the results reported here, a minimal composting time of 3.3 days for the phase I process was calculated. Further suggestions are made to reduce the time necessary for the production of a substrate for A. bisporus considerably.     

Corrinoids from Methanosarcina barkeri: Structure of the α-ligand

5-Hydroxybenzimidazole is the only base detected in cobamide compounds from methanol-grown Methanosarcina barkeri. 5-Hydroxybenzimidazolylcobamide accounted for about 83 and 90% of the total corrinoids of whole cells and cell-free extracts, respectively. Probably, the rest of the corrinoids are base-less.     

Novel replication-incompetent vector derived from adenovirus type 11 (Ad11) for vaccination and gene therapy: low seroprevalence and non-cross-reactivity with Ad5

A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (DeltaE1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV(+)) individuals is as low as that among HIV(-) individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.     

Improved adenovirus vectors for infection of cardiovascular tissues

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.     

Measurement of exhaled hydrogen peroxide from rabbit lungs

Exhaled H2O2 is considered an indicator of lung inflammatory and oxidative stress. Moreover, H2O2 may be involved in signal transduction processes. It is not fully elucidated to what extent (i) H2O2 escapes from the intravascular compartment, and (ii) pulmonary H2O2 generation and nasopharyngeal H2O2 generation contribute to exhaled H2O2. We investigated H2O2 concentrations in breath condensate from isolated buffer-perfused and ventilated rabbit lungs, and from both intubated and spontaneously breathing rabbits with a horseradish peroxidase/2',7'dichlorofluorescin assay. For the perfused lungs, a H2O2 concentration of 58 +/- 19 nM was found. Addition of H2O2 to the buffer fluid resulted in only minute appearance in the exhaled air (<0.001%). Levels of exhaled H2O2 in intubated rabbits and perfused lungs were virtually identical. Nearly ten-fold higher levels were detected in spontaneously breathing rabbits. Decreasing the inspired oxygen concentration from 21% to 1% resulted in a tendency toward decreased H2O2 exhalation in perfused lungs. In contrast, phorbol-12-myristate-13-acetate (PMA) prompted a approximately 4-fold increase in H2O2 exhalation. We conclude that the horseradish peroxidase/2',7'dichlorofluorescin assay is a feasible technique to measure H2O2 in exhaled breath condensate in rabbits. When collecting exhaled air via the tracheal tube, the signal represents pulmonary H2O2 generation with the contribution of the remaining body being negligible.