Immunocytochemical localization of CDW60 antigens on human peripheral T-cells

About 25 - 35% of human T cells display the CDw60 ganglioside (9-O-acetyl-GD3) antigen at the cell surface [E.P. Rieber, in W. Knapp, B. Dörken, W.R. Gilks, E.P. Rieber, R.E. Schmidt, H. Stein, A.E.G.K. von dem Borne (Eds.), Leucocyte Typing IV, Oxford University, Oxford, 1989, p. 361.]. Other leucocytes do not express this antigen on the cell surface. This led us to investigate its presence by flow cytometry and immunoelectron microscopy (IEM). Flow cytometric analysis of isolated peripheral T cells showed 26% of the cell population to have the CDw60 antigen expressed on the cell surface whereas 74% did not. Similarly, IEM analysis of 262 random T cells by the preembedding immunogold labeling technique revealed CDw60 surface expression to be tetrapartite: (a) the majority of 63.7% of the T cells did not show any surface associated gold label; (b) 19.5% were of low CDw60 surface exposition, corresponding to a linear density of 0.05–2.0 gold markers per ?m; (c) about 13.4% showed a medium surface exposition with a linear density of 2.1–4.5 gold markers per ?m; and (d) a high exposition, ranging from 4.6 to 9.0 gold markers per ?m, was seen at 3.4% of the T cells. From postembedding label experiments, which additionally make access to the antigen localized within the cytoplasm, it was found that nearly all T cells contained low levels of intracellular CDw60. Most of it was found to be associated with the cytoplasmic membrane or vesicles, derived from the Golgi. Immunogold conjugates associated with the cytoplasmic membrane showed a linear density up to 0.6 gold markers per µm. The asymmetric expression of the CDw60 antigen on human T cells and its occurrence in nearly all T cells suggests that its surface presentation is tightly regulated

Thiobaca trueperi gen. nov., sp nov., a phototrophic purple sulfur bacterium isolated from freshwater lake sediment

Two strains of a novel species of phototrophic micro-organism were isolated from the sediments of a shallow, freshwater, eutrophic lake. Both strains grew photolithoheterotrophically with sulfide as an electron donor, transiently accumulating intracellular sulfur globules. Photolithoautotrophic growth was not observed. One strain was designated BCHT (the type strain) and was studied in most detail. Cells contained bacteriochlorophyll a, and the dominant carotenoid was lycopene. Cell suspensions were brown. The photosynthetic membranes had a vesicular arrangement. Acetate, propionate, pyruvate, succinate and fumarate were each used as electron donors and carbon sources in the presence of sulfide and bicarbonate. In the presence of light, growth did not occur with hydrogen, thiosulfate or iron(II). The optimum temperature for growth was between 25 and 30 degreesC, the maximum being 36 degreesC. The G+C content of the genomic DNA of strain BCHT was 63 mol %. Analysis of the 16S RNA genes showed that both strains belonged to the gamma-subclass of the Proteobacteria but were phylogenetically distinct from any described phototrophic organisms within the Chromatiaceae. On the basis of phylogenetic and physiological differences from other phototrophic microorganisms, strain BCHT is described as a novel species of a new genus, Thiobaca trueperi gen. nov., sp. nov

Thiobaca trueperi gen. nov., sp nov., a phototrophic purple sulfur bacterium isolated from freshwater lake sediment

Two strains of a novel species of phototrophic micro-organism were isolated from the sediments of a shallow, freshwater, eutrophic lake. Both strains grew photolithoheterotrophically with sulfide as an electron donor, transiently accumulating intracellular sulfur globules. Photolithoautotrophic growth was not observed. One strain was designated BCHT (the type strain) and was studied in most detail. Cells contained bacteriochlorophyll a, and the dominant carotenoid was lycopene. Cell suspensions were brown. The photosynthetic membranes had a vesicular arrangement. Acetate, propionate, pyruvate, succinate and fumarate were each used as electron donors and carbon sources in the presence of sulfide and bicarbonate. In the presence of light, growth did not occur with hydrogen, thiosulfate or iron(II). The optimum temperature for growth was between 25 and 30 degreesC, the maximum being 36 degreesC. The G+C content of the genomic DNA of strain BCHT was 63 mol %. Analysis of the 16S RNA genes showed that both strains belonged to the gamma-subclass of the Proteobacteria but were phylogenetically distinct from any described phototrophic organisms within the Chromatiaceae. On the basis of phylogenetic and physiological differences from other phototrophic microorganisms, strain BCHT is described as a novel species of a new genus, Thiobaca trueperi gen. nov., sp. nov

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers

Werner A, Duvar S, Müthing J, et al. Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers. BIOTECHNOLOGY AND BIOENGINEERING. 2000;68(1):59-70.Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 mu g/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 mu g/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary meta-belie functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system. (C) 2000 John Wiley & Sons, Inc

Immobilization of uranium in biofilm microorganisms exposed to groundwater seeps over granitic rock tunnel walls in Olkiluoto, Finland

In an underground rock characterization facility, the ONKALO tunnel in Finland, massive 5-10-mm thick biofilms were observed attached to tunnel walls where groundwater was seeping from bedrock fractures at a depth of 70 m. In laboratory experiments performed in a flow cell with detached biofilms to study the effect of uranium on the biofilm, uranium was added to the circulating groundwater (CGW) obtained from the fracture feeding the biofilm. The final uranium concentration in the CGW was adjusted to 4.25 x 10(-5) M, in the range expected from a leaking spent nuclear fuel (SNF) canister in a future underground repository. The effects were investigated using microelectrodes to measure pH and E-h, time-resolved laser fluorescence spectroscopy (TRLFS), energy-filtered transmission electron microscopy (EF-TEM), and electron energy-loss spectroscopy (EELS) studies and thermodynamic calculations were utilized as well. The results indicated that the studied biofilms constituted their own microenvironments, which differed significantly from that of the CGW. A pH of 5.37 was recorded inside the biofilm, approximately 3.5 units lower than the pH observed in the CGW, due to sulfide oxidation to sulfuric acid in the biofilm. Similarly, the E-h of +73 mV inside the biofilm was approximately 420 mV lower than the E-h measured in the CGW. Adding uranium increased the pH in the biofilm to 7.27 and reduced the E-h to -164 mV. The changes of E-h and pH influenced the bioavailability of uranium, since microbial metabolic processes are sensitive to metals and their speciation. EF-TEM investigations indicated that uranium in the biofilm was immobilized intracellularly in microorganisms by the formation of metabolically mediated uranyl phosphate, similar to needle-shaped autunite (Ca[UO2](2)[PO4](2)center dot 2-6H(2)O) or meta-autunite (Ca[UO2](2)[PO4](2)center dot 10-12H(2)O). In contrast, TRLFS studies of the contaminated CGW identified aqueous uranium carbonate species, likely (Ca2UO2[CO3](3)), formed due to the high concentration of carbonate in the CGW. The results agreed with thermodynamic calculations of the theoretically predominant field of uranium species, formed in the uranium-contaminated CGW at the measured geochemical parameters. This investigation clearly demonstrated that biological systems must be considered as a part of natural systems that can significantly influence radionuclide behavior. The results improve our understanding of the mechanisms of biofilm response to radionuclides in relation to safety assessments of SNF repositories. (C) 2012 Elsevier Ltd. All rights reserved

Preferential attachment of Escherichia coli to different particle size fractions of an agricultural grassland soil.

This study reports on the attachment preference of a faecally derived bacterium, Escherichia coli, to soil particles of defined size fractions. In a batch sorption experiment using a clay loam soil it was found that 35% of introduced E. coli cells were associated with soil particulates >2 μm diameter. Of this 35%, most of the E. coli (14%) were found to be associated with the size fraction 15-4 μm. This was attributed to the larger number of particles within this size range and its consequently greater surface area available for attachment. When results were normalised with respect to estimates of the surface area available for bacterial cell attachment to each size fraction, it was found that E. coli preferentially attached to those soil particles within the size range 30-16 μm. For soil particles > 2 μm, E. coli showed at least 3.9 times more preference to associate with the 30-16 μm than any other fraction. We report that E. coli can associate with different soil particle size fractions in varying proportions and that this is likely to impact on the hydrological transfer of cells through soil and have clear implications for our wider understanding of the attachment dynamics of faecally derived bacteria in soils of different compositions

Gulosibacter molinativorax ON4T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase ▿ ‡

A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4T has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal