Influence of power input and aeration method on mass transfer in a laboratory animal cell culture vessel

Large-scale animal cell operation is costly both in terms of facilities and consumables. Hence developmental studies with animal cells normally start at laboratory scale, often using small stirred tanks. In order to better optimise cell performance, it is necessary to know the physical conditions under which the cells are grown. In this study a laboratory-scale vessel (2 dm³ working volume) with two large-bladed paddle impellers was characterised hydrodynamically. Three different aeration methods (surface, sparging and membrane aeration) were investigated and compared. Power input and oxygen transfer rates to culture medium were determined as a function of agitation and gas flow rates. Non-dimensional correlations were established for each case, which can be useful for scale-up purposes. The results obtained indicate that power input is quite dependent on the vessel accessories: for the same agitation rate, the maximum power is required for the membrane structure and the minimum for surface aeration, with the addition of the sparger leading to an intermediate situation. Predictions found in the literature can be used for simple vessels, but may not be applicable when accessories are added to the vessel structure; in such cases, the use of experimental relationships are required. Oxygen transfer rate was dependent on the aeration method and working conditions (agitation and gas flow rates), particularly for sparger aeration. Membrane aeration gave larger oxygen transfer but higher gas pressure and flow rates were required. Surface aeration was the least effective method, nevertheless requiring gas flow rates similar to those used for membrane aeration. The aeration method of choice depends upon the culture and work specificities: surface aeration is limited to small cell concentrations and low oxygen consumption rates. For higher cell concentrations and oxygen consumption rates, both membrane and sparger aeration methods can be applied: the use of the sparger is limited to cells that are not affected by the presence of bubbles or the addition of surfactants, whereas the membrane aeration basket should not be used when a hydrodynamically controlled stirred tank is required.     

The origin of the Australasian marsupial fauna and the phylogenetic affinities of the enigmatic monito del monte and marsupial mole

Alternative hypotheses in higher-level marsupial systematics have different implications for marsupial origins, character evolution, and biogeography. Resolving the position of the South American monito del monte (Order Microbiotheria) is of particular importance in that alternate hypotheses posit sister-group relationships between microbiotheres and taxa with disparate temporal and geographic distributions: pediomyids; didelphids; dasyuromorphians; diprotodontians; all other australidelphians; and all other marsupials. Among Australasian marsupials, the placement of bandicoots is critical; competing views associate bandicoots with particular Australasian taxa (diprotodontians, dasyuromorphians) or outside of a clade that includes all other Australasian forms and microbiotheres. Affinities of the marsupial mole are also unclear. The mole is placed in its own order (Notoryctemorphia) and sister-group relationships have been postulated between it and each of the other Australasian orders. We investigated relationships among marsupial orders by using a data set that included mitochondrial and nuclear genes. Phylogenetic analyses provide support for the association of microbiotheres with Australasian marsupials and an association of the marsupial mole with dasyuromorphs. Statistical tests reject the association of diprotodontians and bandicoots together as well as the monophyly of Australasian marsupials. The origin of the paraphyletic Australasian marsupial fauna may be accounted for by (i) multiple entries of australidelphians into Australia or (ii) bidirectional dispersal of australidelphians between Antarctica and Australia.     

Beiträge zur galvanisierung von polymeren, 11. Metallisierung von mischungen aus epoxidharzen und acrylnitril-butadien-copolymeren

The metallization of mixtures containing epoxy resins and acrylonitrile-butadiene copolymers depends on taking the suitable ratio of components and on the hardener, which was brought to reaction with the epoxy prepolymer in presence of the copolymer. Using triethylenetetramine as curing agent the highest values of peel strength have been reached and have been increased up to 150 N/25 mm by short-time-treatment with ozone. Longer ozonization times lead to lower values of peel strength and impairment of the material. Caverns occur only in mixtures which are rich on epoxy resin, and increase in size with increasing time of ozone treatment. These samples have relatively low values of peel strength.     

Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3. We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2. Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5779-5783). By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly. These results suggested the presence of two polypeptides in this protein. Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB). The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences. The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A. These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases. The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus. Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases. The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.     

Formation and analysis of shallow arsenic profiles

Shallow arsenic implants were activated by furnace and rapid thermal annealing (RTA). Comparisons of junction depths measured by secondary ion mass spectrometry (SIMS) and spreading resistance (SR) showed SIMS values 50-90 nm deeper than SR values, due to ion knock-on during SIMS profiling     

Gas transfer in supported Langmuir-Blodgett films of polymeric lipids

Asymmetric membranes were fabricated by depositing Langmuir-Blodgett (LB) films of polymeric lipids on porous supports. Three polymeric lipids were used for deposition. Up to 50 Y-type layers were deposited on one side of the porous supports of polypropylene (Celgard) and polytetrafluoroethylene (GORE-TEX) membranes. A marked decrease in gas transfer with increasing number of LB layers was observed for the asymmetric membranes fabricated with the porous polypropylene. Gas transfer in the asymmetric membranes using porous polytetrafluoroethylene remained unchanged with the number of polymeric layers. The LB multilayers on polypropylene showed no evidence for pores or cracks as determined by scanning electron microscopy (at a magnification of 40 000x), but the LB multilayers on polytetrafluoroethylene revealed large cracks. The gas permeabilities of nitrogen, methane and carbon dioxide in the polypropylene-based asymmetric membranes were a function of the molecular weight of the gas for two of the three polymers which suggests that micropores are present in the LB films. Heat annealing decreased the gas permeabilities from 0% to 40%, but only one polymer showed a change in selectivity.     

Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo

The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in vitro evidence, there has been no in vivo demonstration that blockade of Kv1.3 will attenuate an immune response. The difficulty is due to species differences, as the channel does not set the membrane potential in rodent peripheral T cells. In this study, we show that the channel is present on peripheral T cells of miniswine. Using the peptidyl Kv1.3 inhibitor, margatoxin, we demonstrate that Kv1.3 also regulates the resting membrane potential, and that blockade of Kv1.3 inhibits, in vivo, both a delayed-type hypersensitivity reaction and an Ab response to an allogeneic challenge. In addition, prolonged Kv1.3 blockade causes reduced thymic cellularity and inhibits the thymic development of T cell subsets. These results provide in vivo evidence that Kv1.3 is a novel target for immunomodulation.