NMR spectroscopy of recombinant chinese hamster ovary (CHO) cells in a packed bed reactor

Summary Several clones of CHO cells, including recombinant cell lines expressing Hepatitis B surface antigen, were grown in macroporous collagen microspheres. These provided sufficient cell density in a packed bed recirculation system for phosphorus-31 nuclear magnetic resonance spectroscopic estimation of metabolite concentration. Intracellular nucleoside triphosphate as well as nucleoside tri- plus diphosphate levels were higher in the methotrexate-selected clones compared to the dhfr^– cell line.     

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60)

The nuclear sugar-binding components (i.e., lectinlike molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.     

Zooplankton impacts on chlorophyll and transparency in Onondaga Lake,New York,USA

The transparency of polluted, hypereutrophic Onondaga Lake, New York, USA has improved substantially in the late 1980's as a result of reductions in phytoplankton biomass, in the absence of significant reductions in external phosphorus loading. Much of this improvement has been due to the occurrence of clearing events, e.g. sudden and dramatic increases in transparency. Field measurements, laboratory experiments, and modelling analyses were utilized to identify processes regulating phytoplankton standing crop during the spring to fall interval of 1987. Changes in the zooplankton community documented over the past decade support the conclusion that increased zooplankton grazing has contributed to improvements in transparency. Herbivores now represent a greater fraction of the zooplankton population and more efficient cladocerans are present in greater numbers. Biomanipulation practices, e.g. reestablishment of piscivorous species, designed to reduce the abundance of planktivorous fish species in Onondaga Lake, may serve to reduce pressure on the grazing community and thus result in further improvements in transparency.     

Effect of ammonium ion and extracellular pH on hybridoma cell metabolism and antibody production

The effects of NH(4)Cl addition on batch hybridoma cell growth at different external pH values (pH(e)) were investigated in a bioreactor at constant pH and dissolved oxygen concentration. In agreement with measurements in flasks, changes in pH(e) over the range 6.8-7.6 had minor effects on growth. Addition of 3 mM NH(4)Cl had little effect on cell growth while 10 mM NH(4)Cl caused a substantial growth inhibition, Measurements of the effects of pH(e) and NH(4)Cl concentration on cell metabolism gave similar results for cells grown in flasks in an incubator and in the bioreactor. As pH(e) decreases, the integral cell yield on glucose increases. There is a correlation between the effects of pH(e) on glycolysis and previous measurements of its effects on intracellular pH (pH(i)). Increases in NH(4)Cl concentration were previously determined to decrease pH(i) and are shown here to decrease the integral cell yield on glucose. At all pH(e) values in the absence of NH(4)Cl, glutamine is depleted at the time the maximum cell density is reached. Both pH(e) decreases and NH(4)Cl concentration increases lead to decreases in the integral cell yield on glutamine. Changes in pH(e) and in the NH(4)Cl concentration that cause growth inhibition have no effect on the specific antibody production rate for cells grown in flasks in an incubator or in the bioreactor. Changes in the NH(4)Cl concentration have no effect on the quality of the antibody produced, to a first level of characterization.     

Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling

Efficient expression of a foreign protein product by the yeastSaccharomyces cerevisiaerequires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromereplasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, evenin a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that amaximum of 4-6 centromere plasmids per cell can be stably partitionedat cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.     

Electrochemical cells for voltammetry, coulometry, and protein activity assays of small-volume biological samples

Cell designs, experimental protocols, and results for electrochemical investigation of small quantitites of biological materials under anaerobic conditions are reported. Three types of electrochemical experiments are considered: (i) cyclic voltammetry of 20- to 100-microliters samples; (ii) direct coulometry of 0.5- to 1.5-ml samples; and (iii) an electrochemically initiated protein activity assay which includes provision for analysis of gaseous reaction products and correlation with electron flux. The first two procedures are illustrated by measurement of the formal electrode potential (E0') and number of electrons transferred (n) in redox reactions of small quantities of biological and inorganic materials. The third procedure is illustrated by assaying the activity of the MoFe protein plus Fe protein complex from Azotobacter vinelandii nitrogenase for reduction of C2H2 to C2H4.     

A circular-dichroism study of epidermolytic toxins A and B from Staphylococcus aureus.

The far-u.v. circular-dichroism spectra of the two epidermolytic toxins was analysed into fractional contributions of 0.09 helix and 0.46 beta-sheet to each toxin structure. Trifluoroethanol perturbation caused an initial increase in dichroic absorption at 205 nm and then a change characterized as a beta-sheet-to-alpha-helix transition. The intense near-u.v. spectra suggested that the toxins have unusually rigid, though different, aromatic-side-chain arrangements.