Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas

Quantitative estimates of intracellular fluxes and measurements of intracellular concentrations were used to evaluate the effect of dissolved oxygen (DO) concentration on CRL 1606 hybridoma cells in batch culture. The estimates of intracellular fluxes were generated by combining material balances with measurements of extracellular metabolite rates of change. Experiments were performed at DO levels of 60% and 1% air saturation, as well as under oxygen-limited conditions. Cell extracts were analyzed to evaluate the effect of DO on the intracellular concentrations of the glutamate dehydrogenase reactants, as well as the redox state of the pyridine nucleotides in the cytosol and mitochondria. The relationship between cell density and pyridine nucleotide redox state was also investigated. Dissolved oxygen concentration had a significant effect on nitrogen metabolism and the flux through glutamate dehydrogenase was found to reverse at low DO, favoring glutamate formation. The NAD in the cytosol and mitochondria was more reduced under low DO conditions while the cytosolic NAD was more oxidized at low DO. Cytosolic NAD was reduced at higher cell densities while the redox states of cytosolic NADP and mitochondrial NAD did not exhibit significant variation with cell density. These results point to the fundamental role of the intracellular oxidation/reduction state in cell physiology and the possibility of controlling physiological processes through modulation of the dissolved oxygen level or the oxidation/reduction potential of the culture.     

Degradation of aromatic amino acids by Rhodococcus erythropolis

A Gram-positive Rhodococcus erythropolis strain S1 was shown to assimilate aromatic amino acids such as L-phenylalanine, L-tyrosine, L-tryptophan, D-phenylalanine, D-tyrosine and D-tryptophan, which were utilized not only as the sole carbon source but also as a suitable nitrogen source. The highest growth on these aromatic amino acids occurred at a temperature of 30°C. L-Phenylalanine, L-tyrosine and L-tryptophan degradative pathways would appear to be independent, and to be induced alternatively. The strain S1 also showed the ability to assimilate peptides which consisted of only L-phenylalanine and L-tyrosine.     

Conserved nonplanar heme distortions in cytochromesc

A nonplanar distortion of the heme ofc-type cytochromes is conserved in the proteins isolated from diverse species based upon a comprehensive analysis of available highresolution X-ray crystal structures. This distortion is induced through the cysteine thioether linkages between the porphyrin pyrrole groups and the polypeptide and results in an asymmetric pyrrole distortion. This asymmetry in the heme distortion is also conserved. For other heme proteins which lack these covalent bonds, nearly planar porphyrins are observed. Resonance Raman evidence indicates that nonplanar distortion of porphyrins containing metals, like iron, with large core sizes (?2.00 Å) is energetically unfavorable and can occur only in the presence of significant environmental perturbations. Further, energy minimization and dynamics calculations on the ferric form of yeast iso-1-cytochromec, starting from the crystallographic coordinates and using a molecular mechanics force field which accurately reproduces nonplanar distortions in metalloporphyrins, suggest that this distortion is indeed maintained by the protein tertiary structure. It is proposed that this protein-linked heme distortion modulates electron transfer function through modification of redox potentials of the porphyrin ring and the protein binding properties ofc-type cytochromes.     

Phorbol ester-stimulated superoxide production by murine bone marrow-derived macrophages requires preexposure to cytokines

Murine resident peritoneal macrophages (RPM) generate superoxide (O2-) in response to stimulation with PMA or zymosan. Murine bone marrow-derived macrophages (BMM) generate O2- in response to zymosan but not PMA. However, the ability to generate O2- in response to PMA could be induced in BMM by pre-exposing the cells to certain cytokines, including granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, and, to a lesser extent, IL-1 alpha. Bacterial LPS also induced the ability to respond to PMA. These same agents were also shown to prime RPM for enhanced PMA-induced respiratory burst. In contrast to GM-CSF, CSF-1 did not enhance the ability of BMM or RPM to generate O2- in response to PMA. Pretreatment with GM-CSF or TNF-alpha did not significantly affect the zymosan-induced release of O2- by BMM. These results suggest that unprimed BMM have a deficiency in the PMA-dependent signaling pathway that is corrected by exposure to selected cytokines. The results also raise the possibility that the basal ability of tissue macrophages to generate a respiratory burst in response to PMA may be a reflection of in vivo exposure to cytokines.