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.     

Scheduling algorithms for a two-machine flexible manufacturing system

The flexible manufacturing system (FMS) considered in this paper is composed of two CNC machines working in series—a punching machine and a bending machine connected through rollers acting as a buffer system of finite capacity. The main difference between the present problem and the standard two-machine flow shop problem with finite intermediate capacity is precisely the buffer system, which in our problem consists of two stacks of parts supported by rollers: the first stack contains the output of the punching machine, while the second stack contains the input for the bending machine. When the second stack is empty, the first stack may be moved over. Furthermore, the capacity of each stack depends on the particular part type being processed. The FMS can manufacture a wide range of parts of different types. Processing times on the two machines are usually different so that an unbalance results in their total workload. Furthermore, whenever there is a change of the part type in production, the machines must be properly reset—that is, some tools need to be changed or repositioned. A second important difference between the present problem and the usual two-machine flow shop problem is the objective. Given a list ofp part types to be produced in known quantities, the problem considered here is how to sequence or alternate the production of the required part types so as to achieve various hierarchical targets: minimize the makespan (the total time needed to complete production) and, for instance, compress the idle periods of the machine with less workload into a few long enough intervals that could be utilized for maintenance or other reasons. Although Johnson's rule is optimal in some particular cases, the problem addressed in the paper isNP-hard in general: heuristic procedures are therefore provided.     

Northern practices offer a challenge but few MDs willing to make the move.

Manitoba''s Norway House offers a ruggedly beautiful location and a challenging medical practice, but the physical and professional appeal has not been strong enough to entice physicians to make a long-term commitment to the community, which has needed medical service since September 1994. A physician who left there this spring said concerns about practice restrictions convinced him to move south.     

Antigens Associated with N- and L-Type Calcium Channels in Lambert-Eaton Myasthenic Syndrome

Abstract: In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (¹²⁵I-ω conotoxin GVIA) and L-type ([³H]PN200-110) calcium channels. Sera with a high antibody titer (>3 n M ) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.     

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.