Human tissue-type plasminogen activator (t-PA) contains a variably occupied glycosylation site at Asn-184 in naturally produced t-PA and in t-PA produced in recombinant Chinese hamster ovary (CHO) cells. The presence of an oligosaccharide at this site has previously been shown to reduce specific activity and fibrin binding. In this report, the site occupancy of t-PA is shown to increase gradually over the course of batch and fedbatch CHO cultures. Additional cell culture factors, including butyrate and temperature, are also shown to influence the degree of glycosylation. In each of these cases, conditions with decreased growth rate correlate with increased site occupancy. Investigations using quinidine and thymidine to manipulate the cell cycle distribution of cultures further support this correlation between site occupancy and growth state. Comparison of the cell cycle distribution across the range of cell culture factors investigated shows a consistent relationship between site occupancy and the fraction of cells in the G 0 /G 1 phase of the cell cycle. These results support a correlation between growth state and site occupancy, which fundamentally differs from site occupancy trends previously observed and illustrates the importance of the growth profile of CHO cultures in producing consistently glycosylated recombinant glycoproteins.

The lung is frequently exposed to particulate material that can potentially stimulate release of factors that attract polymorphonuclear neutrophils (PMN). However, few PMN are noted in the airways of normal subjects, suggesting there is some mechanism to dampen influx of these cells. We have isolated from bronchial lavage a peptide that inhibits PMN chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP). In the present study we examined effects of this molecule on 1) chemotaxis to other agonists, 2) FMLP-stimulated PMN superoxide production, 3) PMN calcium fluxes, and 4) binding of FMLP. Our results show that purified inhibitor attenuates PMN chemotaxis to C5a and leukotriene B4. This molecule also inhibits PMN superoxide release in response to FMLP. Exposure to this inhibitor causes an abrupt rise in cytosolic calcium concentration due to a pertussis toxin-sensitive shift of intracellular calcium and attenuates subsequent influx of extracellular calcium in response to FMLP. Binding studies demonstrate the inhibitor induces increased FMLP binding at 37 degrees C but has no effects at 4 degrees C. Inhibition of chemotaxis and increased FMLP binding mediated by this molecule are attenuated by buffering PMN calcium transients. These studies suggest an inhibitor of neutrophil function present in the bronchial environment alters PMN through effects on calcium homeostasis.