Characterization of bimodal cell death of insect cells in a rotating‐wall vessel and shaker flask

In previous publications, we reported the benefits of a high‐aspect rotating‐wall vessel (HARV) over conventional bioreactors for insect‐cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker‐flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first‐order term. Cell‐cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell–cell interactions, waste accumulation, and mass transport. These studies may benefit insect‐cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 14–26, 1999.     

Conformations of bound nucleoside triphosphate effectors in aspartate transcarbamylase. Evidence for the London-Schmidt model by transferred nuclear Overhauser effects

Transferred nuclear Overhauser effects were used to determine the conformations of ATP, CTP, and ITP bound to the regulatory site of aspartate transcarbamylase. The results are in accord with the predictions of the London-Schmidt model [London, R. E., & Schmidt, P. G. (1972) Biochemistry 11, 3136] and show that ATP and CTP bind in the anti conformation while ITP binds in the syn conformation.     

Trichosanthin interacts with acidic ribosomal proteins P0 and P1 and mitotic checkpoint protein MAD2B.

Trichosanthin is a ribosome-inactivating protein with multiple pharmacological properties. By a yeast two-hybrid system, ribosomal phosphoproteins P0 and P1 and a putative mitotic checkpoint protein, MAD2B, were found to interact with an active-site mutated trichosanthin (TCS). The interactions were verified by an in vitro binding assay of recombinant wild-type TCS and target proteins. The interaction domain of P0 was mapped to amino acids 220-273, which had been previously reported to be involved in the interaction with P1 and P2 in yeast. Consistent with our previous finding that the last seven residues of TCS are not essential for an active conformation, the same deletion did not affect the interaction with P0. Our present study suggests that TCS may disrupt the binding of elongation factors to the P-complex, in addition to the well-known N-glycosidase activity for ribosome inactivation.     

Inhibition of serine proteases by functionalized sulfonamides coupled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold.

A challenge associated with drug design is the development of selective inhibitors of proteases (serine or cysteine) that exhibit the same primary substrate specificity, that is, show a preference for the same P(1) residue. While these proteases have similar active sites, nevertheless there are subtle differences in their S and S' subsites which can be exploited. We describe herein for the first time the use of functionalized sulfonamides as a design and diversity element which, when coupled to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold yields potent, time-dependent inhibitors of the serine proteases human leukocyte elastase (HLE), proteinase 3 (PR 3) and cathepsin G(Cat G). Our preliminary findings suggest that (a) appending to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold recognition and diversity elements that interact with both the S and S' subsites of a target protease may result in optimal enzyme selectivity and potency and, (b) functionalized sulfonamides constitute a powerful design and diversity element with low intrinsic chemical reactivity and potentially wide applicability.     

COMPARISON OF GROWTH AND REPRODUCTION BETWEEN TWO POPULATIONS OF SARGASSUM SILIQUASTRUM IN PING CHAU, HONG KONG

In Ping Chau Island of Hong Kong, two populations of Sargassum siliquastrum are present. One is in the shallow water of about 1 to 3 m Chart Datum (CD), and the other one in deeper water of 5 to 10 m CD. These two populations are separated by an extensive sand patch. Individuals of the "shallow" water population increased their size from a mean length of 8.3 ± 3.6 (SD) cm in Aug 1998 to a maximum of 48.2 ± 29.9 cm in early Jan 1999 before they started to die back. In the following year, they attained a minimum of 6.1 ± 3.8 cm in May 1999 and a maximum of 56.1 ± 23.6 cm in Dec 1999. Their reproductive period lasted for two to three months from Jan to Feb 1999, and again from Nov 1999 to Jan 2000. The "deep" water individuals increased their size to a maximum of 123 ± 50.8 cm at the end of Jan 1999 and started to die back in Feb, 1999. They again reached their maximum mean length in Jan 2000. Their reproductive period lasted for five to six months from Sept 1998 to Feb 1999 and again from Sept 1999 to Jan 2000. The "deep" water individuals tended to be longer in size and they attained their maximum growth a month later than the "shallow" water individuals. Their reproductive season tended to start earlier and lasted longer than those in the "shallow" water. These differences in the phenology of the two populations may be related to the temperature differences (up to 5° C difference in summer) between the two depths. Sargassum siliquastrum is likely to be a cold adapted species such that warmer temperature in the shallow water has compressed and shortened their rapid growth and reproductive period to within the few colder months in fall and winter.