Characterization of a transferrin-diphtheria toxin conjugate

We report here the synthesis and properties of a hybrid toxin prepared by covalently coupling diphtheria toxin to transferrin. The purified material contained two major hybrid protein species and was highly cytotoxic to mouse LMTK- cells in culture, reducing protein synthesis by 50% in 24 h at a concentration of 1 ng/ml. Cytotoxic activity was completely abolished in the presence of exogenous transferrin or anti-transferrin or anti-diphtheria toxin, thus demonstrating that the hybrid toxin was intoxicating cells via their transferrin receptors and that both the diphtheria toxin and transferrin components of the conjugate were necessary for activity. NH4Cl, a drug that elevates the pH within acidic intracellular vesicles, also blocked cytotoxic activity, suggesting that a low intravesicular pH was required for activity. The inhibitory effect of NH4Cl could be abolished by exposing toxin-treated cells to acidic culture medium, further implicating an acid-dependent step in the mechanism of the hybrid toxin action. Studies on the kinetics of intoxication also implied that endocytosis and exposure to a low pH within vesicles were necessary for cytotoxicity. Altogether, the results suggest that the transferrin-diphtheria toxin conjugate binds to transferrin receptors and is internalized into acidic endocytic vesicles. The enzymatic moiety of diphtheria toxin then apparently enters the cytosol in response to the low pH and subsequently arrests protein synthesis.     

The transition point for protein synthesis in late S-G2 may be delayed by genome bromosubstitution without affecting the time of entry into mitosis

Bromosubstitution for most of the S period in synchronous populations of Allium cepa L. meristematic cells resulted in a delay in the late S-G2 transition point where protein synthesis is needed for later mitotic entrance to occur. This retardation in the position of the transition point was not accompanied by the expected delay in the entrance into mitosis, suggesting that such protein synthesis is a requisite, but not a timer for prophase triggering.     

An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.     

Analysis of Flow Phenomena in Gastric Contents Induced by Human Gastric Peristalsis Using CFD

This paper uses computational fluid dynamics to simulate and analyze intragastric fluid motions induced by human peristalsis. We created a two-dimensional computational domain of the distal stomach where peristalsis occurs. The motion of the gastric walls induced by an antral contraction wave (ACW) on the wall of the computational domain was well simulated using a function defined in this study. Retropulsive flow caused by ACW was observed near the occluded region, reaching its highest velocity of approximately 12 mm/s in the narrowest region. The viscosity of the model gastric contents applied in this study hardly affected the highest velocity, but greatly affected the velocity profile in the computational domain. The shear rate due to gastric fluid motion was calculated using the numerical output data. The shear rate reached relatively high values of approximately 20 s⁻¹ in the most occluded region. The shear rate profile was almost independent of the fluid viscosity. We also simulated mass transfer of a gastric digestive enzyme (pepsin) in model gastric content when peristalsis occurs on the gastric walls. The visualized simulation results suggest that gastric peristalsis is capable of efficiently mixing pepsin secreted from the gastric walls with an intragastric fluid.