PubMed · 7312217
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1981. Systemic carnitine deficiency.. https://doi.org/10.1111/j.1753-4887.1981.tb06726.x
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The low pH triggered membrane insertion of the T domain of diphtheria toxin is a critical step in the translocation of the C domain of the toxin across membranes in vivo. We previously established that the T domain can interact with membranes in two distinct conformations, one in which the TH8/TH9 helical hairpin lies close to the bilayer surface and a second in which it inserts more deeply and appears to be transmembraneous. The loss of charge on residues E349 and D352 due to protonation at low pH has been proposed to be a critical step in transmembrane insertion, because they are within a loop connecting TH8 and TH9, and must cross the membrane upon transmembrane insertion. In this report, the role of these residues was examined by measuring the effect of the double substitution E349K/D352K on the conformation of the TH8/TH9 hairpin through a fluorescent group attached to TH9. At pH 4.5, there was shallower insertion of TH8/TH9 of the E349K/D352K mutant relative to T domain with wild-type residues at 349 and 352. In addition, smaller and/or fewer pores were obtained with the E349K/D352K mutant relative to the wild-type. On the other hand, high T domain concentrations, or further decreasing pH, allowed transmembrane insertion of both the wild-type and the 349K/352K mutant as well as induction of larger and/or more numerous pores. Furthermore, the transmembrane insertion process was rapid for both the mutant and wild-type. This shows that the mutant has the capacity to form a transmembrane structure similar to that of the wild-type T domain and, thus, that introduction of charged groups in membrane-penetrating regions of a protein does not introduce an insurmountable barrier to transmembrane movement. The linkage between the ability of the T domain to form the transmembrane conformation and pores suggests that the effects of these mutations in inhibiting pore formation are likely to partly result from the inability to insert properly. Additionally, the observation that decreasing pH allows the 349K/352K mutant to insert deeply indicates that there are residues other than E349 and D352 whose protonation promotes transmembrane insertion.
In the present study, we investigated the mechanism by which sphingosine and its analogues, dihydrosphingosine and phytosphingosine, inhibit polymorphonuclear leukocyte (PMN) phagocytosis of IgG-opsonized erythrocytes (EIgG) and inhibit ERK1 and ERK2 phosphorylation. We used antibodies that recognized the phosphorylated forms of ERK1 (p44) and ERK2 (p42) (extracellular signal-regulated protein kinases 1 and 2). Sphingoid bases inhibited ERK1 and ERK2 activation and phagocytosis of EIgG in a concentration-dependent manner. Incubation with glycine, N,N'-[1, 2-ethanediylbis(oxy-2, 1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-bis[ (acetylox y)methyl]ester (BAPTA,AM), an intracellular chelator of calcium, failed to block either phagocytosis or ERK1 and ERK2 phosphorylation, consistent with the absence of a role for a calcium-dependent protein kinase C (PKC) in ERK1 and ERK2 phosphorylations. Western blotting demonstrated that sphingosine inhibited the translocation of Raf-1 and PKCdelta from PMN cytosol to the plasma membrane during phagocytosis. These data are consistent with the interpretation that sphingosine regulates ERK1 and ERK2 phosphorylation through inhibition of PKCdelta, and this in turn leads to inhibition of Raf-1 translocation to the plasma membrane. Consistent with this interpretation, the sphingosine-mediated inhibition of phagocytosis, ERK2 activation, and PKCdelta translocation to the plasma membrane could be abrogated with a cell-permeable diacylglycerol analog. The increase in the diacylglycerol mass correlated with the translocation of PKCdelta and Raf-1 to the plasma membrane by 3 minutes after the initiation of phagocytosis. Additionally, the diacylglycerol analog enhanced phagocytosis by initiating activation of PKCdelta and its translocation to the plasma membrane. Because PMN generate sufficient levels of sphingosine by 30 minutes during phagocytosis of EIgG to inhibit phagocytosis, it appears that sphingosine can serve as an endogenous regulator of EIgG-mediated phagocytosis by downregulating ERK activation.
The viability of boron neutron capture therapy depends on the development of tumor-targeting agents that contain large numbers of boron-10 (10B) atoms and are readily taken up by cells. Here we report on the selective uptake of homogeneous fluorescein-labeled nido-carboranyl oligomeric phosphate diesters (nido-OPDs) by the cell nucleus and their long-term retention after their delivery into the cytoplasm of TC7 cells by microinjection. All nido-OPDs accumulated in the cell nucleus within 2 h after microinjection. However, nido-OPDs in which the carborane cage was located on a side chain attached to the oligomeric backbone were redistributed between both the cytoplasm and nucleus after 24 h of incubation, whereas nido-OPDs in which the carborane cage was located along the oligomeric backbone remained primarily in the nucleus. Furthermore, cell-free incubation of digitonin-permeabilized TC7 cells with the nido-OPDs resulted in nuclear accumulation of the compounds, thus corroborating the microinjection studies. Our observation of fluorescence primarily located in the cell nucleus indicates that nuclear-specific uptake of sufficient amounts of 10B for effective boron neutron capture therapy ( approximately 10(8)-10(9) 10B atoms/tumor cell) via nido-OPDs is achievable.