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Abstract In previous chlorpromazine pharmacokinetic studies a dramatic elevation in blood plasma levels of this drug was observed when taken in combination with oral contraceptives. Different mechanisms have been postulated to explain this observation. The aim of the study was to investigate whether oral contraceptives such as ethinyloestradiol and progesterone enhance the absorption of chlorpromazine by means of inhibiting P-glycoprotein (P-gp) and if this effect is mainly due to ethinyloestradiol or progesterone or their combination. The Caco-2 cell line was used as an in vitro model to study the effects of these compounds on the transport of chlorpromazine. Both apical to basolateral (AP-BL) and basolateral to apical (BL-AP) transport studies were done on chlorpromazine in combination with different compounds. Ethinyloestradiol enhanced the AP-BL cumulative transport of chlorpromazine by 11.5% compared to the control group, which was also statistically significantly higher than the effect caused by progesterone (0.8%). A combination of these two steroidal hormones enhanced the cumulative transport of chlorpromazine by only 2.0% compared to the control group. This indicates the possible existence of separate drugbinding sites for these two hormones and chlorpromazine on P-gp. The drug-binding site (or receptor) for progesterone probably interacts allosterically with the binding site for ethinyloestradiol and thereby decreasing its transport enhancing effects on chlorpromazine.

As a result of transcriptional editing, Ebola virus (EBOV) produces multiple soluble products from its glycoprotein gene, the primary product of which is the secreted glycoprotein (sGP), in addition to the membrane-bound viral spike protein GP1,2. A lack of leukocyte infiltration is observed during EBOV infection, which is thought to allow virus replication to proceed unchecked and thus represents a significant role in the immunopathology of the disease. Currently the only know function of sGP is that it has an anti-inflammatory effect on endothelial cells treated with TNF-α, an effect that has been hypothesized to interfere with recruitment or extravasation of leukocytes. To better characterize this anti-inflammatory function, a link between sGP structure and function was sought. Mass spectrometry (MS) analysis of recombinant sGP demonstrated that it is a parallel-orientated disulphide-linked homodimer that contains Cys53-C53’ and Cys306-C306’ intermolecular disulphide bonds. In addition to being glycosylated with complex N-glycans, sGP also contained a novel post-translation modification, termed C-mannosylation. C-mannosylation was not required for the anti-inflammatory function of sGP; however, glycine mutations at amino acids 53 and 306 resulted in the complete loss of the anti-inflammatory effect on TNF-α treated endothelial cells. Thus, a specific structure mediated by intermolecular disulphide bonds is required for the proposed anti-inflammatory function of sGP, suggesting that this effect is the result of a specific interaction. The spike protein GP1,2, also contains C-mannosylation motifs. MS analysis of GP1,2 indicated that GP1 was C-mannosylated, while two adjacent motifs in the membrane proximal region (MPER) of GP2 were not. The infectious virus-like particle (iVLP) assay, a system for investigating virus particle assembly and entry, was utilized to determine the functional importance of these conserved tryptophans. Elimination of the C-mannosylation motif, which resides in an external loop region of GP1, increased reporter activity, suggesting that particle entry is enhanced and this region may interact with the cell surface despite being outside of the receptor binding site. Decreased reporter activity was observed for all MPER mutants, with multiple MPER tryptophan mutations resulting in decreased GP1,2 incorporation. These data place the MPER tryptophan residues in an important role for glycoprotein incorporation and particle entry. Given the tryptophan content and location is similar to the MPER of HIV gp41, where these residues are required for glycoprotein incorporation and fusion, the MPER of EBOV GP2 may function similarly.

No description available.

As a result of transcriptional editing, Ebola virus (EBOV) produces multiple soluble products from its glycoprotein gene, the primary product of which is the secreted glycoprotein (sGP), in addition to the membrane-bound viral spike protein GP1,2. A lack of leukocyte infiltration is observed during EBOV infection, which is thought to allow virus replication to proceed unchecked and thus represents a significant role in the immunopathology of the disease. Currently the only know function of sGP is that it has an anti-inflammatory effect on endothelial cells treated with TNF-α, an effect that has been hypothesized to interfere with recruitment or extravasation of leukocytes. To better characterize this anti-inflammatory function, a link between sGP structure and function was sought. Mass spectrometry (MS) analysis of recombinant sGP demonstrated that it is a parallel-orientated disulphide-linked homodimer that contains Cys53-C53’ and Cys306-C306’ intermolecular disulphide bonds. In addition to being glycosylated with complex N-glycans, sGP also contained a novel post-translation modification, termed C-mannosylation. C-mannosylation was not required for the anti-inflammatory function of sGP; however, glycine mutations at amino acids 53 and 306 resulted in the complete loss of the anti-inflammatory effect on TNF-α treated endothelial cells. Thus, a specific structure mediated by intermolecular disulphide bonds is required for the proposed anti-inflammatory function of sGP, suggesting that this effect is the result of a specific interaction. The spike protein GP1,2, also contains C-mannosylation motifs. MS analysis of GP1,2 indicated that GP1 was C-mannosylated, while two adjacent motifs in the membrane proximal region (MPER) of GP2 were not. The infectious virus-like particle (iVLP) assay, a system for investigating virus particle assembly and entry, was utilized to determine the functional importance of these conserved tryptophans. Elimination of the C-mannosylation motif, which resides in an external loop region of GP1, increased reporter activity, suggesting that particle entry is enhanced and this region may interact with the cell surface despite being outside of the receptor binding site. Decreased reporter activity was observed for all MPER mutants, with multiple MPER tryptophan mutations resulting in decreased GP1,2 incorporation. These data place the MPER tryptophan residues in an important role for glycoprotein incorporation and particle entry. Given the tryptophan content and location is similar to the MPER of HIV gp41, where these residues are required for glycoprotein incorporation and fusion, the MPER of EBOV GP2 may function similarly.