Sphingomyelinase inhibitory and free radical scavenging potential of selected Nigerian medicinal plant extracts

Ceramides from sphingolipid breakdown, and other sphingolipid metabolites, mediate cellular signalling in infectious and other diseases. Therefore, inhibitors of sphingomyelinases (SMases), hold promise as prospective therapeutic agents. Considering the potential therapeutic utility, this in vitro study explored the sphingomyelinase inhibitory, and free radical scavenging potential of five Nigerian medicinal plant leaf extracts, purported to have efficacy against diseases, including HIV/AIDS. The extracts\u2019 sphingomyelinase inhibitory potencies were assessed colorimetrically and theirfree radical scavenging capabilities were assayed by the ability to quench 2,2\u2010diphenyl\u20101\u2010picrylhydrazyl (DPPH) radical and superoxide anion (O2.\u2010) radical. Considering their IC50 (\u3bcg/ml) values, the extracts inhibited the biochemical activity of sphingomyelinase in a dose-dependent manner, relative to imipramine the standard inhibitor (IC50 38.5 \ub1 2.4 \u3bcg/ml). With Aloe vera as least inhibitory, inhibition increased as follows: Aloe vera (Asphodelaceae) (1132 \ub1 10.8) < Senna siamea (Fabaceae) (992.2 \ub1 11.2) < Azadirachta indica (Meliaceae) (984 \ub1 7.4) < Landolphia owariensis (Apocynaceae) (146.3 \ub1 9.4) < Stachytarpheta angustifolia (Verbenacae) (100.3 \ub1 8.7). DPPH radical scavenging relative to ascorbic acid standard increased as: A. indica < A. vera < S. siamea < S. angustifolia < L. owariensis; and superoxide anion quenching, relative to standard rutin increased as: A. vera < S. angustifolia < L. owariensis < S. siamea < A. indica.These results showed thatthe most potent SMase inhibitor was S. angustifolia; whereas, for DPPH radical scavenging and superoxide inhibition, the most potent of the five extracts were L. owariensis and A. indica respectively.These extracts deserve further investigation into their biological effects

Role of the E1(∧)E4 Protein in the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 31

The most highly expressed protein in the productive life cycle of human papillomaviruses (HPVs) is E1(∧)E4, but its function is not well understood. To investigate the role of E1(∧)E4, we undertook a genetic analysis in the context of the complete HPV type 31 (HPV31) genome. A mutant HPV31 genome (E4M9) was constructed that contained a stop codon in the E4 open reading frame at amino acid 9 and was silent in the overlapping E2 coding sequence. Wild-type and mutant genomes were transfected into normal human foreskin keratinocytes (HFKs) and selected for drug resistance, and pooled cultures were examined for effects of E1(∧)E4 on viral functions. Southern blot analyses of transfected HFKs demonstrated that cells carrying the E4M9 mutant genomes were maintained as episomes at copy numbers similar to those in keratinocytes transfected with wild-type HPV31. Both sets of cells grew at similar rates, exhibited comparable extensions of life spans, and had equivalent levels of early transcripts. Following suspension of the cells in a semisolid medium, differentiation-dependent genome amplification and late gene expression were significantly decreased in cells maintaining the E4M9 mutant genome compared to those with wild-type HPV31. One explanation for these effects could be a reduction in the number of cells harboring mutant genomes that enter S phase upon differentiation. An analysis of cells containing E4M9 mutant genomes in organotypic raft cultures indicated a reduction in bromodeoxyuridine incorporation in differentiated suprabasal cells compared to that seen in wild-type rafts. Our results indicate that the HPV31 E1(∧)E4 protein plays a significant role in promoting HPV genome amplification and S phase maintenance during differentiation

Human Papillomavirus Type 31 E5 Protein Supports Cell Cycle Progression and Activates Late Viral Functions upon Epithelial Differentiation

The function of the E5 protein of human papillomaviruses (HPV) is not well characterized, and controversies exist about its role in the viral life cycle. To determine the function of E5 within the life cycle of HPV type 31 (HPV31) we first constructed HPV31 mutant genomes that contained an altered AUG initiation codon or stop codons in E5. Cell lines were established which harbored transfected wild-type or E5 mutant HPV31 genomes. These cell lines all maintained episomal copies of HPV31 and revealed similar phenotypes with respect to growth rate, early gene expression, and viral copy number in undifferentiated monolayer cultures. Following epithelial differentiation, genome amplification and differentiation-dependent late gene expression were observed in mutant cell lines, but at a rate significantly reduced from that observed in cells containing the wild-type genomes. Organotypic raft cultures indicated that E5 does not effect the expression of differentiation markers but does reduce expression of late viral proteins. Western analysis and immunofluorescence staining for cyclins during epithelial differentiation revealed a decreased expression of cyclin A and B in E5 mutant cells compared to HPV wild-type cells. Using a replating assay, a significant reduction in colony-forming ability was detected in the absence of E5 expression when cells containing wild-type or E5 mutant HPV genomes were allowed to proliferate following 24 h in suspension-induced differentiation. This suggests that HPV E5 modifies the differentiation-induced cell cycle exit and supports the ability of HPV31-positive keratinocytes to retain proliferative competence. In these studies, E5 was found to have little effect on the levels of the epidermal growth factor receptor (EGFR) or on its phosphorylation status. This indicates that EGFR is not a target of E5 action. Our results propose a role for high risk HPV E5 in modulation of late viral functions through activation of proliferative capacity in differentiated cells. We suspect that the primary target of E5 is a membrane protein or receptor that then acts to alter the levels or activities of cell cycle regulators

Transport of the Intracisternal A-Type Particle Gag Polyprotein to the Endoplasmic Reticulum Is Mediated by the Signal Recognition Particle

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum (ER) of rodent cells. The enveloped particles are produced by assembly and budding of IAP Gag polyproteins at the ER membrane. In this study, we analyzed the specific ER transport of the Gag polyprotein of the IAP element MIA14. To this end, we performed in vitro translation of Gag in the presence of microsomal membranes or synthetic proteoliposomes followed by membrane sedimentation or flotation. ER binding of IAP Gag occurred mostly cotranslationally, and Gag polyproteins interacted specifically with proteoliposomes containing only signal recognition particle (SRP) receptor and the Sec61p complex, which form the minimal ER translocation apparatus. The direct participation of SRP in ER targeting of IAP Gag was demonstrated in cross-linking and immunoprecipitation experiments. The IAP polyprotein was not translocated into the ER; it was found to be tightly associated with the cytoplasmic side of the ER membrane but did not behave as an integral membrane protein. Substituting the functional signal peptide of preprolactin for the hydrophobic sequence at the N terminus of IAP Gag also did not result in translocation of the chimeric protein into the ER lumen, and grafting the IAP hydrophobic sequence onto preprolactin failed to yield luminal transport as well. These results suggest that the N-terminal hydrophobic region of the IAP Gag polyprotein functions as a transport signal which mediates SRP-dependent ER targeting, but polyprotein translocation or integration into the membrane is prevented by the signal sequence itself and by additional regions of Gag

Sphingomyelinase inhibitory and free radical scavenging potential of selected Nigerian medicinal plant extracts

Ceramides from sphingolipid breakdown, and other sphingolipid metabolites, mediate cellular signalling in infectious and other diseases. Therefore, inhibitors of sphingomyelinases (SMases), hold promise as prospective therapeutic agents. Considering the potential therapeutic utility, this in vitro study explored the sphingomyelinase inhibitory, and free radical scavenging potential of five Nigerian medicinal plant leaf extracts, purported to have efficacy against diseases, including HIV/AIDS. The extracts’ sphingomyelinase inhibitory potencies were assessed colorimetrically and theirfree radical scavenging capabilities were assayed by the ability to quench 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical and superoxide anion (O2.‐) radical. Considering their IC50 (μg/ml) values, the extracts inhibited the biochemical activity of sphingomyelinase in a dose-dependent manner, relative to imipramine the standard inhibitor (IC50 38.5 ± 2.4 μg/ml). With Aloe vera as least inhibitory, inhibition increased as follows: Aloe vera (Asphodelaceae) (1132 ± 10.8) < Senna siamea (Fabaceae) (992.2 ± 11.2) < Azadirachta indica (Meliaceae) (984 ± 7.4) < Landolphia owariensis (Apocynaceae) (146.3 ± 9.4) < Stachytarpheta angustifolia (Verbenacae) (100.3 ± 8.7). DPPH radical scavenging relative to ascorbic acid standard increased as: A. indica < A. vera < S. siamea < S. angustifolia < L. owariensis; and superoxide anion quenching, relative to standard rutin increased as: A. vera < S. angustifolia < L. owariensis < S. siamea < A. indica.These results showed thatthe most potent SMase inhibitor was S. angustifolia; whereas, for DPPH radical scavenging and superoxide inhibition, the most potent of the five extracts were L. owariensis and A. indica respectively.These extracts deserve further investigation into their biological effects