Positive and Negative Regulatory Elements in the HIV-1 5'UTR Control Specific Recognition by Gag

Biological Sciences (The Ohio State University Denman Undergraduate Research Forum)The 5ʹ untranslated region (5ʹUTR) of the human immunodeficiency virus type 1 (HIV-1) genomic RNA (gRNA) contains a structured RNA element (termed Psi) that is specifically recognized by the HIV-1 Gag polyprotein, ensuring that two strands of gRNA are packaged into newly assembled virions. However, the mechanism by which Gag recognizes gRNA over other cellular RNAs and spliced viral RNAs is not well understood. A recent study suggested that a negative regulatory element upstream of Psi reduces high-affinity Gag binding, and a positive regulatory element downstream of Psi counteracts the upstream element and restores high-affinity binding. The aim of this study is to determine how these elements affect the specificity and mode of Gag binding. Using a fluorescence anisotropy-based salt-titration binding assay, the electrostatic and nonelectrostatic (i.e., specific) components of binding were measured. We have previously shown that Gag interacts with a 109-nucleotide (nt) Psi RNA construct that lacks the putative regulatory elements with high specificity and relatively few electrostatic interactions. Using a 356-nt RNA construct that includes the negative regulatory element in addition to Psi, we observed a loss in Gag binding specificity and an increase in electrostatic interactions. Interestingly, a 400-nt construct that contains the positive and negative elements flanking Psi restored highly specific binding and reduced the electrostatic interactions made with the RNA. Furthermore, a construct wherein the 40-nt positive regulatory element was appended to Psi, demonstrated the same specificity as Psi alone. Taken together, these data are consistent with a mechanism whereby the negative and positive regulatory elements flanking Psi modulate Gag binding mode and specificity.Undergraduate Education Summer Research FellowshipArts & Sciences Undergraduate Research ScholarshipSOLAR Research GrantNo embargoAcademic Major: Biochemistr

Cellular mechanisms by which proinsulin C-peptide prevents insulin-induced neointima formation in human saphenous vein

AIMS/HYPOTHESIS: Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide. METHODS: Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance. RESULTS: Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days). CONCLUSIONS/INTERPRETATION: Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients

Genome-Wide Transcriptional Profiling in a Histidine Kinase Mutant of Helicobacter pylori Identifies Members of a Regulon

To identify putative members of a regulon controlled by the H. pylori sensory histidine kinase HP0164 (HK0164), we constructed HK0164 null mutant H. pylori strains and analyzed bacterial gene transcription using DNA arrays. Seven genes were differentially expressed in multiple HK0164 mutant strains compared to their expression in control strains. Strain-dependent variations in differential expression were also detected. These results indicate that the signal transduction circuit utilizing HK0164 controls the transcription of at least seven genes in H. pylori