Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: Sequences of oligonucleotides for qRT-PCR and EMSA Sequences are listed in the 5′ ∧ 3′ direction

Article Snippet: Assays were conducted as previously described [ 11 ], with the ACE2 activity determined as the difference in the hydrolysis rates of 10 μM of the fluorogenic ACE2 substrate (7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH (Mca-APK(Dnp)) (Enzo Life Sciences, Plymouth Meeting, PA) at 37°C in ACE2 reaction buffer in the absence and presence of 10 μM of the ACE2 inhibitor DX600 (Phoenix Pharmaceuticals, Inc., Burlingame, CA).

Techniques:

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: The ACE2 gene promoter is bipartite in humans and mice. (A) Diagram of the human ACE2 promoter with two conserved regions separated by a repetitive Alu element giving rise to two transcript forms encoding the same protein. (B) Design of DPT and PPT assays involves a common reverse primer, unique forward primers, and unique Taqman probes. (C) The DPT and PPT assays were run with different concentration of pCRII-TOPO plasmids containing cloned DPT and PPT amplicons as samples. A water sample was run as a no-template control. The cycle-threshold values (Ct) were recorded. (D) Comparisons of the concentrations of DPT and PPT in mouse tissues. Concentrations are shown as the Ct values obtained for RNA samples diluted to 20 ng/μl. Samples giving no amplification were assigned a Ct value of 40 which is the highest cycle number in the assays. The lung, kidney, heart, and brain were harvested from 3 female mice and the pancreas from 5 male mice. *: p < 0.05 vs. DPT. (E) Sequences of the conserved distal and proximal promoter regions with potential transcription factor binding motifs. Green shading indicates bases conserved among all tested mammalian species. Pink shading indicates bases conserved among the tested placental mammals. Hyphens indicate positions, where other species have additional bases.

Article Snippet: Assays were conducted as previously described [ 11 ], with the ACE2 activity determined as the difference in the hydrolysis rates of 10 μM of the fluorogenic ACE2 substrate (7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH (Mca-APK(Dnp)) (Enzo Life Sciences, Plymouth Meeting, PA) at 37°C in ACE2 reaction buffer in the absence and presence of 10 μM of the ACE2 inhibitor DX600 (Phoenix Pharmaceuticals, Inc., Burlingame, CA).

Techniques: Concentration Assay, Clone Assay, Amplification, Binding Assay

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: Pancreatic islets from humans possess ACE2 activity. The hydrolysis rate of the ACE2 substrate Mca-APK(Dnp) was measured for pancreatic islets from 5 normoglycemic individuals in the absence and presence of the ACE2-specific inhibitor DX600. *: p < 0.05 vs. no DX600.

Article Snippet: Assays were conducted as previously described [ 11 ], with the ACE2 activity determined as the difference in the hydrolysis rates of 10 μM of the fluorogenic ACE2 substrate (7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH (Mca-APK(Dnp)) (Enzo Life Sciences, Plymouth Meeting, PA) at 37°C in ACE2 reaction buffer in the absence and presence of 10 μM of the ACE2 inhibitor DX600 (Phoenix Pharmaceuticals, Inc., Burlingame, CA).

Techniques: Activity Assay

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: HNF1β stimulates ACE2 promoter activity. (A) HEK 293T, βTC3, and 832/13 cells were transfected with pGL3-Basic with or without distal and proximal ACE2 promoter region sequences. An HNF1β expression plasmid or the control plasmid pRcNhe was co-transfected. The HNF1β response is calculated as the luciferase activity for the HNF1β expression plasmid divided by the luciferase activity for the control plasmid. #, ###: p < 0.05, p < 0.001 vs. the HNF1β response for the pGL3-Basic plasmid without inserts. (B) The HNF1β response was determined for 832/13 cells transfected with pGL3-Basic containing the human ACE2 −454/−1 promoter sequence with or without mutations of three putative HNF1 binding sites in the proximal promoter region. ###: p < 0.001 vs. the HNF1β response of the unmutated promoter sequence. n.s.: No significant difference. (C) The HNF1β response was determined for 832/13 cells transfected with pGL3-Basic containing the human ACE2 −1699/−1 promoter sequence with or without mutation of the putative HNF1 binding site in the distal promoter region. (D) Five individual ChIP experiments of untransfected HEK 293T cells and HEK 293T cells transfected with the HNF1β expression plasmid were conducted. **, ***: p < 0.01, p < 0.001 vs. transfected cells with control IgG; a: p < 0.001 vs. the −1301/−1195 amplicon; b: p < 0.001 vs. the exon 18 amplicon.

Article Snippet: Assays were conducted as previously described [ 11 ], with the ACE2 activity determined as the difference in the hydrolysis rates of 10 μM of the fluorogenic ACE2 substrate (7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH (Mca-APK(Dnp)) (Enzo Life Sciences, Plymouth Meeting, PA) at 37°C in ACE2 reaction buffer in the absence and presence of 10 μM of the ACE2 inhibitor DX600 (Phoenix Pharmaceuticals, Inc., Burlingame, CA).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Sequencing, Binding Assay, Mutagenesis, Amplification

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: HNF1α and HNF1β bind to three motifs in the proximal ACE2 promoter region. (A) EMSA with nuclear extracts from 832/13 cells transfected with the HNF1β expression plasmid or the control plasmid pRcNhe. The probes represent the 3 potential HNF1 binding sites in the proximal promoter region (1st, 2nd, and 3rd motif), the potential HNF1binding site in the distal promoter region (4th motif), and the HNF1 binding site from the rat β-fibrinogen promoter. Complex I is a complex generated for cells transfected with the HNF1β expression plasmid. An HNF1β antibody supershifts complex I (right panel). (B) EMSA with nuclear extracts from 832/13 cells transfected with the HNF1α expression plasmid or the control plasmid pCVM6-XL5. Complex II and II are complexes whose intensities increase for extracts from cells transfected with the HNF1α expression plasmid. The endogenous complexes II and III are supershifted with an HNF1 antibody (right panel). (C) EMSA with nuclear extracts from untransfected 832/13 cells. Complex IV is a fast-migrating complex binding to the 1st and 4th motif that can be supershifted with a GATA4 antibody.

Article Snippet: Assays were conducted as previously described [ 11 ], with the ACE2 activity determined as the difference in the hydrolysis rates of 10 μM of the fluorogenic ACE2 substrate (7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH (Mca-APK(Dnp)) (Enzo Life Sciences, Plymouth Meeting, PA) at 37°C in ACE2 reaction buffer in the absence and presence of 10 μM of the ACE2 inhibitor DX600 (Phoenix Pharmaceuticals, Inc., Burlingame, CA).

Techniques: Transfection, Expressing, Plasmid Preparation, Binding Assay, Generated

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: HNF1α and HNF1β induce ACE2 expression in insulinoma cells. (A) Concentrations of ACE2 mRNA normalized to β-actin mRNA were determined for 832/13 cells and βTC3 cells transfected with the HNF1α expression plasmid, the HNF1β expression plasmid, or the corresponding control plasmids. Each panel summarizes results for four independent transfection experiments. (B) Western blotting was performed for 832/13 cells transfected with the HNF1α expression plasmid, the HNF1β expression plasmid, or the corresponding control plasmids. Each blot shows expression of ACE2 and the loading control γ-tubulin in cell lysates from four independent transfection experiments with 50 μg protein loaded per lane for the upper blot and 100 μg protein loaded per lane for the lower blot. To verify expression of HNF1α and HNF1β, membranes were stripped and re-probed with an antibody recognizing both HNF1α and HNF1β (upper blot) and with an antibody against HNF1β (lower blot). (C) ACE2 enzymatic activity was measured in extracts of 832/13 cells transfected with the HNF1α expression plasmid, the HNF1β expression plasmid, or the corresponding control plasmids. Each panel summarizes results for four independent transfection experiments. (D) Concentrations of ACE2 DPT and PPT were determined for βTC3 cells transfected with the HNF1α expression plasmid, the HNF1β expression plasmid, or the corresponding control plasmids. Samples giving no amplification were assigned a Ct value of 40 which is the highest cycle number in the assays. Each panel summarizes results for four independent transfections. **, ***: p < 0.01, p < 0.001 vs. control plasmid.

Article Snippet: Assays were conducted as previously described [ 11 ], with the ACE2 activity determined as the difference in the hydrolysis rates of 10 μM of the fluorogenic ACE2 substrate (7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH (Mca-APK(Dnp)) (Enzo Life Sciences, Plymouth Meeting, PA) at 37°C in ACE2 reaction buffer in the absence and presence of 10 μM of the ACE2 inhibitor DX600 (Phoenix Pharmaceuticals, Inc., Burlingame, CA).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Amplification

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: HNF1α induces ACE2 expression in pancreatic islet cells. (A) Concentrations of mRNA for RAS components and proteins involved in β-cell function relative to β-actin mRNA were determined for primary islet cells 24 and 48 h after being infected with the HNF1α-encoding adenovirus Ad-HNF1α or the control adenovirus Ad-GFP at MOI 100. AGT = angiotensinogen, PRR = (pro)renin receptor, ACE = angiotensin-converting enzyme, AT1a = angiotensin receptor type 1a, GLUT2 = glucose transporter 2, INS1 = insulin 1, INS2 = insulin 2. Concentrations are shown as the cycle-threshold (Ct) values for the target mRNA minus Ct for the normalizer β-actin mRNA obtained for RNA samples diluted to 0.5 ng/μl. Each data set was obtained from islet cells from 4-7 mice. *: p < 0.05 vs. Ad-GFP by pair-wise t-tests with p-values Bonferroni-adjusted by the number of comparisons. (B) For the mRNA targets that were significantly affected by infection with Ad-HNF1α, the Ad-HNF1α responses are calculated as the mRNA concentration for cells infected by Ad-HNF1α divided by the mRNA concentration for cells infected by Ad-GFP. (C) Insulin secretion at 2.8 and 25 mM glucose was determined for primary islet cells 41 h after being infected with Ad-HNF1α or Ad-GFP at MOI 100. Islet cells were from 3 mice, with each treatment combination of an adenovirus and a glucose concentration given to duplicate cell aliquots. (D) The concentration of ACE2 mRNA relative to β-actin mRNA was determined for primary islet cells 48h after being infected with Ad-HNF1α or Ad-GFP at MOI 100. Islet cells were from 3 mice infused with Ang II and 3 mice infused with saline. ***: p < 0.001 vs. Ad-GFP; #: p < 0.05 vs. saline. (E) Concentrations of mRNA for HNF1α, ACE2, and GLUT2 relative to β-actin mRNA were determined for primary islet cells 48 h after being infected with Ad-HNF1α or Ad-GFP at MOI 4 or 20. *: p < 0.05 vs. Ad-GFP by pair-wise t-tests with p-values Bonferroni-adjusted by the number of comparisons; @: p < 0.05 by pair-wise t-tests without adjustment for multiple comparisons. (F) Calculations of the Ad-HNF1α responses of HNF1α, ACE2, and GLUT2 for cells treated with adenovirus at MOI 4, 20, and 100.

Article Snippet: Assays were conducted as previously described [ 11 ], with the ACE2 activity determined as the difference in the hydrolysis rates of 10 μM of the fluorogenic ACE2 substrate (7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH (Mca-APK(Dnp)) (Enzo Life Sciences, Plymouth Meeting, PA) at 37°C in ACE2 reaction buffer in the absence and presence of 10 μM of the ACE2 inhibitor DX600 (Phoenix Pharmaceuticals, Inc., Burlingame, CA).

Techniques: Expressing, Cell Function Assay, Infection, Concentration Assay

Journal: Biochimica et biophysica acta

Article Title: The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

doi: 10.1016/j.bbagrm.2013.09.007

Figure Lengend Snippet: Comparison of the induction of ACE2 by HNF1α in islet cells and insulinoma cells. The mean concentrations of ACE2 mRNA and HNF1α mRNA relative to β-actin determined for islet cells infected for 48h with Ad-HNF1α or Ad-GFP and βTC3 cells transfected with the HNF1α expression plasmid or the control plasmid pCVM6-XL5 were compared. The calculations were based on the Ct values obtained in the quantitative RT-PCR experiments for Figs. 5 and 6.

Article Snippet: Assays were conducted as previously described [ 11 ], with the ACE2 activity determined as the difference in the hydrolysis rates of 10 μM of the fluorogenic ACE2 substrate (7-Methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys(2,4-Dinitrophenyl)-OH (Mca-APK(Dnp)) (Enzo Life Sciences, Plymouth Meeting, PA) at 37°C in ACE2 reaction buffer in the absence and presence of 10 μM of the ACE2 inhibitor DX600 (Phoenix Pharmaceuticals, Inc., Burlingame, CA).

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR

Journal: Viruses

Article Title: Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance

doi: 10.3390/v12111275

Figure Lengend Snippet: DEER distance profiles for HIV-1 DRV-resistant proteases and PI-naïve subtype B.

Article Snippet: Inhibitors were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, and the non-hydrolysable CaP2 substrate mimic (H-Arg-Val-Leu-r-Phe-Glu-Ala-Nle/NH2 (r = reduced)) was purchased from Peptides International (Louisville, KY, USA).

Techniques: Construct

Journal: Viruses

Article Title: Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance

doi: 10.3390/v12111275

Figure Lengend Snippet: Population analysis of normalized DEER distance profiles for each construct unbound, upon addition of non-hydrolysable CaP2 inhibitor or DRV inhibitor. The gray dashed line represents the overall population profile shown in . The curled open conformation is rendered in black with tight forward hashes. The closed population is drawn with red with moderate spaced forward hashes. The semi-open conformation is represented by a green line with tight back hash lines, and the wide-open conformation is in blue with moderately spaced back hash lines. Given the signal-to-noise ratio for collected DEER echo traces, the error for populations is ±3% P(r). Full details of data processing are given in the supporting information and follow the protocol described previously [ , ].

Article Snippet: Inhibitors were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, and the non-hydrolysable CaP2 substrate mimic (H-Arg-Val-Leu-r-Phe-Glu-Ala-Nle/NH2 (r = reduced)) was purchased from Peptides International (Louisville, KY, USA).

Techniques: Construct

Journal: Viruses

Article Title: Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance

doi: 10.3390/v12111275

Figure Lengend Snippet: Summary of the fractional occupancy of the four nominal states from DEER population analysis.

Article Snippet: Inhibitors were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, and the non-hydrolysable CaP2 substrate mimic (H-Arg-Val-Leu-r-Phe-Glu-Ala-Nle/NH2 (r = reduced)) was purchased from Peptides International (Louisville, KY, USA).

Techniques:

Journal: Viruses

Article Title: Darunavir-Resistant HIV-1 Protease Constructs Uphold a Conformational Selection Hypothesis for Drug Resistance

doi: 10.3390/v12111275

Figure Lengend Snippet: ( A ) Ratio of kcat DRV /kcat WT for each construct versus the fractional occupancy of the semi-open conformation in unbound HIV-1 PR. Data are included for the six DRV constructs (black squares) as well as for a series of accumulated mutations in response to nelfinavir (NLV) (gray circles)–taken from . ( B ) Plot of the change in the closed population (∆closed% = closed (inhibitor)–closed (unbound)) for each DRV construct with solid bars being the non-hydrolysable CaP2 inhibitor and slashed bars data for the DRV inhibitor. Numbers above the bar report the fold change in K m values, whereas numbers below the bars report the fold change in K i values (data taken from ). ( C ) Plot of K i value as a function of the fractional occupancy of the ratio of the open-like to closed states for unbound DRV constructs with K i (DRV) plotted as solid squares compared to our earlier investigations of NFV-resistant constructs to K i (NLV) plotted as open circles. The dashed line is a guide for the eyes showing a linear trend. The y-axes differ for the two data sets and are labeled according to the inhibitor.

Article Snippet: Inhibitors were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, and the non-hydrolysable CaP2 substrate mimic (H-Arg-Val-Leu-r-Phe-Glu-Ala-Nle/NH2 (r = reduced)) was purchased from Peptides International (Louisville, KY, USA).

Techniques: Construct, Labeling