Journal: International Journal of Molecular Sciences

Article Title: The Discovery of Highly Potent THP Derivatives as OCTN2 Inhibitors: From Structure-Based Virtual Screening to In Vivo Biological Activity

doi: 10.3390/ijms21197431

Figure Lengend Snippet: ( A ) Binding modes of l -carnitine (white sticks) THP (yellow sticks) resulting from docking simulations. Residues involved in protein‒ligand interactions are drawn as balls and sticks; the receptor hOCTN2 is reported in tan ribbons; ( B ) structural modifications carried out on THP; ( C ) synthesis of TCL 4a – t . (a) HCOH 37%, HCOOH, 60 °C, 16 h; (b) for 3a - j and 3l - t : amine, DCC, HOBT, Et 3 N, CH 2 Cl 2 , or THF, RT, 24 h, for 3k : 4-nitroaniline, PCl 3 , pyridine, 3 h, 40 °C; (c) CH 3 I, acetone, 18 h, RT. Abbreviations: 3-(2,2,2-trimethylhydrazine)propionate, THP; THP structurally related compounds, TCL; hydroxymethylene, HCOH; formic acid, HCOOH; N , N ’- dicyclohexylcarbodiimide, DCC; hydroxybenzotriazole, HOBT; N , N -diethylethanamine, Et 3 N; dichloromethane, CH 2 Cl 2 ; tetrahydrofurane, THF; room temperature, RT; phosphorus trichloride, iodomethane, PCl 3 .

Article Snippet: To obtain STHdh Q111/111 cells (STHdh Q111/111 ) overexpressing OCTN2, STHdh Q111/111 were transfected with plasmid DNA (solute carrier family 22 member 5 (SLC22A5) (NM_003060) Human Untagged Clone) containing hOCTN2 cDNA (pCMV6-XL5/hOCTN2, Origene), and then selected following a procedure already described [ ].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: The Discovery of Highly Potent THP Derivatives as OCTN2 Inhibitors: From Structure-Based Virtual Screening to In Vivo Biological Activity

doi: 10.3390/ijms21197431

Figure Lengend Snippet: ( a , b ) Western blot analysis of hOCTN2 expression in STHdh Q7/7 , STHdh Q111/111 , and transfected STHdh Q111/111 (STHdh Q111/111 /hOCTN2). §§ p < 0.01 vs. STHdh Q7/7 . ( c ) The total accumulation of carnitine (10 µM), THP, or TCL (50 μM) in STHdh Q111/111 cells with or without the heterologous expression of hOCTN2. ( d ) The efficiency of the transport of drugs by hOCTN2 in STHdh Q111/111 cells with the heterologous expression of hOCTN2. ( e ) Accumulation at 37 °C or at 4 °C of carnitine, THP, or TCL in STHdh Q111/111 cells with or without the heterologous expression of hOCTN2. ( f ) Carnitine uptake in OCTN2-transfected STHdh Q111/111 cells in the presence of 50 μM THP, and the newly synthesized compound. The bars represent the mean ± standard deviation ( n = 3). Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CTL.

Article Snippet: To obtain STHdh Q111/111 cells (STHdh Q111/111 ) overexpressing OCTN2, STHdh Q111/111 were transfected with plasmid DNA (solute carrier family 22 member 5 (SLC22A5) (NM_003060) Human Untagged Clone) containing hOCTN2 cDNA (pCMV6-XL5/hOCTN2, Origene), and then selected following a procedure already described [ ].

Techniques: Western Blot, Expressing, Transfection, Synthesized, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Discovery of Highly Potent THP Derivatives as OCTN2 Inhibitors: From Structure-Based Virtual Screening to In Vivo Biological Activity

doi: 10.3390/ijms21197431

Figure Lengend Snippet: The binding modes of the different ligands tested, resulting from docking simulations. The receptor hOCTN2 is depicted by tan ribbons, and all the ligands are sticks. The residues involved in protein‒ligand interactions are drawn as shapes and sticks. ( a , b ) The binding mode for 4i (pink sticks) and 4j (light blue sticks); 4k is in green in ( c ); ( d ) molecule 4t is shown in orange.

Article Snippet: To obtain STHdh Q111/111 cells (STHdh Q111/111 ) overexpressing OCTN2, STHdh Q111/111 were transfected with plasmid DNA (solute carrier family 22 member 5 (SLC22A5) (NM_003060) Human Untagged Clone) containing hOCTN2 cDNA (pCMV6-XL5/hOCTN2, Origene), and then selected following a procedure already described [ ].

Techniques: Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Peripheral Immune Cell Infiltration in the Hippocampus of Sepsis Mice

doi: 10.1111/jcmm.70872

Figure Lengend Snippet: Hub gene selection and correlation with myeloid immune cell infiltration scores. (A) Identification of seven hub genes. (B) Boxplot showing the expression of hub genes in the sham and CLP groups (median ± IQR, **** p < 0.0001). (C) Protein expression of hub genes was elevated in CLP tissues. (D–H) Correlations between hub genes and myeloid immune cell infiltration scores. Fcgr3 and Itgb2 were positively correlated with aDCs and pDCs. Fcer1g, Fcgr2b and Slc11a1 were positively correlated with monocytes and neutrophils. Icam1 and Itgb2 were positively correlated with monocytes (* p < 0.05, **** p < 0.0001). aDCs, activated dendritic cells; CLP, cecal ligation and puncture; pDCs, plasmacytoid dendritic cells.

Article Snippet: The antibodies used in this study are listed as follows: CD45 (Ptprc, 1:400 dilution, Proteintech, 20103‐1‐AP), ICAM1 (1:100 dilution, AiFang Biological, AF0688), CD18 (Itgb2, 1:100 dilution, Affinity Biosciences, DF6896), NRAMP1 (Slc11a1, 1:200 dilution, BOSTER, A02547‐3), CD32 (Fcgr2b, 1:200 dilution, Affinity Biosciences, AF04227), CD64 (Fcgr1g, 1:100 dilution, Affinity Biosciences, AF13220).

Techniques: Selection, Expressing, Ligation

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 2. Structure of the DB3 combining site. (A) Crystal structure of the complex between DB3 and progesterone (Arevalo et al. 1993a) showing some of the contact residues. Light and the heavy chains are in blue and purple, respectively; steroid oxygen atoms in red; steroid A-ring asterisked. (B) Model of site with TrpH100 replaced by arginine. (C) Superposition of A and B.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques:

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 3. Western blot of wild type and mutants of DB3 VH/K. Samples were run on 10% SDS-PAGE and electrotransferred to an Immobilon-P membrane (PVDF); the blot was probed with HRP-coupled sheep anti- mouse k.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques: Western Blot, SDS Page, Membrane

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 4. Antigen-binding and idiotypic properties of periplasmic extracts of E. coli expressing VH/K mutants. (A) Binding activity by ELISA on progesterone-BSA coated wells; (B) Competitive inhibition of binding by free progesterone (100 ng/mL) in ELISA; (C) Competitive inhibition by rabbit polyclonal anti-DB3-idiotype (10 mg/mL). Results are means of duplicates. (d) Deletion; (Co.) negative control.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques: Binding Assay, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Negative Control

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 5. Steroid specificity of DB3 wild-type VH/K and TrpH100Arg mutant. Binding to progesterone-BSA of fixed amounts of wild-type DB3 VH/K (A) and the TrpH100Arg mutant (B) was carried out in the presence of free progesterone, aetiocholanolone, 5a-dihydroprogesterone, 5b-dihydroprogesterone, or testosterone as inhibitors. Results are means of duplicates.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques: Mutagenesis, Binding Assay

Journal: Protein science : a publication of the Protein Society

Article Title: Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3.

doi: 10.1110/ps.062236806

Figure Lengend Snippet: Figure 6. Binding specificity of wild-type DB3 VH/K and the TrpH100Arg mutant for progesterone derivatives. Binding to progester- one-BSA of wild-type DB3 VH/K (A) and the TrpH100Arg mutant (B) was carried out in the presence of free progesterone-3-CMO (C3-CMO), progesterone-6-HMS (C6-HMS), progesterone-11a-HMS (C11a-HMS), or progesterone-21-HMS (C21-HMS). Results are means of duplicates.

Article Snippet: Antibody DB3 engineering JOBNAME: PROSCI 15#9 2006 PAGE: 3 OUTPUT: Saturday August 5 02:47:19 2006 csh/PROSCI/118160/ps0622368 1469896x, 2006, 9, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1110/ps.062236806 by U niversity O f T he Philipines D ilim an M ain L ibrary, W iley O nline L ibrary on [05/04/2024].

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Wnts are endothelial cell-derived PKD1/PKD2-dependent autocrine/paracrine vasodilators

doi: 10.64898/2026.03.17.712518

Figure Lengend Snippet: (A) Representative RT-PCR showing that mRNA for PKD1 is present in isolated endothelial cells from tamoxifen-treated Pkd1 fl/fl but absent in isolated endothelial cells from tamoxifen-treated Pkd1 fl/fl :Cdh5(PAC)-CreERT2 mice. Representative of 5 independent experiments for each genotype. (B) Representative Western blots illustrating PKD1, PKD2, AT1, eNOS, Fzd-7 and actin proteins in mesenteric arteries of Pkd1 fl/fl and Pkd1 ecKO mice. (C) Mean data from experiments shown in panel B. Significance was assessed using Student t-tests, n=5 independent mesenteric arterial lysates for each genotype and each protein. (D) Immunofluorescence images (representative of three mesenteric arteries from three mice for each genotype) illustrating that PKD1 (Alexa Fluor 546) is abolished in endothelial cells of en face mesenteric arteries from Pkd1 ecKO mice. CD31 (Alexa Fluor 488) and DAPI are also shown. Scale bars, 50 μm. (E) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular Wnt9b (3 μg/ml) applied in continuous flow in pressurized (80 mmHg) mesenteric arteries from Pkd1 fl/fl and Pkd1 ecKO mice. (F) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular boiled Wnt9b (3 µg/ml) applied under continuous flow (10 dyn/cm 2 ) in pressurized (80 mmHg) mesenteric arteries from Pkd1 fl/fl and Pkd1 ecKO mice. (G) Mean data from experiments shown in panels E and F, and the modulation of flow and Wnt9b-mediated vasodilation by SRI37892 (Fzd-7 Inh, 5 µM). Significance was assessed using a two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=10 arteries from 7 mice of each genotype for flow, flow+ Wnt9b and Wnt9b. n=7 arteries from 5 mice for Fzd-7 Inh+flow and Fzd-7 Inh+flow+Wnt9b. n=5 arteries from 5 mice for each genotype for flow + boiled Wnt9b. (H) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular Wnt5a (3 µg/ml). (I) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular boiled Wnt5a (3 µg/ml). (J) Mean data from experiments shown in panels H and I and the modulation of flow and Wnt5a-induced vasodilation by SRI37892 (Fzd-7 Inh, 5 μM). Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=8 arteries from 6 mice from Pkd1 fl/fl for flow, flow+ Wnt5a. n=5 arteries from 5 mice from Pkd1 ecKO for flow, flow+ Wnt5a. n=5 arteries from 5 mice of each genotype for flow + boiled Wnt5a. n=8 arteries from 6 mice from Pkd1 fl/fl for flow+ Fzd-7 Inh and flow+ Wnt5a+ Fzd-7 Inh.

Article Snippet: Membranes were blocked with 5% milk or 5% BSA and incubated with one of the following primary antibodies: PKD1 (Polycystic Kidney Disease Research Resource Consortium, Baltimore), PKD2 (Alomone), eNOS (Abcam), p-eNOS (Cell Signaling), Wnt9b (R&D Systems), Wnt5a (R&D Systems), AT1 receptor (Alomone) or actin (Cell Signaling) overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot, Immunofluorescence