Journal: Journal of Biological Chemistry

Article Title: A Eukaryotic Type Serine/Threonine Kinase and Phosphatase inStreptococcus agalactiae Reversibly Phosphorylate an Inorganic Pyrophosphatase and Affect Growth, Cell Segregation, and Virulence

doi: 10.1074/jbc.m212747200

Figure Lengend Snippet: FIG. 7. A, purification of P35p by HPLC. HPLC purification of phos- phorylated [-32P]P35 was performed on soluble fractions of LR114 as described under “Experimental Procedures.” An aliquot of the HPLC- purified fraction containing the highest concentration of phosphoryl- ated P35 was analyzed on an SDS-PAGE along with (rStk1 phospho- rylated) soluble and membrane fractions of LR114, prior to HPLC purification. The gel was stained with Coomassie followed by autora- diography. The left panel shows Coomassie-stained proteins in the molecular mass range of 55–25 kDa, and the corresponding autoradio- graph is shown in the right panel. Lanes 1 and 2 represent soluble and membrane proteins of LR114. The fraction (fraction 28) containing the highest concentration of P35p protein is seen in lane 3. Lanes 4–6 represent the corresponding autoradiograph. Note that P35p is ob- served in lanes 4–6, with the highest intensity in lane 6. B, P35 is phosphorylated at serine residues. Phosphoamino acid analysis was performed on purified P35p. The phosphorylated protein was hydro- lyzed in 6 N HCl and subjected to two-dimensional, ascending thin layer chromatography. The open circles denote the positions of the nonradio- active phosphoamino acid standards identified by ninhydrin staining. pS, phosphoserine; pT, phosphothreonine; pY, phosphotyrosine. The plate was subsequently exposed to autoradiography. The numbers and arrows indicate the first and second dimensions used for separation of the phosphoamino acids.

Article Snippet: The sample was exchanged into 30 mM Tris-HCl, pH 7.5, using a Centriprep YM-10 (Millipore Corp., Bedford, MA), and 5 mg of total protein was injected into a Bio-logic Duoflow HPLC equipped with a UNO Q-1 anion exchange HPLC column (7 35 mm; Bio-Rad).

Techniques: Purification, Concentration Assay, SDS Page, Membrane, Staining, Autoradiography, Phosphoamino Acid Analysis, Thin Layer Chromatography

Journal: Prion

Article Title: A conservative mutant of a proteolytic fragment produced during fibril formation enhances fibrillogenesis

doi: 10.4161/19336896.2014.983745

Figure Lengend Snippet: Chromatograms of reverse-phase HPLC of R peptide (top), WT and R peptide together after 1 month of incubation post-addition of R peptide at 4°C (middle), and WT peptide after 1 month of incubation at 4°C (bottom).

Article Snippet: Reverse-phase HPLC was performed using a BioLogic DuoFlow Pathfinder 20 system (BioRad, USA) and a YMC TriArt C18 column (250*4.6 mm, 5 μm grains) for analysis and YMC C18 (150*10 mm, 10 μm grains) for preparative purposes.

Techniques: Incubation

Journal: Prion

Article Title: A conservative mutant of a proteolytic fragment produced during fibril formation enhances fibrillogenesis

doi: 10.4161/19336896.2014.983745

Figure Lengend Snippet: Mass-spectrometry analysis of the HPLC fraction of pH 2.0 incubated sample.

Article Snippet: Reverse-phase HPLC was performed using a BioLogic DuoFlow Pathfinder 20 system (BioRad, USA) and a YMC TriArt C18 column (250*4.6 mm, 5 μm grains) for analysis and YMC C18 (150*10 mm, 10 μm grains) for preparative purposes.

Techniques: Mass Spectrometry, Incubation

Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: Specific Amyloid Binding of Polybasic Peptides In Vivo Is Retained by β-Sheet Conformers but Lost in the Disrupted Coil and All D-Amino Acid Variants

doi: 10.1007/s11307-017-1063-0

Figure Lengend Snippet: Predicted secondary structure of peptides used in this study. Peptide p5 was predicted to be α-helical whereas p5(sheet) was an extended sheet [13]. Peptide p5(coil) and p5(Pro3) were random coils or disrupted helices. Structures were generated using the online prediction program, iTASSER, and rendered using DeepView/Swiss-PDBViewer v4.0.4 (Swiss Institute of Bioinformatics). Helix content of each peptide was predicted using Agadir.

Article Snippet: Peptides p5 (D) , p5 (sheet) , p5 (coil) , and p5 (Pro3) ( ) were purchased from Keck Laboratories (New Haven, CT) or Anaspec (Fremont, CA) as impure preparations and purified by RP-HPLC (Biologic DuoFlow; BioRad, Hercules, CA) by using a C3 solid phase (Zorbax TM 300SB; Agilent, Santa Clara, CA).

Techniques: Generated

Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: Specific Amyloid Binding of Polybasic Peptides In Vivo Is Retained by β-Sheet Conformers but Lost in the Disrupted Coil and All D-Amino Acid Variants

doi: 10.1007/s11307-017-1063-0

Figure Lengend Snippet: Biodistribution of radiolabeled peptides in WT mice using SPECT/CT imaging. The uptake of iodine-125-labeled peptides p5(D), p5(sheet), p5(coil), and p5(Pro3) was visualized by using SPECT/CT imaging of WT mice euthanized at 2 and 24 h pi. Coronal and axial views are shown, where the axial slice is at the level of the kidneys. Radioactivity is false colored yellow-red. K kidney, L liver, S stomach, T thyroid.

Article Snippet: Peptides p5 (D) , p5 (sheet) , p5 (coil) , and p5 (Pro3) ( ) were purchased from Keck Laboratories (New Haven, CT) or Anaspec (Fremont, CA) as impure preparations and purified by RP-HPLC (Biologic DuoFlow; BioRad, Hercules, CA) by using a C3 solid phase (Zorbax TM 300SB; Agilent, Santa Clara, CA).

Techniques: Single Photon Emission Computed Tomography, Imaging, Labeling, Radioactivity

Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: Specific Amyloid Binding of Polybasic Peptides In Vivo Is Retained by β-Sheet Conformers but Lost in the Disrupted Coil and All D-Amino Acid Variants

doi: 10.1007/s11307-017-1063-0

Figure Lengend Snippet: Biodistribution (%ID/g) of radioiodinated peptides in AA mice (mean ± SD)

Article Snippet: Peptides p5 (D) , p5 (sheet) , p5 (coil) , and p5 (Pro3) ( ) were purchased from Keck Laboratories (New Haven, CT) or Anaspec (Fremont, CA) as impure preparations and purified by RP-HPLC (Biologic DuoFlow; BioRad, Hercules, CA) by using a C3 solid phase (Zorbax TM 300SB; Agilent, Santa Clara, CA).

Techniques: Mouse Assay

Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: Specific Amyloid Binding of Polybasic Peptides In Vivo Is Retained by β-Sheet Conformers but Lost in the Disrupted Coil and All D-Amino Acid Variants

doi: 10.1007/s11307-017-1063-0

Figure Lengend Snippet: Biodistribution (%ID/g) of radioiodinated peptides in WT mice (mean ± SD)

Article Snippet: Peptides p5 (D) , p5 (sheet) , p5 (coil) , and p5 (Pro3) ( ) were purchased from Keck Laboratories (New Haven, CT) or Anaspec (Fremont, CA) as impure preparations and purified by RP-HPLC (Biologic DuoFlow; BioRad, Hercules, CA) by using a C3 solid phase (Zorbax TM 300SB; Agilent, Santa Clara, CA).

Techniques: Mouse Assay

Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: Specific Amyloid Binding of Polybasic Peptides In Vivo Is Retained by β-Sheet Conformers but Lost in the Disrupted Coil and All D-Amino Acid Variants

doi: 10.1007/s11307-017-1063-0

Figure Lengend Snippet: Biodistribution of radiolabeled peptides in mice with AA amyloidosis using SPECT/CT imaging. The uptake of iodine-125-labeled peptides p5(D), p5(sheet), p5(coil), and p5(Pro3) was visualized by using SPECT/CT imaging of AA mice euthanized at 2 and 24 h pi. Coronal and axial views are shown, where the axial slice is at the level of the kidneys. Radioactivity is false colored yellow-red. K kidney, L liver, S stomach, T thyroid.

Article Snippet: Peptides p5 (D) , p5 (sheet) , p5 (coil) , and p5 (Pro3) ( ) were purchased from Keck Laboratories (New Haven, CT) or Anaspec (Fremont, CA) as impure preparations and purified by RP-HPLC (Biologic DuoFlow; BioRad, Hercules, CA) by using a C3 solid phase (Zorbax TM 300SB; Agilent, Santa Clara, CA).

Techniques: Single Photon Emission Computed Tomography, Imaging, Labeling, Radioactivity

Journal: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging

Article Title: Specific Amyloid Binding of Polybasic Peptides In Vivo Is Retained by β-Sheet Conformers but Lost in the Disrupted Coil and All D-Amino Acid Variants

doi: 10.1007/s11307-017-1063-0

Figure Lengend Snippet: Microautoradiographic distribution of [125I]p5(D) and [125I]p5(sheet) in WT and AA mice at 24 h pi. Radiolabeled a p5(D) and b p5(sheet) peptides were visualized in 6-μm-thick, formalin-fixed tissue sections from WT and AA mice by using microautoradiography (ARG). Consecutive tissue sections were stained with Congo red (CR) to show the presence of birefringent amyloid. ARG and CR images were taken using objective magnifications of ×5 and ×10, respectively, except for images of the stomach that were acquired with a ×10 objective magnification. The CR image area is noted by the black box in the AA-ARG image.

Article Snippet: Peptides p5 (D) , p5 (sheet) , p5 (coil) , and p5 (Pro3) ( ) were purchased from Keck Laboratories (New Haven, CT) or Anaspec (Fremont, CA) as impure preparations and purified by RP-HPLC (Biologic DuoFlow; BioRad, Hercules, CA) by using a C3 solid phase (Zorbax TM 300SB; Agilent, Santa Clara, CA).

Techniques: Staining

Journal: Journal of Translational Medicine

Article Title: Evaluation of the effect of d- amino acid incorporation into amyloid-reactive peptides

doi: 10.1186/s12967-017-1351-0

Figure Lengend Snippet: Primary structure of peptides

Article Snippet: Peptides AQA p5 (d) , aqa p5 and AQA p5 (Table ) were purchased from Anaspec (Fremont, CA) as crude preparations and were purified by reverse-phase high performance liquid chromatography (RP-HPLC; Biologic DuoFlow; BioRad, Hercules, CA) using a C3 matrix (ZorbaxTM 300SB; Agilent, Santa Clara, CA).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Evaluation of the effect of d- amino acid incorporation into amyloid-reactive peptides

doi: 10.1186/s12967-017-1351-0

Figure Lengend Snippet: Peptides exhibit an α-helical secondary structure. a Circular dichroism spectra of peptides aqa p5, AQA p5 (d) and AQA p5 adopt a helical secondary structure in the presence of low molecular weight heparin as evidenced by the change in mean residue ellipticity at 222 nm. b The heparin-mediated change in ellipticity at 222 nm yielded midpoints of 0.33, 0.15, and 0.19 mg for peptides aqa p5, AQA p5 (d) and AQA p5, respectively

Article Snippet: Peptides AQA p5 (d) , aqa p5 and AQA p5 (Table ) were purchased from Anaspec (Fremont, CA) as crude preparations and were purified by reverse-phase high performance liquid chromatography (RP-HPLC; Biologic DuoFlow; BioRad, Hercules, CA) using a C3 matrix (ZorbaxTM 300SB; Agilent, Santa Clara, CA).

Techniques: Circular Dichroism, Molecular Weight, Residue

Journal: Journal of Translational Medicine

Article Title: Evaluation of the effect of d- amino acid incorporation into amyloid-reactive peptides

doi: 10.1186/s12967-017-1351-0

Figure Lengend Snippet: Biodistribution of radiolabeled peptide in healthy WT mice. The tissue distribution, expressed as percent inject dose per gram (%ID/g) of tissue, of radioiodinated peptides AQA p5 (d) , aqa p5 and AQA p5 was determined in WT mice by measuring tissue radioactivity at 1 h ( a ), 4 h ( b ) and 24 h ( c ) post injection. The data are expressed as mean ± SD (n = 3) and were analyzed using ANOVA with multiple comparisons (**** p < 0.0001)

Article Snippet: Peptides AQA p5 (d) , aqa p5 and AQA p5 (Table ) were purchased from Anaspec (Fremont, CA) as crude preparations and were purified by reverse-phase high performance liquid chromatography (RP-HPLC; Biologic DuoFlow; BioRad, Hercules, CA) using a C3 matrix (ZorbaxTM 300SB; Agilent, Santa Clara, CA).

Techniques: Radioactivity, Injection

Journal: Journal of Translational Medicine

Article Title: Evaluation of the effect of d- amino acid incorporation into amyloid-reactive peptides

doi: 10.1186/s12967-017-1351-0

Figure Lengend Snippet: Microdistribution of radiolabeled peptides revealed liver and kidney retention of AQA p5 (d) but not of the other two peptides. a Peptide AQA p5 (d) was observed microautoradiographically, as evidenced by the presence of black silver grains, in the liver and renal cortex at 1, 4, and 24 h post injection. Peptides aqa p5 ( b ) and AQA p5 ( c ) were apparent in the renal cortex at 1 h post injection during catabolism, but their presence decreased at 4 and 24 h post injection. There was no evidence of retention of either peptide in the liver at any time point

Article Snippet: Peptides AQA p5 (d) , aqa p5 and AQA p5 (Table ) were purchased from Anaspec (Fremont, CA) as crude preparations and were purified by reverse-phase high performance liquid chromatography (RP-HPLC; Biologic DuoFlow; BioRad, Hercules, CA) using a C3 matrix (ZorbaxTM 300SB; Agilent, Santa Clara, CA).

Techniques: Injection

Journal: Journal of Translational Medicine

Article Title: Evaluation of the effect of d- amino acid incorporation into amyloid-reactive peptides

doi: 10.1186/s12967-017-1351-0

Figure Lengend Snippet: SPECT/CT imaging of radioiodinated aqa p5 and AQA p5 in AA and healthy mice. a Radiolabeled aqa p5 was observed in the liver (L), spleen (Sp), pancreas (P) and intestine of mice with AA amyloid which persisted for more than 24 h post injection. In contrast, in WT mice, [ 125 I] aqa p5 was observed in the hepatic blood pool and kidney (K) at 1 h but was excreted and not visible at 4 h post injection. b [ 125 I] AQA p5 was observed in the liver, spleen, pancreas and intestine of AA mice; whereas, in WT mice free radioiodide liberated during catabolism was observed in the stomach (St) and thyroid (T)

Article Snippet: Peptides AQA p5 (d) , aqa p5 and AQA p5 (Table ) were purchased from Anaspec (Fremont, CA) as crude preparations and were purified by reverse-phase high performance liquid chromatography (RP-HPLC; Biologic DuoFlow; BioRad, Hercules, CA) using a C3 matrix (ZorbaxTM 300SB; Agilent, Santa Clara, CA).

Techniques: Single Photon Emission Computed Tomography, Imaging, Injection

Journal: Journal of Translational Medicine

Article Title: Evaluation of the effect of d- amino acid incorporation into amyloid-reactive peptides

doi: 10.1186/s12967-017-1351-0

Figure Lengend Snippet: Specific amyloid binding of radiolabeled peptides aqa p5 and AQA p5 in AA mice. Both [ 125 I] aqa p5 ( a ) and [ 125 I] AQA p5 ( b ) localized with AA amyloid in the spleen, liver, intestine, and pancreas as evidenced microautoradiographically, by the presence of black silver grains at sites of amyloid deposition. c The characteristic distribution of amyloid in the organs was evidenced by the presence of green–gold birefringent amyloid in Congo red-stained tissue sections

Article Snippet: Peptides AQA p5 (d) , aqa p5 and AQA p5 (Table ) were purchased from Anaspec (Fremont, CA) as crude preparations and were purified by reverse-phase high performance liquid chromatography (RP-HPLC; Biologic DuoFlow; BioRad, Hercules, CA) using a C3 matrix (ZorbaxTM 300SB; Agilent, Santa Clara, CA).

Techniques: Binding Assay, Staining