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Rapid Publication

Proteins Linked to a Protein Transduction Domain Efficiently Transduce Pancreatic Islets

  1. Jennifer Embury,
  2. Dagmar Klein,
  3. Antonello Pileggi,
  4. Melina Ribeiro,
  5. Sundararajan Jayaraman,
  6. R. Damaris Molano,
  7. Christopher Fraker,
  8. Norma Kenyon,
  9. Camillo Ricordi,
  10. Luca Inverardi and
  11. Ricardo L. Pastori
  1. Diabetes Research Institute, University of Miami School of Medicine, Miami, Florida
    Diabetes 2001 Aug; 50(8): 1706-1713. https://doi.org/10.2337/diabetes.50.8.1706
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    • FIG. 1.
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      FIG. 1.

      Transduction of pancreatic β-cells with TAT/PTD–β-galactosidase. A: 106 βTC-3 cells were transduced for 3 h with 800 μg of TAT/PTD–β-galactosidase or β-galactosidase (minus TAT/PTD) and analyzed for β-galactosidase activity using X-Gal staining. B: βTC-3 were transduced for 30 min with 800 μg TAT-PTD–β-galactosidase/Alexa-Flour 488. The cells were washed twice with DMEM and incubated in a medium free of TAT/PTD-fusion protein at 37°C for 0, 2.5, 10, 24, and 48 h. The cells were analyzed by flow cytometry. The mean fluorescence values of all samples are shown.

    • FIG. 2.
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      FIG. 2.

      Transduction of islets with TAT/PTD-β-galactosidase. A: Rat islet cells were transduced with 0, 1, and 2 μg of the Alexa-Fluor 488-labeled fusion protein per islet (panels 1, 2, and 3, respectively) and incubated for 2 h. After incubation, islets were dissociated and evaluated by flow cytometry. B: β-galactosidase activity in transduced islets assessed by X-Gal staining. Rhesus monkey islets were transduced with 1.25 and 2.5 μg of TAT/PTD-β-galactosidase (panels I and II, respectively) or 2.5 μg of β-galactosidase (minus TAT/PTD; panel III) per islet. After transduction, islet cells were spread uniformly on the slide using cytospin and then fixed and stained by X-Gal.

    • FIG. 3.
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      FIG. 3.

      Inhibition of TNF-α–induced caspase activity by anti-apoptotic proteins expressed as TAT/PTD-fusion proteins. A: βTC-3 cells were transduced with 30 μg of TAT/PTD–Bcl-XL, 25 μg of TAT/PTD–PEA-15, or 90 μg of TAT/PTD–β-galactosidase. After 3 h, cultures were treated with a combination of TNF-α (500 U/ml) and CHX (10 μg/ml) for 18 h. Controls remained untreated during this period. Cells were harvested and lysed. Caspase proteolytic activity was measured using the fluorogenic Ac-DEVD-AFC substrate in the presence or absence of caspase-3 inhibitor, and the activity was then adjusted by protein content. Data represent the means ± SD of five (Bcl-XL–PEA-15) or three (TAT/PTD–β-galactosidase) separate experiments. *P < 0.01. B: βTC-3 cells nontransduced or transduced with TAT/PTD–Bcl-XL (panel I) or TAT/PTD-β-galactosidase (panel II) for 3 h were treated with TNF-α/CHX for 18 h. Controls remained untreated all this time. Pretreatment of pancreatic cells with zVAD-FMK (50 μmol/l) completely inhibited TNF-α–induced apoptosis (marked as control in panel II). Cells were resuspended and incubated in 10 μmol/l of FITC-VAD-FMK in situ marker and evaluated by flow cytometry.

    • FIG. 4.
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      FIG. 4.

      Increased viability of pancreatic β-cells transduced with TAT/PTD anti-apoptotic proteins. βTC-3 cells were transduced with 30 μg of TAT/PTD–Bcl-XL and 25 μg of TAT/PTD–PEA-15 for 3 h and treated with TNF-α/CHX as described in the legend for Fig. 3. Cells were harvested and stained with FluoroQuench stain (acridine orange plus ethidium bromide) and examined by fluorescence microscopy. Viable cells are stained green, whereas dead or dying cells appear yellow and orange. Pictures are representative from three experiments. Pictures were captured by a charge-coupled device camera (Lei-750; Leica) using VideoVixen software and analyzed with Photoshop version 5.5. The bars below each picture represent the percentage of viable (green) and dead (orange) cells in each experiment.

    • FIG. 5.
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      FIG. 5.

      Transduction of islets with TAT/PTD fusion proteins does not affect insulin secretion capability. A: Reversal of hyperglycemia in diabetic immunodeficient mice with pancreatic islets transduced with TAT/PTD–Bcl-XL. Lewis rat islets were transplanted under the kidney capsule of chemically induced diabetic CB17-scid mice. Animals received islets cultured for 24 h either in the presence (triangles) or absence (circles) of TAT-fusion protein (day −1). TAT-treated islets restored normoglycemia (blood glucose <200 mg/dl) in the transplanted animals as well as in control islets 1 day after implantation (day 0). The arrow on day 29 indicates the removal of the kidney bearing the graft. TAT1 and TAT2 represent two independent experiments performed with the same fusion protein. B: Static glucose challenge of islets transduced with TAT/PTD–Bcl-XL fusion proteins. Rat islets were incubated with 0.4 μg TAT/PTD–Bcl-XL per islet and subsequently cultured overnight before being harvested for the static glucose challenge test. The experiment shown is representative of five independent experiments performed in duplicate with islets isolated from different pools of animals. Bars with pattern are control islets; empty bars represent transduced islets. L1, low glucose; H, high glucose; L2, second low-glucose incubation.

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    Proteins Linked to a Protein Transduction Domain Efficiently Transduce Pancreatic Islets
    Jennifer Embury, Dagmar Klein, Antonello Pileggi, Melina Ribeiro, Sundararajan Jayaraman, R. Damaris Molano, Christopher Fraker, Norma Kenyon, Camillo Ricordi, Luca Inverardi, Ricardo L. Pastori
    Diabetes Aug 2001, 50 (8) 1706-1713; DOI: 10.2337/diabetes.50.8.1706

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    Proteins Linked to a Protein Transduction Domain Efficiently Transduce Pancreatic Islets
    Jennifer Embury, Dagmar Klein, Antonello Pileggi, Melina Ribeiro, Sundararajan Jayaraman, R. Damaris Molano, Christopher Fraker, Norma Kenyon, Camillo Ricordi, Luca Inverardi, Ricardo L. Pastori
    Diabetes Aug 2001, 50 (8) 1706-1713; DOI: 10.2337/diabetes.50.8.1706
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