Expression of transcobalamin/oleosin chimeric proteins in transfected N1E-115 cells.

<p>A: Transgenic expression in N1E-115 cells at 48 h after transfection using lipofectamine. The cells were transfected with one of the following plasmids: pCMV-TCII-OLEO coding for transcobalamin-oleosin (lane 1), pCMV-OLEO-TCII coding for oleosin-transcobalamin (lane 2), pCMV-TCII coding for transcobalamin II (lane 3), pCMV-OLEO coding for oleosin (lane 4), pCDNA3 (lane 5), and pCMV-GFP-TCII-OLEO coding for GFP-TCII-OLEO (lane 6). The housekeeping gene was β-actin. The size in bp of the amplified products was 1347 for transcobalamin-oleosin (TCII-OLEO), 1240 for oleosin-transcobalamin (OLEO-TCII), 551 for transcobalamin II (TCII), 275 for oleosin (OLEO), and 349 for β-actin. B: Western blotting of homogenate of N1E-115 cells transfected with the various plasmids. From lane 1 to 6: homogenates from cells transfected with pCMV-TCII-OLEO, pCMV-OLEO-TCII, pCMV-TCII, pCMV-OLEO, empty plasmid, pCMV-GFP-TCII-OLEO, respectively. C: Vitamin B12 binding capacity in transfected cells. ⁵⁷Co-labeled Cobalamin (Cbl, ∼300 µCi per µg) was incorporated into culture medium (30,000 dpm/mL) for three days. The total amount of radioactivity taken by each cell lines was measured in pellets and supernatants. Mean and S.E.M. are indicated. D: Indirect immunofluorescence of TCII in N1E-115 cells transiently transfected with lipofectamine. The four constructs and the empty plasmid were tranfected in N1E-115 cells (1–5). The immunofluorescence was done with a goat polyclonal antibody to TCII and a donkey antigoat IgG fluorescein labeled. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 10 µm. E: Confocal analysis showing co-localization of the protein GFP-TCII-OLEO with endoplasmic reticulum in transfected N1E-115 cells. The cells were transfected with the plasmid pCMV-GFP-TCII-OLEO coding for GFP-transcobalamin-oleosin (GFP-TCII-OLEO), using lipofectamine. Cell nuclei were counterstained with Hoechst 33258 (1, 5). Co-localization was evidenced with fluorescence from GFP (2, 6), immuno-fluorescence with a mouse monoclonal antibody to the human golgin-97 (3) or a rabbit polyclonal antibody to calreticulin (7) and merge fluorescence (4,8). The secondary antibodies include a donkey IgG anti-mouse TRITC labeled or a donkey IgG anti-rabbit TRITC labeled. Calibration bars = 20 µm.</p

Viability and apoptosis of N1E-115 cells stably transfected with different plasmids.

<p>The plasmids were pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO), pCMV-OLEO-TCII coding for oleosin-transcobalamin (OLEO-TCII), pCMV-TCII coding for transcobalamin II (TCII), pCMV-OLEO coding for oleosin (OLEO), and pCDNA3. A: Cell viability and apoptosis among growth delay in proliferated state was monitored by the absorbance of formazan dye resulting from enzymatic metabolism of MTT by the mitochondrial dehydrogenase. The no reagent blank was MTT alone (0.5 mg/mL in culture medium). Western blot analysis of the temporal course of p53 in cells transfected with pCMV-TCII-OLEO was made by using a mouse anti-p53 antibody and a mouse anti-actin antibody, which were revealed with a goat anti-mouse IgG coupled to peroxidase. Cleaved Caspase-3 was identified by a rabbit anti-caspase-3 polyclonal antibody and a donkey anti-rabbit secondary antibody (labeled with peroxidase). B: Western blotting of cleaved Caspase-3 at Day 7 of proliferate state. The mean±S.E.M. were obtained from three independent experiments made in triplicate. Two-way ANOVA and Bonferoni post-test analyzed statistical differences from the control groups. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Expression of transcobalamin II/oleosin (TCII/OLEO) chimeric proteins in rats 60 days after transfection with the NTS-polyplex.

<p>A: RT-PCR from the plasmid transcripts in the <i>substantia nigra</i> of rats. A group of rats (n = 3) was transfected with the plasmid pCMV-TCII-OLEO and another (n = 3) with the plasmid pCMV-OLEO-TCII. RT-PCR amplified a fragment of 380 bp for TCII-OLEO, a fragment of 394 for OLEO-TCII, and a fragment of 349 for β-actin, the internal control. Lane 1 corresponds to the amplified fragment from the plasmid (positive control). Lane 2 is a PCR in the absence of plasmid or cDNA (negative control). The amplified product from the transfected substantia nigra of each rat corresponds to the lanes 3, 5, and 7, and the lanes 4, 6, and 8 show the RT-PCR outcome from the non-transfected side. B: GFP immunofluorescence in the rat <i>substantia nigra</i> transfected with pCMV-GFP-TCII-OLEO. The pCMV-GFP-TCII-OLEO encodes for the fusion protein green fluorescent protein-transcobalamin-oleosin (GFP-TCII-OLEO). The immunofluorescence was done with a mouse monoclonal antibody to GFP and a donkey antimouse IgG fluorescein labeled. Representative micrographs of coronal section of control substantia nigra (1) and transfected substantia nigra (2) of the same rat are presented. Calibration bars = 100 µm. C: Double immunofluorescence against TCII and tyrosine hydroxylase (TH) in the <i>substantia nigra</i> of rats. The neurons were transfected with NTS-polyplex with pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO). Slices from mesencephalon (40 µm) were immunostained at 7-day after transfection. The primary antibodies were a goat polyclonal anti-TCII and a mouse monoclonal anti-TH. The secondary antibodies were a donkey antigoat IgG fluorescein labeled and a donkey antimouse IgG rhodamine labeled. Representative micrographs of coronal section of control <i>substantia nigra</i> (1–3) and transfected <i>substantia nigra</i> (4–6) of the same rat are presented. Calibration bars = 50 µm.</p

Apoptosis of tyrosine hydroxylase (TH) immunoreactive cells in the <i>substantia nigra</i> of rats transfected with several plasmids.

<p>A: TH-immunoreactive neurons after transfection. The neurons were transfected with NTS-polyplex with one of the following plasmids, pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO, 1), pCMV-OLEO-TCII coding for oleosin-transcobalamin (OLEO-TCII, 2), pCMV-TCII coding for transcobalamin II (TCII, 3), pCMV-OLEO coding for oleosin (OLEO, 4), and the pCDNA3, the empty plasmid (5). Mesencephalon slices (40 µm) were immunostained at 2-month after transfection with a mouse monoclonal antibody to TH and a donkey antimouse IgG fluorescein labeled. Representative micrographs of sagital section of the rat mesencephalon are presented. Calibration bars = 200 µm. B: Apoptosis in TH-immunoreactive neurons after transfection with the plasmid pCMV-TCII-OLEO. Representative micrographs of the <i>substantia nigra</i> (with double immunostaining at 15-day after transfection) are presented. The primary antibodies were a mouse monoclonal antibody to TH, and a rabbit polyclonal antibody to cleaved Caspase-3. The secondary antibodies included a donkey anti-mouse IgG FITC labeled (1 and 4), and a donkey anti-rabbit IgG rhodamine labeled (2 and 5). Representative micrographs of coronal section of control <i>substantia nigra</i> (1–3) and transfected <i>substantia nigra</i> (4–6) of the same rat are presented. Scale bars = 50 µm. C: Apoptosis in TH immunoreactive neurons expressing the TCII-OLEO chimera. Representative micrographs of the <i>substantia nigra</i> with triple immunostaining at 15-day after transfection. The primary antibodies were a mouse monoclonal antibody to TH, a goat polyclonal antibody to TCII, and a rabbit polyclonal antibody to cleaved Caspase-3. The secondary antibodies were a donkey anti-mouse IgG AMCA labeled (1 and 5), a donkey antigoat IgG fluorescein labeled (2 and 6), a donkey anti-rabbit IgG rhodamine labeled (3 and 7). Representative micrographs of coronal section of control <i>substantia nigra</i> (1–4) and transfected <i>substantia nigra</i> (5–8) of the same rat are presented. Scale bars = 50 µm.</p

Analysis of apoptosis in N1E-115 cells stably transfected with different plasmids.

<p>The plasmids were pCMV-TCII-OLEO coding for transcobalamin-oleosin (TCII-OLEO), pCMV-OLEO-TCII coding for oleosin-transcobalamin (OLEO-TCII), pCMV-TCII coding for transcobalamin II (TCII), pCMV-OLEO coding for oleosin (OLEO), and pCDNA3. The immunofluorescence was done with a rabbit polyclonal antibody to cleaved Caspase-3 and a donkey antirabbit IgG fluorescein labeled. Before fixation, cells were incubated with 4 µM propidium iodide for 10 min. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 100 µm.</p

Methamphetamine-induced turning behavior in rats transfected with several plasmids.

<p>The plasmids were pCMV-TCII-OLEO coding for transcobalamin-oleosin (TO), pCMV-OLEO-TCII coding for oleosin-transcobalamin (OT), pCMV-TCII coding for transcobalamin II (T), pCMV-OLEO coding for oleosin (O), and pCDNA3 (P). The values are the mean±SEM of 3 animals per group. ** = Significantly different from control groups. <i>P</i><0.01, repeated-measures two-way ANOVA and Bonferroni post-test.</p

Figure 6

<p><b>(A) Intracellular conversion of exogenous [⁵⁷Co]-labeled B12 (cyano-cobalamin, CN-Cbl)</b> into methyl-cobalamin (Me-Cbl) and ado-cobalamin (Ado-Cbl) in the cytosolic and mitochondria enriched fractions. <b>(B)</b> Homocysteine (Hcy) and methylmalonic acid (MMA) concentrations. <b>(C)</b> S-adenosylmethionine/S-Adenosylhomocysteine ratio (SAM/SAH) in TO and wild type NIE-115 cells. <b>(D)</b> Activity of methionine synthase in TO transfected and wild type NIE-115 and Caco2 cells in exponential growth. Mean±S.D. of sextuplet assays are given. ****: p<0.0001, **: p<0.001, *: p<0.001.</p

Figure 2

<p><b>(A)</b> Characterization of the recombinant oleosin (top) and transcobalamin-oleosin (bottom) chimeric proteins; SDS-PAGE (12.5%) (lanes 1–3, 10) and Western blot (WB) (lanes 4–9) of subcellular fractions (pellets and supernatant from differential centrifugation) from <i>Sf</i>9 cells infected with recombinant baculovirus expressing either peanut oleosin or the transcobalamin-oleosin chimeric protein. Lanes 1, 4, 7: 900 g pellet, lanes 2, 5, 8: 100 000 g supernatant, lanes 3, 6, 9: 100 000 g pellet, lane 10: 85 ng purified peanut oleosin and lane 11: autoradiography of [¹⁴C]-leucine-labeled oleosin expressed in rabbit reticulocyte lysate. <b>(B)</b> Saturation curve of vitamin B12 binding of transcobalamin-oleosin (TC-O) in membrane fraction of lysed sf9 insect cells and corresponding Scatchard plot (inlet).</p

Figure 5

<p><b>(A)</b> Confocal microscopic examination of transcobalamin in COS-7 cells transiently transfected with lipofectamine. The cells were transfected with one of the following pCDNA3-plasmids: TC-O coding for transcobalamin-oleosin (TC-O), O-TC coding for oleosin-transcobalamin (O-TC), TC coding for transcobalamin (TC), O coding for oleosin (O), and the empty plasmid (pCDNA3). The immuno-fluorescence was done with a goat polyclonal antibody to TC and a donkey antigoat IgG fluorescin labeled. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 20 µm. <b>(B and C)</b> Co-localization of the protein GFP-TC-O with endoplasmic reticulum in transient transfected Cos-7 cells using lipofectamine. The cells were transfected with the plasmid GFP-TC-O coding for GFP-transcobalamin-oleosin (GFP-TC-O). The immuno-fluorescence was done with a mouse monoclonal antibody to the human golgin-97 or a rabbit polyclonal antibody to calreticulin. The secondary antibodies were a donkey IgG anti-mouse TRITC labeled or a donkey IgG anti-rabbit TRITC labeled. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 20 µm.</p

Figure 3

<p><b>(A)</b> Vitamin B12 binding capacity (cyano[⁵⁷Co]Cbl) of membrane fraction (100 µg proteins per assay) from Caco2 cells, 72 hrs after transient transfection with various recombinant pcDNA3 plasmids expressing transcobalamin (TC), oleosin (O) and the chimeric proteins transcobalamin-oleosin (TC-O) and oleosin-transcobalamin (O-TC). <b>(B)</b> Vitamin B12 ([57Co]-labeled) binding capacity of Caco2 cells of the intact and lyzed (Caco2 cells were lysed prior to the addition of vitamin B12 or used intact), at days 5–15 of culture, after stable transfection with a recombinant pcDNA3 plasmid expressing the chimeric protein transcobalamin-oleosin. Exponential and stationary phases were evaluated at days 5–7 and days 10–15, respectively. Bars from left to right for each time: non-transfected intact cells, non-transfected lysed cells, TC-O transfected intact cells, TC-O transfected lysed cells.</p