Overexpression of the pumpkin (<i>Cucurbita maxima</i>) 16 kDa phloem protein CmPP16 increases tolerance to water deficit

<div><p>The phloem plays an important role in the delivery of nutrients and signals between photosynthetic to heterotrophic tissues. Proteins and RNAs in the phloem translocation stream may have an important role in maintaining the integrity of the sieve tube system, as well as in long-distance signaling. CmPP16 is a pumpkin phloem protein, which has been shown to bind RNA in a non-sequence specific manner, and move it cell-to-cell and conceivably, long-distance. The protein and RNA are found in both companion cell (CC) and sieve elements (SE). However, a more precise function for this protein is not known. In this work we report the overexpression of CmPP16 fused to GFP via transformation of pumpkin (<i>Cucurbita maxima</i> cv. Big Max) plants in the cotyledonary stage by direct inoculation of <i>Agrobacterium tumefaciens</i> and <i>Agrobacterium rhizogenes</i>. Plants overexpressing <i>CmPP16</i> did not show an obvious phenotype. However, these plants displayed higher photosynthetic capacity during drought than wild-type (WT) pumpkin or transformed with another construct. These results suggest that CmPP16 may be involved in the response to stress through long-distance signaling.</p></div

Effect of recombinant TCTP on B cell proliferation.

<p>Splenic B cells (2×10⁶cells/well) were plated with RPMI 1640 supplemented with 10% FBS and incubated with 10 µg/mL of recombinant HsTCTP or PfTCTP labeled with Oregon Green 488 (green) fluorochrome for 24–120 h at 37°C and 5% CO₂. LPS (1 µg/mL) was used as positive control. Untreated and 20 mM phosphate buffer-treated B cells were used as negative controls. Viable B cell density was measured with a Countess Automated Cell Counter. Results are representative of two independent experiments and are expressed as the mean ±SD of duplicate cultures. *, <i>p</i><0.05 vs 10 µg/mL labeled PfTCTP treated B cells.</p

Effect of recombinant TCTP in the progression of cell cycle from B cells.

<p>Splenic B cells (2×10⁶cells/well) were plated with RPMI 1640 supplemented with 10% FBS and incubated with 10 µg/mL of recombinant HsTCTP or PfTCTP labeled with Oregon Green 488 (green) fluorochrome for 24–120 h at 37°C and 5% CO₂. The percentage of cell cycle distribution and incorporation of fluorescently-labeled of PfTCTP and HsTCTP into splenic B cells was analyzed by flow cytometry (FACS). Results (%) are expressed as mean ± SD of duplicate determinations. One of two similar experiments is shown. Incorporation, percentage of incorporation of fluorescently-labeled protein into B cells.</p><p>*, <i>p</i><0.05 vs 10 µg/mL labeled HsTCTP treated B cells</p><p>**, <i>p</i><0.01 vs 10 µg/mL labeled HsTCTP treated B cells</p

Localization of incorporated TCTP in B cells.

<p>Confocal microscopy analysis of negatively isolated B cells (2×10⁵cells/well) incubated 30 min with 50 ng/mL of recombinant HsTCTP labeled with Oregon Green 488 dye (green) and 50 ng/mL of recombinant PfTCTP labeled with Alexa Fluor 594 dye (red). Representative images are shown. A. Bright field. B. DAPI staining C. Green fluorescence. D. Red fluorescence. E. Merge.</p

Expression and purification of recombinant <i>Bacillus stearothermophilus</i> DNA polymerase (Bst) and reverse transcriptase (RT) from Moloney Murine Leukemia Virus.

Coomassie blue-stained 8% Tricine-SDS-PAGE electrophoresis gel analysis. (A) Expression of recombinant Bst enzyme and Ni2+-IMAC purification. Lane 1: clarified supernatant loaded; Lane 2: flow-through fraction; Lane 3–6: washing step fractions; Lane 7–9: eluted fractions containing Bst. (B) Heparin column purification of recombinant Bst. Lane 1: desalted sample loaded; Lane 2: flow-through fraction; Lane 3: washing step fraction; Lane 4–6: elution fractions; Lane 7–8: concentrated Bst-containing fractions. (C) Final Bst formulations from three different purification batches (B1, B2, B3). (D) Expression of recombinant RT enzyme and Ni2+-IMAC purification. Lane 1–3: flow-through fractions; Lane 4–5: washing step fractions; Lane 6–10: elution fractions containing RT. (E) Cation exchange column purification of recombinant RT. Lane 1: IMAC elution fraction; Lane 2: desalted sample loaded; Lane 3–5: flow-through fractions; Lane 6–7: washing step fractions; Lane 8–13: elution fractions. (F) Final RT formulations from three different purification batches (B1, B2, B3). M: molecular weight marker; C+: previously purified Bst or RT enzyme, employed as control positive; I: insoluble fraction; S: soluble fraction; C: clarified supernatant. Violet or green arrows indicate the expected size for Bst and RT enzymes, respectively.</p

Effect of additives in colorimetric end-point RT-LAMP assay performance.

(A) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set and 40 mM of guanidine hydrochloride (GuHCl) in the reaction buffer. The figure shows the colorimetric determination of each reaction (upper panel) and the electrophoretic profile of the amplification reaction products (lower panel). (B) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set in absence of GuHCl in the reaction buffer. The figure shows the colorimetric determination of each reaction (upper panel) and the electrophoretic profile of the amplification reaction products (lower panel). (C) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set and 0.8M of betaine in the reaction buffer. The figure shows the electrophoretic profile of the amplification reaction products. (D) Colorimetric RT-LAMP reactions under optimized conditions using N1 primer set in absence of betaine in the reaction buffer. The figure shows the electrophoretic profile of the amplification reaction products. C+: 1x104 copies of N1 in vitro transcript used as positive control; NTC: non-template control; M: DNA molecular weight marker 1 Kb Plus DNA Ladder (Invitrogen). (TIF)</p

Chromatographic profiles of the purification steps of Bst and RT by fast protein liquid chromatography (FPLC).

(A) Chromatogram of Bst purification by Ni+2-IMAC. (B) Chromatogram of RT purification by Ni+2-IMAC. (C) Chromatogram of the desalting step of the Bst-containing fractions. (D) Chromatogram of the desalting step of the RT-containing fractions. (E) Chromatogram of the second purification step by heparin affinity chromatography for RT. (F) Chromatogram of the second purification step by cation exchange chromatography for RT. Values expressed in mAU are shown in purple (Bst) or green (RT). The dotted lines correspond to the concentration of the elution buffer used in each case: EB-AI (A), EB-BI (B), DB-A (C), DB-B (D), EB-AII (E), EB-B-II (F). Black arrows indicate the peaks of the chromatograms selected for the following purification steps. (TIF)</p