In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter-1

<p><b>Copyright information:</b></p><p>Taken from "In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter"</p><p>http://www.virologyj.com/content/4/1/88</p><p>Virology Journal 2007;4():88-88.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2034561.</p><p></p>es -748 to +284 bp); EGFP, enhanced green-fluorescent protein; BGHpA, bovine growth hormone poly adenylation sequence. (b) (Left panel), Representative histogram obtained by flow cytometry 48 h after transduction of BUVEC-E6E7-1 cells with 100 MOI of BacFLT-GFP and 5 mM of butyrate. The percentage of GFP+ cells is reported in the inset, and was calculated by subtracting the background from mock transduced cells (see Methods). (Right panel) Levels of expression obtained with BacCMV-GFP and BacFLT-GFP in BUVEC-E6E7-1 cells, representative of four independent experiments, mean ± SD. (c) Mean fluorescence intensity (MFI) measured 48 h after transduction with BacFLT-GFP relative to BacCMV-GFP. The percentage of GFP+ cells are indicate above of each bar. Cells were transduced with 100 MOI of BacFLT-GFP or BacCMV-GFP. Data are from four independent experiments ± SD

In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter-2

<p><b>Copyright information:</b></p><p>Taken from "In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter"</p><p>http://www.virologyj.com/content/4/1/88</p><p>Virology Journal 2007;4():88-88.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2034561.</p><p></p>FP and treated with increasing concentrations (shown in the figure) of butyrate or trichostatin A (TSA). The percentage of GFP+ cells (shown inside green rectangles) is reported in the insets, and was calculated by subtracting the background from mock transduced cells. (b) Fold induction in GFP expression mediated by BacFLT-GFP with increasing concentrations of butyrate or TSA. (c) Fold induction of the expression of GFP in transfected cells with the transfer plasmid pBlueFLT-GFP (see Methods) and treated with increasing concentrations of butyrate or TSA. Results from four independent experiments ± SD. NS = non significant

In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter-3

<p><b>Copyright information:</b></p><p>Taken from "In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter"</p><p>http://www.virologyj.com/content/4/1/88</p><p>Virology Journal 2007;4():88-88.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2034561.</p><p></p>ye vitreous chamber. Three days after the virus injection, the rats were sacrificed and the retinas processed for immunohistochemistry using an antibody against von Willebrand factor (red) and DAPI (blue) to highlight nuclei (see Methods). Representative retinas from eyes injected with 1 × 10pfu of BacFLT-GFP (a, b, c, d) or vehicle (PBS) (e, f, g, h). The same sections were evaluated by phase-contrast microscopy (a, e) and by anti-von Willebrand factor antibodies coupled to TRITC (red) and GFP green fluorescence. Merge images illustrate the colocalization of red and green fluorescence (in yellow) in capillary-like structures (big white rectangles in c, d, g) obtained from the areas in small white rectangle insets from b, f. Confocal three-dimensional reconstruction images of retinas injected with vehicle (h) or with BacFLT-GFP (i). In red is shown the localization of von Willebrand factor (red) within characteristic blood vessel structures. The colocalization of red (vWF) and green (GFP) fluorescence is shown in yellow (white arrowheads in i). (j) Percentage of vWF and GFP pixel co-localization in retinas injected with BacFLT-GFP, (= 10 animals). Abbreviations: RPE, retinal pigment epithelium GCL, ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer; ILM inner limiting membrane

In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter-4

<p><b>Copyright information:</b></p><p>Taken from "In vivo transcriptional targeting into the retinal vasculature using recombinant baculovirus carrying the human flt-1 promoter"</p><p>http://www.virologyj.com/content/4/1/88</p><p>Virology Journal 2007;4():88-88.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2034561.</p><p></p> of BacCMV-GFP. Abbreviations: CMV, cytomegalovirus immediately-early promoter/enhancer (nucleotides -655 to +106); EGFP, enhanced green-fluorescent protein; BGHpA, bovine growth hormone poly adenylation sequence. (b) Representative histograms obtained by flow cytometry after 48 h post-transduction. The percentage of GFP+ cells is reported in the insets, and was calculated by subtracting the background obtained with mock transduced cells (see Methods). Numbers in the inset refer to the percentage of GFP+ cells ± SD without (above) or with (below) treatment with butyrate. (c) Percentage of GFP+ cells determined by FACS analysis in either the presence or absence of butyrate. *< 0.01, **< 0.05, ***= 0.7244 versus cells non-treated with butyrate. NS = non significant. d) Levels of expression of GFP and induction ration (indicated on the right) as determined by the mean fluorescence intensity in the presence or absence of butyrate. Values are means ± SD of four independent experiments

N-terminal PRL fragments content in the anterior pituitary varies along the estrous cycle.

<p>A: Recombinant 23 kDa PRL (r23-PRL), recombinant 16 kDa prolactin (r16-PRL) or pituitary protein extracts treated with β-mercaptoethanol (P+β, reducing conditions) or without β-mercaptoethanol (P, non-reducing conditions) were incubated with anti-recombinant rat PRL antibody (anti rrPRL, 1∶25000). The antibody recognizes both PRL forms. B: Anterior pituitaries from rats euthanized at diestrus I or proestrus were processed for western blot. Upper panel: Each column represents the mean ± SE of the relative increment of N-terminal PRL fragments content with respect to diestrus I. Data of each column were normalized to β-actin expression (n = 4–7 animals per group). *p<0.01 vs. diestrus I, Student's t test. Lower panel: Representative blot of pituitary proteins from rats euthanized at diestrus I or proestrus.</p

16 kDa PRL inhibits anterior pituitary cell proliferation.

<p>A and B: Anterior pituitary cells from OVX rats were incubated for 24 h with VEH and FSK, and with or without 16 kDa PRL (10 nM) for the last 4 h. D and E: Anterior pituitary cells from OVX rats were incubated for 24 h with E2, then for 1 h with E2 and FSK, and for the last 4 h in the same media with or without 16 kDa PRL (10 nM). Proliferation was determined by BrdU incorporation (3 h) and fluorescence microscopy. A and D: Each column represents the percentage ± CI (95%) of BrdU-positive cells (n≥1000 cells/group, left) or BrdU-positive lactotropes (n≥300 cells/group, right), representative of four independent experiments. Data were analyzed by χ². **p<0.01 vs. respective control without 16 kDa PRL. B and E: Representative images of anterior pituitary cells showing immunoreactivity for prolactin (red) counterstained with DAPI (blue) and BrdU (green). Arrowheads indicate proliferating lactotropes. Scale bar: 50 µm. C and F: Anterior pituitary cells in culture were incubated with VEH (C) or E2 (F) for 24 h, and with or without 16 kDa PRL (10 nM) for the last 4 h. Cell cycle was analyzed by flow cytometry using PI. Left: Each column represents the mean ± SE of the percentage of S-phase cells (n≥4 wells/group), representative of three independent experiments. Data were analyzed by Student's t test. *p<0.05 vs. respective control without 16 kDa PRL. Right: Representative histograms of DNA content of anterior pituitary cells incubated in the presence or absence of 16 kDa PRL.</p

Estradiol increases the secretion and content of N-terminal PRL fragments from anterior pituitary cells in culture.

<p>Anterior pituitary cells from OVX rats were incubated in the presence of VEH or E2 for 24 h. A: Culture media and B: cells were obtained and processed for western blot analysis. Each column represents the mean ± SE of the relative increment of N-terminal PRL fragments with respect to VEH. In B, data of each column were normalized to β-actin expression (A, n = 6 wells/group; B, n = 6 wells/group), representative of three independent experiments. *p<0.05, **p<0.01 vs. VEH without E2, Student's t test. Lower panels: Representative blots of media (A) or cells (B) cultured with VEH or E2.</p

16 kDa PRL induces apoptosis of anterior pituitary cells.

<p>A and B: Anterior pituitary cells from OVX rats were incubated with VEH or E2 for 24 h and with or without 16 kDa PRL (10 nM) for the last 4 h. A: Each column represents the percentage ± CI (95%) of TUNEL-positive cells (n≥1500 cells/group, left) or TUNEL-positive lactotropes (n≥300 cells/group, right), representative of four independent experiments. Data were analyzed by χ². **p<0.01 vs. respective control without 16 kDa PRL. ∧∧p<0.01, ∧p<0.05 vs. respective control without E2. B: Representative images of anterior pituitary cells showing immunoreactivity for prolactin (red) counterstained with DAPI (blue, upper panels) and DNA fragmentation determined by TUNEL method (green, lower panels). Arrowheads indicate apoptotic lactotropes. Scale bar: 50 µm. C: Anterior pituitary cells from OVX rats were incubated with E2 for 24 h, and with or without 16 kDa PRL (10 nM) for the last 4 h. The percentage of hypodiploid cells was determined by flow cytometry using PI. Left: Each column represents the mean ± SE of the percentage of sub-G1 cells (n = 3 wells/group), representative of three independent experiments. Data were analyzed by Student's t test. **p<0.01 vs. respective control without 16 kDa PRL. Right: Representative histograms of fluorescence intensity of DNA content of anterior pituitary cells incubated in the presence or absence of 16 kDa PRL.</p

DataSheet_1_Immunometric and functional measurement of endogenous vasoinhibin in human sera.pdf

IntroductionCirculating levels of the antiangiogenic protein vasoinhibin, a fragment of prolactin, are of interest in vasoproliferative retinopathies, preeclampsia, and peripartum cardiomyopathy; however, it is difficult to determine the circulating levels of vasoinhibin due to the lack of quantitative assays. MethodsThis study used human serum samples to assess the concentration and bioactivity of vasoinhibin using a novel enzyme-linked immunosorbent assay (ELISA) for human vasoinhibin, which employs an anti-vasoinhibin monoclonal antibody, a human umbilical vein endothelial cell (HUVEC) proliferation assay, and a chick chorioallantoic membrane (CAM) angiogenesis assay. ResultsSerum samples from 17 pregnant women without (one group) and with preeclampsia and pregnancy induced hypertension (another group) demonstrated endogenous vasoinhibin concentrations in the range of 5–340 ng/ml. Immunoactive vasoinhibin levels were significantly higher in preeclampsia serum compared to healthy pregnancy serum (mean 63.09 ± 22.15 SD vs. 19.67 ± 13.34 ng/ml, p = 0.0003), as was the bioactive vasoinhibin level as determined by the HUVEC proliferation assay (56.12 ± 19.83 vs. 13.38 ± 4.88 ng/ml, p DiscussionThese results demonstrate the first quantitation of endogenous vasoinhibin in human sera and the elevation of it levels and antiangiogenic activity in sera from women with preeclampsia. The development and implementation of a quantitative assay for vasoinhibin overcomes a long-standing barrier and suggests the thorough clinical verification of vasoinhibin as a relevant biomarker.</p