Twisted gastrulation (Twsg1), a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs) and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7) in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of  Podocytes have recently emerged as an early injury site in many types of kidney disease. Podocyte loss correlates with severity of glomerular injury and degree of proteinuria, and leads to glomerular sclerosisBone morphogenetic protein 7 (Bmp7) is a member of the Bmp family within the TGF-β superfamily, and plays pivotal roles in the development of the kidneys and eyesBmp7 transgenic miceRecently, several groups have demonstrated that systemically administered Bmp7 retards the progression of glomerular diseasesuterine sensitization–associated gene-1 (USAG-1) as a Bmp antagonist, which is by far the most abundantly expressed in the kidney, and demonstrated that USAG-1 negatively regulates the renoprotective activity of Bmp7 in many types of kidney diseaseThe local activity of endogenous Bmp is controlled by certain classes of binding molecules that act as positive or negative regulators of Bmp signalingTwisted gastrulation (Twsg1) is a Bmp modulator that synergistically interacts with chordin or chordin-like molecules to regulate Bmp activityHere, we demonstrate that Twsg1 plays an essential role in the progression of podocyte injury, possibly by antagonizing the renoprotective function of Bmp7.Twsg1-lacZ reporter mice (+/lacZTwsg1 mice)LacZ/LacZTwsg1 pups were significantly fewer than expected in C57BL/6 background, possibly due to the embryonic lethality. To obtain enough number of LacZ/LacZTwsg1 mice, we utilized the mixed background between C57BL/6 and 129/Svj strains in LacZ/+Twsg1 mice. To minimize the background effect, all experiments were performed with +/+:NEP25Twsg1 littermates and LacZ/LacZ:NEP25Twsg1 littermates. All animal studies were approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University, and were strictly in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.Heterozygote 3/ml and grown at 33°C in collagen I-coated culture dishes in DMEM containing 10% FCS and 10 U/ml of IFN-γ. The podocytes from Professor Shankland were seeded at a concentration of 2×104/ml and grown at 33°C in collagen I-coated culture dishes in RPMI 1640 containing 10% FCS and 50 U/ml of IFN-γ. To induce differentiation, podocytes were incubated at 37°C without IFN-γ for 7–14 days. While only the results obtained using the podocytes from Professor Mundel are shown in the figures, the expression pattern of Bmp and Bmp antagonists in the two groups of podocytes was similar.Conditionally immortalized murine podocytes were kind gifts from Professor Mundel  and Professor Shankland . Podocytes were cultured and differentiated as previously describedFor a study of the effect of Bmps on podocyte differentiation, podocytes were incubated under non-permissive conditions. Recombinant proteins (300 ng/ml) were added to the culture medium at 24 h and on day 7, then incubated for an additional 7 days before analysis. We utilized recombinant human Bmp4, Bmp7, and Twsg1 . For a study of dorsomorphin, podocytes were incubated with 0.5–2 µM dorsomorphin  or dimethyl sulfoxide  in the presence or absence of Bmp7, and were harvested 7 days later.For a study of Bmp signaling pathways, podocytes treated with Bmp and Twsg1 were subjected to western blotting utilizing antibodies against pSmad1/5  and GAPDH .Cell surface area was analyzed in 30 cells in each group. Podocytes cultured under non-permissive condition for 7 days were subjected for real-time RT-PCR analysis n = 3 in .2 cells/well and were incubated under non-permissive conditions. Recombinant proteins were administered after 24 hours, and podocytes were incubated for additional 5 days. We utilized recombinant Bmp7 (300 ng/ml), Bmp4 (300 ng/ml), and Twsg1 (300 ng/ml). After the incubation, BrdU was administered and the cells were re-incubated for additional 24 hours. After removing culture medium, the cells were fixed and analyzed according to the manufacturer's instruction. Cell proliferation was measured using BrDU incorporation assay with Cell Proliferation ELISA, BrDU (colorimetric) kit  according to the manufacturer's instruction. Briefly, murine podocytes were seeded at a concentration of 2.4×10Real-time RT-PCR was performed with a 7700 Sequence Detection System  as previously describedThe sequences of the primers were as follows:GGAATATGCACGGGTGAAAGGChrdl1:TGGTTTACTGTGTGAACTGCATCTG, TTGCTGTGGCTCCATCACAChrdl2:GATATACTGCGTGCGCTGTACCT, TCGACCATGGTGGCCGGGACCCG, TGTTCCCGGAAGATCTGGAGTBmp2: CCGAGCCAACACTGTGAGGAGTTTCC, GGATGCTGCTGAGGTTGAAGAGGBmp4: GGATGCTGCTGAGGTTGAAGAGG, TCAGGTGCAATGATCCAGTCCBmp7: TTGACCAGTCCCAAAGCTCTG, CACGCCATTTACCCATCCATBmpR1a: CCTGATGCGGCATGAGAATA, TTTGCGTCT AAGGTGGTGGATBmpR1b: CATAAGGCGACTATCAAAACAGCTAABmpR2:CAACACCACTCAGTCCACCTCAT, CAGTCAAAGCGTCCTTCAAGCC, TTCATGTCACTGACTCCGGCCTPodocalyxin: AACCACCATGAAGCGCCTCTT, GGTCACTGCATCTAAGGCAACCPodocin: TTCGTCTTGTCGTCCGATTTG, CCCCATTTTTGGTCCAAGTGANephrin: Primer sequences for Bmp antagonists were shown in the previous articleCells were grown on collagen I-coated glass coverslips and fixed with acetone. Nonspecific binding was blocked with 3% bovine serum albumin plus species-control IgG. The primary antibody utilized was anti-podocalyxin antibody, which was generated according to the method described previouslyNEP mice to induce podocyte injury as previously describedNEP mice were collected at day 0, day 3 and day 7 of LMB2 administration utilizing the magnetic bead methodRecombinant LMB2 (1.25 ng/g body weight) was intraperitoneally administered to NEP mice were euthanized, and tissues and blood samples were collected for further analysis+/+:NEP25Twsg1 mice, 24 LMB2-injected LacZ/LacZ:NEP25Twsg1 mice and 13 control mice. Kidney samples of 15 LMB2-injected +/+:NEP25Twsg1 mice, 9 LMB2-injected LacZ/LacZ:NEP25Twsg1 mice and 6 control mice were subjected to real-time PCR analysis of nephrin, podocin and podocalyxin.Four days after the injection, urine was collected and analyzed for albumin excretion. Seven days after the injection, LacZ staining was performed as described previously+/+:NEP25Twsg1 mice and 17 LacZ/LacZ:NEP25Twsg1 mice.The mice were placed in metabolic cages, and urine was collected over a 24-hour period as described+/+:NEP25Twsg1 mice and 23 LacZ/LacZ:NEP25Twsg1 mice by experienced pathologists who were blinded to the genotypes. The numbers of glomeruli counted in each mouse were 90±14 in +/+:NEP25Twsg1 mice, and 97±16 in LacZ/LacZ:NEP25Twsg1 mice, and the numbers were not significantly different between two genotypes.The kidneys were fixed in Carnoy's solution and embedded in paraffin. Sections (2-µm thick) were stained with PAS for routine histological examination, and the degree of morphological change was determined for 26 Glomerular injuries were classified into four categories according to the percentage of hemorrhagic glomeruli, sclerotic glomeruli, and cellular crescents: No lesion, 0%; mild, <10%; moderate, 10%–25%; severe, >25%. Tubulointerstitial injuries were also classified into four categories according to the percentage of injured area: No lesion, 0%; mild, <10%; moderate, 10%–25%; severe>25%.t test for two-group comparisons. P<0.05 is considered significant. For the histological analysis of podocyte injury model, statistical significance was assessed using the Kruskal-Wallis test.Data are presented as means ± SD. Statistical significance was assessed by Student's Expression of Bmps, Bmp receptors and Bmp antagonists was first examined using murine immortalized podocyte cell lines (kind gifts from Professors P. Mundel and S.J. Shankland)The podocyte cell lines stably express the temperature-regulated tsA58 mutant T antigen, and proliferate under permissive conditions (33°C supplemented with IFN-γ). Under growth restrictive conditions (37°C without IFN-γ), podocytes stop replicating within 14 days, substantially increase in size, and begin to express podocyte markers such as podocalyxinThe administration of Bmp4 and Bmp7 to the cultured podocytes increased cell size, whereas the administration of Twsg1 decreased cell size  and a part of distal nephron, but not in glomerular cells We further induced podocyte injury in Twsg1LacZ/+:NEP25 mice injected with LMB2, LacZ staining emerged within glomerular cells in some glomeruli, whereas LacZ staining in other glomeruli was still confined in PECs . We further analyzed the number of podocytes and the expression of podocyte differentiation markers, and did not find significant differences between both genotypes at the baseline .To examine the role of Twsg1 in the progression of podocyte injury, we generated Twsg1 null mice by crossing Twsg1LacZ/LacZ:NEP25 mice) along with the control littermates (Twsg1+/+:NEP25 mice) and administered the abovementioned immunotoxin to both genotypes. While Twsg1+/+:NEP25 mice exhibited severe nephrotic syndrome characterized by massive proteinuria, hypoalbuminemia and hyperlipidemia, Twsg1LacZ/LacZ:NEP25 mice showed milder hypoalbuminuria and hyperlipidemia .Next we generated NEP25 transgenic mice deficient in Twsg1 gene . Furthermore, the expression of podocyte markers in the kidneys of Twsg1+/+:NEP25 mice was reduced by the administration of the immunotoxin, whereas this reduction was significantly attenuated in Twsg1LacZ/LacZ:NEP25 mice .Histological analysis demonstrated glomerular injury and tubulointerstitial damage in Twsg1Taken together, these findings show that Twsg1 null mice were more resistant to podocyte injury than their control littermates were.Twsg1 is known for its context-dependent anti- and pro-Bmp functions: Twsg1 inhibits Bmp activity, either directly or indirectly, in some contextsIn this manuscript, we demonstrated that the administration of Bmp7 induces the differentiation and inhibits the proliferation of podocytes, whereas the simultaneous administration of Twsg1 antagonizes these effects of Bmp7. Although the mechanisms determining the direction of Twsg1 function are unclear, the results of this study support the idea that Twsg1 functions as an anti-Bmp factor in podocytes. Although Twsg1 is considered to form a trimer including Bmp and Chordin, Chrdl1, Chrdl2 in some contextWe further showed that the stabilization of podocyte differentiation by Bmp is, at least in part, mediated through pSmad1/5/8 signaling, and that this pathway is partially blocked by Twsg1. However, the inhibitory effect of Twsg1 on the phosphorylation of Smad is moderate compared to the inhibitory effect of Twsg1 on podocyte differentiation. This discrepancy indicates the contribution of other signaling pathways in the inhibitory effect of Twsg1 on podocyte differentiation.LacZ/+:NEP25Twsg1 mice might be the migrating PECs, which originally express Twsg1/β-galactosidase. Another possible explanation is that injured podocytes begin to express Twsg1/β-galactosidase. This idea is supported by the fact that cultured podocytes expressing Twsg1. In either way, it is plausible that the expression of Twsg1 in the glomeruli accelerates podocyte injury by inhibiting the renoprotective action of Bmp signaling. Reduction of Bmp7 expression in the glomeruli of NEP mice should also contribute to the attenuation of Bmp signaling in the glomeruli , and additionally expressed in glomerular cells in a podocyte injury model, although the cell type expressing Twsg1 in injured glomeruli is still unclear due to weak LacZ staining. Several groups recently reported that glomerular parietal epithelial cells (PECs) migrate onto glomerular tufts and differentiate into podocytes after glomerular injuryTwsg1 null mice are resistant to podocyte injury, possibly due to the enhanced Bmp signaling in podocytes. Although Twsg1 is also known to inhibit T cell activation and plasma cell productionWe further demonstrated that uterine sensitization-associated gene-1 (USAG-1), a novel Bmp antagonist expressed in the distal tubules of the kidney, plays a critical role in modulating the renoprotective action of Bmp, and that inhibition of USAG-1 is a promising potential novel treatment for various renal diseasesAlthough several reports have indicated that pharmacological doses of Bmp7 inhibit renal injury in animal models, the systemic administration of Bmp7 causes undesired side effects in other tissues, because Bmp receptors are widely expressedFigure S1(A) Podocyte numbers were analyzed by counting WT1-positive podocytes in both groups. 38 glomeruli were analyzed in wild-type kidneys, whereas 42 glomeruli were analyzed in Twsg1 null kidneys. (B) The expression of nephrin, podocin, and podocalyxin were analyzed in both groups. Data were normalized by GAPDH and were shown in relative to those of wild type littermates. Data are presented as means ± SD. n = 4. NS, no significant difference.(TIF)Click here for additional data file.Figure S2Serum creatinine and BUN in the mice of (TIF)Click here for additional data file.Figure S3Real-time PCR of hCD25 in the samples of (TIF)Click here for additional data file.Figure S4(A) Representative immunostaining of podocalyxin in Twsg1 wild-type and null mice. (B) Representative immunostaining of podocalyxin in Twsg1 +/+:NEP mice and Twsg1−/−:NEP mice with or without LMB2 administration. Scale bars: 100 µm.(TIF)Click here for additional data file.


New quantitative data show that the Bicoid morphogen gradient is generated from a dynamic localized source and that protein gradient formation requires protein movement along the anterior-posterior axis. Drosophila embryo and serves as a paradigm of morphogen-mediated patterning. The simplest models of gradient formation rely on constant protein synthesis and diffusion from anteriorly localized source mRNA, coupled with uniform protein degradation. However, currently such models cannot account for all known gradient characteristics. Recent work has proposed that bicoid mRNA spatial distribution is sufficient to produce the observed protein gradient, minimizing the role of protein transport. Here, we adapt a novel method of fluorescent in situ hybridization to quantify the global spatio-temporal dynamics of bicoid mRNA particles. We determine that >90% of all bicoid mRNA is continuously present within the anterior 20% of the embryo. bicoid mRNA distribution along the body axis remains nearly unchanged despite dynamic mRNA translocation from the embryo core to the cortex. To evaluate the impact of mRNA distribution on protein gradient dynamics, we provide detailed quantitative measurements of nuclear Bicoid levels during the formation of the protein gradient. We find that gradient establishment begins 45 minutes after fertilization and that the gradient requires about 50 minutes to reach peak levels. In numerical simulations of gradient formation, we find that incorporating the actual bicoid mRNA distribution yields a closer prediction of the observed protein dynamics compared to modeling protein production from a point source at the anterior pole. We conclude that the spatial distribution of bicoid mRNA contributes to, but cannot account for, protein gradient formation, and therefore that protein movement, either active or passive, is required for gradient formation.The Bicoid morphogen gradient directs the patterning of cell fates along the anterior-posterior axis of the syncytial  Drosophila embryos. This gradient is the classic example of morphogen-mediated patterning of a developing metazoan and serves as a major topic for mathematical modeling. Accurate modeling of the gradient requires a detailed account of the underlying bicoid mRNA distribution. The classic model holds that mRNA protein gradient arises via protein diffusion from mRNA localized at the anterior of the developing egg. In contrast, recent proposals suggest that an mRNA gradient generates the protein gradient without protein movement. In this study, we introduce a novel mRNA quantification method for Drosophila embryos, which allows us to visualize each individual mRNA particle accurately in whole embryos. We demonstrate that all but a few mRNA particles are confined to the anterior 20% of the egg, and consequently that the protein must move in order to establish a gradient. We further report that the mRNA distribution is highly dynamic during the time of protein synthesis. In numerical simulations, we show that incorporating realistic spatial locations of the individual source mRNA molecules throughout the developmental period is necessary to accurately model the experimentally observed protein gradient dynamics.The Bicoid protein gradient plays a crucial role in determining the anterior body pattern of  Accurate development of metazoan embryos requires precise production, reception, and interpretation of patterning cues along appropriate spatial axes on realistic timescales. Many embryonic patterning events utilize graded spatial distributions of patterning molecules, or morphogens, whose activities rely fundamentally on molecular interactions susceptible to environmental fluctuations and stochasticity in gene expression Drosophila melanogaster offers an optimal context in which to study the basis of developmental accuracy in embryonic patterning by morphogens. In the Drosophila embryo, anterior-posterior (AP) axial patterning originates with maternal cues deposited into the developing egg bcd mRNA is believed to commence upon fertilization, after which the embryo undergoes 13 rapid nuclear mitotic cycles (n.c.) without cytokinesis. By the start of interphase 14 about 2 h after egg deposition (AED), the embryo consists of a syncytial blastoderm layer of about 6,000 nuclei at the cortical surface, surrounding the interior core of yolk and vitellogenic nuclei. During the blastoderm stage Bcd protein distributes along the AP axis as an exponentially decaying gradient The wealth of molecular and genetic tools available in Recently, quantitative analysis of living embryos expressing Bcd-GFP revealed that the protein gradient remains nearly unchanged subsequent to the arrival of nuclei to the cortex at n.c. 10 (about 85 min AED), and therefore that the nuclear gradient achieves stability within only about 80 min at 25°C bcd mRNA. Females carrying altered genetic dosages of bcd produce gradients of altered amplitude, resulting in mispatterned embryos bcd mRNAs into discrete ribonucleoprotein (RNP) complexes, and their subsequent localization to the anterior oocyte cortex, requires a suite of maternal factors . Subsequently, bcd mRNA is relocalized to the egg cortex during cortical nuclear migration , during which bcd particles tend to dissociate without bcd mRNA degradation. Despite the dynamic behavior of bcd particles, at all times >90% of mRNA is localized within the anterior 20% of the embryo, a distribution which is insufficiently extended to produce the observed protein gradient in the absence of large-scale protein redistribution along the anterior-posterior axis.To address these questions, we developed two novel quantitative measurement approaches. First, we adapted a method of fluorescent in situ hybridization to the Second, we measured nuclear Bcd-GFP levels during presyncytial stages using GFP fluorescence in fixed embryos. We discovered that Bcd-GFP does not begin accumulating in nuclei until interphase 6, about 45 min AED, reaching peak levels 50 min later in n.c. 11 and 12. Unexpectedly, in fixed samples we found that the nuclear gradient declines between n.c. 13 and n.c. 14, a result which differs from prior analysis of living embryos. We present evidence that attributes this difference in part to delayed fluorescence maturation of GFP in living embryos.bcd mRNA distribution impacts protein gradient formation, we incorporated its observed spatio-temporal dynamics into numerical simulations of protein production and movement. We found that models utilizing the actual mRNA distribution result in improved predictions of protein gradient dynamics, compared to models employing a single point source at the anterior pole. Therefore, we conclude that although the spatially extended mRNA localization contributes to the protein gradient, mRNA localization alone cannot account for protein gradient dynamics. Our results demonstrate that protein movement, whether active or passive, is a necessary component of Bcd protein gradient formation.Finally, to examine whether the bcd mRNA distribution in fixed embryos, we adapted a fluorescent in situ hybridization (FISH) protocol bcd open reading frame  structure of nd noise  and 2A–Darticles  and 2E–Farticles , owing tbcd mRNA particles, their localization, and fluorescence intensities, we designed custom image analysis software to determine the spatial positions, radii, and fluorescence intensities of bcd mRNA particles  and leveling off at nearly zero particle density by 40% EL  throughout embryogenesis In midsagittal planes at n.c. 4, regions near the embryo surface contain fewer particles than the center of the embryo , showing that Bcd-GFP has not accumulated to high enough levels for visualization in nuclei. Tissue autofluorescence may preclude detection of lower levels of nuclear localized Bcd-GFP; therefore, Bcd-GFP accumulation begins during n.c. 6 at the latest. Delayed nuclear accumulation would be consistent with previous work suggesting that the translation rate of bcd may be relatively low during the first hour of development before polyadenylation of bcd mRNA The earliest time at which we can detect nuclear Bcd-GFP is about 45 min AED, when Bcd-GFP begins to accumulate in the most anteriorly positioned nuclei during the first 2 min of the sixth interphase , coincido 60% EL . In contBetween the sixth mitosis and interphase 10, nuclei migrate toward the egg cortex as an expanding, elliptical shell, while continuing to accumulate Bcd-GFP . A gradiTo quantify the Bcd gradient at these early stages, we extracted nuclear fluorescence intensities from embryos expressing both Bcd-GFP and Histone-RFP. The latter provides a nominally uniform fluorescence signal in all nuclei and serves as a reference to normalize Bcd-GFP values at different optical depths in the sample see . Correctbcd mRNA distribution, which at no time resembles an exponentially decreasing gradient reaching to >75% EL, such as observed for Bcd-GFP from n.c. 9 onward. Bcd-GFP is visible in nuclei positioned at 50% EL and beyond from n.c. 8 onward, yet at these positions we observe essentially no mRNA particles. Instead, the protein gradient can only be accounted for if protein moves towards the posterior from anteriorly localized mRNA.These observations of gradient dynamics differ sharply from the th mitosis By live imaging, nuclear Bcd-GFP levels appear relatively stable after the 10To determine the effect of fixation on Bcd-GFP fluorescence, we imaged living Bcd-GFP and Histone-RFP embryos at blastoderm stages, then fixed the embryos within 3 min of live imaging, and subsequently re-imaged under the same microscopy settings . We founA maturation-based explanation supports the idea of protein movement away from anterior mRNA. With delayed maturation, immature fluorescent protein synthesized at the anterior would tend to attain fluorescence while en route toward the posterior. As a result, in a live embryo, the fraction of immature eGFP would be higher in the anterior than the posterior, and allowing additional time for eGFP to attain fluorescence would then disproportionately increase anterior fluorescence compared to posterior, as we observe by fixation.bcd RNA, we asked whether that distribution is a necessary component of gradient establishment by performing numerical reaction-diffusion simulations of gradient formation. We compared our measured nuclear gradients to protein distributions predicted to arise from either a realistic mRNA distribution or an anterior point source. We find that the combination of a non-monotonic time course for nuclear gradient amplitude  move closer to the detection threshold, which could, in principle, lead to a significant number of missed particles. Therefore our counting results for late embryos should be strictly interpreted as lower bounds on the actual number of particles in late embryos. However, the fact that the total particle light is approximately conserved  mediating mRNA particle movement during embryogenesis are as yet unknown. Previous work has shown that the reorganization of the cortical actin cytoskeleton upon egg activation allows the dispersion of bcd particles containing more mRNAs tend to be localized closer to the anterior than weakly fluorescent particles. Possibly, brighter bcd RNP complexes possess greater numbers of protein components required for establishment and/or maintenance of anterior localization, such as Staufen It is interesting that bcd RNA which failed to localize to the anterior during oogenesis, although we cannot formally rule out that such RNAs were in fact initially correctly localized and became posterior later. The fraction of bcd mRNA in the posterior 60% is near zero and thus contributes only negligibly to the protein gradient. Nevertheless, the embryo possesses a failsafe mechanism of suppressing bcd translation via Nanos activity in the posterior bcd.We observed weakly fluorescent particles in the posterior at all nuclear cycles. Such particles likely represent bcd mRNA are similar to the originally observed, steeply graded mRNA distributions of St. Johnston et al. bcd mRNA movement at the cortex accounts for the gradient of Bcd protein bcd mRNA on the embryo surface does not extend far beyond ∼5% EL  as measured in the blastoderm cytoplasm by photobleaching 2/s, which in principle would be rapid enough to establish a stable gradient prior to n.c. 9 Our observations present a revised description of protein gradient dynamics in which along the entire AP axis, nuclear Bcd concentrations reach their maximum values by n.c. 11. Previous work cited the difficulties of a simple SDD-based model of gradient formation given the apparent slow diffusion coefficient D of Bcd-GFP . For high resolution imaging, we used a 100× HCX PL APO CS NA 1.46 oil immersion objective on a Leica SP5 laser-scanning confocal microscope with 514 nm excitation wavelength at 2048×2048 pixels (linear dimension of 75.7 nm) and z-spacing of 420 nm, and obtained one anterior and one posterior stack of each embryo. Low resolution images of the midsagittal plane were taken with a 20× HC PL APO NA 0.7 oil immersion objective at 1024×500 pixels.OregonR embryos were fixed as described 5 small image patches from each posterior z-slice, computed the mean and variance in raw intensity from each patch, and fit a linear relation between the two; if noise in the image were purely uncorrelated Poisson shot noise, the slope would yield a conversion factor between the raw microscope signal and the photon counts. We then normalized every z-slice by this factor, which changed slowly and systematically with z. Neither realignment nor normalization crucially affect our reported results.To correct for drift of the microscope stage in the xy-plane, we first realigned the z-slices by starting at the midsaggital plane (z = 0) and shifted successive slices such that the pixel-wise cross-correlation in raw intensity between slice z and z+1 was maximized. To normalize for changes in optical medium with depth z, we selected 10bcd mRNA particle, and found the local maxima in the filtered images that exceed the detection threshold. These are candidate particles. If the detection threshold is too low, the number of candidate particles in the posterior stack rapidly increases due to false detections of noise and autofluorescence, as shown in We next filtered each z-slice by a balanced circular difference-of-Gaussians filter with center radius size of 1.5 pixels and surround size 2.5 pixels, roughly matched to the size of single c, y−yc, rx, ry, theta), where xc and yc are the light intensity center coordinates, rx and ry are orthogonal ellipse axes, with rx inclined by angle theta to the horizontal. If within the 9×9 patch other candidates are identified, we clip out a larger patch and simultaneously fit all nearby candidates to properly resolve their radii and amplitudes .Each candidate particle is then processed in turn: starting with the brightest unprocessed candidate C in the z-stack, successive nearby z-slices are examined for other candidates that appear at the same x-y location due to the axially extended PSF of the microscope ; these cplitudes . Each cax, ry be above 0.7 and below 2.5 pixels, (ii) the amplitude to exceed the background offset, and (iii) the number of shadows be at least 1. Detection threshold and number of shadows are the most stringent criteria  rcriteria ; their ic, yc location of each particle detected at 100× into the projection along AP axis. Using image manipulation, we register the 20× and 100× images of midsaggital plane and extract automatically the coordinates of the AP axis, of length L, from 20× images. This allows us to compute for each particle its x/L relative coordinate, and to construct distributions in To extract the spatial distributions, we must translate the xyw egfp-bcd; His2Av-mRFP1; E1bcd females , while the gradient amplitude increases by ∼3-fold from n.c. 8 to the time it reaches its peak in n.c. 12, and decreases to about 3/4 of the value in n.c. 14. Simultaneously satisfying both constraints, i.e. preserving the decay length and properly scaling the gradient amplitude as a function of time, is challenging in the framework of such extended SDD models. Decrease in gradient amplitude at later times can be generated in models in which the source turns off at n.c. 12, but if diffusion still remains active, the gradients are very flat at low x/L, inconsistent with the data. Models in which the decrease in gradient amplitude after n.c. 12 is generated by turning on degradation later yield gradients that match the data reasonably at low x/L, but do not preserve the exponential profile with a conserved decay constant across developmental time; even models in which degradation is present during the whole developmental time, but can accelerate to a higher value in blastoderm stages, do not fit the data well. Furthermore, to account for low gradient amplitudes at n.c. 7 and 8, the source needs to be turned on late, i.e. for most of the models around n.c. 5 or 6.bcd mRNA translation, to find the set of parameters minimizing 2/s) decreases sharply to 0 at n.c. 11–12 . The With these observations in mind we carried out an exhaustive search for D, τ, and the start and end of g  error ; the souc. 11–12  , interphase 6 (B), mitosis 6 (C), n.c. 7 (D), n.c. 11 (E), and n.c. 13 (F) and n.c. 14 . Green, bcd mRNA; blue, DAPI. Scale bar: 50 µm.Low magnification (2.22 MB TIF)Click here for additional data file.Figure S2Example particle detection with custom analysis software. (A) A single well isolated particle detected as a local maximum of light intensity. Crosshairs indicate major and minor axes. (A′) Fitted elliptical Gaussian intensity distribution of the particle detected in (A). (B) Two overlapping spots within the red square are distinguished by software and represented by fitted Gaussians in (B′). (C) z-slices from 0.4 µm above (top) to 1.2 µm below (bottom) the image in panel (B) (red boxed panel). The position of the particle denoted by crosshairs in (B) is marked by boxed red dot. Cyan dots indicate all detected particles. The particle shown in (B) and its shadows on neighboring z-planes are indicated by the blue circled dot.(0.18 MB TIF)Click here for additional data file.Figure S3µm diameter fluorescent beads suspended in acrylamide. (B) Particle diameter (in pixels) of 345 beads measured by detection algorithm.Point spread function. (A) Sequential z-slice confocal images of diffraction-limited, 200 (0.13 MB TIF)Click here for additional data file.Figure S4x-axis correspond to particle candidates at very high contrast and thus high signal-to-noise ratio; low values correspond to dim particle candidates barely different from the background. Each line denotes analysis of one embryo. Red lines: the histograms for embryo posteriors. Essentially no candidates are detected for which the filter value exceeds ∼150 . For smaller values of the threshold the number of candidates would explode as the detection algorithm starts picking up noise . Blue lines: the histograms for embryo anteriors. For low filter values, the same explosion in noise detections is observed as in the posteriors. However, there is a clear excess of signal at high filter values due to true mRNA particle detections (the peak in the histograms above ∼150). Cyan lines: the histogram of filter values for embryo anteriors for only those particle candidates that are detected on at least 2 z-slices. This is one of the additional criteria used in our particle detection procedure  filter values used to detect candidate particles see  from n.cdure see  that cle(0.28 MB TIF)Click here for additional data file.Figure S5giant. Scale bars: (A) 50 µm, (B) 25 µm, (C) 2 µm.FISH background fluorescence. (A–C) Wild-type (Oregon-R) embryo at n.c. 5 processed for FISH without probes. (D–F) Embryo at the second mitosis processed for FISH with probes against the purely zygotically expressed gene (4.03 MB TIF)Click here for additional data file.Figure S6bcd mRNA particle number as a function of position along the AP axis. Shown are probability distributions of particle counts (A) and the cumulative distribution of particles (B) as a function of fractional distance along AP axis and nuclear cycle (color). The dataset and color code are equivalent to that shown in (0.23 MB TIF)Click here for additional data file.Figure S7bcd mRNA particle distribution at n.c. 13. (A–F) n.c. 13 embryo near the midsagittal plane at the anterior (A) and posterior (F). Shaded boxes indicate magnified views (B–E) corresponding to cortex (upper) or core (lower). (F inset) Boxes indicate regions shown in (A) and (F). (G–L) Same embryo as (A–F), imaged at the nuclear layer. (H–K) Selected z-slices of boxed regions indicated in (G) and (L) showing apical (upper panels) and basal (lower panels) planes surrounding the nuclear layer (middle panels). Arrowheads indicate selected faint particles. Scale bars: 25 µm (A), 2 µm (B).(2.47 MB TIF)Click here for additional data file.Figure S8bcd mRNA; blue, DAPI. Scale bar: 50 µm.Low magnification FISH with multiple oligonucleotides in n.c. 3–6. Low magnification confocal images of midsagittal  or coronal (D–E) sections. Green, (1.49 MB TIF)Click here for additional data file.Figure S9bcd mRNA particle 3d distribution at early and late nuclear cycles. Locations of identified mRNA particles from early  and late  embryos were used to construct average 3d particle density profiles and typical cross-sections. (A–D) Sections of the transverse , coronal , and midsagittal  planes of the 3d distribution (D) of early embryos show a wedge-shaped distribution of mRNA with a high concentration at the anterior pole A; redder color indicates higher particle concentrations. (E–H) In late embryos, bcd mRNA distribution resembles a cup covering the anterior pole, extending slightly farther into the posterior. Note the high concentration of particles in the cortex evident from the sections (E–G).Schematic representation of (0.50 MB TIF)Click here for additional data file.Figure S10Nuclear accumulation of Bcd-GFP is not evident at interphase 5. Shown are maximum z-projections to display nuclei in multiple focal planes. (A) Bcd-GFP, (B) DAPI. Boxed regions in (A) and (B) are shown in (C) and (E), respectively. (D) Outlines of nuclear DAPI staining superimposed onto the image in (C). Scale bars: 50 µm (A), 20 µm (C).(0.88 MB TIF)Click here for additional data file.Figure S11Estimated total Bcd during blastoderm stages. Nuclear volume (red curve) was calculated from previous measurements of nuclear diameter (0.10 MB TIF)Click here for additional data file.Figure S12Confocal and two photon microscopy yield similar Bcd-GFP gradients in fixed embryos. (A) Example image obtained using custom built two-photon microscope as described (0.45 MB TIF)Click here for additional data file.Figure S13Bcd-Venus and Bcd-mRFP live and fixed gradients. Bcd-Venus (0.83 MB TIF)Click here for additional data file.Figure S14Simulated GFP maturation. The simulation of (0.10 MB TIF)Click here for additional data file.Figure S152–errors of fits of the model to measured mean Bcd protein gradients for n.c. 7–14; darker values correspond to smaller χ2 and better fits. (A) The starting and stopping times of bcd mRNA translation, TSrcoff and TSrcon , are varied for 4 scenarios, where diffusion is either always on or drops to zero in n.c. 11, 12, or 13 as denoted in each quadrant of (A). For each choice of these three timing parameters, we look for the best values for parameters D and τ. (B) D and τ search for TDiffoff = 12, TSrcon = 7, TSrcoff = 13. The best fits are obtained with D = 3.1 µm2/s and τ = 100 min; these values are used in Exploration of parameter space for extended SDD modeling. Shown in grayscale are χ(0.16 MB TIF)Click here for additional data file.Figure S16Bcd-GFP levels in yolk nuclei at blastoderm stages. Individual data points indicate mean attenuation-corrected intensities for individual nuclei, compared to binned means of dorsal cortical nuclei at n.c. 11 (black line).(0.15 MB TIF)Click here for additional data file.Movie S1bcd probes. Each image represents one z-slice with interval of 0.42 µm; total z thickness 5 µm. One pixel represents 75×75 nm.Confocal stack of an embryo at n.c. 4 labeled with (10.07 MB AVI)Click here for additional data file.Movie S2z-slice with interval of 0.42 µm; total z thickness 5 µm. One pixel represents 75 × 75 nm.Confocal stack of an embryo at n.c. 13. Each image represents one (10.07 MB AVI)Click here for additional data file.Table S1bcd antisense oligonucleotides.Sequences of (0.02 MB XLS)Click here for additional data file.


Food allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.Using the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.Two new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Alignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients' diet.The biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention. Such claim seems to fit perfectly in the allergy field where the clinical phenotype is tightly linked to the IgE immune recognition and each patient seems to display a different clinical picture when compared to another. Such personalized approach, formerly almost impossible because of the number of tests to be performed in each patient, is now becoming increasingly feasible because of the biotechnology/microtechnology combination supported by information technology tools recently made available for the routine work Plant LTPs are widely distributed, structurally related, small proteins involved in defense mechanisms. Although their lipid-binding ability has been well reported, the biological function of LTPs is still largely unknown. The plant LTP family includes two subfamilies according to their molecular masses: the 9-kDa LTP1 and the 7-kDa LTP2. Although LTP1 and LTP2 share a common compact fold consisting of four α-helices stabilized by four disulfide bridges, the pairing partners of cysteines are not completely conserved between the two subfamilies, that also display a low overall sequence similarity (about 30% identity) www.allergome.org, accessed September 12, 2011).Up to now 63 LTPs have been characterized as allergens, being 46 of them expressed in edible parts of plants, almost all of them belonging to the LTP1 protein subfamily, and just two, having very preliminary reported data, to the LTP2 subfamily , we sought to compare the LTP biochemical, immunochemical, and clinical features in order to define the extent of their heterogeneity. The study, leading first to the full characterization of KF LTPs, brought us to abandon the former interpretation of “one molecule fits all” and to search for as much differences as possible among allergenic proteins facing each allergic patient, in order to increase the quality of personal decision making he or she deserves.The study received the approval of the Institutional ethical committee of the Istituto Dermopatico dell'Immacolata, Rome, Italy (66/CE/2006). Patients or caregivers signed an informed consent when undergoing tests not in the routine workup. Patients' demographical and clinical data, namely respiratory and ingestion-related symptoms on KF exposure, as well as all the in vivo and in vitro diagnostic data, were recorded for all patients by an allergy specialist, or transferred real time from the laboratory, into the InterAll software, a customized allergy electronic record for diagnostic and clinical data storing . KF specific clinical information were collected using the standard questionnaire reported in a previous study A subset of 259 consecutive subjects, tested nPru p 3 positive on the Immunosolid phase Allergen Chip (ISAC) 103 , was tested on the ISAC Exp96 . To verify the presence of LTP IgE positive subjects beside the nPru p 3 in the subset reported above, a control population of 744 consecutive allergic subjects tested positive to any allergen on ISAC 103 has been created. Overall 1,003 subjects underwent IgE testing with the ISAC Exp96 microarray.ST were performed and recorded as wheal areas using a standard methodology as already reported KF and peach DBPCFC have been performed as reported in literature in enrolled subjects when they did not report a recent anaphylaxis episode Total and specific IgE using a singleplex system were determined in patients' sera from venous blood samples obtained at the time of enrolment by the ImmunoCAP system . To verify the IgE binding capability of the new KF LTPs, biotinylated nAct d 10 and nAct c 10 (see below) have been coupled on a streptavidin-CAP following the standard methodology as suggested by the manufacturer (Phadia) and reported in literature www.allergome.org) Specific IgE for allergenic molecules were detected by ISAC 103 microarray test (PMD); ISAC tests were performed as previously reported IgE inhibition experiments have been performed applying the SPHIAa as previously reported Actinidia chinensis) and green KF (Actinidia deliciosa) were purchased at a local market. KFs were peeled, homogenized in a household blender after addition of 1 M NaCl (1∶1 w/v) and stirred at 4°C for 2 h. After centrifugation at 12,500 x g for 60 min, the supernatant representing the total protein extract was collected, dialyzed against water, aliquoted and stored at −20°C until used.Gold KF  and stirred at 4°C for 2 h. Seeds were crushed by pestle in a mortar until a smooth powder was obtained. Seed proteins were extracted in 0.5 M NaCl, at 4°C for 2 h. After protein extraction, pulp and seed samples were centrifuged at 12500 x The protein concentration in the extracts was estimated by the BIO-RAD Protein Assay , using calibration curves made with bovine serum albumin.RP- HPLC of KF extracts was performed on a Vydac  C8 column (0.21×25 cm), using a Beckman System Gold apparatus . Elution was accomplished by a multistep linear gradient of eluant B (0.08% TFA in acetonitrile) in eluant A (0.1% TFA) at a flow rate of 1 ml/min. The eluate was monitored at 220 and 280 nm. The separated fractions were manually collected and analyzed.KF LTPs, namely nAct c 10 and nAct d 10, were extracted from seeds and purified from the seed extracts using the procedure described by Ciardiello et al. The molecular mass of purified proteins was estimated by MALDI-TOF mass spectrometry measurements carried out on a PerSeptive Biosystems  Voyager-DE Biospectrometry Workstation. Analyses were performed on pre-mixed solutions prepared by diluting samples  in 4 volumes of matrix, namely 10 mg/ml α-ciano-4-hydroxycinnamic acid in 60% acetonitrile containing 0.3% TFA.In vitro simulated gastric digestion of nAct d 10 and nPru p 3 was performed as described by Bublin et al. www.expasy.org).Amino acid sequencing of LTP N-terminal region was performed with an Applied Biosystems Procise 492 automatic sequencer . Denaturation and alkylation of LTP sulfhydryl groups with 4-vinylpyridine was carried out as already described Purified nAct c 10 and nAct d 10 preparations  were incubated with 5 times molar excess of biotin N-hydroxysuccinimide ester  dissolved in DMSO (Sigma-Aldrich). After 3.5 hours at room temperature, the excess reagents were removed by gel-filtration chromatography using a PD10 column  equilibrated in PBS. The biotinylated preparations were then used for coupling with the streptavidin-CAP as described above.-µm filter , in a sterile horizontal laminar flow hood.Dialyzed protein extracts and purified LTP samples in deionized water were mixed with sterile glycerin in a 1∶1 ratio. The final protein concentration was 0.5 mg/ml. The LTP solutions were sterilized by membrane filtration through a 0.22e-record and underwent descriptive statistics including prevalence and median value for quantitative variables. Prevalence comparisons have been carried out using the χ2 test followed by pair-wise comparison using the Tukey post hoc test. Significances in inter-group value correlation comparisons were evaluated by the Spearman test. Specific IgE and ST results were expressed using median value, with interquartile ranges (5th percentile-95th percentile) (IQR). Serum IgE values were analyzed using the Kruskal-Wallis One-Way ANOVA by Ranks and the pair-wise comparison with a Mann-Whitney test with Bonferroni correction. Serum IgE percent value distribution obtained after the inhibition assay were compared using the Kruskal-Wallis One-Way ANOVA by Ranks, followed by the post hoc test Nemenyi-Damico-Wolfe-Dunn. The χ2 or Fisher's exact tests were applied to contingency tables and used when appropriate depending on the number of observations. All tests were used with two-sided options and significance level was set at a p value<0.05. Statistical analysis and graphical visualization of data have been performed using the R software (www.r-project.org) and Graphpad Prism .All diagnostic and experimental data were stored into the InterAll Seed and pulp extracts of green and gold KF were prepared, fractionated by RP-HPLC and the separated protein components were manually collected and analyzed by N-terminal amino acid sequencing. The analysis of a peak eluted at a retention time very similar to that observed for nPru p 3 allowed the identification of the LTP, namely Act c 10 and Act d 10, in the seed extracts from the gold and the green KFs, respectively, rather than from the pulp (see below). Act c 10 was eluted as a single peak, whereas Act d 10 was eluted in three overlapping peaks suggesting the presence of some isoforms. Direct protein sequencing of the purified protein allowed the identification of two different isoforms of Act d 10 (see below). Possible additional isoforms were not identified probably because of the low yield. Conversely, the LTP peak was absent in the RP-HPLC profiles obtained for the pulp extracts of the two KF species.−1 cm−1). Purity of the protein preparations was checked by SDS-PAGE, RP- HPLC and N-terminal amino acid sequencing.The recovery of the pure protein was about 0.4 mg per gram of green and gold KF seeds. The pulp was not used for LTP preparation. Protein concentration was estimated on the basis of the molar extinction coefficient at 280 nm , and encoded 5735 and 5737, respectively, in the Allergome database .The direct sequencing of the N-terminal region of native nAct d 10 produced 17 identifiable amino acid residues, AVSCGQVDTALTPCLTY . A smallThe analyses by MALDI-TOF mass spectrometry of purified nAct d 10 provided two values, 9.464 (±20) and 9.484 (±20) kDa, in good agreement with the mass values deduced from the amino acid sequence of the two isoforms having alanine (9.458 kDa) or treonine (9.488 kDa) as N-terminal residues, respectively. A molecular mass of 9.460 (±20) kDa was obtained for Act c 10 by mass spectrometry.The analysis by SDS-PAGE and RP-HPLC of the LTP samples subjected to digestion in SGF showed that nAct c 10 and nAct d 10, similarly to nPru p 3, are resistant to gastric digestion. Moreover, in line with the results reported by Cavatorta et al. www.expasy.org) showed the sequence identity between Act d 10 and other already known allergenic LTPs to be not very high, ranging between 55% with Ara h 9 (isoform Ara h 9.0201) and 35% with Par j 2. Identities among the full length amino acid sequence of the six allergenic LTPs under study  are comprised in the 42–70% range  confirming the positive IgE result on ISAC 103 used for selection, and replicated on ISAC Exp96 , without statistically significant differences when compared to ISAC 103 results. A slightly not statistically significant different skin reactivity was recorded when considering nAct c 10  and nAct d 10   . To set 2.6–159) , panel A2.6–159) , panel B2.6–159) , panel COverall the findings reported above preliminary marked immunological differences between the LTPs from peach and KFs, thus suggesting that the shared LTP reactivity recorded in the first set of patients and leading to the identification of KF LTPs could not be extended to all nPru p 3 positive subjects.As from the previous section findings, showing heterogeneity between LTPs from two allergenic sources in that small study group, a larger study was set using the routine testing approach by the ISAC microarray, in order to define any possible subsets related to different LTP IgE recognition. A comparative evaluation of seven LTPs was undertaken using the ISAC 103 and ISAC Exp96 microarrays in parallel.versus KF LTPs, peanut and mulberry ones. Absence of nAct d 10 exclusively IgE positive subjects statistically differed to all the remaining LTPs rather than nAct c 10 (data not shown). Raw prevalence are reported in 2 test has been obtained taking together the seven prevalence, whereas not all prevalence were statistically different when compared each other. For instance, nPru p 3 was anyway the most prevalent sensitization with no difference only when compared to nMor n 3. IgE sensitization to nAct c 10 and nAct d 10 had statistically significant higher prevalence than the mugwort and the hazelnut LTPs, but less prevalent than nMor n 3 and nPru p 3, though proven by statistics just for nPru p 3. The less prevalent IgE reactivity have been recorded for the mugwort and the hazelnut LTPs being statistically different from all other LTPs. Detailed statistics are reported in Unless the original two population were defined on the basis of being nPru p 3 IgE positive or negative as tested on ISAC 103, additional nPru p 3 positive subjects were found in the control population using the ISAC Exp96 microarray bearing a different nPru p 3 preparation, thus raising the number of nPru p 3 positive subjects from 259 to 296. The same control population revealed a number of subjects being nPru p 3 IgE negative but positive to at least one of the other LTPs, increasing the overall number of subjects being positive to at least one of the LTPs from 296 to 431. nAct c 10, nAct d 10, rAra h 9, nArt v 3, rCor a 8, and nMor n 3 were positive when nPru p 3 was negative in 48, 41, 17, 56, 43, and 17 cases, respectively. The number of subjects positive to one LTP as listed above and negative to all others was relatively low, being 5, 0, 9, 25, 10, 9, 17. These prevalence were statistically different only when considering nArt v 3 values To visualize exclusive sensitization to one LTP compared to others, a series of Venn diagrams showing all possible logical relations between our finite collections of IgE values obtained by the two microarray testing, have been generated and reported in A cluster analysis performed using all subjects having IgE positive results to at least one of the seven LTPs generated the heat map shown in Beside the evaluation of positive/negative results reported above and clearly showing non-overlapping behavior among LTPs, we sought to verify whether detected IgE value distributions were different comparing the seven LTPs under study. A Kurskal-Wallis test gave statistically significant results (p<0.0001), and, when comparing the paired groups using the Mann-Whitney test several statistically different findings were recorded at different p values . Differiversus nAct d 10 correlation was taken, having the highest Spearman r (0.94) and χ2 (851.6) values  (2 (107.2) when comparing nAct c 10 and nArt v 3  values , panel A= 761.8) , panel VnArt v 3 , panel EnArt v 3 , panel OnArt v 3 , panel SnArt v 3 , panel SnArt v 3 , panel OTo define immunological relationships in terms of IgE binding among LTPs, the SPHIAa assay was run using nAct c 10, nAct d 10, and nPru p 3 as inhibitor on the seven LTPs. The assay was performed using ten individual sera selected for being IgE positive to the three inhibitors as shown in To verify whether the heterogeneous immunological behavior between the two KF LTPs and the peach LTP could lead to define a subset of patients who are also not clinically reactive to all LTPs, we first performed a series of experiments to define the LTP distribution within the KF tissues, namely the pulp and the seeds, based on biochemical, immunochemical and clinical methods.in vivo skin testing with green KF seed and pulp preparations in 21 selected subjects. As reported in As reported in the first paragraph of the in vivo clinical reactivity to green KF in well characterized peach allergic patients. We thus recruited patients having different diagnostic profiles. All enrolled ones had to be nPru p 3 positive with either a positive DBPCFC to peach or a recent severe generalized reaction on peach ingestion. All underwent the tests as reported in in vivo or in in vitro tests, whereas among the KF tolerant in the majority of the cases the KFs LTP were tested with negative results, but with exceptions, unless positive values were in the lowest range for both ISAC and ST .We herein report for the first time the identification of two new LTPs from the gold and the green KFs and their characterization as clinically relevant allergens. The primary structure of nAct d 10, the one from green KF, has been fully elucidated, being the one of nAct c 10 from gold KF very similar, mainly on the basis of the almost overlapping findings described throughout the present study. The identification process was straight forward as, using a panel of other KF allergens we selected patients being IgE negative for them and having just a positive IgE result for a potentially homologous molecule, namely nPru p 3. This process is highly suggested as leads to a secured evaluation of the newly identified structure, without overlapping positivity as sometimes reported in literature Unless we started the identification using subjects having the double clinical reactivity to peach and KF, the first evidence that we were dealing with new allergens not being part of a tight homogeneous group of molecules came by the comparative evaluation of primary structures and the preliminary extended parallel comparative testing with the peach LTP on the 44 peach allergic subjects. The former is the biochemical evidence that Act d 10 is not closely related to anyone of the already known allergenic LTPs. In fact, the observed sequence identities are never higher than 55% and generally fall in the narrow range of 40–55%, whereas more closely related LTPs display higher values. For instance, identities of 97, 91, 88, 87 and 79% are obtained when Pru p 3 sequence is compared with Pru du 3, Pru ar 3, Pru d 3, Pru av 3, Mal d 3 ones, respectively About comparative testing, our data on a huge population where just one LTP, nPru p 3, was tested did not arise such suspicion Regarding molecule to molecule immunological relationships, some studies reported on the need of studying conformational epitopes shared or not by different LTPs In the need of profiling patients reporting KF allergy, we used in collaboration with other research groups, Pru p 3 as representative LTP also for KF LTP in vivo/in vitro results as herein reported.Overall we can describe the clinical relevance of our findings as follows: we found non-symptomatic subjects as in previous studies After all, it remains that LTP represent a cross-sectional group of allergens having a patient's related IgE co-recognition. What should be avoided is the interpretation of LTP as part of a given allergenic source in the so called component-resolved diagnosis Last important finding of our study is the original distribution of LTP within the KF tissues. LTP is generally reported to be found on the surface of fruits, as in the case of peach In conclusion, we suggest the use of the microarray testing system bearing panels of homologous molecules as it adds much to our comparative knowledge on biochemical, immunochemical and clinical differences between closely related structures. Clinical allergists might take advantage of missing IgE recognition of one or the other LTP for keeping the specific food in patient's diet, avoiding useless exclusion, generally leading to deterioration of patient's quality of life.Furthermore, we herein document the need of having the microarray-based testing available for routine allergy diagnosis workup, as it creates the conditions for generating a wealth of data without selection biases and on cohorts otherwise not approachable, as we cannot exclude a different behavior of patients living in different geographical areas. As preliminary depicted in a review by us


Like many other G-protein coupled receptors (GPCRs), they undergo agonist-dependent phopshorylation and desensitization by GPCR Kinases (GRKs), a phenomenon recently shown to play a major role in the sympathetic overdrive that accompanies and aggravates chronic heart failure. A deletion polymorphism in the human α2B-AR gene (Glu301-303) causes impaired agonist-promoted receptor phosphorylation and desensitization in heterologous cell lines. Given the importance of α2-ARs in regulation of catecholamine secretion from chromaffin cells, we sought to investigate, in the present study, the desensitization properties and the sympatho-inhibitory activity of this variant in a chromaffin cell line. For this purpose, we expressed this variant and its wild type counterpart in the well-established chromaffin cell line PC12, and performed receptor phosphorylation and desensitization studies, as well as in vitro catecholamine secretion assays.α2B-AR are significantly impaired in PC12 cells, resulting in enhanced signaling to inhibition of cholinergic-induced catecholamine secretion in vitro.Both the agonist-induced phosphorylation and agonist-dependent desensitization of the human Glu301-303 deletion polymorphic α2B-AR gene polymorphism (Glu301-303 deletion) might confer better protection against conditions characterized and aggravated by sympathetic/catecholaminergic overstimulation in vivo.This α This assay is widely used to measure the G-protein coupling efficiency of GPCRs [35S] GTPγS binding assay was performed on these membranes using again 10 μM UK 14304 as agonist. As shown in Table 35S] GTPγS binding . However, the agonist-pretreated WT α2B-AR displays a significantly decreased agonist-stimulated [35S] GTPγS binding , compared to the Control WT α2B-AR, which reflects a significant functional desensitization of this receptor (~53%). In contrast, the agonist-pretreated Del α2B-AR shows almost the same agonist-stimulated [35S] GTPγS binding  as the Control Del α2B-AR, indicating that the Del α2B-AR fails to undergo desensitization in PC12 cells.We next examined the impact of this impaired phosphorylation on the functional desensitization, i.e. on the agonist-induced G-protein coupling, of the Del αof GPCRs . Again, 50 of Epi to stimulate GTPγS binding through the WT α2B-AR, whereas Epi pretreatment did not induce significant changes on the EC50 for the Del α2B-AR  in the EC2B-AR in PC12 cells, and, more specifically, the impact on the ability of the receptor to inhibit CA secretion. Physiologically in chromaffin cells, CA secretion is tonically stimulated by acetylcholine acting on nicotinic cholinergic receptors endogenously expressed in chromaffin cell membranes  Guanosine 5'-O-(3-thiotriphosphate), [35S] GTPγS was from Amersham Pharmacia Biotech .Guanosine diphosphate (GDP), Guanosine 5'-O-(3-thiotriphosphate) (GTPγS), nicotine, epinephrine, 5-bromo-6-(2-imidazoline-2-ylamino)-quinoxaline (UK14304), and oxymetazoline were from Sigma-Aldrich . The anti-phosphoserine (anti-P-Ser) rabbit polyclonal antibody was from Chemicon . The anti-α2B-AR  was modified (Del322-325) using polymerase chain reaction-mediated mutagenesis . PC12 cells were purchased from American Type Culture Collection  and transfected with the constructs of interest (i.e. wild type or Del α2B-AR) via the Lipofectamine method . For comparison of the properties of the two receptors, cell lines with comparable expression levels were chosen, as determined by ligand binding with the α2AR-specific antagonist [3H]-rauwolscine . The culture of PC12 cells was performed, as described previously  GTPγS binding was measured as described previously  GTPγS) with or without agonist (10 μM UK 14304) in a total volume of 100 μl. The incubation was terminated by dilution with 4 volumes of ice-cold 10 mM Tris-HCl buffer, pH 7.4, and vacuum filtration through Whatman GF/B glass fiber filters, which were then placed in scintillation vials for counting in a liquid scintillation counter (Beckman counter). Non-specific binding was measured in the presence of 10 μM GTPγS. For desensitization experiments, cells were pretreated with 10 μM UK 14304 or 10 μM epinephrine for 30 min at 37°C or with vehicle, placed on ice, and washed three times with ice-cold phosphate-buffered saline prior to membrane preparation.Cell membranes were prepared by centrifugation and agonist-induced stimulation of  GTPγS: [35S] Guanosine 5'-O-(3-thiotriphosphate)The authors declare that they have no competing interests.35S] GTPγS binding and in vitro catecholamine secretion assays, and helped to draft the manuscript. TK participated in the receptor phosphorylation experiments. AL conceived of the study, designed it, performed the transfections to establish the PC12 clones and the receptor phosphorylation experiments, and assisted in drafting and editing the manuscript. All authors read and approved the final manuscript.KN carried out the [


BackgroundThe cell type(s) mediating the maternal influence on allergic disease in children remain unclear. We set out to define the relationship between maternal allergy and frequencies of cord blood (CB) basophils, and plasmacytoid dendritic cells (pDCs); to characterize surface-bound IgE and FcεRI expressions on these cells; and to investigate the association between maternal and CB serum IgE levels with surface-bound IgE and FcεRI expressions.MethodsOne hundred and three mother/infant dyads were recruited prenatally, and maternal allergic history was recorded. Maternal blood was collected prior to delivery, and CB was collected after birth. Flow cytometry was used to identify CB basophils and pDCs and to determine surface-bound IgE and FcεRI expressions.ResultsFrequencies of CB basophils and pDCs were low and not related to maternal history of allergy. Percentages of CB basophils with surface-bound IgE were significantly higher in infants of allergic mothers compared with infants of non-allergic mothers . IgE on CB basophils correlated with CB IgE levels , but not with maternal IgE levels . IgE on CB pDCs was low and not significantly associated with maternal or CB IgE levels. Similarly, FcεRI expression by CB basophils and pDCs was not significantly associated with maternal or CB IgE levels.ConclusionsFrequencies of CB basophils and pDCs are not influenced by maternal allergy. CB basophils and pDCs have surface-bound IgE and express FcεRI; however, only IgE on CB basophils appears influenced by maternal allergy. Recruiting strategy and eligibility criteria are described in the electronic repository (ER).Pregnant women were divided into two groups based on the absence or presence of allergic disease defined by a physician’s diagnosis of asthma, allergic rhinitis, atopic dermatitis, or food allergy, and associated symptoms  within the past 12 months. The study was approved by the Institutional Review Board at Hartford Hospital, and informed written consent was obtained from all prospective mothers and on behalf of their infants.Upon admission to Labor and Delivery, maternal blood was obtained by venipuncture. Following delivery, CB samples were obtained from the umbilical cord-placenta unit by means of needle puncture of the umbilical cord vein cleansed with alcohol. Total serum IgE concentrations were determined by Phadia  using the ImmunoCap method. The detection limit for IgE in maternal and CB specimens was 2.00 KU/l and 0.10 KU/l, respectively. As a surrogate marker for maternal blood contamination, sera from CB samples were analyzed for total IgA levels using a commercial ELISA kit . The detection limit for IgA was 0.50 μg/ml (based on a serum dilution of 1:100), and CB samples containing ≥10 μg/ml IgA were excluded from the study Cord blood was obtained for isolation and cryopreservation of CB peripheral blood mononuclear cells (CBMCs) using a procedure adapted from the Immune Tolerance Network (PBMC CPT v.009) online protocol and a published protocol by Basford et al. +high BDCA-2- and CD123+high BDCA-2+ cells, respectively Cord blood basophils and pDCs were identified as the CD123In some experiments, CBMCs were pre-incubated with 50 μg monoclonal human IgE (HE1)  or 400 IU WHO IgE preparation  for 1 h at 18–25°C prior to staining. This was carried out to investigate the ability of CB basophils and pDCs to bind exogenous IgE and whether or not bound IgE blocked binding of the FcεRI detection antibody.To determine the primary outcome of the relationship between maternal allergy and levels of CB basophils and pDCs, CB samples containing a minimum of 200 basophils and 200 pDCs were analyzed. Frequencies of CB basophils and pDCs were expressed as percentages of total CBMCs. To determine secondary outcomes, CB basophils and pDCs were analyzed for surface-bound IgE and FcεRI expressions. Percentages of CB basophils and pDCs identified by flow cytometry with bound IgE or that expressed FcεRI were compared between infants of allergic and non-allergic mothers. In addition, the association between maternal and CB serum IgE levels with surface-bound IgE and FcεRI expressions was investigated.Statistical analyses were performed using Prism 4 . For measured variables, statistical comparisons were performed by the nonparametric Mann–Whitney test. Chi-square was used to compare the distribution of weight gain between allergic and non-allergic mothers, and the Fisher’s exact test was applied to compare the remaining categorical variables between groups. Correlation analyses were performed using Pearson’s correlation. Statistical significance was defined as a p-value ≤ 0.05.The number of mother/infant dyads recruited, enrolled, and excluded is described in Pregnant women with a history of allergic disease demonstrated higher levels of serum total IgE than pregnant women without a history of allergic disease  . CB IgE Frequencies of CB basophils and pDCs for the different groups of infants are shown in We first determined the ability of CB basophils and pDCs to bind exogenous IgE. CBMC preparations were pre-incubated in the absence or presence of exogenous IgE. Following pre-incubation, cells were stained to identify CB basophils and pDCs, and to define each cell type’s capacity to bind IgE or level of FcεRI expressions. CB basophils and pDCs from a patient with low levels of endogenous surface-bound IgE were capable of binding monoclonal human IgE (HE1) or polyclonal WHO IgE . FurtherWe next determined the percentages of CB basophils and pDCs with bound IgE or FcεRI expression from study subjects. The percentages of CB basophils with bound IgE were significantly greater in infants of allergic mothers compared with infants of non-allergic mothers  . There wTo examine the association between maternal and CB serum IgE levels with bound IgE and FcεRI expressions by CB basophils or pDCs, data from the allergic and non-allergic mother/infant dyads were grouped together. There were no significant correlations between maternal serum IgE levels and percentages of CB basophils or pDCs with bound IgE  (data not shown). In contrast, there was a significant correlation between CB IgE levels and percentages of CB basophils with bound IgE  of total IgE in the fetal compartment. Screening for cell-bound IgE, in addition to other parameters such as CB IgE


Inhibitory synapses can be organized in different ways and be regulated by a multitude of mechanisms. One of the best known examples is provided by the inhibitory synapses formed by Golgi cells onto granule cells in the cerebellar glomeruli. These synapses are GABAergic and inhibit granule cells through two main mechanisms, phasic and tonic. The former is based on vesicular neurotransmitter release, the latter on the establishment of tonic γ-aminobutyric acid (GABA) levels determined by spillover and regulation of GABA uptake. The mechanisms of post-synaptic integration have been clarified to a considerable extent and have been shown to differentially involve α1 and α6 subunit-containing GABA-A receptors. Here, after reviewing the basic mechanisms of GABAergic transmission in the cerebellar glomeruli, we examine how inhibition controls signal transfer at the mossy fiber-granule cell relay. First of all, we consider how vesicular release impacts on signal timing and how tonic GABA levels control neurotransmission gain. Then, we analyze the integration of these inhibitory mechanisms within the granular layer network. Interestingly, it turns out that glomerular inhibition is just one element in a large integrated signaling system controlled at various levels by metabotropic receptors. GABA-B receptor activation by ambient GABA regulates glutamate release from mossy fibers through a pre-synaptic cross-talk mechanisms, GABA release through pre-synaptic auto-receptors, and granule cell input resistance through post-synaptic receptor activation and inhibition of a K inward-rectifier current. Metabotropic glutamate receptors (mGluRs) control GABA release from Golgi cell terminals and Golgi cell input resistance and autorhythmic firing. This complex set of mechanisms implements both homeostatic and winner-take-all processes, providing the basis for fine-tuning inhibitory neurotransmission and for optimizing signal transfer through the cerebellar cortex. The fundamental anatomical and functional organization of the cerebellar cortex was defined since the ‘60s, thanks to improvements in anatomical and physiological techniques applied to the brain tissue Eccles, . Golgi cAt the present state, the granular layer inhibitory circuit appears to be organized as follows. The mossy fibers, which have been shown to convey protracted discharges or short high-frequency bursts   in granule cells  anion channel implicated in glutamate release from astrocytes  around 0.2 μM shows amongst the highest GABA affinities  curve through an additive process . Signals traversing the core are transmitted more fastly, with higher gain and over a broader frequency-band than in the surround. Moreover, the center tends to make LTP and the surround LTD. The superposition of multiple Mexican-huts can generate various logical operations including AND and XOR. Thus, inhibition can sculpt granular layer activity patterns over a 4D spatio-temporal domain.Since inhibition can shape the activity of neuronal networks defining their functional state and modifying signal transmission and coding, it is of extreme interest to understand how inhibition is regulated in turn. General regulation mechanisms include the turn-over and membrane expression of the different GABA receptor subunits. A major role has been proposed for gephyrin, which controls GABA receptor anchoring to the plasma membrane . In these conditions, the massive release of GABA activates post-synaptic GABA-B receptors determining downstream effects through G-protein-dependent pathways on granule cell membrane channels. A first mechanism is the long-lasting reduction of synaptic inhibition observed following intense Golgi cell-granule cell synaptic transmission, which reflects downregulation of GABA-A receptor-dependent activity . This leads to a long-lasting reduction of Golgi cell firing dependent on granule cell activity  GABA release from pre-synaptic Golgi cell terminals, (ii) granule cell GABA-A receptor-mediated IPSCs, (iii) granule cell inward rectifier, (iv) glutamate release from mossy fiber terminals. Thus, it seems that the glomerulus employs all available mechanisms to counterbalance an excess of inhibition, which would severely damage information transfer through the mossy fiber-granule cell pathway. The glutamate system appears to operate on a different principle. In the feedback loop, the increase of glutamate released by active granule cells can activate Metabotropic glutamate receptors (mGluRs) at the parallel fiber-Golgi cell synapses, enhancing the inward rectifier current and hyperpolarizing the Golgi cell. In the glomerulus, a glutamate increase reduces GABA release from mossy fiber terminals. In both cases, Golgi cell inhibition is reduced implementing a “winner-take-all” mechanism. Thus, the final effect of inhibition on the granular layer circuit will depend on a variety of local and external factors. This aspect deserves future experimental investigation.The inhibitory circuit could be regulated also by other intrinsic and extrinsic factors, including general neuromodulators. For example, serotonin activates Lugaro cells  regulation of timing, number and frequency of granule cell spikes, (ii) regulation of granular layer oscillations and resonance, (iii) regulation of center-surround responses and logical operations in the granular layer, (iv) regulation of long-term synaptic plasticity at the mossy fiber-granule cell relay. These effects should be eventually integrated and converge toward the optimization of granular layer functions. The most attractive perspective is now to understand the intricate balance of mechanisms that concur to generate efficient transmission along the mossy fiber pathway, to ensure an efficient inhibition determining sparseness, and to prevent that excessive inhibition blocks transmission degrading information transfer. This investigation may be aided by mathematical models resolving the intricate network of interactions regulating glomerular and inhibitory circuit functions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hepatoma-derived growth factor (HDGF) is a nuclear protein that is a mitogen for a wide variety of cells. Mass spectrometry based methods have identified HDGF as a phosphoprotein without validation or a functional consequence of this post-translational modification.We found that HDGF in primary mouse aortic vascular smooth muscle cells (VSMC) was phosphorylated. Wild type HDGF was phosphorylated in asynchronous cells and substitution of S103, S165 and S202 to alanine each demonstrated a decrease in HDGF phosphorylation. A phospho-S103 HDGF specific antibody was developed and demonstrated mitosis-specific phosphorylation. HDGF-S103A was not mitogenic and FACS analysis demonstrated a G2/M arrest in HDGF-S103A expressing cells, whereas cells expressing HDGF-S103D showed cell cycle progression. Nocodazole arrest increased S103 phosphorylation from 1.6% to 29% (P = 0.037).Thus, HDGF is a phosphoprotein and phosphorylation of S103 is mitosis related and required for its function as a mitogen. We speculate that cell cycle regulated phosphorylation of HDGF may play an important role in vascular cell proliferation. NM_004494] is a heparin binding protein originally isolated from conditioned media of a human hepatoma (HuH-7) cell line. HDGF was subsequently shown to be a mitogen for many cell types with nuclear localization necessary for its mitogenic activity . PrevioIn the present study, we detail that HDGF is indeed a phosphoprotein, identify S103 as a significant phosphorylation site and demonstrate that phosphorylation of S103 plays a critical role in regulating HDGF mitogenic function.2. For proliferation experiments VSMC were serum starved for 36 hours, then incubated overnight with BrdU . For cell cycle arrest studies, MDA-MB231 cells were seeded at 105 cells/ml in 6 well dishes containing a cover slip and DMEM with 10% serum. After 8 h cells were left in serum free (0.5% serum) media for overnight. Next morning cells were re-stimulated with 10% FCS. After 8 h cells were treated with or without 200 nM nocodazole for next 16 h. Next morning cells were briefly washed with ice cold PBS and fixed with 4% formaldehyde in DPBS.HEK-293T, MDA-MB231 and COS-7 cells were obtained from ATCC . Low passage mouse primary aortic vascular smooth muscle cells (VSMC) were isolated as previously described  orthophosphate for 2 hours. The tagged proteins were recovered by anti-GFP immunoprecipitation and as shown in Figure Previously we found multiple forms of HDGF by 2D gels and suspected this was due to a post-translational modification such as phosphorylation that could change the pI of the protein. The NetPhosK 1.0 computer algorithm  using stTo further explore HDGF S103 phosphorylation we developed a phospho-S103-HDGF antibody. Western blotting of enriched COS-7 phosphoproteins demonstrated detection of S103-HDGF phosphorylation Figure . The phoHDGF is a potent mitogen for VSMC . To examTo test for a possible role of phosphorylation in HDGF function, S103 was mutated to an aspartic acid (S103D) as a phospho-mimic residue. The effect of HDGF-S103D on cell proliferation was tested with asynchronous HEK-293 cells transfected to express, GFP fused to wild type HDGF, HDGF S103A or S103D and cell cycle analysis performed by FACS on the GFP positive cells and the GFP negative cells from the same sample as a control. As shown in Figure HDGF is an abundant nuclear protein with activity as a mitogen, in that it stimulates cell cycle progression. In this study we demonstrate phosphorylation of S103 during mitosis and show this phosphorylation is required for HDGF mitogenic activity. Our study of HDGF phosphorylation in vivo was suggested by the computer search engine NetPhosK 1  that matWe found that phosphorylation of HDGF-S103 has a significant effect on HDGF mitogenic activity. A substitution mutation in HDGF to S103A to prevent phosphorylation nullified HDGF mitogenic activity, whereas a S103D phospho-mimic mutation was constitutively active, resulting in an increased mitogenic activity relative to wild type HDGF. This data would suggest that one model of VSMC proliferation is that activation of mitotic kinases results in phosphorylation of S103-HDGF, leading to increased cell proliferation. As the impact of the S103 mutants on the cell cycle was much more profound than the wild type protein, this would suggest that HDGF mitogenic function is dependent on phosphorylation and not just dependent on the amount of HDGF present.Although the mechanism of phospho-S103-HDGF function during mitosis is unclear, it is of interest that another HDGF family member LEDGF, demonstrates metaphase chromatin binding, requiring cooperative interaction of the PWWP and AT-hook domains. Although HDGF does not contain AT-hook domains, it does bind DNA directly requiring a large 36 bp recognition sequence and requires the PWWP domain for DNA binding . It is uIt is of great interest that a S282P mutation in the DNA methyltransferase 3b  gene results in the ICF syndrome  . This seHDGF is a mitotic phosphoprotein and phosphorylation of S103 plays an important role in regulating the proliferation of cells and the mitogenic function of HDGF.ADE conceived the experiments and wrote the manuscript. JY made the phospho HDGF mutants, generated the in vitro phosphorylation data and drafted that experimental section. MR performed nocodazole and cell sorting experiments and drafted the experimental results. PD performed cell transfections and immunohistochemical analyses. DLB edited the draft and contributed significantly to experimental design. All authors have read and approved the final manuscript.


Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2.Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β Activated integrins can bind to extracellular matrix components with high affinity and mediate, through ‘outside-in’ signaling events, many vital cellular processes such as adhesion, migration, and proliferation Integrins are a family of transmembrane, heterodimeric glycoproteins composed of alpha and beta subunits. Each integrin subunit contains a large extracellular ligand-binding portion, a single membrane-spanning region, and a short cytoplasmic tail devoid of any enzymatic activity 33 phosphorylated at Y747 with a higher affinity and thus, by replacing talin, favors the latent state of the receptor IIbβ3, the biochemical studies suggest that phosphorylation of both tyrosines (Y747 and Y759) is required for the recruitment of myosin, while phosphorylation of Y759 alone is sufficient for interaction with the adapter protein Shc 747, Y759) on the β3CT 3 is involved in the regulation of αVβ3 integrin-dependent adhesion, specifically under conditions of cell activation, and that Y747F mutation diminished the stimulation-induced cell adhesion to vitronectin (VN), suggesting that phosphorylation is necessary for a fully functional αvβ3vβ3-VN interaction were shown to be dependent on phosphorylation of β3CT vβ3 mediated adhesion to fibronectin was shown to be abolished with increased β3 phosphorylation 3 integrin might support different aspects of integrin function. Consequences of integrin phosphorylation might be further modulated by interactions between integrin and intracellular mediators, the presence of which depends upon the cell type and the stage of cell adhesion and spreading.The β subunits' CTs include two tyrosine phosphorylation sites, located within NPxY and/or NPxY-like motifs, and are known to interact with phosphotyrosine binding (PTB) domains of intracellular signaling mediators vβ3 function on the endothelium depends on its cross-talk with VEGF type receptor (VEGFR2) vβ3 and VEGFR2, as well as a conformational change of αvβ3 to a high affinity state 3CT on Y747 and Y759, which are located within the NPxY and NxxY motifs, respectively 3 integrin and VEGF-induced angiogenic responses 3 appears to be of a reciprocal nature, as β3 phosphorylation also leads to enhanced phosphorylation and activation of VEGFR2 3 expression, the interplay between the two receptors regulates a number of cellular responses underlying angiogenesis, including EC adhesion, migration, and formation of endothelial tube networks 747F-Y759F substitutions diminish adhesion to VN and integrin activity, we suggest that phosphorylation of these residues must play a pivotal role in regulation of integrin function 3 motifs corresponding to its membrane-proximal region, we have shown that tyrosine-phosphorylation promotes the above interaction between β3 and VEGFR2 CTs, and we have proved physiological significance of this interaction by confirming that β3 derived Y747 containing inhibitory peptide diminishes EC tube formation and angiogenesis. Overall, these data provide novel molecular and mechanistic insights into phosphorylation dependent cross-talk between integrins and VEGF receptors.We have previously demonstrated that α3 tyrosine phosphorylation is crucial for VEGF-induced tyrosine phosphorylation of VEGFR2 3CT and how tyrosine phosphorylation affects this interaction. Unlabeled synthetic peptide VpepA (sequence shown in 15N-labeled β3NP (non-phosphorylated CT) and 15N-labeled β3MP (mono-Y747-phosphorylated CT). Associated chemical shift perturbations were monitored , which might represent the primary binding site for VEGFR2. However, upon phosphorylation, in addition to the above region, maximal perturbations were recorded near the Y747 phosphorylation site (744NPLYKEA750). This finding represents the second binding site and, possibly, increased affinity. Although the chemical shift perturbations were rather modest, they were reproducible and concentration dependent, and reached saturation at a peptide to protein ratio of 3 to 1, indicating the low affinity but specific nature of the observed interaction. Our attempts to calculate the dissociation constants from these titration series were unsuccessful due to low solubility and high tendency of the both VpepA and β3CT to precipitate while forming the complex.Based on our findings that VEGFR2 regulates integrin activation and signaling onitored . Chemica3, we have employed an additional trNOE-based approach. β3CT was coupled to glutathione S-transferase (GST-β3) and two smaller VEGFR2 peptides were synthesized . The patterns of additional peaks observed for both VpepB and VpepC peptides when mixed with GST-β3 were similar  and thus we can confidently use this system for structural characterization of VEGFR2-derived peptides.Thus, to further confirm and characterize the weak binding of VEGFR2-CTderived peptides to non-phosphorylated β3. VpepB exhibits a well defined C-terminal region (801YLSIV805), forming an α-helical turn and an additional loop surrounding two Gly residues (792ANGGE796), whereas the remaining parts are unstructured. 801YLSI804. While the structural analysis was reported for extracellular ligand binding 3CT interacts with VEGFR2, and this interaction involves the membrane proximal region within β3CT and 801YLSI804 motif within VEGFR2. β3 Y747 phosphorylation generates an additional VEGFR2 binding site within the 744NPLYKEA750 region of β3CT, which is not susceptible to structural characterization using the approach employed for the non-phosphorylated β3 (as we were unable to purify GST-fused mono-Y747-phosphorylated β3CT) and awaits the development of novel strategy for further investigation.Collectively, our data demonstrate that non-phosphorylated β747 might stabilize the integrin activated state, and the data presented above show that Y747 phosphorylation also promotes β3CT binding to VEGFR2, we next sought to assess the physiological consequences of β3CT phosphorylation in ECs. To this end, we utilized a phosphopeptide containing a 16 amino acid sequence from β3CT encompassing the 744NPLpY747 motif 3MP, including the VEGFR2 cytoplasmic domain. Previous studies have utilized the mutations of Y747 and Y759 to phenylalanines in order to simulate the non-phosphorylated state of β3CT. Therefore, in addition to a peptide containing nonphosphorylated 744NPLY747 motif  as controls in our studies  VEGF and peptides was injected subcutaneously in C57BL/6 (wild type) mice. Seven days later, matrigel implants were removed, sectioned, and blood vessels were stained using CD31 antibody. The pY747 peptide inhibited VEGF-induced vascularization by more three-fold, while the control F747 or Y747 peptides had no effect , no obvious defects have been observed 3.Previous studies using cell systems with “activatable” α747 phosphorylation in β3CT appears to promote a complex formation with VEGFR2, and this complex is not present in DiYF mutant cells 3-VEGFR2-CTs complex, but also the key regulatory role of the Y747 phosphorylation site in this complex formation. Furthermore, β3CT-derived Y747-phosphorylated peptide was able to disrupt VEGF-induced signaling, EC tube formation, and angiogenesis, in contrast to the unphosphorylated control. Thus, our in vitro data supports in vivo observations that β3CT phosphorylation is crucial for the interaction and cross-talk with VEGFR2 747 likely increases the affinity of β3 for VEGFR2-derived, membrane-proximal peptides, additional phosphorylation of Y759 might diminish this effect (data not shown). This finding may indicate a key role for the NPLY747 motif in positive regulation of the cross-talk between VEGFR2 and β3747 vs. Y759 phosphorylation in response to VEGF in ECs Y3 integrin and VEGFR2 in vitro, structurally characterized one of the VEGFR2 binding motifs, and confirmed that the above interaction is further enhanced by Y747 phosphorylation of β3 integrin. We also showed in vivo that pY747 affects β3 integrin cross-talk with VEGFR2 resulting in suppressed VEGF-induced signaling, endothelial tube formation, and angiogenesis. These findings identify important regulatory elements controlling the activity of β3 integrins in ECs, which underlie a number of more complex responses, including thrombosis/haemostasis and pathological angiogenesis.To conclude, we have shown direct complex formation between cytoplasmic tails of β786LRTVKRANGGELKTGYLSIVMDPDELPLDE815), VpepB (786LRTVKRANGGELKTGYLSIV805), VpepC (791RANGGELKTGYLSIVMDPD809), and membrane-permeable corresponding to β3CT peptides, pY747(YGRKKRRQRRRDTANNPLpYKEATSTFT),Y747 (YGRKKRRQRRRDTANNPLYKEATSTFT), and F747 (YGRKKRRQRRRDTANNPLFKEATSTFT) were synthesized in the Cleveland Clinic Molecular Biotechnology Core laboratory byFmoc chemistry using solid phase Omega 396 synthesizer . Quality analysis of the peptides was performed by HPLC on an analytical reverse phase C-18 column and by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS).Short peptides corresponding to the membrane proximal region of VEGFR2, VpepA . Tyrosine phosphorylation of β3CT has been achieved in vivo by using TKB1 bacterial cell line from Stratagene following the manufacture's protocol for the recombinant protein induction as described elsewhere Cloning, expression, and purification of β1H-15N Heteronuclear single quantum correlation (HSQC) titration experiments were performed in water at 25°C on Varian Inova 600 MHz equipped with inverse-triple resonance cryoprobe. Chemical shifts assignments have been determined previously 1H-1H TOCSY and NOESY spectra 1H-1H NOESY experiment were used for the structure calculations. The structures were calculated based on the hybrid distance geometry-dynamical simulated annealing method using the X-PLOR-NIH http://psvs-1_3.nesg.org/).Sequence-specific assignments of integrin tails are described elsewhere 3-integrin, and mouse monoclonal anti-phospho tyrosine antibodies were purchased from Santa Cruz Biotechnology, Inc. . Anti-ERK1/2, Anti-p-ERK1/2, and anti-phospho-VEGFR2 were from Cell Signaling Technology . VEGF was purchased from R&D Systems  and matrigel was obtained from BD Biosciences . DiI was obtained from Invitrogen . Drabkin's reagent was from Sigma. C57BL/6 (wild type), β3 knock-out (C57BL/6 background), and DiYF knock-in  were grown in DMEM:F12 media supplemented with 15% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 90 µg/mL heparin sulfate, and 90 µg/mL endothelial cell growth factor. Lung ECs: Mouse lungs were excised, minced, and digested using a collagenase-dispase reagent . Digests were strained and the resulting cell suspension was plated on flasks coated with 10 µg/mL fibronectin in HUVEC growth media.The aortic ring assays were performed as described previously The formation of vascular tube-like structures by HUVEC was performed as described previously Matrigel angiogenesis assays were done as described HUVEC were grown in monolayer on glass slides and then treated with fluorescein-labeled peptides for the indicated time periods. The cells were further stained with the lipophilic tracer DiI to stain the cell membrane following the manufacturer's instructions, then fixed with 2% paraformaldehyde for 10 min, washed, mounted with cover slips, and analyzed by confocal microscopy (Leica).Network analysis of tube forming HUVEC was performed in an automated fashion using customized visual basic macros developed within Image-Pro Plus . Bright-field images were imported into Image-Pro one-by-one, in batch mode. A high-pass spectral filter was applied to each image to enhance/equalize intensity; enabling application of a fixed threshold to segment the cell network (will be referred to as the “tube mask”). Since branches were sometimes thin (1 pixel thick), node to node branches were often disconnected following segmentation, even though visual observation confirmed continuity. To resolve this issue, a second approach was applied to preserve continuity. A top-hat morphological filter was applied to the original image to enhance the appearance of low intensity signal, and segmented using a fixed intensity and area threshold. This image was then “added” to the tube mask and inverted . Using Euclidean distance map (EDM)-based background clustering, the lumen of each tube was segmented such that no lumen was in contact with another. This image was then inverted and “skeletonized” to create continuous single pixel-width medial lines along the entire tube network. To avoid errors due to the “skeletonization” process, an additional algorithm was applied to cluster nodes that were within a given distance . These nodes were then classified as 3−, 4−, or 5+ branch nodes and summed for each category for output to Microsoft Excel. In addition to incorrect node classification, the skeletonization process can also produce spurious branches. To eliminate these, a “pruning” filter was applied to remove branches of a predefined length connected to a single node. The total number of branches was then summed and exported to Excel. To determine mean node and branch thickness, a Euclidean distance map was generated from the tube mask and “multiplied” by the node and skeletal branch masks respectively. Thickness values were calculated by summing the resulting pixels values in each of these images, multiplying these values by a factor of two and then by the pixel resolution, and lastly dividing by the total number of node pixels or skeletal branch pixels.Values were expressed as mean plus or minus standard deviations (SD). P values were based on the paired t-test. All the experiments were repeated at least three or more times. Results were considered statically significant with P value less than 0.05.Figure S1Sequential connectivity map for VpepB. Figure is produced by CCPN Analysis 2.1.1.(JPG)Click here for additional data file.Figure S23CT derived peptides by EC. Peptides were labeled with fluorescein (green) at the N-termini and added to HUVEC growth media for the indicated times.Uptake of β The plasma membranes were labeled with Dil (red) and nuclei stained with DAPI (blue). The cells were fixed and analyzed by confocal microscopy.(JPG)Click here for additional data file.Figure S3bFGF induced signaling events are not affected by F747 or pY747 peptides. HUVEC cells pretreated with peptides as indicated and stimulated with bFGF for 30 min. Total cell lysates were immuno-probed with anti phospho-erk antibodies.(JPG)Click here for additional data file.