Self-organization of the vascular system in plant leaves: inter-dependent dynamics of auxin flux and carrier proteins

The vegetative hormone Auxin is involved in vascular tissues formation throughout the plant. Trans-membrane carrier proteins transporting auxin from cell to cell and distributed asymmetrically around each cell give to auxin a polarized movement in tissues, creating streams of auxin that presume future vascular bundles. According to the canalization hypothesis, auxin transport ability of cells is thought to increase with auxin flux, resulting in the self-enhancement of this flux along auxin paths. In this study we evaluate a series of models based on canalization hypothesis using carrier proteins, under different assumptions concerning auxin flux formation and carrier protein dynamics. Simulations are run on a hexagonal lattice with uniform auxin production. A single cell located in the margin of the lattice indicates the petiole, and acts as an auxin sink. The main results are: (1) We obtain branching auxin distribution patterns. (2) The type of self-enhancement described by the functional form of the carrier proteins regulation responding to the auxin flux intensity in different parts of a cell, has a strong effect on the possibility of generating the branching patterns. For response functions with acceleration in the increase of carrier protein numbers compared to the auxin flux, branching patterns are likely to be generated. For linear or decelerating response functions, no branching patterns are formed. (3) When branching patterns are formed, auxin distribution greatly differs between the case in which the number of carrier proteins in different parts of a cell are regulated independently, and the case in which different parts of a cell compete for a limited number of carrier proteins. In the former case, the auxin level is lower in veins than in the surrounding tissue, while in the latter, the auxin is present in greater abundance in veins. These results suggest that canalization is a good candidate for describing plant vein pattern formation.     

Bone quality and healing in a swine vascularized bone allotransplantation model using cyclosporine-based immunosuppression therapy

Although vascularized bone and joint allotransplantation is a promising new treatment option for reconstructing large bone defects, the need for immunosuppressive agents to prevent rejection in these procedures poses a major problem. This problem stems from the fact that several of these agents can cause harmful side effects, such as alterations in bone quality and healing. Therefore, the purpose of this study was to determine what effect the commonly used immunosuppressant regimen cyclosporine A-based combination therapy has on bone quality and healing. In 10 pigs, vascularized bone allografts with skin and muscle components (osteomyocutaneous free flaps) were transplanted from size-matched donor animals. Recipient animals received oral cyclosporine A/mycophenolate mofetil/prednisone therapy for 90 days. Bone quality was studied before and after transplantation by measuring the bone's acoustic velocity and density and calculating the bone's elastic coefficient. Bone healing was assessed using radiographic analysis. Four animals were lost as a result of graft rejection or immunosuppression-related complications before the 90-day endpoint of the study. Although bone specimens taken from the six animals that completed the 90-day protocol had histological signs of rejection, they all seemed to have normal bone healing. Posttransplant bone density values were significantly decreased (p < 0.05) (1544.7 +/- 47.5 kg/m3) as compared with pretransplant values (1722.7 +/- 44.1 kg/m3). Results of the acoustic velocity and elastic coefficients measurements showed a significant decrease (p < 0.05) in posttransplant values (from 3503.0 +/- 165.1 meters/sec to 2963.0 +/- 54.6 meters/sec and from 21.6 +/- 2.2 GPa to 13.6 +/- 0.5 GPa, respectively), indicating diminished bone quality. The findings indicate that cyclosporine A/mycophenolate mofetil/prednisone combination therapy is ineffective in preventing bone rejection, that it decreases bone quality, and that it is associated with systemic toxicity, suggesting that this immunosuppressive regimen at the doses used in this study is not ideal for vascularized bone allotransplantation procedures.     

Internalization and trafficking of the human and rat growth hormone-releasing hormone receptor

Internalization and intracellular trafficking of the growth hormone-releasing hormone receptor (GHRH-R) were studied in rat anterior pituitary and human (h) and rat (r) GHRH-R-transfected BHK cells, with the GHRH agonist, [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2), Ala(8), Ala(15), Lys(22)]hGHRH(1-29)NH(2) (Fluo-GHRH). Time- and temperature-dependent internalization of stimulated GHRH-R was blocked by phenyl arsine oxide (PAO) in both cell types. In anterior pituitary and rGHRH-R-transfected BHK cells, only filipin III and cerulenin blocked receptor-mediated internalization of Fluo-GHRH while in hGHRH-R-transfected BHK cells, only hyperosmolar sucrose inhibited this process. These results suggest that hGHRH-R internalization is clathrin-dependent, while fatty acid acylation of rGHRH-R appears to be a prerequisite to caveolin-dependent internalization. Experiments in anterior pituitary using Bodipy-FL-C(5) ganglioside GM1, a specific marker of lipid rafts such as caveolae, confirmed this latter pathway. Co-localization of Fluo-GHRH with LysoTracker indicated that Fluo-GHRH was directed to acidic organelles in both cell types. Finally, studies using cycloheximide and monensin showed that upon stimulation with GHRH, an optimal concentration of functional GHRH-R was maintained at the plasma membrane due to de novo synthesis and recycling in pituitary cells and to de novo synthesis solely in hGHRH-R-transfected BHK cells. This first study on the dynamics of the GHRH/GHRH-R complexes using fluorescence imaging in a native environment compared to cell system models, revealed that both receptor primary structure and concentration at the plasma membrane play important roles in internalization and trafficking of specific G-protein-coupled receptors (GPCR).     

The enhancer-blocking activity of the Fab-7 boundary from the Drosophila bithorax complex requires GAGA-factor-binding sites

In the work reported here we have analyzed the role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C). One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and we show that it recognizes sequences from HS1 in vitro. Using two different transgene assays we demonstrate that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. We show that distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. We also show that the enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations in the gene encoding the GAGA factor Trithorax-like.     

Influence of colloid, preservation medium and trimetazidine on renal medulla injury

In organ transplantation, preservation injury is an important factor which could influence short-term and long-term graft outcome. The renal medulla is particularly sensitive to oxidant stress and ischemia-reperfusion injury (IRI). Using an autotransplant pig kidney model, we investigated renal function and medullary damage determined between day 1 and week 2 after 24- or 48-h cold storage in different preservation solutions: University of Wisconsin solution (UW), Hopital Edouard Herriot solution (a high Na+ version of UW), ECPEG (high Na+ preservation solution with PEG) and ICPEG (a high K+ version of ECPEG) with or without trimetazidine (TMZ). TMZ improved renal preservation and increased renal function when added in each preservation solution (particularly HEH and ECPEG). Medullary damage led to the early appearance of trimethylamine-N-oxide (TMAO) followed by 1H-NMR in urine and plasma. TMZ and ECPEG is the most efficient association to reduce medullary damage. This study clarifies the role of colloid and polarity solution and the role of mitochondrial protection by TMZ.     

Identification of a trafficking motif involved in the stabilization and polarization of P2X receptors

Extracellular ATP-gated channels (P2X receptors) define the third major family of ionotropic receptors, and they are expressed widely in nerve cells, muscles, and endocrine and exocrine glands. P2X subunits have two membrane-spanning domains, and a receptor is thought to be formed by oligomerization of three subunits. We have identified a conserved motif in the cytoplasmic C termini of P2X subunits that is necessary for their surface expression; mutations in this motif result in a marked reduction of the receptors at the plasma membrane because of a rapid internalization. Transfer of the motif to a reporter protein (CD(4)) enhances the surface expression of the chimera, indicating that this motif is likely involved in the stabilization of P2X receptor at the cell surface. In neurons, mutated P2X(2) subunits showed reduced membrane expression and an altered axodendritic distribution. This motif is also present in intracellular regions of other membrane proteins, such as in the third intracellular loop of some G protein-coupled receptors, suggesting that it might be involve in their cellular stabilization and polarization.