Piggy-BACing the human genome II. A high-resolution, physically anchored, comparative map of the porcine autosomes

Using the INRA-Minnesota porcine radiation hybrid panel, we have constructed a human-pig comparative map composed of 2274 loci, including 206 ESTs and 2068 BAC-end sequences, assigned to 34 linkage groups. The average spacing between comparative anchor loci is 1.15 Mb based on human genome sequence coordinates. A total of 51 conserved synteny groups that include 173 conserved segments were identified. This radiation hybrid map has the highest resolution of any porcine map to date and its integration with the porcine linkage map (reported here) will greatly facilitate the positional cloning of genes influencing complex traits of both agricultural and biomedical interest. Additionally, this map will provide a framework for anchoring contigs generated through BAC fingerprinting efforts and assist in the selection of a BAC minimal tiling path and assembly of the first sequence-ready map of the porcine genome.     

Corticosterone-dependent metabolic and neuroendocrine abnormalities in obese Zucker rats in relation to feeding

The obese Zucker (fa/fa) rat is characterized by hyperphagia, hyperinsulinemia, an increase in fat deposition, and a hyperactivity in the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis in fa/fa rats is hypersensitive to stressful experimental conditions. Food deprivation even leads to a stress reaction in obese fa/fa rats. The present study was conducted to investigate the role of corticosterone in obese rats on the basal, fasting, and postprandial metabolic rate as well as on the central expression of the thyrotropin-releasing hormone (TRH) in these conditions. In addition, the study was aimed at clarifying whether the high levels of corticosterone in obese rats are responsible for the induction of the stress reaction to food deprivation in these animals. The present results demonstrate that whole body fat oxidation and postprandial metabolic responses in obese Zucker rats were improved by adrenalectomy (ADX). At the level of the central nervous system, ADX reversed a decrease in TRH mRNA expression in the paraventricular hypothalamus (PVH) detected in fasting animals. Considering all feeding conditions, the obese rats demonstrated lower TRH mRNA levels compared with lean animals. ADX resulted in an enhanced postprandial activation of the parvocellular PVH. In contrast, the magnocellular part of the PVH was less responsive to refeeding in ADX animals. Finally, ADX failed to prevent the stress response of obese rats to food deprivation. The present results provide evidence that the removal of adrenals resolve some of the metabolic defects encountered in obese Zucker rats. They also demonstrate that not all the abnormalities of the obese Zucker rats are attributable to the hyperactivity of the HPA axis.     

Identification of the cytoplasmic domains of CXCR4 involved in Jak2 and STAT3 phosphorylation

The chemokine SDF-1alpha transduces G(i)-dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a G(i)-independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1alpha binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr(135) in the DRY motif at the N terminus of ICL2 nor the Tyr(65) and Tyr(76) in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1alpha binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling.     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

Galig, a novel cell death gene that encodes a mitochondrial protein promoting cytochrome c release

Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector. Furthermore, Bcl-xL co-transfection significantly reduced the release of cytochrome c induced by galig expression, suggesting a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Bcl-xL. This antagonism was not observed upon co-transfection of Bcl-2 and galig. Galig encodes a mitochondrial-targeted protein named mitogaligin. Structure-activity relationship studies showed that the mitochondrial addressing of mitogaligin relies on an internal sequence that is required and sufficient for the release of cytochrome c and cell death upon cell transfection. Moreover, incubation of isolated mitochondria with peptides derived from mitogaligin induces cytochrome c release. Altogether, these results show that galig is a novel cell death gene encoding mitogaligin, a protein promoting cytochrome c release upon direct interaction with the mitochondria.     

High levels of endogenous nitric oxide produced after burn injury in rats arrest activated T lymphocytes in the first G1 phase of the cell cycle and then induce their apoptosis

Major physical traumas provoke a systemic inflammatory response and immune dysfunction. In a model of thermal injury in rats, we previously showed that an overproduction of nitric oxide (NO) was responsible for the collapse of lymphoproliferative responses. In the present work, we performed a time-course analysis of cell proliferation and cell death parameters in order to establish the sequence of events triggered by the high NO output in Wistar/Han rat splenocytes activated with Con A, 10 days after burn injury. We demonstrate that activated T cells from burned rats never divided whereas normal T cells underwent four division cycles. However, T cells from both burned and normal rat entered the G1 phase as shown by increase of cell size, mitochondria hyperpolarization, and expression of cyclin D1. Burned rat T cells progressed to the late G1 phase as shown by expression of the nuclear Ki-67 antigen, but they never entered the S phase. They underwent apoptosis as shown by morphological parameters, disruption of transmembrane mitochondrial potential, and DNA fragmentation. Persistent accumulation of the p53 protein accompanied these phenomena. NO synthase inhibitors antagonize alterations of cell proliferation and cell death parameters in burned rat T cells and accelerated p53 turnover.     

Sustained inhibitory effect of Agouti Related Protein on the ACTH-induced cortisol production by bovine cultured adrenal cells

The adrenal gland is the second tissue after hypothalamus exhibiting high expression level of Agouti Related Protein (Agrp) mRNA, which suggests that this peptide may control adrenal cell functions. However, its role in this tissue remained to be determined. In this report, we studied the effects of a long-term treatment (24 h) of cultured bovine adrenal cells by Agrp on the (Nle4, d-Phe7)-alphaMSH (NDP-alphaMSH)- or ACTH-induced cortisol release. We showed that Agrp inhibited, in a dose-dependent manner, the 10(-9) M NDP-alphaMSH-induced cortisol production through its antagonistic properties towards MSH at the level of MC4-R. Surprisingly, we found that Agrp in the same conditions of cell treatment also induced a strong inhibition of the ACTH-induced cortisol release. These effects were stronger using low concentrations of Agrp and disappeared for higher concentrations resulting in U-shaped curve data. There was no effect of SHU9119 in the same conditions of stimulation of the cells. Our data confirmed that Agrp is not an antagonist of ACTH at the level of MC2-R and that its sustained effect on ACTH-induced steroidogenesis did not involve its antagonistic properties at the level of MC4-R. The hypothesis would be that Agrp is acting on adrenal steroidogenesis through an alternate mechanism.