VEGF is required for growth and survival in neonatal mice.
We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development. (+info)
Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own.
Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners. (+info)
Bone resorption induced by parathyroid hormone is strikingly diminished in collagenase-resistant mutant mice.
Parathyroid hormone (PTH) stimulates bone resorption by acting directly on osteoblasts/stromal cells and then indirectly to increase differentiation and function of osteoclasts. PTH acting on osteoblasts/stromal cells increases collagenase gene transcription and synthesis. To assess the role of collagenase in the bone resorptive actions of PTH, we used mice homozygous (r/r) for a targeted mutation (r) in Col1a1 that are resistant to collagenase cleavage of type I collagen. Human PTH(1-34) was injected subcutaneously over the hemicalvariae in wild-type (+/+) or r/r mice four times daily for three days. Osteoclast numbers, the size of the bone marrow spaces and periosteal proliferation were increased in calvariae from PTH-treated +/+ mice, whereas in r/r mice, PTH-induced bone resorption responses were minimal. The r/r mice were not resistant to other skeletal effects of PTH because abundant interstitial collagenase mRNA was detected in the calvarial periosteum of PTH-treated, but not vehicle-treated, r/r and +/+ mice. Calcemic responses, 0.5-10 hours after intraperitoneal injection of PTH, were blunted in r/r mice versus +/+ mice. Thus, collagenase cleavage of type I collagen is necessary for PTH induction of osteoclastic bone resorption. (+info)
DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model.
Myotonic dystrophy (DM) is the most common form of muscular dystrophy and is caused by expansion of a CTG trinucleotide repeat on human chromosome 19. Patients with DM develop atrioventricular conduction disturbances, the principal cardiac manifestation of this disease. The etiology of the pathophysiological changes observed in DM has yet to be resolved. Haploinsufficiency of myotonic dystrophy protein kinase (DMPK), DM locus-associated homeodomain protein (DMAHP) and/or titration of RNA-binding proteins by expanded CUG sequences have been hypothesized to underlie the multi-system defects observed in DM. Using an in vivo murine electrophysiology study, we show that cardiac conduction is exquisitely sensitive to DMPK gene dosage. DMPK-/- mice develop cardiac conduction defects which include first-, second-, and third-degree atrioventricular (A-V) block. Our results demonstrate that the A-V node and the His-Purkinje regions of the conduction system are specifically compromised by DMPK loss. Importantly, DMPK+/- mice develop first-degree heart block, a conduction defect strikingly similar to that observed in DM patients. These results demonstrate that DMPK dosage is a critical element modulating cardiac conduction integrity and conclusively link haploinsufficiency of DMPK with cardiac disease in myotonic dystrophy. (+info)
Predicting insecticide resistance: mutagenesis, selection and response.
Strategies to manage resistance to a particular insecticide have usually been devised after resistance has evolved. If it were possible to predict likely resistance mechanisms to novel insecticides before they evolved in the field, it might be feasible to have programmes that manage susceptibility. With this approach in mind, single-gene variants of the Australian sheep blowfly, Lucilia cuprina, resistant to dieldrin, diazinon and malathion, were selected in the laboratory after mutagenesis of susceptible strains. The genetic and molecular bases of resistance in these variants were identical to those that had previously evolved in natural populations. Given this predictive capacity for known resistances, the approach was extended to anticipate possible mechanisms of resistance to cyromazine, an insecticide to which L. cuprina populations remain susceptible after almost 20 years of exposure. Analysis of the laboratory-generated resistant variants provides an explanation for this observation. The variants show low levels of resistance and a selective advantage over susceptibles for only a limited concentration range. These results are discussed in the context of the choice of insecticides for control purposes and of delivery strategies to minimize the evolution of resistance. (+info)
Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA.
Inheritable mutations in nucleotide excision repair (NER) genes cause cancer-prone human disorders, such as xeroderma pigmentosum, which are also characterized by symptoms of accelerated ageing. To study the impact of NER deficiency on mutation accumulation in vivo, mutant frequencies have been determined in liver and brain of 2-16 month old NER deficient XPA-/-, lacZ hybrid mice. While mutant frequencies in liver of 2-month old XPA-/-, lacZ mice were comparable to XPA+/-, lacZ and the lacZ parental strain animals, by 4 months of age mutant frequencies in the XPA-deficient mice were significantly increased by a factor of two and increased further until the age of 16 months. In brain, mutant frequencies were not found to increase with age. These results show that a deficiency in the NER gene XPA causes an accelerated accumulation of somatic mutations in liver but not in brain. This is in keeping with a higher incidence of spontaneous liver tumors reported earlier for XPA-/- mice after about 15 months of age. (+info)
Inward rectification in KATP channels: a pH switch in the pore.
Inward-rectifier potassium channels (Kir channels) stabilize the resting membrane potential and set a threshold for excitation in many types of cell. This function arises from voltage-dependent rectification of these channels due to blockage by intracellular polyamines. In all Kir channels studied to date, the voltage-dependence of rectification is either strong or weak. Here we show that in cardiac as well as in cloned KATP channels (Kir6.2 + sulfonylurea receptor) polyamine-mediated rectification is not fixed but changes with intracellular pH in the physiological range: inward-rectification is prominent at basic pH, while at acidic pH rectification is very weak. The pH-dependence of polyamine block is specific for KATP as shown in experiments with other Kir channels. Systematic mutagenesis revealed a titratable C-terminal histidine residue (H216) in Kir6.2 to be the structural determinant, and electrostatic interaction between this residue and polyamines was shown to be the molecular mechanism underlying pH-dependent rectification. This pH-dependent block of KATP channels may represent a novel and direct link between excitation and intracellular pH. (+info)
The Gab1 PH domain is required for localization of Gab1 at sites of cell-cell contact and epithelial morphogenesis downstream from the met receptor tyrosine kinase.
Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses. (+info)