NAB2, a corepressor of EGR-1, inhibits vascular endothelial growth factor-mediated gene induction and angiogenic responses of endothelial cells

In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGF-induced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.     

Borna disease virus accelerates inflammation and disease associated with transgenic expression of interleukin-12 in the central nervous system

Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4(+) and CD8(+) T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.     

Magnetic phagosome motion in J774A.1 macrophages: influence of cytoskeletal drugs

The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-microm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 microM), Colchicine (CoL, 10 microM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 +/- 0.01 after 5 min versus 0.34 +/- 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 +/- 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10(-18) Joules and corresponds to a force of 7 pN on a single 1.3-microm phagosome.     

Immunohistochemical distribution of connexin 43 in the cartilage of rats and mice

Using fluorescence immunohistochemistry, the distribution of connexin 43 was examined in hyaline cartilage and in the perichondrium of mouse and rat knee joints. In addition, rat chondrocytes were shown to be coupled in dye transfer studies with Lucifer Yellow. Connexin 43 was detected between chondrocytes in the outer layer of knee joint cartilage, between chondrocytes of the growth plate and between fibrocartilage-like cells at tendon and ligament insertions and in the tendons and ligaments proper. However, in the hyaline cartilage of the hind limbs of mature rats, the degree of connexin 43 immunoreactivity was diminished. These data suggest a possible involvement of connexins in cartilage development. © 1998 Chapman & Hall     

Cytochemical and X-ray microanalytical studies of calcium in the extending zone, particularly in the rhizodermis of Zea mays roots

Calcium was detected in CaCl (10 m M )-pretreated roots of Zea mays L. (cv. LG 11) by means of electron probe X-ray microanalysis with energy-dispersive spectrometry (EDS) using embedded samples, fixed by the antimonate staining procedure. A high level of calcium was found where large amounts of antimonate precipitates were observed by light or transmission electron microscopy. In the elongation zone, after 20 h in humid air following a 2 h CaCl₂ pretreatment, the level of calcium was higher in trichoblasts than in atrichoblasts. In these cells it was detected mainly in the walls and nucleus, and antimonate staining was observed in the walls. Abundant precipitates containing calcium were associated with the nucleus, vacuoles, mitochondria and endoplasmic reticulum of non-differentiated cells, whereas they were confined to the walls of these cells just after the CaCl₂ pretreatment. The involvement of calcium in the formation of root hairs is discussed.     

Immunogenetic Variation and Differential Pathogen Exposure in Free-Ranging Cheetahs across Namibian Farmlands

Background

Genes under selection provide ecologically important information useful for conservation issues. Major histocompatibility complex (MHC) class I and II genes are essential for the immune defence against pathogens from intracellular (e.g. viruses) and extracellular (e.g. helminths) origins, respectively. Serosurvey studies in Namibian cheetahs (Acinonyx juabuts) revealed higher exposure to viral pathogens in individuals from north-central than east-central regions. Here we examined whether the observed differences in exposure to viruses influence the patterns of genetic variation and differentiation at MHC loci in 88 free-ranging Namibian cheetahs.

Methodology/Principal Findings

Genetic variation at MHC I and II loci was assessed through single-stranded conformation polymorphism (SSCP) analysis and sequencing. While the overall allelic diversity did not differ, we observed a high genetic differentiation at MHC class I loci between cheetahs from north-central and east-central Namibia. No such differentiation in MHC class II and neutral markers were found.

Conclusions/Significance

Our results suggest that MHC class I variation mirrors the variation in selection pressure imposed by viruses in free-ranging cheetahs across Namibian farmland. This is of high significance for future management and conservation programs of this species.     

Metabolic heterogeneity of muscle fibers classified by myosin ATPase.

Muscle fibers are commonly classified histochemically into three types by the staining intensity for myosin ATPase combined with those for metabolic enzymes. Preincubation at pH 4.6 gives rise to three staining intensities of myosin ATPase which are also used for fiber typing. The two classification systems were compared by computer analysis of the individual staining profiles of over 2,500 fibers, and found not to be equivalent. The analysis showed metabolic heterogeneity among the fiber groups distinguished according to their differences in myosin ATPase.     

K-induced alkalinization in all cell types of rabbit gastric glands: A novel K/H exchange mechanism

Digital image processing of the pH-sensitive dye BCECF was used to examine the effects of high [K] media on cytoplasmic pH (pHi) of individual cells within isolated rabbit gastric glands. When cells were acidified to pHi 6.5 from the resting pHi of 7.2-7.3 and then exposed to solution containing 77 mM K plus amiloride (to block Na/H exchange), recovery to pHi 7.0 was observed. This K-induced alkalinization occurred in all cell types of the gland, including cells within antral glands that were devoid of parietal cells (PC). This process was independent of extracellular Na and Cl and was unaffected by: 5 mM Ba or 200 microM bumetanide, or acute treatment with either 500 microM ouabain or 100 microM cimetidine, histamine or carbachol. SCH28080, which inhibits the PC H/K-ATPase when used in the low microM range of concentrations, blocked the K effect on pHi at 100 microM but was ineffective at 1 microM. A similar pHi recovery was also stimulated by Li, Cs (both 72 mM), and Tl (10 mM), in the order Li greater than K greater than Cs greater than Tl (all in the presence of amiloride), and these alkalinizations were also blocked by 100 microM SCH28080. Parallel experiments were performed to test the effect of these ions on 14[C]-aminopyrine accumulation, an index of acid secretion by the H/K-ATPase at the lumenal membrane of the PC. There was no correlation between the rates of cation-induced pHi recovery from an acid load and H secretion as measured by the accumulation of aminopyrine. We conclude that the K- (and Cs- and Li-) dependent pHi recovery is mediated by a novel cation/H exchange mechanism that is distinct from the PC H/K-ATPase.