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Isoform-Specific Effect of Apolipoprotein E on Cell Survival and β-Amyloid-Induced Toxicity in Rat Hippocampal Pyramidal Neuronal Cultures
Joaquı́n Jordán, Marı́a F. Galindo, Richard J. Miller, Catherine A. Reardon, Godfrey S. Getz and Mary Jo LaDu
Journal of Neuroscience 1 January 1998, 18 (1) 195-204; DOI: https://doi.org/10.1523/JNEUROSCI.18-01-00195.1998
Joaquı́n Jordán
Departments of Pharmacological and Physiological Sciences and
Marı́a F. Galindo
Richard J. Miller
Catherine A. Reardon
Pathology, University of Chicago, Chicago, Illinois 60637
Godfrey S. Getz
Mary Jo LaDu
Although the genetic link between the ε4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established, the isoform-specific activity of apoE underlying this correlation remains unclear. To determine whether apoE influences the neurotoxic actions of β-amyloid (Aβ), we examined the effect of native preparations of apoE3 and E4 on Aβ-induced toxicity in primary cultures of rat hippocampal pyramidal neurons. The source of apoE was conditioned medium from HEK-293 cells stably transfected with human apoE3 or E4 cDNA. ApoE4 (10 μg/ml) alone was toxic to the cultures, whereas apoE3 had no effect. ApoE3 treatment prevented the toxicity induced by 10 μm Aβ(1–40) or Aβ(25–35). The apoE3 protective effect appears to be specific to Aβ-induced toxicity, because apoE3 did not protect against the cytotoxicity produced by NMDA or staurosporine, nor did apoE3 affect the increase in intracellular calcium induced by either NMDA or KCl. ApoE3 had no effect on the toxicity produced by Aβ in the presence of receptor-associated protein, an inhibitor of apoE receptors, particularly the LDL-receptor-related protein. Interaction with apoE receptors may not mediate the toxic actions of apoE4, because receptor-associated protein did not affect apoE4-induced neurotoxicity. Consistent with our previous biochemical experiments, analysis of the culture medium revealed that SDS-stable apoE3:Aβ complex is present in greater abundance than apoE4:Aβ complex. Thus, the protection from Aβ-induced neurotoxicity afforded by apoE3 treatment may result from clearance of the peptide by apoE3:Aβ complex formation and uptake by apoE receptors.
β-amyloid
primary hippocampal neuron cultures
LDL receptor-related protein
receptor-associated protein
Apolipoprotein (apoE), a 35 kDa component of several classes of lipoproteins, acts as a ligand for lipoprotein receptors, thus regulating plasma lipid transport and clearance. In humans, apoE has three major isoforms: E2 (Cys112, Cys158), E3 (Cys112, Arg158), E4 (Arg112, Arg158), products of alleles at a single gene locus. Although not fully understood, apoE also appears to play a role in both normal and pathologic brain function. The ε4 allele is linked genetically to Alzheimer's disease (AD) (Saunders et al., 1993; Strittmatter et al., 1993). Immunostaining of normal human brain shows apoE in neurons; in AD brain, apoE colocalizes with β-amyloid (Aβ) to senile plaques (Namba et al., 1991; Metzger et al., 1996). In addition, the density of Aβ deposition in cerebral plaques of patients with late-onset AD correlates with the ε4 allele (Rebeck et al., 1993; Schmechel et al., 1993). In vitro, Aβ has been shown to be neurotoxic (Yankner et al., 1990; Pike et al., 1993; Simmons et al., 1994). In spite of this evidence that apoE may interact with Aβ, the isoform-specific role of apoE in the pathogenesis of AD remains unclear. In vitro data suggest that apoE may have an isoform-specific effect both directly on neuronal development and on survival and via modulating the toxic actions of Aβ. Using several different cell culture models, lipid-associated ("native") apoE3 has been shown previously to increase neurite outgrowth, whereas apoE4 may reduce neurite outgrowth and causes depolymerization of tubulin (Nathan et al., 1994, 1995; Bellosta et al., 1995; Holtzman et al., 1995; Fagan et al., 1996). Purified apoE4, including full length, a 22 kDa thrombin cleavage fragment, and various peptides, has been shown also to be neurotoxic (Marques et al., 1996, 1997; Tolar et al., 1997). In the B12 rat neuronal cell line, apoE exhibited an isoform-specific protective effect (E2 > E3 > E4) on the cytotoxicity induced by oxidative stress, including treatment with Aβ peptides (Miyata and Smith, 1996). In human cortical neurons, apoE4 increased the neurotoxicity of Aβ peptides (Ma et al., 1996). Because the hippocampus is the region of the brain most affected by AD, we studied the effect of apoE3 and E4 on cell viability and Aβ-induced toxicity in primary cultures of rat hippocampal neurons. We have shown previously that Aβ(1–40) and Aβ(25–35) induce neurotoxicity via apoptotic pathways in this culture system (Jordan et al., 1997). Because apoE requires a lipid particle-associated conformation to be biologically active, we used native preparations of apoE3 and E4 (conditioned media), in which the protein is associated with small, dense particles (LaDu et al., 1995a). In biochemical experiments, this native apoE3 formed an SDS-stable complex with Aβ that was 20-fold more abundant than apoE4:Aβ complex (LaDu et al., 1994, 1995b). We hypothesize that this complex formation allows for the clearance of the peptide via apoE receptors. Thus, we predict that apoE3 will protect preferentially against Aβ-induced neurotoxicity in a receptor-mediated process.
Materials. Aβ(1–40) (Bayer, Wuppertal, Germany), Aβ(25–35) (Sigma, St. Louis, MO), and staurosporine (Sigma) were dissolved in dimethylsulfoxide (DMSO, Sigma). NMDA (Sigma) was dissolved in water. Stock solutions (1000×) for each reagent were prepared and stored at −80°C for no more than 1 month.
Hippocampal cultures. Pyramidal neurons were prepared from the hippocampi of fetal rats at 17 d of gestation (Scholz and Palfrey, 1991). Hippocampi were dissected in Ca2+/Mg2+-free HBSS and incubated in 0.1% trypsin for 15 min. The hippocampi were triturated by aspirating 7–10 times with a normal-bore Pasteur pipette and 7–10 times with a flame-narrowed Pasteur pipette. Cells were plated (2 × 105 cells/ml) in DMEM (Life Technologies, Grand Island, NY) plus 10% horse serum (Life Technologies) on poly-l-lysine (Sigma; 0.5 mg/ml in borate buffer, pH 8.0)-coated 15 mm round glass coverslips (0.2 ml/slip) and allowed to adhere. After 2–4 hr, cells were transferred to 60 mm dishes containing the supporting astrocytes attached to the bottom of the culture dish. Astrocytes were prepared from the cerebral hemispheres of newborn rats (Brorson et al., 1995). Cytosine-β-d-arabinofuranoside (5 μm) was added to each plate 2 d later to inhibit non-neuronal cell proliferation.
Toxicity treatments. Aβ(1–40) or (25–35) was added to the hippocampal pyramidal cultures on day in vitro (DIV)-5 to a final concentration of 10 μm, and cell viability was analyzed 5 d later. Staurosporine was added on DIV-5 to a final concentration of 0.3 μm, and cell viability was analyzed 24 hr later. For NMDA-induced toxicity, DIV-5 cultures were incubated for 20 min in 100 μm NMDA in 2 mmCa2+/Na+ and 10 μmglycine in Mg2+-free saline solution, culture medium was replaced, and cell viability was analyzed 24 hr later. A final concentration of 0.2% DMSO alone, the vehicle in which Aβ and staurosporine were initially dissolved, did not affect the general health of the cultures (data not shown). For fura-2 video imaging, neuronal cultures (DIV-5) were treated with apoE3 or apoE4 (10 μg/ml) for 5 d. On DIV-10, cells were perfused with 50 μmNMDA for 3 min in Mg2+-free saline solution or with 50 mm KCl for 1.5 min. Cells were then washed in balanced salt solution and loaded with 2 μm fura-2 AM, as described below.
ApoE preparations. Serum-free conditioned medium from HEK-293 cells stably transfected with human cDNA encoding apoE3 or E4 was prepared as described (LaDu et al., 1994). Medium was harvested, concentrated, dialyzed (10 kDa cutoff membrane) in PBS, and sterilized by passage through 0.22 μm syringe filters. Samples were diluted serially, run on reducing SDS-PAGE, and visualized by Coomassie blue staining, and the approximate apoE concentration was determined by densitometry (Molecular Dynamics, Sunnyvale, CA) and comparison with a purified apoE standard. ApoE in concentrated medium was 500 μg/ml on average. Control cultures were treated with conditioned medium from HEK cells stably transfected with the neomycin resistance expression vector alone and were not significantly different from untreated cultures (data not shown).
Cell viability. Cell death was analyzed using the fluorescein diacetate/propidium iodide double-staining procedure (Favaron et al., 1988). Living and dead cells were counted on adjacent fields of each coverslip for a total of 300–450 cells. The percentage of neurons surviving was determined on three to four coverslips for each condition and normalized to parallel controls. Each coverslip was treated as a single observation. Cell viability was determined from at least three separate experiments using different batches of both peptide and apoE-conditioned media with n ≥ 10. Results are expressed as means ± SE, and significance was determined by Student's t test.
Western blot analysis. Media samples were added to 2× nonreducing Laemmli buffer (Laemmli, 1970) (4% SDS, no βME) and frozen at −20°C. Samples were boiled 5 min and electrophoresed on 10–20% SDS-tricine gels, transferred to Immobilon-P membrane (Millipore, Bedford, MA), and probed with antibodies to Aβ (monoclonal antibody 4G8 to amino acids 17–24; Senetec, St. Louis, MO) or apoE antiserum. ApoE antiserum was obtained by immunizing rabbits with apoE purified from human serum. Proteins on Western blots were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Fura-2 video imaging. Cells were loaded with 2 μm fura-2 AM using a balanced salt solution (standard buffer) of the following composition (in mm): NaCl 159.0, KCl 5.0, MgSO4 0.4, MgCl2 0.5, KH2PO4 0.64, NaHCO3 3.0, HEPES 20.0, glucose 5.0, Na2HPO4 0.33, CaCl2 2.0, and BSA 0.2% (330 mOsm/kg), pH-adjusted to 7.35. The cells were incubated with fura-2 for 30 min at room temperature to avoid probe compartmentalization and then incubated for an 30 min at room temperature to allow de-esterification of the fura-2 dye. Coverslips were mounted on a coverslip chamber for fluorescence measurements. All measurements were made at room temperature, as described previously (Meucci and Miller, 1996), using standard buffer supplemented with 10 μm glycine. Each cell in the image was analyzed independently for each time point in the captured sequence. All individual cell [Ca2+]itraces shown are representative responses for a given field of cells. For the calibration of fluorescent signals, we used cells loaded with fura-2; Rmax andRmin, ratios at saturating and zero Ca2+, respectively, were obtained by perfusing cells with standard buffer containing 10 mm CaCl2 and 4 μm ionomycin and, subsequently, with a Ca2+-free solution containing 10 mmEGTA. The values of the obtained Rmax andRmin, expressed as gray-level mean, were used to calculate the calibration curve by TARDIS software. The [Ca2+]i was determined according to the equation of Grynkiewicz and associates (1985).
LRP Immunocytochemistry. Cultures (DIV-5) were fixed by incubating with 4% paraformaldehyde in culture medium at 37°C for 15 min. After washing three times in 0.1 m PBS, pH 7.4, cells were incubated for 1 hr in blocking medium (0.1% Tween 20, 4% BSA, in 0.1 m PBS) at room temperature. Incubations were performed overnight at 4°C, using rabbit antisera for rat LDL receptor-related protein (LRP) (1:1000, kindly provided by Dudley Strickland, American Red Cross, Rockville, MD), diluted in blocking medium. Cultures were rinsed three times with blocking medium, incubated with a biotinylated secondary antibody for 1 hr, and then incubated for 30 min with streptavidin conjugated with indocarbocyanine (1:500, Jackson Immunoresearch Lab, Inc, West Grove, PA). Coverslips were mounted using an aqueous mounting solution (10% glycerol in PBS) containing 2.5% (w/v) of 1,4-diazabicyclo[2.2.2.]octane (Sigma). Cell fluorescence was detected using a standard epiillumination fluorescence microscope (Olympus Optical, Tokyo, Japan).
Receptor-associated protein (RAP) treatments. Rat RAP was generously provided by Guojun Bu (Washington University, St. Louis, MO). RAP was generated as a recombinant glutathione S-transferase fusion protein and purified as described previously (Warshawsky et al., 1993). Before use, RAP was dialyzed against cell culture medium, as was a comparable molar concentration of BSA to serve as a control. RAP was added on DIV-5 for a final concentration of 1.0 μm. Additional RAP (0.5 μm) was added on DIV-7 and DIV-9, and cell viability was assayed on DIV-10. The BSA-only control had no effect (data not shown).
ApoE4 is toxic to hippocampal pyramidal neurons
Although the concentration of apoE in the extracellular space of the brain is unknown, the concentration of apoE in normal human CSF is 1.4–2.9 μg/ml, and the plasma concentration is 50 μg/ml (Pitas et al., 1987; Gelman et al., 1988). Primary hippocampal neuronal cultures were treated on DIV-1 with either control or apoE3- or apoE4-conditioned HEK cell medium (final concentration of 1, 10, and 100 μg apoE/ml), and cell viability was assayed after 5 and 10 d by the fluorescein diacetate/propidium iodide double-staining procedure (see Fig. 2). ApoE4 at 10 and 100 μg/ml significantly reduced cell viability in cultures treated for 5 d (data not shown; also, see Fig. 3C,D) and 10 d (Fig.1, also see Fig. 3A), whereas apoE3 and control media had no effect. ApoE4 toxicity resembled the apoptotic cell death induced by Aβ treatment, with propidium iodide labeling of condensed and fragmented nuclei in affected cells (Fig.2C).
ApoE4 is toxic to primary rat hippocampal neuron cultures. Dose–response. Cultures were treated on DIV-1 with 1, 10, or 100 μg apoE/ml culture medium, and cell viability was assayed on DIV-10. Source of apoE: conditioned medium from HEK cells stably transfected with human cDNA encoding apoE3 (•) or E4 (▴). Control cultures (▪) were treated with conditioned medium from cells stably transfected with the neomycin expression vector alone. Significance: versus control (*p < 0.05; **p < 0.001). Both 10 and 100 μg/ml apoE4 were also significantly neurotoxic after 5 d in culture (p < 0.05 and p < 0.01, respectively; data not shown).
Fluorescence staining of cells treated with apoE3 and E4 in the presence and absence of Aβ. Primary rat pyramidal hippocampal cultures (DIV-1) were treated with control medium (A, D), apoE3 (B,E), or apoE4 (C, F) (10 μg apoE/ml). On DIV-5, 10 μm Aβ(1–40) was added to D–F, and cells were stained with fluorescein diacetate/propidium iodide on DIV-10. Source of apoE as described (Fig. 1). Typical micrographs are shown with fluorescein diacetate (yellow/green)- labeled intact cells and propidium iodide (orange)- labeled condensed or fragmented nuclei of compromised cells (arrows in C, D,F). Scale bar, 10 μm.
Treatment with apoE3 protects hippocampal pyramidal neurons against β-amyloid-induced death
Aβ(25–35) has been shown previously to be toxic to primary rat hippocampal pyramidal neuron cultures (Loo et al., 1993; Prehn et al., 1996; Jordan et al., 1997). In the current study, 10 μm Aβ(25–35) and Aβ(1–40) treatment of DIV-5 cultures for 5 d produced a significant 30% decrease in cell viability (Fig.3). The involvement of apoptotic pathways in Aβ-induced neuronal toxicity has been shown previously by DNA laddering (Loo et al., 1993), Hoechst 33342 chromatin staining (Jordan et al., 1997), and TUNEL staining (Prehn et al., 1996). In the present study, dying neurons exhibited several of the hallmarks of apoptosis, including cell shrinkage and condensation of the cytoplasm, and chromatin aggregation and fragmentation in situ visualized with propidium iodine staining (Fig. 2D).
ApoE3 protects against Aβ-induced neurotoxicity. Primary rat pyramidal hippocampal cultures were treated on DIV-1 (A, B) or DIV-5 (C,D) with control, apoE3, or E4 (10 μg/ml, white bars). On DIV-5, 10 μm Aβ(1–40) (A, C) or Aβ(25–35) (B,D) was added (shaded bars), 1 hr after apoE treatment to C and D. Cell viability was analyzed on DIV-10 (see time line). Source of apoE as described (Fig. 1). Significance: versus control without Aβ (a1 , p < 0.05;a2 , p < 0.001); versus control with Aβ (b, p < 0.01).
To determine the effect of native apoE3 and E4 on Aβ-induced neurotoxicity, we treated DIV-1 cultures with apoE (10 μg/ml) for 5 d before the addition of peptide on DIV-5 and assayed cell viability on DIV-10 (Fig. 3A,B). Only apoE3 protected against the Aβ-induced neurotoxicity. Whereas apoE4 alone was toxic, the toxicity of apoE4 and Aβ was not additive, suggesting that a subset of neurons may be vulnerable to cytotoxic treatments. Because apoE has been reported to affect neuronal development (Holtzman et al., 1995; Nathan et al., 1995; Puttfarcken et al., 1997), DIV-5 cultures were treated with apoE for just 1 hr before the addition of either Aβ(25–35) or Aβ(1–40) (Fig.3C,D). Again, only apoE3 protected against Aβ-induced neurotoxicity.
ApoE3 neuroprotection appears to be specific for β-amyloid-induced death
To determine whether the neuroprotective effect of apoE3 was specific to Aβ-induced toxicity, cultures were treated with staurosporine and NMDA (Fig. 4). Staurosporine is a protein kinase C inhibitor that induces apoptosis in a variety of cultured cells, including neurons (Jordan et al., 1997), whereas NMDA is a glutamate receptor agonist that produces an increase in intracellular calcium, triggering excitotoxic cell death. DIV-1 cultures were treated with either control or apoE3- or apoE4-conditioned HEK cell medium (10 μg apoE/ml) for 5 d before treatment with the toxic agent on DIV-5, and cell viability was assayed after 24 hr. ApoE3 was unable to protect against the 35% decrease in neuronal cell viability produced by both staurosporine (0.3 μm) and NMDA (100 μm) (Fig.4A,B, respectively). In addition, apoE3 had no effect on the increase in intracellular calcium induced by NMDA or KCl (Table 1).
ApoE3 does not protect against other neurotoxic stimuli. Primary rat pyramidal hippocampal cultures were treated with apoE3 or E4 (10 μg/ml) alone (white bars) or 5 d before the addition of 0.3 μm staurosporine (A) or 100 μm NMDA (B) (shaded bars), as described in Methods. ApoE was added on DIV-1, drug treatment was on DIV-5, and cell viability was analyzed on DIV-6 (see time line). Source of apoE as described (Fig. 1). Significance: absence versus presence of drug (a, p < 0.001).
ApoE isoforms do not effect Ca2+ homeostasis
ApoE3:Aβ complex is more abundant than apoE4:Aβ complex in medium of treated neuronal cultures
In previous biochemical studies, native apoE3 formed an SDS-stable complex with Aβ that was 20-fold more abundant than apoE4:Aβ complex (LaDu et al., 1994, 1995b, 1997; Zhou et al., 1996; Yang et al., 1997). To determine whether this complex forms in the medium of neuronal cultures treated with Aβ and apoE, we used SDS-PAGE and Western blot analysis for Aβ and apoE on medium sampled at several time points over the incubation period (Fig.5). Monoclonal antibody 4G8 (Fig.5A) detected an SDS-stable 45 kDa apoE3:Aβ complex in greater abundance than apoE4:Aβ, as well as Aβ monomer (4 kDa), dimer and small aggregates. In addition, a complex with apoE3 dimer and Aβ was also detected. ApoE antiserum detected full-length apoE immunoreactive species corresponding to apoE3 and E4 monomer (35 kDa) and apoE3 dimer even after 10 d of incubation (Fig.5B). ApoE antiserum was unable to detect apoE:Aβ complex at the exposures of the immunoblots possible using this tissue culture medium.
ApoE3:Aβ ≫ apoE4:Aβ in the medium of treated neuronal cultures. Western blot analysis of the medium from primary rat pyramidal hippocampal cultures. DIV-5 cultures were treated with 10 μg of apoE/ml of apoE3 (lanes 1-4), apoE4 (lanes 5-8), or control (lanes 8-12) medium 1 hr before the addition of 10 μm Aβ(1–40). Source of apoE as described (Fig. 1). Aliquots of the culture medium were taken after the addition of apoE but before the addition of Aβ (lanes 1,5, 9), 2 hr after the addition of Aβ (lanes 2, 6, 10), on DIV-7 (lanes 3, 7, 11), and on DIV-10 (lanes 4, 8, 12). To visualize SDS-stable complex formation between apoE and Aβ, samples were run in nonreducing Laemmli buffer on 10–20% SDS-tricine gels, transferred to Immobilon-P membrane, and probed for Aβ with 4G8 antibody (A) and for apoE with antiserum (B).
RAP has no effect on apoE4 toxicity but abolishes the protective effect of apoE3 on Aβ-induced toxicity
It has been our working hypothesis that apoE:Aβ complex is cleared by apoE receptors. In vivo, apoE receptors in the brain are localized cell-specifically with neurons immunopositive for LRP (Moestrup et al., 1992; Wolf et al., 1992; Rebeck et al., 1993,1995) and glial cells for the LDL receptor (Pitas et al., 1987; Rebeck et al., 1993). Primary rat hippocampal neurons in vitro are LRP-immunopositive (Fig. 6). Our source of apoE3 and E4 is conditioned medium from HEK-293 cells stably expressing human apoE3 or E4, which is secreted associated with small, dense particles (LaDu et al., 1995a). These particles are taken up by U87 cells, human glioblastoma cells that express abundant LRP (Bu et al., 1994), in a RAP-inhibitable manner (M. J. LaDu and G. Bu, unpublished observations), demonstrating that the apoE in this preparation can serve as a ligand for LRP. RAP was used to inhibit apoE receptor-mediated uptake to determine whether this pathway plays a role in either the toxic actions of apoE4 alone (Fig.7) or the protective effect of apoE3 on Aβ-induced toxicity (Fig. 8). Cultures were treated with RAP on DIV-5 for a final concentration in the cell culture medium of 1.0 μm and additionally with 0.5 μm RAP on DIV-7 and DIV-9. This concentration of RAP is well in excess of the 3.3 nm Kd for LRP (Iadonato et al., 1993) as well as the 250 nm Kd for the LDL receptor (Medh et al., 1995). ApoE4 (10 μg/ml) was added 4 hr after the initial RAP treatment on DIV-5, and cell viability was assayed on DIV-10. ApoE4 treatment produced a 20% decrease in cell viability that was not affected by the addition of RAP (Fig. 7). In contrast, addition of RAP abolished the protective effects of apoE3 on Aβ-induced neurotoxicity. On DIV-5, apoE3 (10 μg/ml) was added 4 hr after the initial RAP treatment and 1 hr before the addition of Aβ (10 μm). Cell viability was assayed on DIV-10. Whereas apoE3 protected against the 25% decrease in cell viability induced by Aβ, RAP inhibited this neuroprotective effect of apoE3 (Fig. 8). In addition, RAP did not effect the neurotoxicity of Aβ in the absence of exogenous apoE, suggesting that in the culture system used here, Aβ is not being cleared by other LRP ligands.
Primary rat hippocampal neurons are LRP-immunopositive. Cultures grown on glass coverslips for 5 d were fixed in 4% paraformaldehyde, incubated with LRP antiserum, and visualized using streptavidin conjugated with indocarbocyanine (CY3). Representative photomicrograph is shown. Magnification, 2000×.
RAP has no effect on apoE4-induced neurotoxicity. Primary rat pyramidal hippocampal cultures (DIV-5) were treated with 1 μm purified rat RAP 4 hr before the addition of apoE4 (10 μg/ml). Additional RAP (0.5 μm) was added on DIV-7 and DIV-9, and cell viability was assayed on DIV-10 (see time line). Source of apoE as described (Fig. 1). A comparable concentration of BSA was used as a control for RAP and had no effect either alone or with apoE4 (data not shown). Significance: versus control (*p < 0.05).
RAP abolishes the protective effect of apoE3 on Aβ-induced neurotoxicity. Primary rat pyramidal hippocampal cultures (DIV-5) were treated with 1 μm RAP 4 hr before the addition of apoE3 (10 μg/ml). Aβ (10 μm) was added 1 hr later. Additional RAP (0.5 μm) was added on DIV-7 and DIV-9, and cell viability was assayed on DIV-10 (see time line). Source of apoE as described (Fig. 1). A comparable concentration of BSA was used as a control for RAP and had no effect alone, with apoE3, or with apoE3 and Aβ (data not shown). Significance: versus control (**p < 0.01).
In this paper, we have confirmed the neurotoxicity of the soluble Aβ peptides Aβ(25–35) and Aβ(1–40) and have shown that this neurotoxicity can be attenuated by the presence of apoE3 but not E4 in primary rat hippocampal neuron cultures. The viability of these cells is reduced by apoE4 even in the absence of Aβ. The toxic effects of both the Aβ peptides and apoE4 appear to be mediated by an increase in programmed cell death. The apoE3 protective effect may be specific to Aβ, because apoE3 did not protect the cultures from the neurotoxic effects of either NMDA or staurosporine. Furthermore, we demonstrate that the abrogation of the toxicity of Aβ by apoE3 is dependent on the participation of LRP or related receptors, whereas the toxicity of apoE4 appears to involve a different pathway.
The findings reported in this paper support the hypothesis that apoE3 facilitates the clearance of those Aβ species that are neurotoxic. We demonstrate that apoE3, unlike apoE4, forms readily detectable SDS-stable complex with Aβ(1–40) in the hippocampal cell culture medium under the conditions of these experiments. Although apoE3 almost completely abolishes the toxicity of Aβ, the molar ratio of Aβ and apoE3 in the medium in which the cells are incubated is ∼30:1. The SDS-stable complex between apoE3 and Aβ contains one molecule of Aβ per apoE3, as judged from the size of the complex observed by SDS-PAGE (Fig. 5). Clearly, there is not enough apoE3 present to complex with a major portion of the available Aβ. Therefore, either apoE3 protects against Aβ neurotoxicity by a mechanism that is not mediated by SDS-stable complex formation or only a portion of the Aβ present is actually neurotoxic and these molecules more readily form complex with apoE3. We favor the latter possibility for several reasons. First, the manifestations of Aβ cytotoxicity require prolonged incubation (5 d), during which cytotoxic species of Aβ could be generated. The precise nature of this species is unknown. Possible candidates are fibrillar forms of the peptide (Yankner et al., 1990; Pike et al., 1993; Simmons et al., 1994) or small soluble aggregates (Oda et al., 1995; Roher et al., 1996). Second, the attenuation of Aβ toxicity by apoE3 is reversed in the presence of RAP at concentrations that would block apoE binding to LRP and related receptors, including the LDL receptor. LRP is abundant on the cultured hippocampal neurons used in these experiments. The attenuation of the protective effect of apoE3 by RAP is consistent with the possibility that apoE3 protects against Aβ-induced toxicity by clearing the peptide via this neuronal LRP. RAP has no effect on Aβ-induced toxicity in the absence of exogenous apoE, suggesting that the peptide is not being cleared by other LRP ligands. It is also possible that apoE:Aβ complex is being cleared by astrocytes via the LDL receptor. Of the other candidate apoE receptors for Aβ clearance, gp330 is found only in ependymal cells in the CNS (Zheng et al., 1994), scavenger receptors are present on microglia (Christie et al., 1996; El Khoury et al., 1996), and the receptor for advanced glycation end products, although expressed by neurons, is not thought to be influenced by either apoE or RAP (Yan et al., 1996).
The hypothesis that apoE3 protects against Aβ-induced neurotoxicity by binding and clearing the peptide is also supported by the specificity of the apoE3 protective effect. ApoE3 does not protect against the neurotoxicity of staurosporine, which does not involve a cell surface receptor, or of NMDA, which does not involve an apoE-type receptor. In addition, apoE had no effect on the intracellular Ca2+ spikes induced by either KCl or NMDA. This suggests that apoE3 may be interrupting the Aβ toxicity cascade at a point before the initiation of cellular events and is consistent with a mechanism of action in which extracellular apoE3 interacts physically with the peptide to form a complex that is subsequently cleared by apoE receptors. Miyata and Smith (1996)reported that apoE exhibits an isoform-specific protective effect (E2 > E3 > E4) on the cytotoxicity induced by oxidative stress, including treatment with Aβ peptides. They propose that the protective action of apoE2 and E3 is attributable to the general antioxidant property of apoproteins. Our results do suggest that apoE may interact with Aβ extracellularly. However, because apoE receptors mediate the protective effects of apoE3, the antioxidant effects of apoE would have to occur within the endosomal compartment after the proteins are internalized. Such a mechanism is unknown.
The precise basis for the toxic effects of apoE4 alone remains unclear. It has been shown previously that apoE4 is associated with microtubule depolymerization, suggesting that apoE4 may actively promote neuronal destabilization (Nathan et al., 1995). Whether this contributes to the apoptosis induced by apoE4 in our experiments is not clear. However, the toxicity induced by apoE4 in the current experiments resembles programmed cell death and was not blocked by the addition of RAP. This suggests that apoE4 may signal an apoptotic cascade that is independent of apoE receptor-mediated uptake and subsequent intracellular localization. This is consistent with the neurotoxic effects of both purified full-length and a 22 kDa thrombin cleavage fragment of apoE4, species that have little receptor-binding activity (Marques et al., 1996, 1997). Our observation that apoE receptors do not mediate the neurotoxic actions of apoE4 is in apparent contrast to recent data from Tolar and associates (Tolar et al., 1997). Using chick sympathetic neurons, they report that the toxicity induced by a 22 kDa fragment and various peptides to the receptor-binding domain of apoE4 is abolished by RAP and other agents that disrupt the interaction between apoE and LRP. Although intriguing, the physiologic relevance of this profound toxicity observed with apoE4, toxicity that approached 80% within 24 hr of exposure, to a disease for which symptoms take decades to develop is unclear. In addition, it is not clear how purified apoE4 peptides can serve as ligands for LRP, the preferred substrate for which is apoE-enriched β-VLDL (Kowal et al., 1990).
The effects of Aβ and/or apoE on neural cultures appear to vary depending on the preparation of Aβ and apoE, the duration of exposure, the species and type of neuronal cultures, the developmental status of the neurons, the precise culture conditions, and the specific assay used to quantitate the effects. For example, Aβ can be either neurotrophic or neurotoxic, depending on the physical state and concentration of the peptide, as well as on the maturity of the cells (Whitson et al., 1989; Yankner et al., 1990; Pike et al., 1991, 1993). The effect of the apoE isoforms on neural cultures is also variable. Neurite outgrowth is enhanced by the addition of apoE3 to a variety of culture systems, including primary rat hippocampal neurons and several immortalized CNS cell lines (Holtzman et al., 1995; Nathan et al., 1995; Puttfarcken et al., 1997). However, apoE4 has been observed to inhibit (Bellosta et al., 1995; Nathan et al., 1995), have no effect (Holtzman et al., 1995; Fagan et al., 1996), or even enhance neurite outgrowth (Puttfarcken et al., 1997), apparently depending on the cell type and culture conditions. In addition, as also reported here, apoE4 may be toxic under some conditions (Marques et al., 1996,1997). Thus, the combined effects of apoE and Aβ promise to be no less complicated than their separate actions.
We have reported previously that both apoE3 and E4 are neurotrophic and protect primary rat hippocampal neurons from the toxicity produced by aged Aβ(1–42) (Puttfarcken et al., 1997), a peptide preparation with considerable secondary structure that induces neurotoxicity after as few as 24 hr of exposure (Puttfarcken et al., 1996). In these experiments, because the apoE was added at plating with cells in a minimally supplemented, serum-free medium with no glial feeder layer, we interpreted the results to mean that both apoE isoforms were acting as growth factors in these developing cultures (Puttfarcken et al., 1997). It has also been suggested that apoE modulates the activity of other growth-promoting agents (Mahley, 1988). Indeed, apoE has been shown recently to potentiate the activity of CNTF (Gutman et al., 1997). Thus, the combination and availability of other growth-promoting agents may modulate the neurotrophic actions of apoE, masking these trophic effects in culture models such as the one used here. For the current experiments, we have used primary rat hippocampal cells cultured in the presence of cerebral astrocytes. The glial feeder layer provided basic metabolic support to the neurons, allowing for experiments of longer duration and the possible detection of other apoE functions beside its growth-promoting activity. This longevity is also important when using soluble forms of Aβ, because the peptides manifest their neurotoxicity only after 4–5 d in culture at the concentrations used (current data) (Loo et al., 1993; Prehn et al., 1996; Jordan et al., 1997). The peptide preparation used in this study was dissolved in DMSO immediately before addition to a culture medium already containing apoE3 or E4. Thus, apoE was available during any aging or aggregation process affecting Aβ.
This paper describes an in vitro model in which apoE3 appears to protect against Aβ-induced neurotoxicity by forming an SDS-stable complex with the peptide that is subsequently cleared by apoE receptors. In addition, apoE4 alone induces neurotoxicity via a mechanism that is independent of apoE receptor-mediated uptake. Clearly, additional work is required to confirm and further elucidate both the neuroprotective effects of apoE3 and the neurotoxic actions of apoE4. Recently developed transgenic models expressing the human apoE isoforms in either neurons or glia will facilitate both the in vitro and in vivo study of these issues.
This work was supported in part by Public Health Service Grants DA02121, DA02575, and MH40165; American Health Assistance Foundation Grant 95100; National Institutes of Health Grant 1F32 HL08833-01 (M.J.L.); and a fellowship from the Minister of Education and Science of Spain (J.J). We thank Dudley Strickland for the LRP antibody (American Red Cross, Rockville, MD), Guojun Bu for RAP (Washington University, St. Louis, MO), John Lukens for technical assistance, and Alessandro Fatatis for expertise in Ca2+imaging.
J.J. and M.F.G. contributed equally to this work.
Correspondence should be addressed to Dr. Mary Jo LaDu, Department of Pathology, M/C 6079, University of Chicago, 5841 South Maryland Avenue, Chicago IL 60637.
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Joaquı́n Jordán, Marı́a F. Galindo, Richard J. Miller, Catherine A. Reardon, Godfrey S. Getz, Mary Jo LaDu
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In Episode 5 of the new podcast, Talking My Book, Daniel Gross, executive editor of strategy+business, interviews Sebastian Mallaby, a senior fellow at the Council on Foreign Relations, contributing columnist at the Washington Post, and author of The Man Who Knew: The Life and Times of Alan Greenspan (Penguin Press, 2016).
Listen to the conversation for insights on the career of former Federal Reserve Chairman Alan Greenspan, perhaps the most influential central banker of the 20th century, and on the ways in which we inhabit the financial world Greenspan helped forge. Listen, too, for the opportunity to receive a copy of The Man Who Knew.


text:
Confusion over Nottingham city centre road as thousands received bus lane fines
Some think more road signs are needed
Ellie Danemann
Canal Street in Nottingham where thousands of bus lane fines have been handed out (Image: Nottinghamshire Live/Marie Wilson)
Confusion over signs and out of date sat navs are among the reasons cited by drivers for them getting caught out in Nottingham's bus lane fine hot spot.
A total of 36,545 fines have been issued to drivers in Canal Street this year.
But Nottingham City Council says more than 99 percent of drivers are managing to observe the signs and travel around the area without being fined.
Canal Street, from the junction with Greyfriar Gate to Middle Hill, can only be used by buses and authorised vehicles, while regular traffic is re-routed towards Castle Boulevard or Wilford Road and onto the southern relief route.
The bus lane was installed in September 2020 as part of the pedestrianisation of Nottingham's southern gateway.
The breakdown exact locations on the road where the 36,545 drivers were caught is as follows: 30,328 fines at Canal Street near Albion Street East bound, 5,817 at Canal Street near Greyfriar Gate, 399 at Canal Street near Middle Hill had (East) and one at Canal Street near Middle Hill (West).
Cara Walters, 22, student, received a bus lane fine on Canal Street last month.
She said: "I was following my sat nav on my phone and the system hadn't updated to inform car users tat the road had become bus and taxi only.
"Frustratingly I saw other cars using the road so I presumed it was a normal route for drivers but I later realised it wasn't.
"Then I got a ticket in the post and a £60 fine.
"I've seen multiple other road users receive a fine in the same place, road signs indicating bus only are poor an scarce in the particular area.
"My main issue is the navigation - I think that needs to be sorted.
Bus lane enforcement at Canal Street in Nottingham (Image: Nottinghamshire Live/Marie Wilson)
"Road signs need to be a lot clearer - the area is so confusing and it is hard to focus on everything at once."
Mark Golds-Jones, 55, Lowdham, was driving in the city on April 8 for the first time since changes were made to the road network in September 2020, bringing in a bus-only lane on the busy Canal Street.
The fine was overturned on appeal.
Mr Golds-Jones said: "It's still chaos down there.
"That many signs and that many things that have changed when you're trying to drive and process it all, you can't it is too much.
"Signs are always good but its just the amount and positioning of them,
"It's a very weird situation.
"By the time I realised where I was it was too late to turn back or go anywhere.
"People should definitely appeal it.
"Most people will pay it because they're frightened not to."
A driver who wished to remain anonymous, also received a bus lane fine at Canal Street.
She said: "The layout of the road is incredibly confusing.
"I didn't see the road signs as I was too focused on the lay out of the road, I was just driving home.
"It was an honest mistake but I can definitely see why other people also received fines there."
But Nottingham City Council insists the majority of drivers don't have any issue with the new route.
A City Council spokesperson said: "We have removed or reduced traffic in central parts of the Broad Marsh area as part of a complete transformation of the area – with an exciting vision for the former shopping centre site only just announced. A safe and pleasant pedestrian-friendly destination for people to enjoy is being created by restricting access to pedestrians, buses, taxis and cyclists.
"This has involved moving traffic onto a route further out of the centre which is better suited for major traffic movements across the city. It may take time for people to adjust to the new road layouts, but we have put an appropriate amount of signage in place to alert motorists.
"We have put up some additional large electronic signs to provide further alerts about the bus lane. More than 99 percent of drivers are managing to observe the signs and travel around the area without being fined."
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text:
Bombing won't stop Redwood Summer - Issue 334, Summer, 1990 - Fifth Estate Magazine
Bombing won't stop Redwood Summer
by Luba
Fifth Estate # 334, Summer, 1990
(Ieft) Judi Bari and Darryl Cherney, victims of an assassin's bomb, playing earlier at a Redwood Summer benefit. (right) The car they were driving when the bomb exploded.
In a doubly bizarre set of circumstances, two California environmentalists experienced an assassination attempt followed by their arrest for "possessing" the device that almost killed them.
Judi Bari and Darryl Cherney, members of Earth First!, were victims of a car bombing Thursday, May 24, in Oakland, California. They were en route to Santa Cruz to continue preparations for the "Mississippi Summer in the Redwoods," a planned season of civil disobedience aimed at disrupting logging of old growth forests.
Long-time activists, the pair of Earth First! folksingers had been among the most committed and visible promoters of the summer action intended to be the environmental equivalent of the 1964 voter registration campaign in Mississippi. They worked to focus attention on the threatened thousand-year-old trees on the North Pacific coast. Only five percent or less of original growth forests remain in the lower 48 states of the U.S.
Darryl Cherney co-founded the Garberville chapter of Earth First! in 1986. He was also known for his performances and song writing.
Judi Bari, whose background was union organizing, joined Earth First! two years ago. She had worked as a carpenter, but most recently had organized an Industrial Workers of the World chapter at Georgia Pacific.
Bari was the driving force behind an alliance between Earth First! and local timber workers which created storms of controversy and denunciation in both camps. Many environmentalists were shocked by Bari's and others' repudiation of a well-known EF! tactic, tree spiking.
Tree spiking was originally conceived as a way to prevent cutting in spiked groves. More important than the actual spiking was the notifying of loggers (via letters. posted signs, etc.) that certain areas were spiked and unsafe for logging. It was not anticipated that logging companies would knowingly cut in spiked groves.
When the logging companies began sending their workers to cut down spiked trees, Bari questioned the utility of this tactic. A press release renouncing tree spiking, issued in April by the Northern California Earth First!, reflected Bari's view that " the real conflict is not between us and the timber workers, it is between the timber corporations and our entire community." Thus, a small but remarkable alliance between radical environmentalists and loggers was catalyzed by Bari.
The May 24th Bombing
Bari and Cherney were driving by the Oakland High School on Park Street when their car exploded.
Later, the Oakland Police bomb squad said it was "a crude device." It was made of two-inch pipe, using an ordinary safety pin to prevent accidental explosion and an alarm clock timer. They said it was an "anti-personnel" device, meaning it was strong enough to destroy flesh and bone, but not large buildings.
Recalling the incident, Cherney said his first thought was that another car had struck them. "But something didn't make sense. I had my seat belt on and had blood coming down from my head. I heard voices from outside, 'It's a bomb.' Then I realized someone tried to kill us."
It had been placed on the floor behind the driver's seat where Judi Bari sat. It fractured her pelvis, damaged her abdominal organs, and destroyed the soft tissues in between. She also sustained facial lacerations. She required emergency surgery and several days of intensive care.
Bari was barely conscious the night of the blast when she was placed under house arrest at Highland hospital.
Cherney, who was sitting in the passenger seat, suffered a head laceration and an abrasion to the cornea of one eye as well as perforated ear drums and hearing loss. That night, after release from the hospital, he was taken to Oakland city jail where he was interrogated by police and the FBI. He was held over 24 hours without access to legal counsel.
Both Bari and Cherney were charged with possession and transportation of explosives.
Cherney and Bari had attended the Bay area meeting with members of the Seeds of Peace collective, another group actively preparing for Redwood Summer. Without a search warrant, the police cordoned off two of their residences and entered. Eight collective members were handcuffed, searched and taken into custody.
After their release, the Seeds found their home ransacked. One member stated, "The contents of entire rooms were piled up in the middle of the floor. Let's put it this way: the earthquake last year did not do as much damage as the bomb squad." Files and notebooks were missing. When police left, every door in the house was left open. Cherney's van, parked by the Seed's residences, was also searched. Police took two boxes and exploded them in the street. Hundreds of feet of cassette recording tape were left hanging down nearby utility poles.
Later, on the north coast of California, friends prevented authorities from searching Bari's and Cherney's homes without warrants. Explosives were not found in any of the searches. The most suspicious evidence obtained consisted of several rolls of duct tape and a sack of finishing nails.
Policeman Mike Sims kept the media informed on their "investigation": "The evidence is strong that they were transporting the device. We're assuming the bomb was placed in the car by the occupants" (emphasis added).
Sims was on somewhat firmer ground when he later told reporters "We're making the assumption that if the bomb was built, it was meant to be used."
In essence, there was no investigation. As far as the police were concerned, the victims were "obviously" the guilty parties. The police found themselves with the opportunity to search the Seeds of Peace collective without a warrant and to arrest some activists.
If Bari and Cherney's guilt was "obvious" to the police, their innocence seemed equally obvious to themselves and scores of people who knew them. Cherney stated, "I'm innocent. I'm scared to death of explosives. I've never even lit a firecracker in my life." Bari has likewise consistently denied any knowledge of the bomb and all involvement with violent activities.
Bari's attorney, Susan Jordon said, "This is clearly attempted murder by someone who did not want them to do nonviolent protest in the Redwoods this summer. This charge is insane. There's a person out there who attempted murder. Why aren't they looking for that person?"
Indeed, there appears to be no one personally acquainted with the pair who finds any credibility in the police assertion that the bomb belonged to Bari and Cherney. During the entire four days and nights that Cherney was held in jail, a vigil of supporters waited outside on the street. Immediately after the bombing, many groups declared their support for Bari, Cherney and Redwood Summer.
Another Assassination Attempt
The lack of evidence linking the couple to the bomb, and the testimonials by acquaintances were not enough to cause the police and FBI investigators to question the source of the bomb. Nor were the many and varied death threats that Bari and Cherney had received in the last year.
Things had started getting rough last summer when a logging truck rammed Bari's car. Inside the car were Bari, her two children and another activist.
The police characterized it as "a typical traffic accident." Bari described how her car was hit "with no warning whatsoever: there was no squeal of the brakes, nothing. We were hit full force by a logging truck." A photo taken the day before shows the same truck unable to pass an EF! blockade in which Bari had participated.
On April 10, Bari received a letter stating, "Get out and go back where you came from. We know everything. YOU WON'T GET A SECOND WARNING." Two weeks later a photo of Bari with a gun site's cross hairs drawn on her head was pinned to the door of the Environmental Center at Ukiah. When Bari reported the incidents to the Mendocino County sheriff's office, she was told, "We don't have the manpower to investigate death threats like this. If you turn up dead, we'll investigate." The sheriff remains true to his word; no investigation has been started.
It seems that we must either consider a conspiracy theory, or be willing to believe that the authorities are very, very stupid. Both explanations have some merit. Adding even further to the confusion is a letter received on May 30 by the Santa Rosa Press Democrat. The three-page single-spaced typed document details the structure of the bomb. It opened by saying, "I built with these hands the bomb that I placed in the car of Judi Bari….Now all who would come to the forest and worship the trees like gilded idols have been Warned. They see the fate that awaits them." The letter's author wanted to "strike down" Bari after encountering her at an Operation Rescue anti-abortion counter-picket.
Many local activists believe the letter to be another fraud. The FBI called it "interesting evidence."
We may very well never know who tried to kill Cherney and Bari.
Judi Bari Still Hospitalized
Judi Bari remains under guard at Highland Hospital. If her pelvis heals well, she will require around six weeks of traction. She is still heavily medicated because of constant severe pain. She has no feeling in part of her leg and it is unknown if this will be permanent. She will have to undergo many more months of hospitalization and rehabilitation before it will be known if she will ever walk again.
In an interview on June 4th from her hospital bed, Bari appealed, "I hope people don't lose sight of what this is really about. It isn't about Judi Bari. It's about the state of the forests and the communities that are dependent on them."
Support Redwood Summer
Redwood Summer actions are starting now to defend the last of the old growth forests in the Northwest. Major actions are scheduled for June 20 at the export dock of the Louisiana Georgia lumber company near Eureka, Calif.; July 21 at the Georgia Pacific lumber company in Ft. Bragg; July 30 in the Sequoia National Forest in Southern California; and a concert—"Redwoodstock"—on a date to be announced.
Smaller actions will occur throughout the summer. Those interested in participating may write for an information packet at: SF EF!, POB 411233, San Francisco CA 94141 or call (415) 824-3841.
Donations to support the summer activities and for medical and legal expenses are desperately needed. Please send them to Redwood Summer, Sonoma County EF!, c/o Pam Davis, POB 7544, Santa Rosa CA 95407. Make checks payable to Redwood Summer.
"Redwood Summer: Anatomy of an Action," FE #335, Winter, 1990-9
"FBI Still Stalling on EF! Bombing Case," FE #352, Winter, 1999
Judi Bari website: http://www.judibari.org/
Fifth Estate's I.W.W. Resource Page.


Our bakery offers loaves of fresh-baked breads, (including low carb bread), rolls, desserts, pastries, and our signature cakes.
Our cafe offers a variety of delicious sandwiches served on our fresh baked bread, soup, cold beverages, organic fair trade coffee, and tea.
We are proud to offer a variety of Keto/Low Carb products!


We asked the VoiceOfHair™ community to help us find their favorite hairstylists in 10 major US cities. Below you'll find the "Best Stylists" in those cities, as voted by the VoiceOfHair™ community.
In addition, 5 randomly selected winners are going to win sponsored products from the brands below. (Winners are being notified via e-mail).


Using the mobile app, you can download single assets to your mobile device from the Widen Collective, then open them in the app they were created in or save them to your camera roll. When saved to your mobile device, assets can be quickly accessed and used when you're not connected to WiFi or the internet.
After your download is complete, the apps that are installed on your device will display, as well as a save option. Choose the app to open the asset or click Save Image and it'll be saved to your camera roll.


Ryse: Son of Rome is an Xbox One exclusive action-adventure game that was developed by Crytek. As a Roman general, you must fight as well as control your legion. The story is described as an epic tale of revenge.


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Q: Membership in two domains I would your suggestions for an effective solution for a person, who needs to access resources in two Windows domains and wants to use one computer. It's about our CEO, who has accepted a second position in another company.
Accessing files and folders isn't big problem. The greatest challenge I see is that he wants to conveniently access Exchange accounts in both companies; he would like to send and receive mail in single Outlook if possible (two profiles?) There is also a challenge with calendars: he would like to have one calendar for all activities from both Exchange accounts.
Creating a POP3 account for accessing second Exchange server is a last resort, because obviously there is a problem with scheduling meetings and other calendar related tasks.
Forwarding and receiving all mail/tasks on primary Exchange server is inconvenient because simple replying to original sender is disabled; and also when manually changing the recepient, he will receive mail from the wrong address.
We were considering Virtualisation, that is setting up an instance of virtual machine inside existing installation and then joining this virtual computer to a second domain. Then installing another MS Outlook. This would of course mean two different Outlook accounts, two different calendars, but would at least enable our CEO to access all information from a single laptop.
Does anyone have any other idea? I know setting up two domains on a single computer is a no-go (without much hacking at least), but effective workarounds are appreciate. The thing I am looking here is high usage/efficiency/productivity, but also as elegant solution from the administration point of view.
Thank you very much (if you managed to read this through, this is a good sign ^_^ )

A: Having to Exchange accounts on one machine is possible if you use 2 separate outlook profiles and he selects the one he wants when opening Outlook. However they are always going to remain 2 separate accounts, he's not going to be able to have 1 calendar for each account, or merge the email from both account if he is using MAPI to connect to both.
You could as you say pull email from one account into the other using POP, but you're then intermixing the email from two companies, and you then become responsible for storing and backing up this email, which is not a great idea and it's only going to solve the email problem, not calendaring. It also means that he loses the ability to access the global address list, and use internal distribution lists, etc.
I would suggest keeping them in 2 profiles and switching between when needed. If he really has to have access to both at the same time, you could get him to use Outlook for one and Outlook Web Access for the other, or set up a VM so he could have 2 copies of Outlook running (all a bit of a kludge really).

A: Two machines (virtual might do fine) would be the way I would suggest, mostly because mixing two different companies' information in one machine seems dangerous and might involve breaching policies and generally losing control (this situation should already be answered in existing IT policies for the two companies)...
Are both companies under the same umbrella with the same IT department and so forth? If not, I would stay away from trying to mix them in a single machine.... wishing for a single machine is one thing, a single machine being a good idea is a totally different question ^^
If it was me, I'd have him use a second laptop issued by the new company and not have it touch anything in the old company except for connecting to the new company through a guest network and VPN when situated at the old office... and vice versa. But then again, I'm a bit paranoid about these things. He'd then possibly grow tired of this at times and just get by using web site access to one or the other company for the simpler stuff like handling e-mail and calendars? ^^

A: Well, in Exchange you can give one user full access rights to another mailbox (from the Exchange Console), then from the local Outlook, you can load that additional mailbox.  However with a cross-domain environment, I think this can only be accomplished by creating a Trust between the domains.  You might have to research that part first.

redpajama_set_name: RedPajamaStackExchange


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Rachel Douglas
Rachel Douglas is currently enrolled at SOAS studying an MSc in the Politics of Conflict, Rights and Justice. She completed her BA in Politics at the University of Leeds in 2015, with an additional year of international study at the Universidad de Guadalajara, Mexico. She wrote her bachelors thesis on the failure of State Building in post invasion 2003 Iraq, linking to the continued violence, destabilisation and ultimately the rise of ISIS in the region.
After her BA, Rachel completed a number of internships including at a small London-based think tank, Project for the Study of the 21st Century, where she coordinated publishing think pieces and panel discussions on social, economic, political and cultural issues.
Prior to coming to SOAS, Rachel spent the last five years at an NGO supporting access to health planning services in East and Southern Africa. She has spent time living in Malawi, Tanzania and Kenya with frequent travel to other countries in the region.
Rachel's key areas of interest include peace building and violence prevention strategies, conflict resolution, and the intersection of these processes with women's and marginalised communities rights.
Contact: 695044@soas.ac.uk
Find out more on Linkedin


Former Florida Supreme Court Justices Against the Environment, Why?
It has been quite an education to serve on Florida's Constitution Revision Commission.
If you follow my blog, you might recall that during the 2017/18 Commission, I sponsored five public-submitted proposals, all environmental in nature: A Right to a Clean and Healthful Environment; the Department of Environmental Protection as a cabinet position; Clarifying the Water and Land Legacy; Florida Wildlife Commission/Wildlife Corridors; and No Oil and Gas Drilling in Florida's Territorial Seas.
Early on in my journey, with an outcry of controversy from the Associated Industries of Florida, the Florida Chamber, and the Florida Agriculture Coalition, I fought. It was a scathing experience, I will never forget.
With regard to A Right for a Clean Environment, former Chief Justice of the Florida Supreme Court, Kenneth Bell, was hired along with lawyers from U.S. Sugar Gunster Law Firm to destroy my proposal, which they did.
In the end, only one of my proposals made it to the ballot, supported by all by but one CRC vote that day. That proposal, now an amendment, is "No Oil and Gas Drilling in Florida's Territorial Seas," our state waters, ten miles off our west coast and three miles off the east. These waters belong to the people of Florida, not to the federal government.
And now Amendment 9 is under attack from yet another former Supreme Court Justice, Harry Lee Anstead! For me, it is of no consequence that my proposal is bundled with another environmental proposal.
I think it is repulsive that these men of influence, who raised their hand to represent fairly all the people of Florida now are spending their "golden years" off the bench getting paid by Big Corporate Interests to partake in the carnage of Florida's environment. Former power and influence should not be allowed to seduce the court.
Shame on you former Supreme Court judges, for getting paid by Big Business, Big Petroleum, Big Tobacco, and Big Gambling to try to influence the Florida Supreme Court to deny taxpayers of Florida the quiet enjoyment of their properties and the waters we all treasure.
More than likely we shall know by September 24 if the Florida Supreme Court decides to place CRC Constitutional Revision 9 on the 2018 ballot or not. In the meanwhile, hang in there and please keep fighting for Florida's precious environment.


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Mesa reports fiscal Q1 loss as Omicron and pilot attrition hit earnings
By Pilar Wolfsteller2022-02-10T01:09:00+00:00
Regional carrier Mesa Airlines reported a loss for the first quarter of its fiscal year as the company struggled with staffing issues stemming from the Omicron variant of Covid-19 as well as pilots leaving for larger carriers.
The Phoenix-based airline, which operates regional routes for United Airlines and American Airlines and flies cargo for DHL, says on 9 February that it lost $14.3 million during the three months that ended in December, the company's fiscal first quarter 2022, compared to a profit of $14.1 in the same quarter a year ago.
Revenue in the quarter was $147.8 million, down 1.7% from $150.4 million the carrier reported in the same period in 2020.
Mesa Airlines posts a loss as Covid-19 and pilot attrition cut into earnings
"Mesa's results reflect the impact of Covid to our quarter's operations and financials," says chief executive Jonathan Ornstein. "Its effect on this quarter was significant and unlike anything we have seen in twenty years. This was further impacted by elevated pilot attrition as the major and national airlines have accelerated hiring."
Pilot attrition is an increasing problem at regional carriers, which fly smaller aircraft and pay lower salaries than the legacy carriers. Since passenger demand began to rebound last summer, demand for pilots has grown across the board, and regional airlines are taking the biggest blows. Pilots are leaving the regionals in greater numbers to fill seats left vacant by pilots who retired from legacy carriers amid the coronavirus-driven aviation slump of 2020-2021.
"Looking ahead, we are cautiously optimistic that we are already seeing a decrease in Covid-related absence rates. Managing through the challenges of pilot attrition in our core regional operation remains our team's top priority," he adds.
"One piece of good news is that demand for our products have never been stronger and our primary challenge will be meeting that demand."
Ornstein is hoping the Omicron wave is now in the rear-view mirror, and the company can normalise its operations. During the peak infection period, Mesa had up to 23% of its employees call in sick, he says. "There were days we had to cancel more flights in a day than in the last 12 months."
As a result, executives say they expect to book 15-20% fewer block hours in the current three-month period, fiscal Q2, than in last quarter. By the end of 2022, block hours will be 5-10% lower than at the end of 2021.
At the end of the quarter that ended in December 49% of the company's total revenue was derived from contracts with United, 45% from American, 1% from DHL, and 5% from leases of aircraft to a third party.
At the end of 2021, Mesa operated a fleet of 167 aircraft, comprising 80 Embraer 175s, 20 MHIRJ CRJ700s, 40 CRJ900s, and two Boeing 737-400F aircraft. 25 aircraft remained in storage. Mesa has approximately 402 daily departures.
Building the company's nascent cargo business will be a priority for the carrier going forward, says Ornstein. Therefore the company will add a third Boeing 737-400F to its fleet this month, which will be operated for DHL.
"The cargo business is a very important growth area," he says. "We have only done it for about a year, we used that to prove ourselves, and it's a good indication of our potential that we can grow that."
On 26 January Mesa said it intends on taking 150 of Elroy Air's Chaparal C1 hybrid-electric vertical take-off and landing heavy-cargo aircraft to support that growth and serve the express parcel and healthcare sectors.
"We are increasingly seeing the demand for same and next-day delivery, but so many rural communities have been cut off from the national transportation system," Ornstein said at the time. "With the Chaparral, we're excited to be able to provide autonomous cargo delivery to help reconnect those communities."
Mesa to sell 18 Bombardier CRJ700 regional jets to United
Mesa Air Group, the parent company of regional carrier Mesa Airlines, will sell 18 Bombardier CRJ700 aircraft to United Airlines for "approximately $50 million".
Mesa posts fiscal Q3 loss amid increasing pilot shortage issues
Mesa Air Group, the parent company of Mesa Airlines, posted a loss for its fiscal third quarter as the company's block hours fell and it continues to suffer from the pilot shortage.
Mesa fiscal second quarter 'very disappointing'
Mesa Airlines, which flies regional jets for United Airlines, American Airlines as well as Boeing 737 freighters for DHL, continues to suffer under pilot attrition, leading it to post a loss for the second quarter of fiscal 2022.
More Strategy
Scott Sheldon steps down as president of Allegiant Air
Allegiant Travel Company, parent of Allegiant Air, has accepted the resignation of co-president and chief operating officer Scott Sheldon, prompting a reshuffling of the airline's executives.
Italy signs letter of intent with Lufthansa to kick-off ITA stake talks
Italy's government has signed a letter of intent with Lufthansa as it opens talks with the carrier over the acquisition of ITA Airways.
Varadi hopeful on improved Wizz operational resilience
Wizz Air chief executive Jozsef Varadi believes air traffic control delays are likely to provide the biggest challenge this summer as the carrier bids to avoid a repeat of the operational disruption it suffered last year.


While many younger patriots might be unaware of who Charlie Daniels is . . . that is not the case for those who proudly serve our great country and fellow citizens in the military. For years this country music legend and his band have been entertaining the troops wherever their tour of duty might take them including Iraq and Afghanistan.
Charlie Daniels does so out of love and pride of country and endearing respect for our brave troops. Nor is it about self, which is a noble trait shared by other living and past celebrity patriots like Bob Hope, Wayne Newton, Ann-Margret, John Wayne, Gary Sinise, Fred Travalena, Lee Ann Womack, Chuck Norris, and others listed in Stars... There For The Troops.
Charlie Daniels and David Bancroft, founder of USA Patriotism!
David Bancroft, Charlie Daniels, and Mike Bancroft, Marine / Iraq War veteran You can easily see it in Charlie Daniels' expressions and hear it in his voice when he talks about the troops. And it is so very evident in the associated DVD that comes with the purchase of The Charlie Daniels Band's CD, Live From Iraq.
In fact, watching the DVD stirred this father of a Marine veteran to write my poem, Patriot Charlie Daniels, which I was able to present a signed copy to Charlie before his concert in Houston, Texas on September 1, 2007.
What made my meeting Charlie Daniels especially memorable was being able to introduce him to my youngest son, who served in Iraq.
Then there is Charlie Daniels, the performer. He and his band are excellent musicians and really know how to entertain . . . You also witness his love of the USA and pride for our treasured troops.
You can learn more about Charlie Daniels' patriotic efforts at his recognition page that includes a link to his website.


The No. 18 Mississippi State Bulldogs are coming to town for what's sure to be a great football matchup, but how will the Wildcats fare against a team that has one of the best defensive lines in college football?
First-year head coach Joe Moorhead leads a talented team that finished 9-4 last season under former head coach Dan Mullen.
The fact that they are one of the biggest and strongest defensive lines K-State will see will likely be enough to stymie K-State's offense, but they also run aggressive and complicated blitzing schemes.
An overlooked part of out-of-conference matchups is the physical makeup of a team's offense and defense. For example, defensive coordinator Dan Shoop (and his predecessor, Todd Grantham) has designed a defense to do well in the SEC.
SEC defenses are built differently than Big 12 defenses because of what SEC offenses are built to do. SEC defensive coordinators see a lot more power running and running spread-type offenses than Big 12 defensive coordinators.
While that difference may not show up as much in actual schemes—there are 4-3 base defenses in the Big 12—it does show up in the types of players that fill those positions.
Mississippi State has big, physical, run-stopping type linebackers and lineman as opposed to the typical Big 12 defense, which is focused on stopping the quick pass-first offenses.
On offense, Mississippi State will run a spread offense with an excellent quarterback at the helm: fifth-year senior Nick Fitzgerald.
Fitzgerald, who sat out last week due to a one-game suspension, is a powerful runner and a more-than-capable passer. K-State head coach Bill Snyder compared him to former Wildcat great Collin Klein, which seems to be pretty accurate.
Fitzgerald is not afraid to tuck the ball and run it, or take a designed run and run a defender over. He is tough to take down and a little shiftier than Klein was. In the passing game, he flashes excellent accuracy and a strong arm.
He's the kind of quarterback that gives conservative defenses fits because if they try to sit back and defend the pass on third and middle distances he will easily just wait for a hole and get the first down with his legs.
K-State will need to get pressure on him, but keep him contained if they want to have a chance to stop him on offense. They cannot let him scramble out of the pocket or sit and tear them apart.
The Mississippi State offense presents a number of issues for K-State. The biggest may be that it is a little different from what Mullen's teams ran and there is almost no film of what it may look like with Fitzgerald under center.
K-State's coaching staff will have trouble evaluating what schemes will be used with Fitzgerald and if they are struggling to predict it this week, they will struggle to stop it this weekend.
What they do know is that they put up nine touchdowns on an overmatched Stephen F. Austin team with backup quarterback Keytaon Thompson in the game.
Further, each coaching staff has unknowns to deal with heading into this game. Moorhead does not know who will play at quarterback or what K-State's offense will look like. Snyder has to prepare for a quarterback and system that there is no film on.
It is possible the Snyder was being intentionally vanilla on offense and defense to limit what was on tape for Moorhead. The head games that have gone into this game will make it an exciting chess match.
K-State offensive line versus Mississippi State defensive line — K-State's offensive line, headed by senior Dalton Risner, was considered one of the best heading into the season, but it looked bad against South Dakota.
This will be an interesting test for K-State's offensive line. K-State's only chance to win is if that offensive line can open up running holes for junior running back Alex Barnes and whoever is playing quarterback at that particular time.
If K-State is not drawing extra defenders to stop its run game, they will struggle to move the ball. The Wildcats do not have a quarterback or receiving corps that is talented enough to move the ball only through the air.
K-State's only chance is if Fitzgerald is rusty and the Wildcats are on fire on offense. They have to avoid all of the mistakes that they made against South Dakota as well. The Wildcats were negative-four in the turnover margin and were penalized for over 100 yards. I do not think any they will be able to turn all of it around and Mississippi State will win this one going away, 35-28.


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40 Days Snake In The Grass
After fourteen days, many who remain are struggling to adapt to their environment and each other. Some consider migrating to try and find new resources, while in other camps tension continues to rise and may lead to our second tap out of the season.
40 Days Jungle Rich
After nearly three weeks in the bush, two survivalists have tapped out, most others are making desperate treks across the savannah in search of food. If they can't find sustenance, they'll be forced out of the biggest challenge of their lives.
40 Days Death March
The 10 remaining survivalists cross the 21-day milestone and enter into uncharted waters. Scarce resources send the survivalists on a collision course. They fight for their lives with a huge electric eel and are visited by a gigantic puma.
40 Days Sacrifice
With 15 days to go in the 40-day challenge, the ten remaining survivalists - dehydrated and protein starved - find themselves converging at dangerous Piranha Lake where they will they compete over scarce food and water.
40 Days Falling Apart
After thirty-one days in the Colombian jungle, the pressure is on to get the nourishment they need to survive extraction, but when one member doesn't play by the rules, the future of one group is thrown into jeopardy.
40 Days Filth and Fury
The final, and most difficult, part of the 40-day challenge begins. Jeff & EJ decide to attempt their extraction alone, and relationships grow contentious between both teams as they navigate the treacherous landscape to try and finish the challenge.
Dirty Dozen Return
The Dirty Dozen survivors from Naked and Afraid XL come together for the first time since their life-changing 40 day challenge. They'll discuss good times, hard times, and try to heal unresolved issues.
Season 2 Preview: Mission Impossible
In a special one-hour preview, 12 Naked & Afraid veterans prepare for the most formidable survival challenge on the planet - surviving 40 days and 40 nights in the sun-scorched bush of South Africa.
Lions at the Gate
12 Naked & Afraid veterans put their lives on the line and begin their 40 day & 40 night challenge in the sun-scorched bush of South Africa during the middle of a drought, while alongside the deadliest predators on the planet. Will even one survive?
On the drought-stricken plains of South Africa, the remaining survivalists enter the second week of their brutal 40-day challenge. A lack of water leads one group to venture off to find others and another group goes hunting again for big game.
Human Prey
Only two weeks in and South Africa is already taking a huge toll on our survivalists, mentally and physically. Frustrations between survivalists explode, deadly leopards circle, and cases of tick-bite fever threaten to take some out of the challenge.
Too Many Chiefs
The Survivalists approach the halfway point of their brutal challenge. With starvation & sickness worse than ever they must come together to help one another, but the battle for Alpha Male distracts them from their strategy and may be their downfall.
Steven fails to return with Darrin from their two-day hunting expedition, leaving the group with no choice but to break camp in a desperate search for answers.
After moving their camp to Pondo Ravine on Day 25, the nine remaining survivalists are plagued by extreme hunger and issues of trust. Meanwhile, tragedy strikes when Ryan separates from the group
The Sickness
In a 24-hour period, the contaminated fruit of a single tree sets off a shocking chain of events. As panic and illness spread through the group it's questionable whether anyone will make it to Day 40.


With over 30 years of accountancy experience, Karen has held a variety of senior positions at the Audit Commission, the body formerly responsible for protecting the public purse.
In January 2013, she was appointed as Comptroller and Auditor General for Jersey and is responsible for providing independent assurance to the State of Jersey that the island's public finances are being regulated, controlled and accounted for in accordance with law.
Non-executive appointments to NHS trusts made by NHS Improvement are subject to the Code of Practice of the Commissioner for Public Appointments. Karen takes up her role of non-executive director with the Trust from 07 January 2019 for a four year term; she is entitled to receive a remuneration of £6,157 per annum. Karen has no declared political activity in the last five years.
This entry was posted on Tuesday, January 15th, 2019 at 3:21 pm	and is filed under News.


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Former President George H.W. Bush named 2016 Shell Legacy Award winner
Atascocita Observer // Atascocita Sports
April 3, 2016 Updated: Oct. 5, 2016 3 a.m.
L-R back: Marvin Odum, Roger Penske, PGA TOUR Commissioner Tim Finchem, Secretary James A. Baker III. L-R front: Former First Lady Barbara Bush and President George H.W. Bush
Shell Oil Company honored former President George H. W. Bush on Tuesday with the 2016 Shell Legacy Award, a recognition that celebrates individuals who share in the Shell Houston Open's legacy of charitable giving. As a noted philanthropist, avid Houston sports fan and longtime supporter of the Shell Houston Open, Bush was a natural choice to receive the award. The occasion was especially significant as this year marks the 25th anniversary of the Shell Houston Open.
Bush served as the 41st president of the United States from 1989-1993. Since leaving office, Bush has tirelessly helped raise hundreds of millions of dollars for a multitude of charities. Locally, Bush is a Life Member of the The University of Texas MD Anderson Cancer Center board of visitors. He also founded national nonprofit Points of Light, where he serves as honorary chairman. Points of Light was created in 1990 as an organization to promote the same spirit of volunteerism that Bush described in his 1989 inaugural address; the group mobilizes people to take action and change the world. Bush often references "one thousand point of light" when speaking on community organizations spreading "like stars" across the nation. He also takes an active role in the George Bush Presidential Library and Museum and the Bush School of Government and Public Service, both located on the campus of Texas A&M University.
Locally, Bush sits as the honorary chair emeritus of The First Tee of Greater Houston, a longstanding beneficiary of the tournament that promotes youth character development. Most recently, Bush serves on the honorary committee for the Gus Wortham Park Golf Course restoration project, a major initiative of the Houston Golf Association to revitalize Houston's East End and support The First Tee.
"I will always be grateful for the many opportunities I have had to serve our wonderful country both in office and, just as importantly, as a private citizen," Bush said. "To be recognized by a company I respect and that does so much to touch and lift lives in our community makes this award very personal and meaningful. I can only hope it was not a close vote for the nominating committee!"
In recognition of his work inside and outside of office, Bush was bestowed the Presidential Medal of Freedom, the highest civilian award in the United States, by President Barack Obama in February 2011. Bush joins the ranks of his wife, former first lady Barbara Bush, who was awarded the Shell Legacy Award in 2009.
"It is such a privilege to recognize an individual who has given so much of himself," said Marvin Odum, president of Shell Oil Company. "Tonight, we truly celebrate all that President Bush has done not only for Houston, but for our country. His legacy is one we can all admire."
The Shell Legacy Award celebrates individuals who have greatly impacted the community. The award was established in 2008 and previous recipients include: the late Dr. Charles Sifford, World Golf Hall of Fame member (2008); Barbara Bush, former first lady (2009); Emmitt Smith, NFL Hall of Fame member (2010); James A. Baker III, former secretary of state (2011); Clay Walker, nationally acclaimed recording artist and humanitarian (2012); George Foreman, two-time world heavyweight champion (2013); Brian Ching, former Houston Dynamo and philanthropist (2014); and Herm Edwards, former NFL coach and player (2015).


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The following is a list of films produced in the Kannada film industry in India in 2003, presented in alphabetical order.

List of Kannada film released

January - June

July - December

References

External links
 Kannada Movies of 2003 at the Internet Movie Database

2003
Lists of 2003 films by country or language
Kannada

redpajama_set_name: RedPajamaWikipedia


Stir-fried thin rice noodles with shrimp, bean curd, bean sprouts, scallions, egg, ground peanuts and tamarind traditional seasoning.
Slices of tender chicken sauteed with peppers garlic, onion and fresh basil leaves.
Crispy roasted semi-boneless duck, served in a special chu-chee curry sauce, flavored with pineapple and basil.
Boldly spiced chicken sauteed with vegetable medley, exotic spices and herbs.
A choice of hot and spicy green curry sauce or a milder red curry with coconut milk, bamboo shoots, green bean and fresh basil with chicken, beef or shrimp.
Stir-fried jumbo rice noodles with onion, tomatoes, basil, and spicy seasonings with chicken, beef, shrimp or seafood.
Fresh Thai basil stir-fried with jasmine rice, bell peppers, onion, garlic then top with crispy basil with chicken, beef or shrimp.
Stir-fried shrimp with fresh mango, ginger, onion, bell peppers, carrots and green beans in brown sauce.
Sautéed seafood medley, shrimp, scallops, and green shell muscles with lemon grass, basil, onion and peppers in a delicious spicy sauce.