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Oligomeric and fibrillar species of β-amyloid (Aβ42) both impair mitochondrial function in P301L tau transgenic mice
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DOI: 10.1007/s00109-008-0391-6
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- Eckert, A., Hauptmann, S., Scherping, I. et al. J Mol Med (2008) 86: 1255. doi:10.1007/s00109-008-0391-6
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We recently provided evidence for a mitochondrial dysfunction in P301L tau transgenic mice, a strain modeling the tau pathology of Alzheimer's disease (AD) and frontotemporal dementia (FTD). In addition to tau aggregates, the AD brain is further characterized by Aβ peptide-containing plaques. When we addressed the role of Aβ, this indicated a synergistic action of tau and Aβ pathology on the mitochondria. In the present study, we compared the toxicity of different Aβ42 conformations in light of recent studies suggesting that oligomeric rather than fibrillar Aβ might be the actual toxic species. Interestingly, both oligomeric and fibrillar, but not disaggregated (mainly monomeric) Aβ42 caused a decreased mitochondrial membrane potential in cortical brain cells obtained from FTD P301L tau transgenic mice. This was not observed with cerebellar preparations indicating selective vulnerability of cortical neurons. Furthermore, we found reductions in state 3 respiration, the respiratory control ratio, and uncoupled respiration when incubating P301L tau mitochondria either with oligomeric or fibrillar preparations of Aβ42. Finally, we found that aging specifically increased the sensitivity of mitochondria to oligomeric Aβ42 damage indicating that oligomeric and fibrillar Aβ42 are both toxic, but exert different degrees of toxicity.
KeywordsAlzheimer's disease Amyloid aggregates Amyloid β-peptide Amyloid toxicity Fibrils Frontotemporal dementia Globulomer Mitochondria Oligomer Protein aggregation Respiration Tau Transgenic mice
β-Amyloid (Aβ)-containing plaques and tau-containing neurofibrillary tangles (NFTs) are hallmarks for brain lesions in both familial and sporadic cases of Alzheimer's disease (AD); however, how these lesions and their proteinaceous components impair cellular functions and ultimately lead to neuronal cell loss is only partly understood [1, 2, 3]. Attempts to gain insight into pathogenic mechanisms, and eventually, to develop therapeutic strategies have been greatly assisted by experimental animal models that express mutant forms of AD-associated genes [4, 5].
In AD, pathogenic mutations have been identified in both the gene encoding the precursor of the Aβ peptide, APP, itself and in the PSEN genes which encode part of the APP–protease complex. No mutations have been identified in the MAPT gene encoding the microtubule-associated protein tau [5]. Although AD is the most prevalent form of dementia at high age, NFTs are, in the absence of Aβ plaques, also abundant in additional neurodegenerative diseases, including frontotemporal dementia (FTD). In familial cases of FTD (FTD with Parkinsonism linked to chromosome 17 (FTDP-17)), exonic and intronic mutations have been identified in the MAPT gene which lead to the formation of tau aggregates in the brain [6]. Gallyas silver impregnation techniques are frequently employed to visualize NFTs in both AD and FTD brains [7].
The P301L tau mutation was among the first to be identified in FTDP-17 [8]. It was expressed by us in neurons of transgenic mice [9]. P301L tau transgenic mice develop NFTs that contain aggregated forms of hyperphosphorylated tau and show age-related memory impairment [10, 11]. Whereas transgenic mice expressing human APP with mutations found in familial cases of AD develop Aβ plaques, they fail to form NFTs. On the other hand, the P301L transgenic pR5 mice model aspects of the tau pathology of AD and FTD, but lacks plaque pathology. Both pathologies have been successfully combined either by crossing the respective transgenic strains or by establishing so-called triple transgenic mice [12] (reviewed in [13]). We addressed the pathogenic relationship of Aβ plaques and NFTs by an alternative approach that is by injecting fibrillar preparations of Aβ42 stereotaxically into the somatosensory cortex and the hippocampus of P301L and wild-type human tau transgenic mice and non-transgenic littermate controls. As shown previously, this caused a fivefold increase of NFTs in the amygdala of P301L transgenic, but not wild-type tau transgenic or control mice [14]. NFT formation was correlated with the phosphorylation of the pS422 and AT100 epitopes of tau [9, 15]. These findings could be confirmed by us in vitro, using neuronally differentiated SH-SY5Y neuroblastoma cells [16].
Subsequently, in addition to transcriptomic approaches [17, 18], we performed a proteomic and functional analysis of P301L tau transgenic mice which revealed a mitochondrial dysfunction together with a reduced electron transport chain complex I activity [19]. We also provided first evidence for an increased vulnerability of P301L tau mitochondria towards fibrillar Aβ insult, suggesting a synergistic action of tau and Aβ pathology on the mitochondria [19, 20, 21].
In recent years, attempts have been undertaken to identify the toxic species of Aβ. The focus of attention has since shifted from fibrillar to oligomeric species as the large, insoluble Aβ deposits which form the amyloid plaques in the limbic and association cortices of AD patients are in equilibrium with small, diffusible Aβ oligomers that appear capable of interfering with hippocampal synaptic function and memory [22]. Thus, in this study, we determined whether oligomeric Aβ42 would already exert a pronounced toxicity towards P301L tau transgenic mitochondria, compared to disaggregated (mainly monomeric) and fibrillar Aβ.
Materials and methods
Transgenic mice
The transgenic mice used in the present study express the human pathogenic mutation P301L of tau together with the longest human brain tau isoform (htau40) under control of the neuron-specific mThy1.2 promoter [9]. The htau40 isoform contains exons 2 and 3 as well as four microtubule-binding repeats (2+3+4R, human tau40). Pronuclear injections were done into C57Bl/6 × DBA/2 F2 oocytes to obtain founder animals that were back-crossed with C57Bl/6 mice to establish transgenic lines as described [23]. P301L tau mice show tau hyperphosphorylation already at 3 months [9]. NFT formation starts at 6 months of age [14]. Consistent with our previous studies [19], the mice were analyzed at 13–15 and 21–22 months of age. The mice were maintained in a 12-h dark–light cycle with pelleted food and water ad libitum. Animals were handled according to the Swiss guidelines for animal care. The experiments were conducted in accordance with international standards on animal welfare as well as being compliant with local regulations.
Monomer, oligomer, and fibril preparation
The Aβ42 synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland) was suspended in 100% 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) at 6 mg/ml and incubated for complete solubilization under shaking at 37°C for 1.5 h as described previously [24].
Monomeric preparation [24]
To obtain the monomeric preparation, 30 mg HFIP-treated Aβ42 was resuspended in 132 μl dimethylsulfoxide (DMSO) at RT for 10 min. A 60 ml buffer (20 mM NaPi, 140 mM NaCl, and 0.1% Pluronic F68 pH 7.4) was added and the mixture stirred for 1 h at RT. Following a 20 min centrifugation at 3,000×g, the supernatant was discarded and the pellet resuspended in 6 ml buffer. Then, 34 ml of H2O was added and the mixture stirred for 1 h at RT. Following a second centrifugation for 20 min at 3,000×g, the supernatant was stored as 5 ml aliquots at −80°C.
Oligomeric preparation [24]
To obtain the oligomeric preparation, HFIP was removed by evaporation in a SpeedVac and Aβ42 resuspended at a concentration of 5 mM in DMSO and sonicated for 20 s as described [16]. The HFIP-pretreated Aβ42 was diluted in phosphate-buffered saline (20 mM NaH2PO4, 140 mM NaCl, pH 7.4) to 400 μM and 1/10 volume 2% sodium dodecyl sulfate (SDS; in H2O) was added (final concentration of 0.2% SDS). An incubation for 6 h at 37°C resulted in the 16/20-kDa Aβ42 globulomer (short form for globular oligomer) intermediate. The 38/48-kDa Aβ42 globulomer was generated by a further dilution with three volumes of H2O and incubation for 18 h at 37°C. This was followed by centrifugation for 20 min at 3,000×g and concentrating the supernatant to 1.8 ml by dialysis against 5 mM NaPi, 35 mM NaCl pH 7.4 overnight at 6°C with a 30-kD centriprep and subsequent centrifugation of the concentrate for 10 min at 10,000×g. The supernatant was then stored in 100 μl aliquots at −80°C.
Fibrillar preparation adapted from previously described methods [16, 25]
To prepare fibrillar Aβ42, the peptide was dissolved in Tris-buffered saline pH 7.4 (TBS) at a concentration of 0.5 mM and stored at −20°C. (All aqueous solutions were prepared with deionized and filtered water (Millipore)). The stock solution was diluted in TBS to a concentration of 50 μM and incubated at 37°C with gentle agitation for 24 h to obtain aged, aggregated preparations of Aβ42.
Visualization of Aβ preparations on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels
The samples were analyzed using Tris-glycine 4–20% gradient SDS-PAGE gels and stained with Coomassie Brilliant Blue R250 according to [24]. Gels were scanned with an Epson 4180 scanner.
Structural characterization of Aβ aggregates
For negative staining analysis, 4 μl of the sample was placed on copper grids covered with formvar and carbon and counterstained with 2% uranyl acetate, using the droplet technique [26]. Specimens were examined in a Zeus 902 transmission electron microscope operated at an acceleration voltage of 80 kV.
Thioflavine T (ThT) spectra were recorded at room temperature with a Shimadzu RF-5301PC fluorimeter, using an excitation wavelength of 482 nm and a cuvette with 5 mm path length. Samples contained a final concentration of 20 μM ThT and 5 μM Aβ42 in 5 mM NaPi/35 mM NaCl, pH 7.4 (globulomers) or TBS, pH 7.4 (fibrils).
Congo Red (CR) absorption spectra were recorded at room temperature, using an Analytik Jena Specord 210 spectrophotometer. Samples contained a final concentration of 10 μM CR and 15 μM Aβ42 in 5 mM NaPi/35 mM NaCl, pH 7.4 (globulomers) or TBS, pH 7.4 (fibrils).
For attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, Aβ42 fibrils were concentrated by centrifugation for 30 min at 500,000×g and 25°C in a Beckman Optima TLX ultracentrifuge, using a Beckman TLA-120.2 rotor. Subsequently, the fibril pellet was resuspended in a small volume of TBS, pH 7.4 by pipetting and vortexing. ATR-FTIR spectra of 1.9 mM Aβ42 globulomers in 5 mM NaPi/35 mM NaCl, pH 7.4 and 1.1 mM Aβ42 fibrils in TBS, pH 7.4 were recorded as described [27].
Brain tissue preparation for mitochondrial analysis
Cellular preparations were obtained to determine the mitochondrial membrane potential (MMP) and mitochondria and to determine respiration rates as previously described [19, 25]. For that, mice (six pairs of 13–15-month-old and six pairs of 21–22-month-old hemizygous P301L tau and WT control mice) were sacrificed by decapitation and brains quickly dissected on ice. The cerebellum and one cortical hemisphere (the other hemisphere was directly used for preparation of isolated mitochondria for mitochondrial respiration) were separately minced into 1 ml of medium I (138 mM NaCl, 5.4 mM KCl, 0.17 mM Na2HPO4, 0.22 mM K2PO4, 5.5 mM glucose, 58.4 mM sucrose, pH 7.35) with a scalpel and further dissociated by trituration through a nylon mesh (pore diameter 1 mm) with a pasteur pipette. The resulting suspension, which contained both neuronal (about 72%) and glial cells (about 26%), was filtered by gravity through a fresh nylon mesh with a pore diameter of 102 μm, and the dissociated cell aggregates were washed twice with medium II (110 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl2·H2O, 1 mM MgCl2·6 H2O, 25 mM glucose, 70 mM sucrose, 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pH 7.4) by centrifugation (400×g for 3 min at 4°C). One hundred microliters of the suspension were used for protein determination. After centrifugation, cells were resuspended in 3 ml DMEM, and then aliquots of 100 μl were distributed per well in a 48-well plate for measurement of the mitochondrial membrane potential. The preparations of cerebellar and cortical cells from P301L tau transgenic mice and WT littermate controls (cross-over design) were made within 2 h under the same conditions and in parallel and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. Viability was found to be >90% using the MTT assay and trypan blue stain exclusion test. Data are expressed as fluorescence units per milligram protein.
Determination of the mitochondrial membrane potential
The membrane potential of the inner mitochondrial membrane was measured using the rhodamine 123 (Molecular Probes, Leiden, Netherlands) dye added to the cell culture medium at a final concentration of 0.4 μM for 15 min. Cells were washed twice with Hank's balanced salt solution (Sigma, Germany), and fluorescence was determined with a Victor2 multiplate reader (Perkin Elmer, Rodgau-Jügesheim, Germany) at 490/535 nm (Ex/Em). Loading capacity of the dye within the membrane decreases when the mitochondrial membrane potential declines after damage, e.g., exposure to Aβ42. For the secondary insult with Aβ42, cells were incubated for 4 h with the different types of preparation as described above. For this purpose, monomeric and oligomeric preparations were handled with specific care to avoid destabilization of conformation. Thus, frozen aliquots were quickly thawed and immediately diluted to the final assay concentration (maximal incubation time at 37°C for 4 h only within the nanomolar concentration range ≤100 nM). Since pre-experiments had shown that a maximum effect of fibrillar Aβ42 with regard to a reduction of MMP was reached at a concentration of 50 nM whereas 100 nM did not further decrease MMP (data not shown), the latter concentration was omitted due to limited brain material.
Preparation of isolated mitochondria
Mice were sacrificed by decapitation and one brain hemisphere was rapidly dissected on ice and washed in ice-cold buffer (210 mM mannitol, 70 mM sucrose, 10 mM HEPES, 1 mM ethylenediamine tetraacetic acid (EDTA), 0.45% BSA, 0.5 mM DTT, protease inhibitor cocktail (Complete tablets, Roche Diagnostics). After removing the cerebellum and one cortical hemisphere for the determination of the membrane potential (see above), the second cortical hemisphere was homogenized in 2 ml buffer with a glass homogenizer (10 to 15 strokes, 400 rpm) followed by centrifugation at 1,400×g for 7 min at 4°C, to remove nuclei and tissue particles. This low speed centrifugation step was repeated once with the supernatant. Then, the supernatant fraction was centrifuged at 10,000×g for 5 min at 4°C to pellet mitochondria. The resulting pellet was resuspended in 1 ml ice-cold buffer and centrifuged again at 800×g for 3 min at 4°C. Finally, the mitochondria-enriched supernatant was centrifuged at 10,000×g for 5 min at 4°C to obtain a mitochondrial fraction. This fraction was resuspended in 100 μl of ice-cold buffer and stored at 4°C until use, followed by determination of protein content.
Mitochondrial respiration
The rate of mitochondrial respiration was monitored at 25°C using an Oxygraph-2k system (Oroboros, Innsbruck, Austria) equipped with two chambers and DatLab software. Mitochondria (0.5 mg) were added to 2 ml of a buffer containing 65 mM sucrose, 10 mM potassium phosphate, 10 mM Tris–HCl, 10 mM MgSO4, and 2 mM EDTA (pH 7.0). State 4 respiration was measured after adding 40 μl malate/glutamate (240 mM/280 mM; assay concentration 4.8/5.6 mM). Then, 10 μl adenosine diphosphate (ADP; 100 mM; assay concentration 0.5 mM) was added to measure state 3 respiration. After determining coupled respiration, 1 μl carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.1 mM; assay concentration 0.05 nM) was added to the reaction chamber, and respiration measured in the absence of a proton gradient. To inhibit complex I activity, a total volume of 3 μl (2 + 1 μl) rotenone (0.1 mM; final concentration 0.15 nM) was added. Then, 10 μl succinate (1 M; final concentration 5 mM) was added and complex II-dependent respiration was measured. Finally, 8 μl KCN (0.5 M; assay concentration 2 mM) was added to inhibit complex IV activity. P301L tau transgenic and WT mitochondria were measured in parallel using the same conditions (cross-over design in the two chamber system). On a routine basis, the intactness of mitochondria was confirmed by addition of cytochrome c (10 μM). To test the effect of Aβ42 on mitochondrial respiration, isolated mitochondria were exposed to different types of Aβ42 preparations (5 μM) or vehicle on ice. Then, the mitochondria suspension was added into the chamber (final concentration of Aβ42 preparation 100 nM).
Statistical analysis
Data are represented as mean ± SEM. For statistical comparison, two-way ANOVA followed by post hoc t test was used. Only p values less than 0.05 were considered statistically significant.
We previously reported a mitochondrial dysfunction in P301L tau transgenic mice that is not caused by alterations in numbers of mitochondria [19]. Furthermore, in aged P301L tau mice, we had found a significantly reduced state 3 respiration, and thus markedly reduced respiratory control ratio compared to age-matched WT mice. First evidence was provided that a secondary insult with Aβ42 caused a higher reduction in membrane potential in P301L tau mitochondria than in WT. Here, we aimed to determine the relative toxicity of oligomeric compared to fibrillar Aβ42 with regards to mitochondrial function, by testing disaggregated (mainly monomeric), oligomeric, and fibrillar preparations of Aβ42.
Characterization of Aβ42 species
The oligomeric preparation with an apparent molecular weight of 50 kDa was generated by a series of concentration, centrifugation, and dialysis steps. Figure 1b shows a Coomassie Blue staining of an SDS-PAGE gel after the initial preparation of Aβ42 (10 μl, lane 2), after concentration (0.5 μl, lane 3), after concentration and dialysis (0.5 μl, lane 4), and the flow through (10 μl, lane 5). After concentrating the sample (lanes 3 and 4), the preparation mainly consisted of oligomeric Aβ42, with monomeric Aβ42 and trimers and tetramers being only a minor contaminant. Negative staining electron microscopy revealed fibril-free preparations of spherical aggregates with a diameter of 2 to 5 nm (Fig. 1c). Aβ42 globulomers bind weaker to the fluorescent amyloid-specific dye thioflavine T (ThT) compared to mature fibrils, as indicated by the smaller fluorescence intensity signal in the ThT spectrum (Fig. 1e,f). Moreover, these aggregate species do not exhibit an increased optical absorption when stained with Congo Red (Fig. 1g). The FTIR spectrum of the Aβ42 globulomers provides further information on their secondary structure (Fig. 1i). The maximum of their amide I′ band is at 1,628 cm−1 and, therefore, within the range that is typically observed for β-sheet-rich amyloid fibrils [28]. However, the FTIR spectrum of the globulomers shows a clearly resolvable peak at 1,693 cm−1, which has also been observed for Aβ40 oligomeric aggregate species and might indicate antiparallel β-sheet structure [29]. For the globulomers, the contribution of the main peak at 1,628 cm−1 to the amide I′ band is smaller than for Aβ42 fibrils, suggesting that they contain less β-sheet structure.
Natural human Aβ oligomers are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell [30]. Previous studies have shown that these Aβ42 globulomers, as they have been also termed, are a persistent structural entity formed independently of the fibrillar aggregation pathway [24]. These preparations are a potent antigen in mice and rabbits eliciting the generation of Aβ42 globulomer-specific antibodies that do not cross-react with the full-length amyloid precursor protein APP, Aβ40, and Aβ42 monomers or Aβ fibrils. Furthermore, Aβ42 globulomers have been shown to bind specifically to dendritic processes of neurons but not glia in primary hippocampal cultures and to completely block long-term potentiation (LTP) in rat hippocampal slices [30].
Fibrillar Aβ42 was obtained by resuspension of Aβ42 in TBS and an incubation at 37°C for 24 h as described [16, 25]. Negative staining electron microscopy clearly shows mature, twisted Aβ42 fibrils with a width of 6 to 12 nm (Fig. 1d). Aβ42 fibrils show amyloid-typical tinctorial properties upon binding to ThT and CR (Fig. 1f,h). The fluorescence intensity of ThT at 482 nm and the CR absorption increase substantially in the presence of Aβ42 fibrils. Moreover, the maximum of the CR absorption is shifted from 497 to 513 nm, as typically observed for amyloid fibrils [29]. The amide I′ band of the fibrils also has its maximum at 1,628 cm−1 and suggests the presence of an amyloid-like extended β-sheet structure in Aβ42 fibrils (Fig. 1j). Within the limits of detection, this preparation was free of oligomers (data not shown).
Both oligomeric and fibrillar Aβ42 cause a decreased membrane potential in P301L tau cortical preparations
We dissected the cerebellum and two cerebral brain hemispheres from 6 P301L transgenic and six non-transgenic control WT littermates at 13–15 months of age. The cerebellum and one cerebral hemisphere were triturated separately to obtain cellular preparations to determine the mitochondrial membrane potential. The second cerebral hemisphere was used to prepare isolated mitochondria to determine the rate of mitochondrial respiration. Brain cells and isolated mitochondria from the same animals were treated in parallel with the different Aβ42 preparations.
We conclude from this that cortical preparations of both WT and P301L tau transgenic mice are susceptible to Aβ42 preparations, be they oligomeric or fibrillar in nature. Importantly, when we looked at cerebellar preparations, they were resistant to all three types of Aβ42 preparations in as much as they showed no impairment of the MMP (Fig. 2d). This finding underscores the concept of selective vulnerability in neurodegeneration. For example, for the Aβ-associated pathology in AD, five phases have been defined, with only the final fifth phase being characterized by Aβ deposition in the cerebellum [31, 32]. Similarly, NFTs are found in the cerebellum only in the final stages of AD, and in FTD, they are also not frequently encountered [33]. The mechanisms underlying selective vulnerability in AD and FTD are largely unknown, and several hypotheses have been put forward. Interestingly, one of the factors determining which cells die first when different types of cells are exposed to the same stress may be variations in mitochondrial composition [34].
Reduced state 3 respiration, respiratory control ratio, and uncoupled respiration in P301L tau mice
We have previously shown a significantly reduced state 3, but not state 4, respiration in aged (24-month-old) P301L tau transgenic compared to age-matched WT mice; this led to a markedly reduced respiratory control ratio [19].
We found in addition, after uncoupling with FCCP, that the respiratory rate in the absence of a proton gradient, i.e., the 'uncoupled respiration', was significantly diminished in 13–15-month-old P301L tau mice after incubation with either oligomeric or fibrillar preparations of Aβ42 (Figs. 3b and 4d) indicating a reduced maximum capacity of the electron transport chain. After complete inhibition of complex I with rotenone, succinate was added as a substrate for complex II. When the succinate-dependent respiration was normalized to the uncoupled complex I-dependent respiration in the respective experiments, there was no significant difference between treated and untreated P301L tau transgenic mitochondria (Fig. 3b). This indicates that neither complex II nor complex III and IV are decisively impaired by Aβ42. In accordance with the respiratory control ratio, we have previously shown that ATP levels of cerebral cells were unchanged in 12-month-old P301L tau transgenic mice, but significantly reduced with aging [19]. Previous studies using homogenates of fresh samples of frontal neocortex from patients with dementia and neurosurgical controls suggested partial mitochondrial uncoupling in disease as the ratio of oxygen uptake rates in the presence and absence of ADP was significantly reduced for the dementia patients compared with controls [35]. These in vitro results indicate that metabolic changes may be relevant to the pathogenesis of AD and related dementias [36].
Together, our results suggest that P301L tau mice exhibit an initial defect in mitochondrial function with reduced complex I activity, which is exacerbated by the presence of either oligomeric or fibrillar Aβ42.
Aging increases the sensitivity of mitochondria to oligomeric Aβ damage
Mitochondrial respiratory chain failure has been implicated as a factor in general aging and is likely to have a greater effect in tissues with a high dependency on energy generated through oxidative phosphorylation such as the brain [37]. We have previously shown that aging contributes to the mitochondrial dysfunction in P301L tau mice [19]. Furthermore, we had found a 62.3% reduction of complex V levels in human FTDP-17 brains obtained from carriers of the P301L mutation, compared to control brains [19]. The decreased levels of complex V in human P301L FTDP-17 brains confirmed our proteomics observation made in the P301L tau transgenic mice and suggested that the P301L mutant tau pathology causes potentially a specific mitochondrial dysfunction in humans as well as in mice [19].
Together, aging increased sensitivity of cortical mitochondria to Aβ damage. This is in agreement with studies in APP transgenic mice [25] as well as in primates, where complex I activity had been shown to be reduced with aging [38]. Thus, aging potentially intensifies complex I activity defects in the P301L tau mice.
Our findings first of all support the notion of a toxic role of Aβ in respiration and, together with our previous findings, antioxidant defense mechanisms [19, 25, 39]. Secondly, they reveal a role for tau in mitochondrial dysfunction. Thirdly, our experiments extend our previous findings that treatment of PC12 cells with extracellular Aβ causes a significant decrease in mitochondrial membrane potential [40]. Finally, our results in P301L tau mice indicate the effects of both fibrillar and oligomeric Aβ on mitochondrial function of cortical brain cells at very low and physiological relevant concentrations within the nanomolar range and within a very short time frame (incubation time 4 h) indicating early and chronic alterations of mitochondrial functions that may impact overall neuronal homeostasis. In a related study, acute treatment of human cortical neurons with oligomeric Aβ (5 μM) for only short periods was shown to be sufficient to activate a mitochondrial apoptotic death pathway [41].
How does Aβ exert its effect on mitochondria? Interaction of Aβ with Aβ binding alcohol dehydrogenase (ABAD), a short-chain alcohol dehydrogenase in the mitochondrial matrix, has been shown to lead to mitochondria failure, e.g., mitochondrial membrane permeability and reduction of the activities of enzymes involved in mitochondrial respiration [42]. ABAD also binds to oligomeric Aβ42 that has been found in cortical mitochondria of APP transgenic mice [43]. Protease sensitivity assays suggest that Aβ gains access to the mitochondrial matrix rather than simply being adsorbed to the external surface of mitochondria [44]. This may explain how Aβ42 affects mitochondrial membrane potential and respiration. In our studies, however, we did not determine whether there is a direct interaction between Aβ and mitochondria, nor whether extracellular Aβ is taken up by the neurons. It is very likely that within the short time frame of our experiments (i.e., 4 h) very little Aβ42 is taken up at all. The effects of this putatively minor fraction cannot be discriminated from that elicited by the majority of Aβ that is not taken up. Therefore, our experimental design does not allow drawing any conclusions concerning the role of intracellular Aβ in mitochondrial dysfunction. In any case, it is generally not understood how Aβ exerts its toxicity in other experimental paradigms, whether it is receptor mediated and if so whether it requires uptake of Aβ by nerve terminals and retrograde transport, whether it is dependent on pore formation and calcium ion influx, or whether it is related to damage to nerve terminals, by interacting with the lipid bilayer [45]. It has been hypothesized that oligomeric Aβ, with its sharp morphology in contrast to monomeric Aβ, has the ability to permeabilize cellular membranes and lipid bilayers thereby entering organelles, such as the mitochondria [46, 47]. Of note, early reports about the action of aggregated Aβ on membranes implicate increased membrane permeability elicited by fibrils [48, 49]. These mechanisms might explain why aggregated Aβ preparations elicit effects on mitochondrial function, but not disaggregated Aβ.
The differences we found between the effects of fibrillar and oligomeric Aβ are subtle. In a related study, their effects have been dissected from monomers. Cell medium containing oligomers and abundant Aβ monomers, but not amyloid fibrils, were microinjected into rat brain and shown to markedly inhibit hippocampal long-term potentiation [30]. Immunodepletion from the medium of all Aβ species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Aβ monomers but not oligomers, did not prevent the inhibition of LTP, indicating a role for Aβ oligomers. These were shown to disrupt synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid, in the absence of monomeric or fibrillar amyloid. When cells were treated with γ-secretase inhibitors at doses which prevented oligomer formation but allowed appreciable monomer production, this no longer disrupted LTP, indicating that synaptotoxic Aβ oligomers can be targeted therapeutically [30, 50]. In Neuro-2A cells, oligomers were shown to induce a tenfold greater increase in neurotoxicity as compared to fibrils [51]. However, whereas LTP seems to be inhibited by oligomeric Aβ only and not by fibrillar Aβ, in a different experimental paradigm, as in our study, the two species seem to have both toxic, yet diverse effects [52]. Using rat astrocyte cultures, oligomeric Aβ42 was shown to induce initial high levels of the pro-inflammatory molecule IL-1β that decreased over time, whereas fibrillar Aβ caused increased levels over time [52]. Oligomeric, but not fibrillar Aβ, induced high levels of iNOS, NO, and TNFα, suggesting that oligomers induce a profound, early inflammatory response, whereas fibrillar Aβ shows less increases of pro-inflammatory molecules, consistent with a more chronic form of inflammation [52]. Similarly, in our experimental paradigm, we found toxic effects of both fibrillar and oligomeric Aβ, but those of oligomeric Aβ were more pronounced.
The presence of the pathogenic P301L mutation of tau severely increased the susceptibility of mitochondria to Aβ preparations. How tau accumulation is mediating these changes is unclear. Overexpression of wild-type tau in cell culture has been shown to impair plus-end-directed axonal transport resulting in a reduction of mitochondria [53]. However, this is unlikely the case for P301L tau mice, as mitochondrial neuritic numbers counted proximal or distal to the cell body, did not vary significantly compared to numbers in WT mice, [19]. Also, the total amount of mitochondria has been shown to be unaltered in the transgenic mice [19]. This is consistent with the finding of similar numbers of mitochondria between NFT and non-NFT-bearing cells in AD [54]. It is likely that tau accumulation has direct effects on the mitochondria as the accumulation of increasingly insoluble ATP synthase α-chain together with NFTs has been shown in AD brains while detergent-soluble levels were reduced [55].
The synergistic effects of Aβ42 on mitochondria isolated from P301L tau transgenic mice may seem subtle, irrespective of whether they are incubated with fibrillar or oligomeric Aβ42, but these effects are of in vivo relevance as AD is a chronic and slowly progressive disease, with time spans of up to several decades between Aβ and tau aggregation and the onset of clinical symptoms.
In conclusion, we found that both fibrillar and oligomeric Aβ42 preparations impaired mitochondrial membrane potential and respiration. Furthermore, we revealed the synergistic effects of Aβ42 and P301L tau (see also: [56]). However, as aged cortical brain mitochondria showed an increased sensitivity to oligomeric compared to fibrillar Aβ, this suggests that oligomeric Aβ may be particularly toxic. Although it is likely that oligomeric Aβ represents the primary toxic insult, further experiments are needed to substantiate this notion. Thus, there is increasing evidence for different modes of toxic actions of Aβ suggesting that in the development of treatment strategies, targeting either oligomeric or fibrillar Aβ may not be sufficient, but the best approach is likely one that either prevents formation of excess Aβ altogether or assists in its rapid clearance.
This research was supported in part by grants from the SNF (Swiss National Science Foundation) #310000-108223 and Eli Lilly International Foundation to AE, and from the NHMRC, the ARC, the New South Wales Government through the Ministry for Science and Medical Research (BioFirst Program), the Medical Foundation (University of Sydney), and the Judith Jane Mason & Harold Stannett Williams Memorial Foundation to JG. JG is a Medical Foundation Fellow. We are grateful to Abbott GmbH & Co KG, Ludwigshafen, Germany, for the kind gift of monomeric and oligomeric Aβ42 preparations. M.F. is supported by grants from BMBF (BioFuture) and DFG.
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Travel Blogs from Sonoma
Wine tasting in Sonoma
The day started with a run for 3 of the boys along the San Francisco bay front. A beautiful morning, a wonderful temperature and so many runners out! After that, we managed to escape for a day of wine tasting in Sonoma and had arranged to be picked up by Valley Wine Tours (who we would definitely recommend) from our hotel at 9.15. To enable us to go tasting, we'd booked a child sitter Lindsey who was tasked with entertaining our 6 10 to 16 year olds. She arrived promptly and after ...
Sonoma, the Napa Valley & Bodega Bay
... navigated well as we drove through the hills and valleys of the vineyards, choosing some ambitious roads for Dave to drive on. We are not sure what they thought in the back! We found a great Italian America restaurant for dinner called Mary's and the food came with great service. We began talking to the waitress and were shocked when she said health insurance for herself, her husband and daughter was over $900 a month and she still had to pay $5000 for ...
The Round
Morning / evening Mashers Well temptation got the better of me today. Having fulfilled my chauffeuring duties early doors, I had no hesitation in making my next decision as to where the Chevy would end up on its next trip. Particularly so given a colleague of the DoP&C for DV&WP (and bar) offered me a set of Pings to use until my TaylorMades arrive next month. With clubs packed and a little taste of what Adobe Creek had to offer yesterday, it was a no brainer that ...
... including walking on the road with the oncoming traffic to contend with, It was safe!
I had always wanted ...
Visit with the Lewis'
... was because of the gold they had found here, but it is the dried out grass on all the hills that turns golden brown in the late summer that gives the state its nickname.
We had to get off I-5 to get to our campground for the night. It seems we had forgotten our new discovery of parking near I-5 and driving in the car over the hills. Anyway, we drove for 2 hours, up, down and around windy hills, sharp switchbacks with ...


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A little illMature
Haven Scott
I fell completely silent when I looked up and realise I had just collided into a highborn soldier. Not only that but it was him. The soldier from before. The one that had told me to go and, that Adaline claims, spared us.
I swallow back a shocked reply and step back curtsying. "I'm sorry" I choke out then rush off again. I can feel his eyes on me and the look of recognition on his face so pure. He was confused almost.
I shook my head and kept walking but slowly I came to a stop and looked back. The soldier was laughing with the other soldier. The other one was different though peering at the seamstress girl's with a look of hunger on his face. My soldier though was calm. Collected. He seemed to keep the whole walk through the quarters as work.
I blushed as I realised what I thought of him. Oh gosh, he wasn't my soldier. I couldn't be thinking that. The soldier was a highborn. Even if he liked playing around with the slaves he'd never be mine No, I had a husband already sorted out for me.
A man named Kieran that was a blacksmith. I was to have 2 children by this man that I hadn't even met. I'd meet him tonight though because he was coming round to dinner.
I swallowed nervously and looked away from the soldiers. I began to walk again towards my home. That was the reason I was heading home so early. To get ready. My marriage letter was only through yesterday and I met my husband today.
I'd have only 2 weeks before we were married. The only thing I hoped for now that he was drastically ugly. I couldn't have to high standards though. I couldn't expect him to be a dashingly handsome as the soldier. It was impossible for a slave to look that good even if they had the natural looks.
Finally, I reached my house. It was a small little thing right on the edge of the quarter. I sighed and closed my eyes against the sight of the simple and mucky home. My mother was there of course. She was retired and had not yet been taken away. They always took the elderly away when they had no use which is why I was so glad that Adaline would still be here after I was married.
Still, there was a worry after when Adaline was married. I wouldn't be able to stop them taking away. It wasn't my choice or hers. It was theirs, the highborns, and they needed people for the science quarter.
I had heard the stories and hoped with every fiber of my being that they weren't true. She didn't want to lose her mother to a fate worse than death.
When I walked in a saw her smiling face though I relaxed. She was still here for now though. "Mother" I said rushing over to hug her tightly.
"Oh oh dear" she laughed. "What's this all about?"
"Nothing" I quickly say. "I just.."
My mothers face hardened knowing exactly what I had just been thinking. "Now, Haven, what have I told you?"
"I can't help but worry about you more though mother" I pleaded for some leeway. "I'll be taken away soon for marriage"
"Taken away sound's very harsh dear for getting married. Now get upstairs and get into some nicer clothes" she snapped waving a spoon at me. "Your future husband will be here any minute"
I sighed and made my way upstairs tugging off my work clothes to pull on a simple blue dress. I'd stitched it myself and it would be beautiful if not for the cheap material. It was as I was pulling back my hair that I heard the know. I swallowed. Kieran. He was here.
I heard voices. My mothers and a very rough one. I took a deep slow breath then headed towards the stairs. I walked down them slowly. He was tall is what I saw first and had strong muscles which were from the forge. Then his darkened skin.
I bit back my horror as I reached the bottom. His face was large and his eyes bulged out like some man in a horror movie that we heard about from the old days. His lips were huge and puffy.
Generally overall I felt a little ill. Oh gosh, this is the man I have to marry?


text:
Research shows that getting kids and their parents in a post-secondary mindset as early as possible -- creating not just hope but an expectation for college attainment -- makes a big difference in whether they get credentials. Minneapolis Mayor R.T. Rybak and his top economic development official, Mike Christenson, without a lot of fanfare, have been working this angle for years. Every year in the fall Mayor Rybak addresses every 9th grade... Read More
The U.S. Census Bureau's report on "Income, Poverty and Health Insurance Coverage in the United States: 2010," is out and it's worth digging in to the spreadsheets showing year-by-year change over the last 25 years for each state. More than 500,000 Minnesotans currently are without health care, even more (560,000) are living below the official poverty level (a ridiculously paltry $22,113 for a family of four) and median income... Read More
Check out the recent column by our friend and ally Joe Nathan at the Center for School Change at Macalester College for some stirring advice on getting kids and parents plugged in to college visits and post-secondary options during the month of August. We all know that our more successful students are prowling college campuses and weighing options by their sophomore or junior years in high school, and Nathan's column provides links to t... Read More
Speakers at an event last week made a powerful and compelling case for equity as a growth strategy. Minnesota stands out for some striking inequities tied to race Read More
Web Development by Creative Arc, a Minneapolis Web Design firm.


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- This event has passed.
Marlborough: MRCC Holiday Shopping Boutique
November 27, 2012Admission is free, but please bring non-perishable food products and/or canned goods.
Event Navigation
The Marlborough Regional Chamber of Commerce is holding a Holiday Shopping Boutique Nov. 27, from 6 to 9 p.m., at the Main St. Caf�, 182 Main St., downtown Marlborough. Gift selections will include jewelry, cosmetics, skin care products, kitchenware, gift baskets, and much more. Cash bar and holiday treats will be on sale at Main St. Caf�. Do you have any "gold" jewelry or other gold pieces you want to sell?? Bring them – even if they are broken, you will receive money right on the spot! Admission is free, but please bring non-perishable food products and/or canned goods to be donated to the Marlborough Community Cupboard. For more information, call 508-485-7746 or email email@example.com.


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Hercules (TUGS)
229,455pages onAdd New Page
this wiki
this wiki
Hercules is a character from TUGS. He is the sixth member of the Star Fleet. He is voiced by Nigel Anthony. In Salty's Lighthouse, he is voiced by Long John Baldry.
His model was bought by the Star Tugs Trust.
- Edward (Thomas the Tank Engine and Friends/The Railway Series)
- Truro (Theodore Tugboat)
- Hodge (Chuggington)
- Mr. Nicholas (Shining Time Station)
- Shadow the Hedgehog or Uncle Chuck (Sonic the Hedgehog)
- Houdini (Rayman)
- Dad Asparagus (Veggie Tales)
- Luigi Mario (Super Mario Bros.)
- Swanky Kong (Donkey Kong Country)
- Jebediah or Doc (The Little Engine That Could)
- Gligar (Pokemon)
- Meta Knight (Kirby)
- Peppy Hare (Star Fox)
- George (Rainbow)
- Peter Perfect (Wacky Races)
- Bo Sheep or Edward R. Furrow (Garfield & Friends)
- Vinnie the Deer (Tiny Toon Adventures)
- Bobby or Dr. Otto Scratchansniff (Animaniacs)
- Ned Flanders (The Simpsons; both wear glasses)
- Spongebob's Dad (SpongeBob SquarePants)
- Howard DeVille (Rugrats)
- Mr. Robert Simmons (Hey Arnold)
- Brian (Doug)
- Brian Griffin (Family Guy)
- Terry Kimple (The Cleveland Show)
- Fancy-Fancy (Top Cat, both are kind-hearted)
- Hank Hill (King of the Hill; both wear glasses)
- Lightning (Teen Titans)
- Brainy Brian (Horrid Henry)
- Mack the Turtle (Dr. Seuss)
- Jimmy's Dad (Jimmy Neutron)
- Wilt or Ivan (Foster's Home for Imaginary Friends)
- Hercules (Hercules, both share the same first name)
- David Read (Arthur)
- Agent Johnson (Fanboy & Chum Chum)
- Trevor (Codename: Kids Next Door)
- Mr. Sparks (Noddy's Toyland Adventures)
- Sam Waldron (Postman Pat)
- Slinky Dog (Toy Story)
- Mr. Soil (A Bug's Life)
- Charlie (Monsters, Inc.)
- Jacques (Finding Nemo)
- Sarge (Cars 1 and 2)
- Roger (Madagascar)
- Crane (Kung Fu Panda)
- Lawrence Fletcher (Phineas and Ferb)
- Derek (Good Luck Charlie)
- Lucius (Bone)
- Goat (Pearls Before Swine)
- Constable Jones (Mary Poppins)
- Mr. Codfish (Bedknobs and Broomsticks)
- Sonic the Hedgehog (Wreck-it Ralph)
- Chop Chop (Skylanders)
- Leadbottom (Planes)
- Toots (Casey Jr. and Friends)
- Gonzo the Great (The Muppet Show)
- Ernie or Bert (Sesame Street)
- Kevin The Crane (Construction Site)
- The Wise Old Goat (Rupert)
- Anatole (Anatole)
- Donatello (Teenage Mutant Ninja Turtles)
- Paul the Ant Colonel (Maya the Bee/Movie)
- Hercules (TUGS)/Thomas
- Hercules (TUGS)/TUGS
- Hercules (TUGS)/The Little Engine That Could
- Hercules (TUGS)/Winnie the Pooh
- Hercules (TUGS)/Shining Time Station
- Hercules (TUGS)/Mario
- Hercules (TUGS)/Sonic
- Hercules (TUGS)/The Simpsons
- Hercules (TUGS)/Family Guy
- Hercules (TUGS)/O.J.
- Hercules (TUGS)/Sunshine
- Hercules (TUGS)/Top Hat
- Hercules (TUGS)/Burke
- Hercules (TUGS)/Blair
- Hercules (TUGS)/Big Mac
- Hercules (TUGS)/Warrior
Hercules talking to OJ in Treasure Hunt


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Emma Davidson works with women in refugee and indigenous communities on understanding their perceptions of need. In this short talk at TEDxCanberra 2011, Emma discusses the need to listen to the women she works with and to understand their issues in the language they themselves use to describe them.
Emma has more than a decade of government department experience working with clients at Centrelink, as well as policy and IT management. Emma volunteers for various women's health and social justice causes, and has authored magazine articles on social justice and environment issues.
Her Bachelor of General Studies combines journalism, law, and IT studies.


text:
Larsson and Jennings
Lugano 40mm Silver-Black Leather Exclusive Watch
Product Actions
- Love this
This item is worth 220 loyalty pointsSign up to Liberty Loyalty to see your purchases converted into vouchers you can spend in-store.
Editor's notes
Exclusive to Liberty, this Larsson and Jennings watch features Liberty engraving on the back of the face. Paying homage to the brand's minimal design, clean lines meet Italian leather in a subtle finish that reflects the brand's commitment to quality.
Product features
- White brass Lader watch
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Leather, White Brass, Crystal Sapphire
Dia: 4cm, Strap: 2cm
Delivery information
UK Delivery
We offer a range of UK delivery options including standard delivery, next day delivery and various weekend options. The available delivery options and prices will vary depending on the delivery location you share with us when checking out.
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We deliver to a number of International countries, our full list of countries is available during checkout. You can change your delivery country at any time using the country selector located in the footer of our site.
Click & Collect
Our Click & Collect Service is quick and simple. Choose collect my items from one UK location during checkout and we'll email you when your order has been processed, it will be ready for collection within 3 working days.
If you are not completely satisfied with your item you may return it within 28 days for a full refund. You can return the item online using our simple service or bring it back to our flagship store in Regent St.


text:
Audi Q3 May Come To United States
While Audi's plans for the U.S. market originally did not include the compact Q3 crossover, but Audi is re-considering the decision to not import the car after seeing the segment expand with offerings like the MINI Countryman, BMW X1 and the Nissan Juke.
While the Q5 starts at $35,200, Audi would likely have to price it closer to the MINI Countryman which retails between $21,650 and $26,950. A sub-$30,000 price tag might be uncomfortable for Audi and its positioning as a premium brand, but with the small crossover segment growing, it could be a chance for Audi to increase their overall volume in North America.
The Q3 would likely be powered by the corporate 2.0T engine, with the possibility of a 2.0L TDI diesel engine as well. The diesel offering could allow the Q3 to stake out a niche within the segment, and remain consistent with Audi's overall diesel strategy.
[Source: Kicking Tires]


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Red Hat Bugzilla – Full Text Bug Listing
|Provide native systemd unit file
|[Fedora] Fedora
|Jóhann B. Guðmundsson <johannbg>
|Robert Scheck <redhat-bugzilla>
|QA Contact:
|Fedora Extras Quality Assurance <extras-qa>
|Docs Contact:
|ADent123, aslanidis, bugzilla, j.orti.alcaine, jrowens.fedora, ken, limburgher, redhat-bugzilla, thomas
|Target Milestone:
|Target Release:
|Fixed In Version:
|Doc Type:
|Bug Fix
|Doc Text:
|Story Points:
|Clone Of:
|Last Closed:
|Mount Type:
|Verified Versions:
|oVirt Team:
|RHEL 7.3 requirements from Atomic Host:
|Cloudforms Team:
|Bug Depends On:
|Bug Blocks:
Description Jóhann B. Guðmundsson 2011-07-04 09:57:27 EDT
Description of problem: https://fedoraproject.org/wiki/Features/SysVtoSystemd Version-Release number of selected component (if applicable): How reproducible: Steps to Reproduce: 1. 2. 3. Actual results: Expected results: Additional info:
Comment 1 Jóhann B. Guðmundsson 2011-07-04 09:58:27 EDT
Created attachment 511195 [details] Native systemd service file for ddclient https://fedoraproject.org/wiki/Packaging:Guidelines:Systemd https://fedoraproject.org/wiki/Packaging:ScriptletSnippets#Systemd http://fedoraproject.org/wiki/Packaging:Tmpfiles.d
Comment 2 Robert Scheck 2011-07-04 10:04:39 EDT
Yes, but I'm not going to write one myself for now. Nevertheless, I'm willing to accept a working and well done systemd service file for inclusion.
Comment 3 Jóhann B. Guðmundsson 2011-07-04 10:07:35 EDT
What the one I submitted is not good enough?
Comment 4 Robert Scheck 2011-07-04 10:19:14 EDT
Sorry, I overlooked your contribution. Will have a look into it the next days.
Comment 5 J. Randall Owens 2011-07-12 06:53:51 EDT
Looks to me like it should perhaps have a "Requisite" or "BindTo" network.target, in addition to the "After". Depends on whether we want ddclient to keep doing its thing when the network goes down, which it is designed to do, after all. I don't see any point to it starting up when there's no network, though. (As long as this doesn't mean it won't work without NetworkManager running and controlling the interface?) Other than that, it looks just about like the one I was working up, and should be good.
Comment 6 J. Randall Owens 2011-07-12 07:01:32 EDT
Also, you might want to hold off on packaging it until you rebuild with the new 3.8.1, which I see just produced Bug #720627. No point in planning to release two versions of the package within a few days of each other.
Comment 7 Jóhann B. Guðmundsson 2011-07-12 07:22:35 EDT
(In reply to comment #5) > Looks to me like it should perhaps have a "Requisite" or "BindTo" > network.target, in addition to the "After". Depends on whether we want > ddclient to keep doing its thing when the network goes down, which it is > designed to do, after all. I don't see any point to it starting up when > there's no network, though. (As long as this doesn't mean it won't work > without NetworkManager running and controlling the interface?) > > Other than that, it looks just about like the one I was working up, and should > be good. Requisite= Would be what you want ( if any ) unless ofcourse you want to take bring up and take down the network when you start and stop this service which I'm pretty sure you dont want..
Comment 8 J. Randall Owens 2011-07-12 07:33:36 EDT
According to systemd.unit(5), "[BindTo is] very similar in style to Requires=, however in addition to this behaviour it also declares that this unit is stopped when any of the units listed suddenly disappears." Which sounds to me like it would mean ddclient ("this unit") would be stopped when the network ("units listed") goes away. But I'm inclined to think that isn't the desired behaviour anyway, and Requisite is the way to go, since ddclient does handle network going away and reappearing, like I said.
Comment 9 Mohammed Arafa 2011-08-06 11:35:21 EDT
i got a box that has a static internal ip and does not use NetworkManager and that is disabled. ddclient is working on that box. i hope the fix wont break that.
Comment 10 Juan Orti 2011-09-08 02:48:24 EDT
I tried the service file provided but had to remove the line: ExecStartPre=/bin/touch /var/cache/ddclient/ddclient.cache because touch create the ddclient.cache file with root owner and the daemon can't write to it.
Comment 11 Jóhann B. Guðmundsson 2011-09-08 04:17:15 EDT
Hum that's odd the submitted file is executed as ddclient user anyway you should be able to add exec start pre line which chowns the file to the ddclient user in that case try this. [Unit] Description=A Perl Client Used To Update Dynamic DNS After=syslog.target network.target [Service] User=ddclient Group=ddclient Type=forking PIDFile=/var/run/ddclient/ddclient.pid EnvironmentFile=-/etc/sysconfig/ddclient ExecStartPre=/bin/touch /var/cache/ddclient/ddclient.cache ExecStartPre=/bin/chown ddclient.ddclient /var/cache/ddclient/ddclient.cache ExecStart=/usr/sbin/ddclient $DDCLIENT_OPTIONS [Install] WantedBy=multi-user.target
Comment 12 Jóhann B. Guðmundsson 2011-09-08 04:19:25 EDT
Oh by the way which version of systemd are you using ( rpm -qa | grep systemd ) I kinda need to know that encase this is an bug.
Comment 13 Juan Orti 2011-09-08 06:30:28 EDT
Let me check it again, because I did some tests and maybe the file was created as root when trying to launch ddclient manually. Problably I messed things up. systemd-26-9.fc15.x86_64 systemd-sysv-26-9.fc15.x86_64 systemd-units-26-9.fc15.x86_64
Comment 14 Juan Orti 2011-09-08 06:37:13 EDT
(In reply to comment #11) > Hum that's odd the submitted file is executed as ddclient user anyway you > should be able to add exec start pre line which chowns the file to the ddclient > user in that case try this. You are right, it's executed as ddclient user, My file already existed with root owner > ExecStartPre=/bin/chown ddclient.ddclient /var/cache/ddclient/ddclient.cache This line isn't necessary. Thank you!
Comment 15 Gwyn Ciesla 2012-02-13 14:54:28 EST
Robert, any objection to my making this change?
Comment 16 Gwyn Ciesla 2012-03-14 12:24:21 EDT
Comment 17 Ioannis Aslanidis 2012-03-29 04:16:44 EDT
Anyone maintaining this package?
Comment 18 Gwyn Ciesla 2012-03-29 07:52:20 EDT
I'll make this change today.
Comment 19 Gwyn Ciesla 2012-03-29 08:19:56 EDT
Done. I'll push systemd to f17, and 3.8.1 to all branches.
Comment 20 Fedora Update System 2012-03-29 08:21:05 EDT
ddclient-3.8.1-1.fc17 has been submitted as an update for Fedora 17. https://admin.fedoraproject.org/updates/ddclient-3.8.1-1.fc17
Comment 21 Fedora Update System 2012-03-29 22:56:57 EDT
Package ddclient-3.8.1-1.fc17: * should fix your issue, * was pushed to the Fedora 17 testing repository, * should be available at your local mirror within two days. Update it with: # su -c 'yum update --enablerepo=updates-testing ddclient-3.8.1-1.fc17' as soon as you are able to. Please go to the following url: https://admin.fedoraproject.org/updates/FEDORA-2012-4858/ddclient-3.8.1-1.fc17 then log in and leave karma (feedback).
Comment 22 Fedora Update System 2012-04-11 23:37:23 EDT
ddclient-3.8.1-1.fc17 has been pushed to the Fedora 17 stable repository. If problems still persist, please make note of it in this bug report.
Comment 23 Kenneth Topp 2012-05-13 23:02:04 EDT
you are missing a file... :::::::::::::: /etc/tmpfiles.d/ddclient.conf :::::::::::::: d /var/run/ddclient 0755 ddclient ddclient -
Comment 24 Gwyn Ciesla 2012-05-14 11:29:03 EDT
Fixed, update for 17 coming.
Comment 25 Fedora Update System 2012-05-14 11:39:38 EDT
ddclient-3.8.1-2.fc17 has been submitted as an update for Fedora 17. https://admin.fedoraproject.org/updates/ddclient-3.8.1-2.fc17
Comment 26 Fedora Update System 2012-05-15 11:40:03 EDT
ddclient-3.8.1-3.fc17 has been submitted as an update for Fedora 17. https://admin.fedoraproject.org/updates/ddclient-3.8.1-3.fc17
Comment 27 Fedora Update System 2012-05-26 03:42:13 EDT
ddclient-3.8.1-3.fc17 has been pushed to the Fedora 17 stable repository. If problems still persist, please make note of it in this bug report.


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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
what do i do now. i have injusry consultants calling me. i didnt see doctor because of no money but have had headache and pain. i owe 1900.00 on my car but its damaged on passenger side. what do i do
You should speak with an experienced Ohio licensed personal injury attorney immediately. An experienced injury attorney will be able to review the facts, your insurance and determine what claims can be pursued. For example, you may have coverage under your own policy to pay for some needed medical treatment. There are many important issues with which an experienced Ohio injury attorney can assist you. I would strongly discourage you from attempting to resolve this matter on your own. Insurance companies are only looking out for their own interests, not yours. Generally, these cases are taken on a contingent fee basis, which means you would not be required to pay any upfront fees to your attorney. Best wishes.Ask a similar question
Contact local and qualified personal injury attorney. If injured, get treatment now. Good luck.
Personal injury cases only; I'm good at it; you be the Judge! All information provided is for informational and educational purposes only. No attorney client relationship has been formed or should be inferred. Please speak with a local and qualified attorney. I truly wish you and those close to you all the best. JeffAsk a similar question
I do not know how it works in Ohio but if you were in Pennsylvania and you had car insurance, at least the first $5000 in medical expense would be covered. If you have coverage and even if you don't you really need to get treatment. First you need to get better and secord if you intend to file a claim you need some documented injury.
Next, get an attorney from your state immediately. A lawyer from your state will give you a free consultation and you will learn exactly what you need to do.
A lot of lawyers on this site can offer their insights including attorneys from Ohio but ultimately you will need to talk to someone personally. Do it now. This site has a find a lawyer feature--use it.
If you feel this is the "best" answer or is "helpful," please indicate. Since I am limited to the information you provide, I cannot guarantee the accuracy of the answer. You should seek the advise of an attorney who can explore all aspects of your question. This communication does not form an attorney client relationship.Ask a similar question
There are many issues that need to be dealt with immediately. First of all if you are hurt get medical treatment as soon as possible. If payment for medical bills is of immediate concern there are several options an experienced injury lawyer can explain to you (blue link below). Time is of the essence and you need to contact a lawyer soon. I'm sure you want to get your car repaired or paid for and you need to get to a doctor. Please don't delay and I hope you feel better. There are simply many things to explain to you in writing on this page. A free phone conference will answer most of your concernsAsk a similar question
If you were injured, call a local personal injury lawyer in your city. Avvo has a terrific "find a lawyer" tool.Ask a similar question
Your primary concern should be to get better. If you're injured, make sure you see your doctor and get proper medical treatment for any injuries you sustained. Depending on your financial situation there are options available to you to get the care you need. You should then turn your attention to finding an experienced local personal injury attorney who can help you deal with the insurance companies to make sure that you are appropriately compensated for your injuries. You should not try to deal with any insurance companies on your own, because insurance companies generally do not have your best interest in mind.
The Law Office of Matthew Hirschfelt provides answers for informational purposes only. Actual legal advice can only be given by an attorney licensed in your jurisdiction, thoroughly familiar with the area of the law in which your concern lies. The responses given to any questions posed on Avvo do NOT establish an attorney-client relationship. Attorney Hirschfelt is available for private hire and consultation for a fee.Ask a similar question
You should obtain needed medical care and treatment immediately and follow the doctor's advice. Do not give any statement to the adverse party or insurance company nor grant them access to any medical records. Photograph the injuries and the damage done to any property. Contact a personal injury attorney in your area as soon as possible so that you can protect your rights. You may also find it helpful to review the Legal Guides I have published on Avvo.com dealing with many of the issues you are now facing. The Guides can be accessed through my profile page on Avvo.com.
Legal Disclaimer:
If this information has been helpful, please indicate below.
Mr. Lundeen is licensed to practice law in Florida and Vermont. The response herein is not legal advice and does not create an attorney/client relationship. The response is in the form of legal education and is intended to provide general information about the matter within the question. Oftentimes the question does not include significant and important facts and timelines that, if known, could significantly change the reply and make it unsuitable. Mr. Lundeen strongly advises the questioner to confer with an attorney in your state in order to ensure proper advice is received.
This ans. does not create an attorney/client relationship.Ask a similar question
You need to get medical assistance for your injuries . Then consult with an experienced personal injury lawyer . The people calling you may be a chiropracter's office.
This is not legal advice. This is just general information.You should always contact a lawyer in your jurisdiction for legal advice. By giving you this information a lawyer client relationship is not created. Although I try to be accurate , you should not rely solely on this information and should get an opinion from a lawyer you speak with personally.Ask a similar question
I would not even return the call of any "consultants" who hunt you down like that. Be VERY wary of going down that road. Do you have any kind of auto insurance that includes medical payments coverage? Check your policy to see...
Not intended as legal advice and no attorney client relationship is formed unless there is a signed agreement between attorney and client.Ask a similar question
You should speak quickly with a personal injury attorney in the state that you were injured in. They will give you the quick do's and dont's in your area. There are issues that are unique to every state.Ask a similar question
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Children & Young People
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What we do has to enable you and your colleagues do what you do better.
Our highly experienced team have a passion for the domain, many of them having been personally involved in front line services themselves. This passion drives our ambition to develop new and innovative systems and services that help you realise the vision for making a difference.
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Grammy Awards
Local News
The Killers are coming to WindsorLas Vegas rockers The Killers are coming back to Caesars Windsor.
Malcolm-Jamal Warner celebrates his recent Grammy win with 'therapeutic' new albumLOS ANGELES, Calif. - Malcolm-Jamal Warner poured his real-life experiences — both in and out of love — into his latest album, "Selfless."
IndyCar notes: Will Power grabs the pole for Detroit Grand PrixCanadian driver James Hinchcliffe is being replaced in the Chevrolet Detroit Belle Isle Grand Prix by Conor Daly.
Kanye West after invading Grammys stage: Beck 'should've given his award to Beyoncé'LOS ANGELES — Kanye West almost pulled another Kanye at the Grammy Awards.
Sam Smith, Pharrell big award winners at GrammysSam Smith was the king of the Grammys, taking home three of the top four awards,
Canadians Drake, Arcade Fire land multiple Grammy Award nominationsThe Grammy Awards once again toasted a few favourite Canadian artists on Friday.
Grammys salute Beatles (with video)On Feb. 9, 1964 — 50 years ago to the day Sunday — 74 million viewers tuned in to The Ed Sullivan Show to watch the Beatles make their U.S. ...
Photos: Grammy's Red CarpetTake note on who graced the red carpet at the 56th Grammy Awards at the Staples Center in Los Angeles, Calif., Jan. 26, 2014.
Grammys by the numbers: A look at how Sunday's awards stack upThe Grammy Awards air this Sunday at 8 p.m. Here's an advance look at how the annual awards show is stacking up.
Paul McCartney, Ringo Starr, Taylor Swift, Macklemore to perform at Grammy AwardsThe Grammys weekend is shaping up to be a Beatles weekend.


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Helping YOU open doors to YOUR Future!
B*Success Academy offers students an opportunity to regain credit toward graduation through non-traditional credit options. Students are able to complete curriculum online in order to recover credits and get back on a path toward graduation.
Students can access B*Success Academy if they have not gained sufficient credits, need initial credit or need EOC review. Students can be served in a variety of ways. Students can attend before/after school, as a scheduled class, or students needing substantial credits attend at the Lighthouse Learning Center. Students work through the course curriculum online at their own pace. In general students who are motivated can complete a semester course credit in approximately 35-40 hours of active time.
Additional Opportunities for Support
Students can work assignments on the online program from home. Quizzes and Tests must be proctored at school. Students can also receive support before (7:30 - 8:30 am)or after school (until 5:45 pm) on their home campuses. Additionally, students can receive support two Saturday mornings a month at the Lighthouse Learning Center. (See attached Saturday schedule.)


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Like cliff diving or ferret ownership, football is an endeavor best watched from the sidelines. Witness acts of bodily bravery with today's GrouponLive deal: for $22, you get one upper-deck ticket for a seat in sections 305–312 (a $30 value) to a University of Maryland football game against the University of Miami, and one official UMD T-shirt (a $15 value) at Capital One Field at Byrd Stadium on Monday, September 5 (a $45 total value). Pregame festivities start at 5 p.m. at the Samuel Riggs IV Alumni Center, and the game begins at 8 p.m. Buyers are encouraged to wear red to the game as part of the Operation 54K effort.
Helmed by new head coach Randy Edsall and steeled by last season's Military Bowl win, the Terrapins plan to shell-shock opponents with cunning strategies and bone-rattling tackles. Groupon getters will help fill the upper deck with red, preventing the groundskeepers from dousing it with ketchup. With 13 starters returning to the team—including sophomore quarterback Danny O'Brien, the Atlantic Coast Conference's 2010 rookie of the year—the Terps hope to add a 25th bowl appearance to their history books. Three hours before kickoff, fans can visit the Samuel Riggs IV Alumni center for a bash filled with giveaways, face painting, and inflatable games. A DJ will prime cheering pipes with a set of festive tunes, and team members will sign Terps T-shirts and the bellies of the refs' pet turtles.
College Park
Stadium Dr. and University Blvd.
College Park, Maryland 20740
800-462-8377Get Directions


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The 64 year old, who was recently conferred with a chieftaincy title by the Obong of Calabar, knows how to enjoy herself o jare...good for her...lol.
Pics thanks to Abiola Aloba
News, Events, Entertainment, Lifestyle, Fashion, Beauty, Inspiration and yes... Gossip! *Wink*
Wow,she's 64? Well partying like this and been happy will sure make her live healthy and longer.But make she slow down o.Sge isn't really on her side.
Chidi Mokeme was there too. Hmmmmm. It was at Charles Ahize's party. Saw the pictures as well on the same blog!
I am not surprised. Who is this new guy? Where is Chidi Mokeme?
Yea, this life is short. It's never a bad idea to have some great fun when you can.
i hear she specializes in very young guys..in fact i know of a family friend she dated..and was constantly sponsoring him and showering him with expensive gifts..what is a 64 yr old doing in a night club..pleeeese cmon now this is way out of line..can you imagine any of your mothers in a night club??? i cant even imagine mine..this is outrageous!!!
Let Mama enjoy o! I hope I'd still be clubbing at 64 too. LOL
This woman still dey claim say she be 64yrs.....Na wah ooooo. Make she go sit her shriveled up 90year old ass somewhere joooor.
Na wa!
Wetin concern bad belle people with wetin she do with her life? Na una body she dey use? Allow the lady to live her life abeg, afterall she is not your mother! BTW, life is very short, we should make the most of the time alloted to us, instead of grumbling about others.
suga mama in the market
Mama bakassi enjoy your time jare no mind these idots sitting on the net and yanning opas. Do the idiots know that you single handedly raised 15 orphaned children? Do these idiots know why the call you Mama Bakassi? Their little minds can never comprehend how much you give to the underprivileged so that is why they sit on their stupid computers looking for whom to abuse. The jump from Bella Naija to Linda Ikeji to other sites drwoning their sorrows in gossip.
if she is 100 and wants to dance naked, at least this woman deserves it.
by the way, whats wrong with her doing this? its not like she does it everyday. we dont even know the context of the event.
there are people whose kids have birthday parties in clubs, they show up very briefly, in the spirit of it, have fun, dance to the music the children of nowadays are dancing to, and leave. Whats wrong with that?
no better way to live younger & longer.
Here we go again..look at dude in black tight top..going in for the kill not even checking those younger chics..he def knows where the bread is buttered..u can't say it was the sexy bod..just keeping it real..
i see you ma'am
Aww this photo made me smile.
She looks so happy and radiant, I love it!
So many things in that country to be bitter& to complain about, so much so that it often reflects in the more morose poses one sees on blogs and mags of much younger Nigerian 'celebs' so when I see genuinely happy people, I love 'em!
I would be lucky if I get to her age and look as radiant and happy as she looks here.
Keep it up madam!!
Is it a crime to dance again? Na wah for we oo! Seriously, if she decided to dance, why not afterall the current nigeria life expectancy is 47 years. Carry go madam, nothing do you.
Come on Linda,
It was a birthday party of a friend of hers and she was probably not the oldest person there.
the amount of female flesh displayed at that party is enough to make a monk go hard!!
Queens, Presidents, First ladies, Governors, anybody can dance in a night club. Same way you attend wedding ceremony or comedy show same way you can go to a night club no big deal. She is not greater than you cos there is a drop of greatness in each and every one of us.
@ Adecool....WoRd!
Abeg, people should free Aunty jo! After all men her age are in the club every weekend and see nothing wrong with it.
Am really surprised at some people"s comment deserving to dance naked and all this bullshit about why they call her Mama Bakassi becos she raised some kids by giving out her toto freely i guess...am sure they will be the first to deny their mama"s if they find her in a nite club. rubbish
Yoruba woman have perfected the act of using owambe and wedding to do night club. you think they invest thousands on jewellery and asoebi for nothing? Na their own night club be that! Dem go dey drink stout and baileys and husband go dey look am as woman drink....no know sey d wifey (Mama Kemi) don shayo dey rock sunny Ade.
We from the Niger Delta are Real.
Abeg Mama Bakassi...nothing! norring do u. Carry go!
Yoruba woman have perfected the act of using owambe and wedding to do night club. you think they invest thousands on jewellery and asoebi for nothing? Na their own night club be that! Dem go dey drink stout and baileys and husband go dey look am as woman drink....no know sey d wifey (Mama Kemi) don shayo dey rock sunny Ade.
We from the Niger Delta are Real.
Abeg Mama Bakassi...nothing! norring do u. Carry go!
Mama,enjoy.2 ave fun once a while aren't bad.
.....what sort of RAZZ looking guys are these abeg??
Pls Linda, get someone to manage responses here. Some ppl write total nonsense, and are very insultive. These types of comments should be removed promptly.
pple shuld be allowed to express themselves however they choose to cc@
last annoymous comment. it wuldnt be a blog then wuld it? And Linda nhun, didnt know bloggers were suppose to take holiday breaks too.. ;)
I have an aunt who is a good ten years older than this woman but looks to be about the same age, she goes to elegant parties with people her age, people with similar interest etc. She is really cool with her friend's children who are in their 20s and 30s, interacts with us her nieces/nephews on all the latest trends HOWEVER you will never catch my aunt at a young folks only soiree UNLESS one of the parents who is a friend were throwing a party for them and people of her age category would be present. She does not hang out in clubs with small boys like Aunt Flo, I mean, look at the guy even dancing with her, he looks like her driver! The guy looks like house boy, are you serious? Were her husband alive would he approve? Heck, if she were my mother which thank God she isn't, I wouldn't be thrilled about my mother going to these sorts of functions.
I have no problem with Mrs. Ita-Giwa having a good time, it is great that she likes to go out and have a good time after all aren't all 15 kids grown now and she is single BUT she does have a tendency to appear too too much among young, young people. I saw her in pictures at Rita Dominick's birthday party in swim wear with all these young folks like Michael Ezerounye, Ini Edo, Kate Henshaw-Nuttal, Susan Peters etc and it was just and ODD look.
In our society, we expect certain behaviors from our elders whether we like it or not, we expect certain conformity and so all you people talking wetin you dey tok na because e no be your mama. You wouldn't be too thrilled if you stopped by the club and your mother is getting down with your best friend! Or your father getting down with one of your girlfriend, if you are a woman to let's call a spade a spade, Aunt Flo needs to take a couple of steps back!
^^^^^^^well written


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Historic Live Music Venue and Event Space Dallas Observer Best Live Music Venue 2007-NOW Best place to see a concert in Dallas. Acts ranging from Wu-Tang Clan to Bob Dylan to MGMT to Black Keys have all graced our stage. Come see a show an you'll fall in love with the place too!
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Sigurður Eysteinsson
Rafael Alves


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BeschreibungThis book presents the latest developments in the major theories of student motivation as well as up-to-date research on the contextual and cultural variables that influence learning motivation in educational settings. An international roster of experts provides ample illustration of the complexities that are revealed when the study of cultural and contextual interactions is combined with motivational and cognitive variables.
1. The Culture, Context of Learning; F. Salili, et al.
Part I: Attribution Theory/Beliefs and Values: Current Status and Research.
2. Intrapersonal, Interpersonal Theories of Motivation from an Attribution Perspective; B. Weiner.
3. Inferences about Responsibility, Values: Implication for Academic Motivation; S. Graham.
4. The Social Functions of Attributional Face Saving Tactics; J. Juvonen.
5. Declining Optimism in Ethnic Minority Students: The Role of Attributions, Self-Esteem; C. van Laar.
6. Chinese Students' Teachers' Inferences of Effort, Ability; Y.-yi Hong.
Part II: Goal Orientation Theory: New Ideas and Recent Research.
7. Cultural Diversity, Student Motivation, Achievement; M.L. Maehr, R. Yamaguchi.
8. Goal Orientation, Self-Regulated Learning in The College Classroom: A Cross-Cultural Comparison; P.R. Pintrich, et al.
9. Contextual Influences on Motivation, Performance: An Examination of Achievement Goal Structures; T. Urdan.
10. Cross-Cultural Response to Failure: Considering Outcome Attributions with Different Goals; H. Grant, C.S. Dweck.
11. The Influence of Culture, Context on Student's Motivational Orientation, Performance; F. Safili, et al.
12. Goals, Motivation of Chinese Students -Testing The Adaptive Learning Model; K. Shi, et al.
Part III: Context of Learning and Classroom Instruction.
13. Classroom Context Effects on Young Children's Motivation; D.J. Stipek.
14. Teaching Across Cultures; J.B. Biggs.
15. Significance of Cultural, Motivation Variables on Students' Attitudes Towards Group Work; S. Volet.
16. Research on Classroom Instruction, Its Effects - Shortcomings, Dead Ends, and Future Perspectives; A. Helmke. Author Index. Subject Index.
Untertitel: The Culture and Context of Learning. 2001. Auflage. Book. Sprache: Englisch.
Verlag: Springer
Erscheinungsdatum: Mai 2001
Seitenanzahl: 392 Seiten
Format: gebunden


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Administrators have always needed a flexible way to search for content simultaneously across multiple mailboxes. Here are a few scenarios requiring cross-mailbox search, and how the 'export-mailbox' command can be used to get the desired results.
Scenario 1: LEGAL DISCOVERY: The administrator needs a way to extract messages from multiple users' mailboxes which contain certain keywords in the subject and/or body for Legal Discovery purposes. In this case, the content found does not need to be deleted, but only extracted to a single target mailbox.
Solution: The administrator will need to use the Export-Mailbox task from the Exchange Management Shell with the –TargetFolder and –TargetMailbox parameters:
PS> get-mailbox –Database DB1 | Export-Mailbox –SubjectKeywords "Discovery Keywords" –TargetMailbox Administrator –TargetFolder 'MyData'
This will search through all mailboxes on the Mailbox Database DB1 for messages containing the words "Discovery" and "Keywords" in the Subject field and copy these to a folder named MyData in the Administrator's mailbox. A folder will be created for each user mailbox which had matching messages under the "MyData" folder, using the following naming convention: "Recovered Data – <MailboxAlias> – <Timestamp>"
Scenario 2: VIRUS THREAT RESPONSE: The administrator needs the ability to rapidly scan a large number of messages across multiple user mailboxes based on specific criteria to identify suspect messages that may be infected by a virus, and perform mass deletion of such email. In this case, the administrator is not required to keep copies of the deleted emails.
Solution: The administrator will need to use Export-Mailbox with the –TargetMailbox,-TargetFolder, and –DeleteContent parameters as follows
PS> get-mailbox | Export-Mailbox –ContentKeywords "Virus message" –TargetMailbox Administrator –TargetFolder 'MyData' –DeleteContent
This will search through all mailboxes in the organization for messages containing the words "Virus message" in the body and copy them to a folder named MyData in the Administrator's mailbox, and delete the original message from the source mailboxes. The administrator can then delete all the messages under "My Data" to ensure that the suspect virus messages are eliminated. In order to delete the items, you will have to use the deletcontent parameter.
NOTE: To run the Export-Mailbox cmdlet on a computer that has the Mailbox server role installed, you must log on by using a domain account that has the permissions assigned to the Exchange Server Administrators group on the source server and the target server. The account must also be a member of the local Administrators group on that computer. You cannot export data from a mailbox in one forest to a mailbox in a different forest. The source and target mailboxes must be in the same forest
For a complete listing of the search criteria that can be used as filters, and other options for the export-mailbox command, please type "Get-help Export-Mailbox" from the Exchange Management Shell. Alternatively, check the Exchange Server 2007 documentation online.


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Integrity - Uncommon Commitment - Superior Performance
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What kind of car does jay leno drive?
I love Oprah Winfrey she is a role model for me and she is the richest woman alive i think. And if she not the richest then who is. I love Oprah and i would love to meet her one day...this is shant...
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What kind of car does Shawn drive in psych?
Shawn doesn't have a car, he drives a motorcycle. Gus's car, however, is a blue 2002 model Toyota Echo hatchback.
What kind of car does Rush Limbaugh drive?
Rush Limbaugh drives a Maybach 57 (a 57-S, I believe). I think he has an Escalade (LWB) in his fleet of a handful of nicer cars which may also include a late model Rolls Royce but I am not certain...
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What kind of car does George Lopez drive?
George Lopez is currently having a 1951 Chevrolet built by a custom shop. The car will be a very traditional lowider or "bomb" and has a metalflake Mexican flag painted on the roof.
What kind of car does Barack Obama drive?
Barack Obama owned a Ford Escape Hybrid before he won the presidency in 2008. As president in 2010, he is chauffeured in a 2009 Cadillac limousine, which is loaded with high-tech security and safet...
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text: Developing & Maintaining Your Own Website Introduction Every day it becomes clearer that the Net is taking its place alongside the other great transformational technologies that first challenged, and then fundamentally changed, the way things are done in the world. - Louis Gerstner, Chairman & CEO of IBM Question… How do you decide whether to create a business website? Question… How do you decide whether to create a business website? Do your competitors have websites? Where are your customers located? What questions are commonly asked? What we'll cover • Website development – – – – – Domain names Content Design Building Hosting • Website maintenance – Monitoring use – Updating Website Development • Requires a plan • Takes time • Will cost money • Hire help or professional assistance if needed Domain Names • Domain name is your internet address/identity • Typically the business name – Ex. Mercer Vu Farms – www.mercervufarms.com • Can be a product name – Ex. Mountain Dew – product of PepsiCo, Inc www.mountaindew.com Domain Names • Two parts Domain Name Registrars – Label – Extension The popular registrars: •www.godaddy.com •www.easydns.com • Mercervufarm.com label •www.networksolutions.com extension • Possible extensions – .com, .net, .org, .edu, .gov, .info, .biz Domain Names • Two options – Stand alone website • Ex. Yourbusinessname.com – Your website is part of a larger website • Ex. Earthlink.com/yourbusinessname • Can register a domain name without creating a website Website Content • Only include pertinent information • Get input & feedback from people familiar with your business • Start simple – Can always add content and features Question… What content should a business's website include? Website Content Content Musts • Business name • Contact information – – – – Address Phone number Fax number Email address • Business description • Product descriptions and/or photos • Service descriptions Website Content Possible content • • • • • • • • Company logo Company vision and/or mission statement Frequently Asked Questions (FAQ) Purchasing Information Special project info Calendar of events Product use ideas/suggestions Forms/links for sending email Website Content • Content must be accurate, clear, and explicit – Don't assume people know what you mean or understand what you're telling them • Must abide by information presented on the website – Updating is essential when things change • Ex. Product prices increase Website Design • Develop content before designing the site • People form instant opinions – Designs should • Be eye-catching, appealing, not too busy • Encourage people to stay & explore • Clean & Professional • Use flow charts or storyboards Website Design • Check out other websites for design elements that you do and don't like. – www.webpagesthatsuck.com Building the Website • Two choices – You • Saves money • Takes time away from other activities • May take longer depending on ability/knowledge – Lose potential sales – Someone else • May be costly • Already possesses the skills/knowledge • Website can be finished more quickly Website Hosting • Websites need a home – called a server • Internal or External hosting – Internal • You house and maintain the server • Typically more expensive • Requires experienced technical knowledge – External • Someone else owns and maintains the server • Multiple websites on the same server • Most internet service providers (ISPs) offer hosting services. Also local hosting businesses. Website Hosting • Hosting Service Considerations – Support for the software and programming language used to design your website – Web space or storage space needed – Bandwidth – speed and amount of data you can transmit – Back-up services – Other software offered • Shopping carts, email, forums, blogs, etc. Brief Review Steps to Build a Website 1. 2. 3. 4. 5. Register a domain name Develop content Develop a design Build the site Find someplace to host the site Website Maintenance • Need to keep your website up to date and error free – You can do this yourself or hire it to be done • People stop visiting websites with incorrect, out of date, or old information Monitoring Use • Monitoring methods – – – – Logs Visits Hits Cookies • Utilize website monitoring software – Provided by hosting services – Purchase your own Monitoring Use • Use information from web monitoring to decide on necessary updates – Is the website being found? • What are you best sources of traffic? – Are people leaving quickly? • No hits on sub-pages Question… How do you know when and what to update? Updating a website • How do you know when and what to update? – Dependent on content • New, Seasonal products? • Monthly newsletter? – Get feedback from visitors • Feedback forms • Email Section Wrap-Up • Website development takes time • Process involved – – – – – Register a domain name Develop content Develop design Build the site Find hosting • Regularly monitor use • Maintain and update as needed • Get assistance!


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Bollywood actress Shilpa Shetty Kundra feels yoga can eliminate stress, and hence makes it a point to perform yoga during elimination round in children's dance reality show "Super Dancer".
According to a source close to the production house, the actress finds it difficult to eliminate children during the audition rounds. She performs yoga during the elimination rounds.
"Shilpa being a mom loves kids and can't see any kid crying or upset during the elimination round. It's very tough for her to go through these rounds and hence she performs yoga on the set to release stress," the source added.
She will be judging the Sony Entertainment Television show along with choreographer Geeta Kapoor and filmmaker Anurag Basu. It will go on air from September 10.