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Cerebral ischemia results in a rapid increase in contractile cerebrovascular receptors, such as the 5-hydroxytryptamine type 1B (5-HT1B), angiotensin II type 1 (AT1), and endothelin type B (ETB) receptors, in the vessel walls within the ischemic region, which further impairs local blood flow and aggravates tissue damage. This receptor upregulation occurs via activation of the mitogen-activated protein kinase pathway. We therefore hypothesized an important role for B-Raf, the first signaling molecule in the pathway. To test our hypothesis, human cerebral arteries were incubated at 37°C for 48 h in the absence or presence of a B-Raf inhibitor: SB-386023 or SB-590885. Contractile properties were evaluated in a myograph and protein expression of the individual receptors and activated phosphorylated B-Raf (p-B-Raf) was evaluated immunohistochemically.
5-HT1B, AT1, and ETB receptor-mediated contractions were significantly reduced by application of SB-590885, and to a smaller extent by SB-386023. A marked reduction in AT1 receptor immunoreactivity was observed after treatment with SB-590885. Treatment with SB-590885 and SB-386023 diminished the culture-induced increase of p-B-Raf immunoreactivity.
B-Raf signaling has a key function in the altered expression of vascular contractile receptors observed after organ culture. Therefore, specific targeting of B-Raf might be a novel approach to reduce tissue damage after cerebral ischemia by preventing the previously observed upregulation of contractile receptors in smooth muscle cells.
Although cerebral ischemia is a leading cause of morbidity and mortality worldwide, few therapeutic advances appear to be of value in the clinic [1]. We have observed an inherent tendency of the vasculature to undergo phenotypic changes as a response to cerebral ischemia [2]. Therefore, the cerebral vessels show transcriptional upregulation of the vasoconstrictive G-protein coupled receptors (GPCRs) 5-hydroxytryptamine type 1B (5-HT1B), angiotensin II type 1 (AT1) and endothelin type B (ETB) after experimental subarachnoid hemorrhage (SAH) or after focal ischemic stroke [3,4]. Identical receptor upregulation has been observed in patients that died of stroke [5]. In both types of experimental stroke, the receptor upregulation is mediated via the mitogen-activated protein kinase pathway MEK/ERK1/2 [6,7]. A similar type of receptor upregulation can be achieved experimentally by incubating isolated arteries in serum-free culture medium at 37°C for 12 to 48 h [8].
The first signaling molecule in the MEK/ERK1/2 pathway, Raf, is a serine/threonine kinase existing in three different isoforms (A-, B-, and C-Raf) with a common activator, Ras, and a single known common substrate, MEK. Even though MEK is the common substrate, experiments on Raf knock-out mice show isoform-specific functions for A-, B-, and C-Raf [9]. B-Raf is the only isoform that is strongly activated by Ras alone [10] and the most active isoform when it comes to phosphorylating MEK in vitro [11]. We therefore designed this study to examine the role of the B-Raf isoform in inducing the observed GPCR alterations seen after cerebral ischemia. Two previously characterized B-Raf selective inhibitors were used in this study, SB-386023 [12] and SB-590885 [13]. The inhibitors are both small ATP competitive inhibitors with high selectivity for B-Raf when tested against a panel of related protein kinases, but are different in that SB-590885 has a higher affinity for B-Raf. We show that culturing human cerebral arteries in the presence of B-Raf inhibitors strongly attenuates 5-HT1B, AT1, and ETB receptor-mediated contractions compared with arteries cultured with vehicle alone. The receptor proteins were evaluated with immunofluorescence and a marked reduction in AT1 receptor immunofluorescence was observed after treatment with SB-590885. Additionally, the observed increase in phosphorylated B-Raf (p-B-Raf) immunoreactivity after incubation was diminished after treatment with the B-Raf inhibitors.
In vitro pharmacology
Initially, the vessel segments were normalized and stretched to 90% of the internal circumference that a fully relaxed vessel under a transmural pressure of 100 mm Hg would have. The mean normalized internal circumference and standard deviation was 725 ± 297 μm. K+-induced contractions did not differ significantly among the three groups; vehicle, SB-386023, and SB-590885 data confirmed that all groups responded similarly to K+, excluding the possibility that the B-Raf inhibitors had an effect on the viability of the vessels. Emax and pEC50 values for each group are presented in Table 1.
Table 1. Contractile responses to 5-CT, Ang II, and ET-1
Contractile responses to 5-carboxamidotryptamine
5-HT1B receptor-mediated contraction was studied using cumulative application of 5-carboxamidotryptamine (5-CT). Vessel segments treated with SB-386023 or SB-590885 both showed attenuated contractile responses to 5-CT and gave rise to reduced Emax values compared with vehicle-treated vessels (Figure 1A). The inhibitory effect was significant for vessels treated with SB-590885, Emax 11.75 ± 3.43% compared with 39.20 ± 12.09% for the vehicle group (P < 0.05, Table 1).
Figure 1. Contractile responses to 5-carboxamidotryptamine (5-CT) (A), angiotensin II (Ang II) (B), and endothelin-1 (ET-1) (C) in human cerebral arteries. Effect of organ culture in the presence of vehicle (n = 5 to 6) and in the presence of SB-590885 (n = 6 to 7) and SB-386023 (n = 6 to 7) are illustrated. The receptor-mediated contractions are clearly reduced by SB-590885. Data are expressed as mean ± s.e.m. ET-1 biphasic concentration-response curve: high-affinity phase refers to the endothelin type B (ETB) receptor-mediated contraction; low-affinity phase refers to the endothelin type A (ETA) receptor-mediated contraction. Statistical analyses are shown for the Emax values where *P < 0.05 and ***P < 0.001 compared with vehicle. Emax values and pEC50 values for respective concentration-response curves are presented in Table 1.
Contractile responses to angiotensin II
Application of angiotensin II (Ang II) induced a concentration-dependent contractile response at lower concentrations (10-12 - 3·10-9 M) and dilatation at higher concentrations (10-8 - 3·10-6 M, data not shown). The maximum contraction was attenuated after treatment with SB-590885 (11.56 ± 2.72%, P < 0.001) and SB-386023 (26.20 ± 4.37%, not significant) compared with 46.43 ± 6.78% for the control vessels treated with vehicle (Figure 1B, Table 1).
Contractile responses to endothelin-1
Endothelin-1 (ET-1) gave rise to a biphasic concentration-dependent response indicating the presence of both ETA (low affinity) and ETB (high affinity) receptors in a manner previously characterized in detail [14]. The high-affinity phase corresponding to ETB receptor-mediated contraction was significantly decreased in the presence of SB-590885 (7.44 ± 2.44%, P < 0.05) while SB-386023 did not have a significant effect (25.60 ± 7.40%) compared with vehicle 36.71 ± 12.09% (Figure 1C, Table 1). ETA receptor-mediated contractions (low-affinity phase) were not significantly altered by the application of either inhibitor (Figure 1C).
Hematoxylin-eosin staining was performed on all specimens. No morphological differences were observed in the smooth muscle cell layers except for two compressed areas from the wires in the in vitro pharmacology experiments (Figure 2). Therefore, these areas were not used for any kind of evaluation or analysis in the immunohistochemical experiments.
Figure 2. The effect of organ culture and in vitro pharmacology experiments on hematoxylin-eosin staining in human cerebral vessel sections. (A) Fresh, non-cultured vessel; (B) Vehicle-cultured vessel followed by in vitro pharmacology experiment. No morphological changes were observed in the smooth muscle cell layer after the organ culture and in vitro pharmacology experiments compared with the fresh vessel sections. Two compressed areas from the wires during the in vitro pharmacology experiments were observed (*). These areas were not used for evaluation or analysis. Data were obtained with light microscopy. Insert: higher magnification, scale bar 50 μm.
Expression of G-protein coupled receptors
Protein expression of individual receptors was evaluated with immunofluorescence using antibodies against the 5-HT1B, AT1 (Figure 3A, B), AT2, ETA (Figure 3C), and ETB receptors. In addition, double immunostaining was performed with 5-HT1B, AT1, and ETB receptors together with actin (smooth muscle cell layer) to determine the localization of the receptors. Double staining revealed that all three receptors were found in the smooth muscle cell layer (AT1, Figure 3A).
Figure 3. Human cerebrovascular receptor expression. (A) Double staining with actin shows expression of angiotensin II type 1 (AT1) receptors in the medial layer. (B) AT1 receptor expression in fresh and in cultured vessel segments in the presence of vehicle, SB-590885, or SB-386023. The increase in AT1 receptor immunoreactivity after culture was diminished significantly after treatment with SB-590885, and diminished slightly after treatment with SB-386023. (C) No difference in endothelin type A (ETA) receptor expression was observed. Autofluorescence was observed in internal elastic lamina. Data were obtained with epifluorescence microscopy. MED - medial layer, ADV - adventitial layer, and IEL - internal elastic lamina.
Fluorescence intensity measurements were performed on all receptor stainings. Because of inter-individual differences and differences in pre-treatment and in vessel size, we did not see a close correlation between immunostaining and the in vitro experiments. However, a marked increase in AT1 receptor immunofluorescence was observed in organ cultured vessels treated with vehicle compared with fresh, non-cultured vessels. The immunofluorescence was reduced in vessels treated with SB-590885, and to a smaller extent after treatment with SB-386023 (Figure 3B), compared with vehicle. No significant differences in 5-HT1B, AT2, ETB, and ETA (Figure 3C) receptor immunoreactivity were detected.
Expression of phosphorylated B-Raf
The protein expression of activated p-B-Raf was evaluated with immunofluorescence. As in the receptor immunofluorescence experiments, inter-individual and segmental differences were observed. Nevertheless, an increase in p-B-Raf immunoreactivity was observed in cultured brain vessels compared with fresh non-cultured vessels. In addition, immunofluorescence intensity was clearly reduced in vessels treated with SB-590885 or SB-386023 (Figure 4).
Figure 4. Phosphorylated B-Raf (p-B-Raf) expression in human cerebral vessels. Cultured vessel with vehicle only shows increased immunoreactivity compared with fresh, non-cultured vessel. This effect was markedly reduced after treatment with SB-590885 and SB-386023. Data were obtained with epifluorescence microscopy. Insert: higher magnification, scale bar 50 μm.
The present study demonstrates for the first time that upregulation of the contractile receptors 5-HT1B, AT1, and ETB in human cerebral arteries is mediated by B-Raf signaling. These receptor subtypes have been characterized in human cerebral arteries in detail in previous studies [15-17]. In human ischemic stroke, we have observed enhanced levels of protein and mRNA of 5-HT1B, AT1, and ETB receptors in middle cerebral artery smooth muscle cells [5]. Presently, the co-incubation of cerebral arteries with two different B-Raf antagonists prevents organ culture-induced upregulation of contractile responses to 5-CT, Ang II, and ET-1. The effect on receptor-mediated contraction was more prominent after treatment with SB-590885, which is to be expected because it has a lower Kd value, and therefore a higher affinity for its ligand, than SB-380623 [12,13].
In vitro pharmacology
The maximum contraction elicited by 5-CT was significantly reduced, as demonstrated by the reduction in Emax after co-incubation with SB-590885 (Figure 1A, Table 1). The receptor responsible for this contraction has been demonstrated to be the 5-HT1B receptor subtype, which is also selectively expressed in human cerebral arteries, as demonstrated by protein 5-HT1B expression and inhibition by a selective 5-HT1B antagonist [17-19]. The role of 5-HT and its receptors in ischemia are not clear; while some studies report a protective role for 5-HT receptor agonists [20], others show increased contractility and improvement with 5-HT receptor antagonists [21]. Guilbert et al. show that 5-HT1B is responsible for the 5-HT aggravation seen in exercise-induced cardiac ischemia in dogs [21]. Additionally, 5-HT1B receptors have been suggested to interact with 20-hydroxyeiscosatetraenoic acid (20-HETE) and thereby contribute to the acute fall in regional cerebral blood flow after SAH [22]. We have previously reported on increased 5-HT1B protein expression and receptor-mediated contraction after SAH and organ culture in cerebral arteries [14,23], which could impair the cerebral blood flow and thereby contribute to ischemic damage. The present study demonstrates that the selective B-Raf inhibitor SB-590885 significantly decreases 5-HT1B receptor-mediated vasoconstriction, suggesting that the elevated contractile response of 5-HT1B receptors observed after organ culture is regulated by the B-Raf/MEK/ERK pathway.
Organ culture of isolated human arteries in the presence of SB-590885 or SB-386023 reduced Ang II-mediated contraction (P < 0.001 for SB-590885 treated vessels, Figure 1B, Table 1). Available data show that cerebral vasoconstriction in response to Ang II is mediated by AT1 receptors, while vasodilatation is mediated by AT2 receptors [24]. The smooth muscle cell AT1 receptors are upregulated and show enhanced contractile responses after experimental cerebral ischemia [25] or in human ischemic stroke [5]. In support, the reduced vasoconstrictor responses observed in the present study after treatment with the B-Raf inhibitor SB-590885 lead to a concomitant reduction of the AT1 receptor protein when examined immunohistochemically. The receptor identity has been confirmed using selective antagonists for the AT2 receptor in human brain vessels [15]. Blockade of the AT1 receptor has been shown to improve injury after transient cerebral ischemia [26,27] and to reduce cardiovascular morbidity and mortality in stroke patients [28].
In agreement with a previous study, the selective ETB receptor agonist sarafotoxin 6c (S6c) did not elicit any vasoconstrictor responses in cultured human cerebral arteries [16]. Therefore, the high-affinity phase of the ET-1 biphasic concentration-response curve, corresponding to ETB receptor-mediated contraction, was studied. The same situation was seen in the rat middle cerebral artery after experimental SAH, detailed pharmacological analysis revealed participation of the ETB receptor [29]. In the present study, we demonstrate a significant reduction of the ETB maximum contraction after co-incubation with SB-590885; SB-386023 had a weaker effect (Figure 1C, Table 1). No effect on the ETA receptor-mediated contraction was observed after treatment with B-Raf inhibitors. It is well known that cerebral vessels have contractile ETA receptors in the smooth muscle cells and relaxant ETB receptors in the endothelium. However, there is a phenotypic change after stroke in both animals and humans, with the appearance of contractile ETB receptors in the smooth muscle cells [5,30]. The effect of selective ETA blockers on infarct volume after experimental stroke is ambiguous, with studies showing both effect and no effect [2]. Results have been similar for the combined ETA and ETB antagonists bosentan and clazosentan. One study using an ETB blocker showed an increase in infarct volume [31]. The administration of an ETB blocker in conjunction with cerebral ischemia causes a blockade of ETB receptor-mediated dilation, which exacerbates the initial vasoconstriction and increases the infarct. The ETB blocker might be beneficial if it is administered after upregulation of the ETB receptor.
ET receptor antagonists are not the best approach for improving cerebral perfusion after ischemia because of the opposing effects of a strong contractile ETA receptor and a dilatory ETB receptor. However, a different approach, whereby the signal transduction of the Raf/MEK/ERK pathway was blocked with the MEK1/2 inhibitor U0126, diminished the upregulated ETB receptor-mediated contraction and reduced stroke volume [7]. Organ culture of rodent and human cerebral arteries is a way to simulate ETB receptor upregulation and to study the molecular mechanisms involved. In the present study, we show that blockade of the MEK/ERK1/2 pathway using upstream B-Raf inhibitors results in attenuated ETB receptor-mediated contraction after organ culture.
When examined with hematoxylin-eosin staining, no morphological changes were observed in the vessels except for the regions where the steel wires used in the in vitro pharmacology experiments have been attached (Figure 2). However, it became obvious in the immunohistochemical examination that the vessels showed considerable inter-individual differences, most likely due to differences between the patients themselves. Some of the patients exhibited more consistent results than others (e.g., repeated immunostainings displayed stable results). These inter-individual differences could explain the inconsistency in the results obtained with the fluorescence intensity measurements.
Immunohistochemical staining using the 5-HT1B antibody showed no differences between the groups. In other studies, 5-HT1B expression in rat cerebral arteries is increased after middle cerebral artery occlusion and SAH [3,4].
AT1 receptor immunoreactivity was reduced after treatment with SB-590885. Previously, increased AT1 receptor immunofluorescence after SAH in rats has been shown to be reduced after application of SB-386023 [32]. In our study, we observed a decrease in AT1 receptor immunofluorescence intensity after application of SB-590885, but only a small decrease after SB-386023 (Figure 3B), results that are in accordance with the in vitro pharmacology experiments.
ETA receptor-mediated contractile responses were not significantly altered by the two B-Raf inhibitors used in the present study (Figure 1C). Immunohistochemical examination disclosed the same pattern; no differences were observed between the groups (Figure 3C).
There was an increase in p-B-Raf immunoreactivity after organ culture and this effect could be reduced considerably in the presence of SB-590885 and SB-380623 (Figure 4). Therefore, the activation of B-Raf protein kinase could be blocked by the application of specific antagonists. We suggest that B-Raf is important for the phenotypic changes of GPCRs observed in the smooth muscle cells of cerebral arteries after organ culture and cerebral ischemia [2]. An interesting question is whether B-Raf functions alone or in a heterodimer in this aspect. There is evidence for B-Raf/C-Raf heterodimerization with highly increased kinase activity compared with the respective homodimers or monomers [33]. Further studies are needed to elucidate whether heterodimerization is important for the regulation of GPCRs in vascular smooth muscle cells after ischemia and organ culture.
In conclusion, we show that selective inhibition of B-Raf using SB-590885 significantly attenuates 5-HT1B, AT1, and ETB receptor-mediated contraction in human cerebral arteries. Therefore, we suggest that B-Raf is important for the altered GPCR expression observed after cerebral ischemia, and that specific blockage might be a novel approach to reduce tissue damage after stroke.
This study was approved by the Regional Ethical Review Board in Lund, Sweden (LU-818-01) and has been performed in accordance with the Declaration of Helsinki.
Tissue collection
Cerebral arteries were obtained from patients undergoing neurological surgery for removal of brain tumor or seizure-producing cortex in severe cases of epilepsy (n = 7, 55 to 77 years old, 4 men and 3 women). Adjacent tissue that was removed contained one or two vessel segments that were used for the experiments. The vessels were dissected during surgery and immediately immersed in cold, sterile Dulbecco's modified Eagle's medium (DMEM, Gibco, Invitrogen, Carlsbad, CA, USA) and transported to the laboratory. The arteries were dissected free from adhering tissue and cut into cylindrical segments approximately 2 mm in length. The outer diameter of the vessels ranged from 250 to 950 μm.
Organ culture
Arterial segments were cultured for 48 h at 37°C in humidified 5% CO2 and air in serum-free DMEM supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml) (Gibco). The method of blood vessel culture and upregulation of contractile receptors has been described previously [34]; the upregulation is not altered with serum present in the medium or by incubation in buffer only, but is a metabolically active process. The segments were cultured in well plates, one segment per well, in the absence or presence of B-Raf inhibitors: SB-386023, 1 μM (Kd B-Raf: 2.4 nM, GlaxoSmithKline, UK); SB-590885, 1 μM, (Kd B-Raf: 0.3 nM, GlaxoSmithKline); or the same volume of vehicle (dimethyl sulfoxide). Incubation was also performed with 10 μM SB-590885, which resulted in non-viable arteries when examined by in vitro pharmacology; therefore arteries incubated in SB-590855 were not used for further experiments. After 24 h of culture, the DMEM was replaced with new fresh medium and inhibitors or vehicle were added as described above. For each patient and group, one to three vessel segments were incubated and examined by in vitro pharmacology and/or immunohistochemistry (see below).
In vitro pharmacology
For contractile experiments, a myograph was used to record the isometric tension in isolated cerebral arteries [35,36]. The cylindrical segments were threaded on two parallel stainless steel wires (diameter, 40 μm) and mounted in a Mulvany-Halpern myograph (Danish Myo Technology A/S, Aarhus, Denmark). One wire was connected to a force displacement transducer attached to an analogue-digital converter unit (ADInstruments, Chalgrove, UK). The other wire was connected to a micrometer screw, allowing adjustments of the distance between the wires and therefore the vascular tone. Measurements were recorded on a computer by use of a Power Lab unit (ADInstruments). The vessel segments were immersed in temperature-controlled tissue baths (37°C) containing a bicarbonate buffer solution of the following composition (mM): NaCl 119, NaHCO3 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5, and glucose 5.6. The buffer was continuously aerated with oxygen enriched with 5% CO2, resulting in pH 7.4. After an equilibration period of ~20 min, each vessel segment was stretched to 90% of the normal internal circumference, which would be the size each vessel would have if relaxed and under a transmural pressure of 100 mm Hg [36]. The normalization procedure makes certain that all vessel segments are set to a normalized internal circumference giving maximal response. Subsequently, the vessels were allowed to stabilize for 20-30 min. The contractile capacity of the vessels was determined by exposure to an isotonic solution containing 63.5 mM K+, obtained by partial change of NaCl for KCl in the above buffer. The contraction induced by K+ was used as reference for contractile capacity [35].
Concentration-response curves were obtained by cumulative application of 5-CT (5-HT1B receptor agonist, concentration range 10-11 to 3·10-5 M, Sigma, St. Louis, USA), Ang II (AT1 and AT2 receptor agonist, concentration range 10-12 to 10-6 M, Sigma) and ET-1 (ETA and ETB receptor agonist, concentration range 10-14 to 3·10-7 M, Alexis Biochemicals, Farmingdale, NY, USA). The contractile responses to S6c, a selective ETB receptor agonist, were tested in a few samples and no contractile responses were observed, in agreement with a previous study [16]. Therefore, the high-affinity phase of the ET-1 concentration-response curve was used to study ETB receptor-mediated contraction.
Immediately after the in vitro pharmacology experiments, the arterial segments were carefully dismantled (n = 4) and embedded in Tissue TEK (Gibco), frozen on dry ice, and kept at -80°C until cryosectioning (10 μm). Additionally, both fresh (non-cultured) and cultured arterial segments (described above) were directly embedded in Tissue TEK without prior in vitro pharmacology experiments (n = 3). These arterial segments were used for the ETA and ETB immunohistochemistry experiments because of the irreversible binding of ET-1 to receptors in the in vitro pharmacology experiments that would likely affect antibody-antigen binding [37].
The sections were collected onto Superfrost(r)Plus (Menzel-Gläser, Braunschweig, Germany) glass slides and stored at -80°C until immunohistochemistry. Sections from the in vitro pharmacology experiments were stained with hematoxylin-eosin to evaluate vessel morphology and the possible effects of the in vitro pharmacology experiments. Thawed sections were fixed for 10 minutes in -20°C acetone and rehydrated in phosphate buffered saline (PBS, pH 7.2) containing 0.25% Triton X-100 (PBST) for 3 × 5 min at room temperature. The sections were then blocked for 1 h at room temperature in blocking solution containing PBS and 5% normal serum (goat or donkey serum, depending on the secondary antibody hosts) to ensure secondary antibody specificity. Subsequently, sections were incubated overnight at 4°C with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA): goat anti-human 5-HT1B 1:100, rabbit anti-human AT1 1:100, rabbit anti-human AT2 1:100, goat anti-human ETB 1:150, and rabbit anti-human ETA 1:50, diluted in PBST containing 1% bovine serum albumin (BSA) and 3% normal serum. Following primary antibody incubation, sections were rinsed in PBS for 2 × 15 min and incubated for 1 h at room temperature with secondary antibodies (FITC-conjugated goat anti-rabbit (Cayman Chemical Co., Ann Arbor, MI, USA) 1:100 or Cy2-conjugated donkey anti-goat (Jackson ImmunoResearch, West Grove, PA, USA) 1:200) in PBST containing 1% BSA and 3% normal serum (goat and donkey serum, respectively). Sections were then rinsed in PBS for 2 × 15 min and mounted in anti-fading mounting medium (Vectashield; Vector Laboratories Inc., Burlingame, CA, USA) or polyvinyl alcohol mounting medium (compatible with Cy2-conjugated secondary antibodies, Sigma). The procedure was repeated on three different occasions to ensure reproducibility. The same experimental procedure was used for negative controls, but primary antibodies were omitted, resulting in no staining in the tissue except for auto-fluorescence in the internal elastic lamina.
Double immunostaining was performed for the 5-HT1B, AT1, and ETB receptors. Antisera against the receptors (described above) were used along with mouse anti-human smooth muscle actin 1:200 (Santa Cruz Biotechnology) and Texas Red-conjugated donkey anti-mouse 1:200 (Jackson ImmunoResearch).
In addition, immunostaining against p-B-Raf was performed on the sections. The protocol mentioned above was used with a primary rabbit anti-human phosphospecific B-Raf antibody 1:25 (Abcam, Cambridge, UK). The secondary antibody used was Cy3-conjugated donkey anti-rabbit 1:400 (Jackson ImmunoResearch).
Immunoreactivity was visualized at the appropriate wavelengths with a light and epifluorescence microscope (Nikon 80i; Tokyo, Japan) and photographed with an attached Nikon DS-2Mv camera. To evaluate colocalization, images from double immunostaining were superimposed in Adobe Photoshop CS (Adobe systems, Mountain View, CA, USA).
Analysis and statistics
Contractile responses from the in vitro pharmacology experiments are expressed as percentage of the contraction induced by 63.5 mM K+. The Emax value represents the maximum contractile response elicited by an agonist and the pEC50 represents the negative logarithm of the drug concentration that elicited half the maximum response. For biphasic responses, Emax (1) and pEC50 (1) describe the high-affinity phase, and Emax (2) and pEC50 (2) describe the low-affinity phase.
The protein expression of the 5-HT1B, AT1, AT2, ETB, and ETA receptors in the smooth muscle cell layer of each vessel were analyzed by fluorescence intensity measurements in four areas within each vessel sample (n = 3 to 4 patients in each group) by use of the ImageJ software http://rsb.info.nih.gov/ij webcite. The four areas were chosen after evaluating the hematoxylin/eosin staining results of all specimens.
Data are expressed as mean ± standard error of the mean (s.e.m.), and n refers to the number of patients. Statistical analyses were performed with Kruskal-Wallis non-parametric test followed by Dunn's multiple comparison test, where P < 0.05 was considered significant.
Study limitations
Human tissue was obtained during neurosurgery for treatment of tumors or epilepsy. Although the surgeons carefully dissected out adjacent non-cancerous or seizure-producing tissue, effects on adjacent tissue cannot be excluded. The collection of human cerebral arteries is performed over a long period of time. Finally, there was great variation in vessel diameter, owing to limited access to human material.
List of abbreviations
5-CT: 5-carboxamidotryptamine; 5-HT1B: 5-hydroxytryptamine type 1 B; Ang II: Angiotensin II; AT1: Angiotensin II type 1; ERK1/2: Extracellular signal-regulated kinase 1 and 2; ET-1: Endothelin-1; ETB: Endothelin type B; GPCR: G-protein coupled receptor; MEK: Mitogen-activated ERK activating kinase
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HA participated in the design of the study, performed the experiments, analyzed the data, and wrote the manuscript. HS and ON performed the surgeries and reviewed the manuscript. LE conceived the study, guided the experimental procedures, and participated in writing the manuscript.
Thanks to Karin Warfvinge and Warfvinge Science Support AB http://www.sciencesupport.se/ for assistance and valuable comments on the manuscript. This work was supported by the Swedish Research Council, the Heart and Lung Foundation, Sweden, and the Lundbeck Foundation, Denmark.
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Prehn JH, Welsch M, Backhauss C, Nuglisch J, Ausmeier F, Karkoutly C, Krieglstein J: Effects of serotonergic drugs in experimental brain ischemia: evidence for a protective role of serotonin in cerebral ischemia.
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Guilbert F, Lainee P, Dubreuil B, McCort G, O'Connor SE, Janiak P, Herbert JM: Serotonin aggravates exercise-induced cardiac ischemia in the dog: effect of serotonin receptor antagonists.
Eur J Pharmacol 2004, 497(1):55-63. PubMed Abstract | Publisher Full Text
Cambj-Sapunar L, Yu M, Harder DR, Roman RJ: Contribution of 5-hydroxytryptamine1B receptors and 20-hydroxyeiscosatetraenoic acid to fall in cerebral blood flow after subarachnoid hemorrhage.
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Hoel NL, Hansen-Schwartz J, Edvinsson L: Selective up-regulation of 5-HT(1B/1D) receptors during organ culture of cerebral arteries.
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Vincent JM, Kwan YW, Chan SL, Perrin-Sarrado C, Atkinson J, Chillon JM: Constrictor and dilator effects of angiotensin II on cerebral arterioles.
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Stenman E, Edvinsson L: Cerebral ischemia enhances vascular angiotensin AT1 receptor-mediated contraction in rats.
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Hosomi N, Nishiyama A, Ban CR, Naya T, Takahashi T, Kohno M, Koziol JA: Angiotensin type 1 receptor blockage improves ischemic injury following transient focal cerebral ischemia.
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Engelhorn T, Goerike S, Doerfler A, Okorn C, Forsting M, Heusch G, Schulz R: The angiotensin II type 1-receptor blocker candesartan increases cerebral blood flow, reduces infarct size, and improves neurologic outcome after transient cerebral ischemia in rats.
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Schrader J, Luders S, Kulschewski A, Berger J, Zidek W, Treib J, Einhaupl K, Diener HC, Dominiak P: The ACCESS Study: evaluation of Acute Candesartan Cilexetil Therapy in Stroke Survivors.
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Hansen-Schwartz J, Hoel NL, Zhou M, Xu CB, Svendgaard NA, Edvinsson L: Subarachnoid hemorrhage enhances endothelin receptor expression and function in rat cerebral arteries.
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Chuquet J, Benchenane K, Toutain J, MacKenzie ET, Roussel S, Touzani O: Selective blockade of endothelin-B receptors exacerbates ischemic brain damage in the rat.
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Ansar S: Protein kinase inhibition in late cerebral ischemia after subarachnoid hemorrhage. Thesis Lund University, Sweden; 2007.
Rushworth LK, Hindley AD, O'Neill E, Kolch W: Regulation and role of Raf-1/B-Raf heterodimerization.
Mol Cell Biol 2006, 26(6):2262-2272. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Adner M, Geary GG, Edvinsson L: Appearance of contractile endothelin-B receptors in rat mesenteric arterial segments following organ culture.
Acta Physiol Scand 1998, 163(2):121-129. PubMed Abstract | Publisher Full Text
Hogestatt ED, Andersson KE, Edvinsson L: Mechanical properties of rat cerebral arteries as studied by a sensitive device for recording of mechanical activity in isolated small blood vessels.
Acta Physiol Scand 1983, 117(1):49-61. PubMed Abstract | Publisher Full Text
Mulvany MJ, Halpern W: Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats.
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Meens MJ, Fazzi GE, van Zandvoort MA, De Mey JG: Calcitonin gene-related peptide selectively relaxes contractile responses to endothelin-1 in rat mesenteric resistance arteries.
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EventsA Night of Worship with Matt Maher
October 17th @5:45pm in Ashburn, VA Read More →tobyMac – This Is Not A Test Tour
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And an NFL game of your choice! Listen to Jerry & Blanca next week at 7:40am. Read More →Seven Days Without
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subset: commoncrawl


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At Riggs Bank, A Tangled Path Led to Scandal
Published: July 19, 2004
(Page 2 of 3)
''Why did the Pinochet accounts have to be closed?'' she asked, according to testimony in the Senate hearing on Thursday by Lester J. Miller, a federal regulator who monitored Riggs accounts and attended the meeting with her.
Bank regulators, who stumbled across the Pinochet funds during a Riggs review in early 2002, could have given Mrs. Allbritton compelling reasons for closing the accounts. Even at a time when the general was detained on charges of human rights abuses and his assets had been frozen by court orders, Riggs helped him disguise millions of dollars in suspect funds and wire the money worldwide, the Senate report says.
''In 1994, top Riggs officials traveled to Chile and asked General Pinochet, a notorious military leader accused of involvement with death squads, corruption, arms sales and drug trafficking, if he would like to open an account at Riggs Bank here in Washington,'' Senator Carl Levin, a Michigan Democrat, observed in the hearing. ''Mr. Pinochet said yes.''
Mr. Allbritton, who secured a controlling stake in Riggs in 1981, coveted the bank's international cachet and made regular business trips abroad, including to Chile. Raymond M. Lund, a former Riggs executive who opened General Pinochet's accounts at the bank in the mid-1990's, said in Senate testimony that Mr. Allbritton had a ''professional business relationship'' with the dictator. Carol Thompson, who oversaw Riggs's Latin American business, told Senate investigators that she occasionally briefed Mr. Allbritton directly on General Pinochet's finances. The Allbrittons have not responded to interview requests.
While General Pinochet's accounts at Riggs may have gone unnoticed by United States regulators until 2002, they were not a secret to the rest of the world. The Associated Press wrote about them as early as 1999, when judicial proceedings began in Spain against General Pinochet. On Dec. 10, 2000, The Observer, a British paper, also mentioned them in connection with possible drug trafficking. Senate records show that the Riggs accounts held $4 million to $8 million from 1994 to 2002.
When regulators asked Riggs in 2000 for a list of its accounts controlled by political figures, the roster provided by the bank did not include General Pinochet's name. Shortly after The Observer article was published, the bank -- in a move that it acknowledged last week was improper -- changed the name on accounts of the general and his wife from ''Augusto Pinochet Ugarte & Lucia Hiriart de Pinochet'' to ''L. Hiriart &/or A. Ugarte,'' ensuring that searches for Riggs accounts named ''Pinochet'' would draw a blank.
General Pinochet was arrested in Chile in early 2001, causing Riggs officers and its board to review whether it was proper to maintain his accounts. The accounts remained open, and the general's arrest was later overshadowed in the news by the terrorist attacks in the United States.
Regulators began vetting American banks more thoroughly after Sept. 11. During a review of Riggs's international operations in April 2002, examiners with the Comptroller of the Currency came across the Pinochet accounts. Overtaxed to complete the international review and a subsequent review of terrorist financing, regulators notified Riggs that they would return in June for a closer look.
On April 8, 2002, Riggs sent $500,000 in cashiers' checks to the general. Later analyses by Senate investigators indicated he cashed them to pay personal expenses. Sometime between the spring and summer of 2002, according to the Senate report and testimony last week, Riggs tried to withhold information about the Pinochet accounts from regulators and then, rather than freeze the accounts as is customary, closed them and returned the money to General Pinochet.
Although Mr. Lund said last week that Riggs executives had documented the source of the general's wealth, Senate investigators say the bank never made any sincere effort to do so, as required by law. Moreover, regulators never considered fining Riggs in 2002 or referring the Pinochet accounts to law enforcement officials, according to Senate records and testimony. The comptroller's lead Riggs examiner at the time, R. Ashley Lee, took an executive position at Riggs later that year.
Mr. Lee said last week that he never made any effort to water down regulatory oversight of Riggs. Senator Levin said that his committee's evidence suggested otherwise and that he planned to ask the Justice Department to investigate.
The Saudi Funds
By October 2002, when Mrs. Allbritton voiced her resentment to regulators about losing General Pinochet's business, another problem was about to engulf Riggs. In November, Newsweek magazine reported that the Federal Bureau of Investigation was examining Saudi Arabian Embassy accounts at the bank in connection with the Sept. 11 attacks.
The F.B.I. investigation, focusing on accounts controlled by the wife of Prince Bandar bin Sultan, Saudi Arabia's longtime ambassador to the United States, was news to regulators. Once again, as the Senate report showed, outside forces were making them examine Riggs more closely and Riggs executives were proving to be, at best, indifferent gatekeepers.


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Cpf1 Discovery Unlikely to Affect the...
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Predictive Diagnostic Tests Are Continuing To...
This video will cheer you up during long hours at the lab this holiday season.
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Google Glass Alamo Drafthouse 'ban' further clarified
Google Glass once again made headlines when a movie theater owner "officially banned" the wearable. The details of the ban were shared by Tim League, who is the theater owner. But while the Glass being banned aspect was caught in many headlines -- it seems League didn't draw a firm line. Not to mention, Glass isn't the only item he bans from use while the movies are playing.


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862 Views 1 Reply Latest reply: Aug 16, 2013 3:03 AM by dominic23
dominic23 Aug 16, 2013 3:03 AM
Re: Charger flashing and the mac not turn on in response to Thapzz
Portables that will not Power on.
Go step by step.
Note: Steps 5 and 6
Reset SMC.
Choose the method for:
"Resetting SMC on portables with a battery you should not remove on your own".
If you plan to visit an Apple Retail Store, make a reservation at the Genius Bar using http://www.apple.com/retail/geniusbar/ .


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Many of you have asked me about several alternative technologies and why they are not used in the Tesla Roadster. These technologies range from other existing battery technologies (e.g. lithium iron phosphate) to technologies that are on the horizon (e.g. ultracapacitors) to impossible technologies that ultimately boil down to perpetual motion. I will try to address as many of these questions as I can this week.
Batteries A123 to Zebra
The bottom line for us is energy density: how many kilowatt hours can we fit in the given space, for the least weight. The amount of energy we can store onboard directly translates to range. For sorted-out, mass produced batteries today, it's lithium ion, no doubt about it. That's why all our handheld goodies use lithium ion.
What's the difference between energy and power?
The amount of energy available from the batteries is like the size of your gas tank. The more energy available, the longer the driving range – all else being equal.
The amount of power available from the batteries is like the size of the fuel line from the tank to the engine – if the battery's power output is too low, it will reduce the horsepower (and therefore acceleration) of the car.
You need to design for both.
Other technologies (e.g. A123's lithium iron phosphate cells) deliver higher power density, but have lower energy density. These are beginning to find applications in power-hungry equipment like cordless drills – and maybe they are the right answer for a shorter-range electric car designed for quick sprints (electric dragsters?).
Some battery chemistries (e.g. Sodium-Nickel Chloride Zebra batteries) have similar energy density to lithium ion batteries, but have much, much lower power density. A 250-mile Tesla Roadster with Zebra batteries would do 0-60 mph in something like 20 seconds. (Zebra batteries also suffer from terrible self-discharge, but that is another subject.) As it turns out, when we designed a battery pack that delivers the desired 250-mile range, it happens also to produce enough power for the kind of acceleration we were after. In this sense, we lucked out with standard lithium ion batteries.
Ultracapacitors are not plug-in replacements for batteries mainly because their energy density is so low. Today's ultracapacitors store around 1% of the energy of today's batteries and the resulting car would have around 1% of the range. They do however deliver very high power density and are excellent for applications needing high power and low energy.
Another difficulty with ultracapacitors is that their voltage drops steeply (exponentially in fact) with state of charge. This means that we would need a fairly sophisticated high-power switching power supply to normalize the voltage to the motor, which isn't impossible, but is definitely tricky and costly.
None the less, I do think that new high-energy ultracapacitors may one day be the right answer for electric cars, just not now. Nobody makes ultracapacitors with anywhere near the energy density or pricing needed. That's why they are not used in any consumer electronics yet. But when ultracapacitors are ready for prime time, we will be all over them.
Something for Nothing
It is tempting to ridicule the various proposals for perpetual motion, but somehow I just can't do that. Some proposals show a lack of understanding of the basic laws of physics, but other schemes are devious enough that the logical flaw is not obvious until you really dig into the proposal.
But listen: you can't get something for nothing. You just can't. If we put generators on the front wheels to charge the batteries while you drive, they will cause the drive motor on the rear wheels to consume additional energy. This additional energy will be more than the generators produce, in all cases, no matter what the particular arrangement of generators, batteries, and motors
The same thing is true for wind turbines: a wind turbine on the car will always increase the aerodynamic drag, and the energy it produces will never be as much as the additional energy needed by the drive motor to overcome the wind turbine's drag.
They're the laws of physics, folks, and there is nothing we can do to overcome them.
I get a couple of free energy proposals every week now – some very simple, some quite involved – and I admit right here that I do not and will not read them. Call me closed-minded, but I've come to have a lot of respect for the basic laws of physics. Push them to their limits, bend them to our needs, take every advantage of the corner cases – but don't count on breaking them. (Oh – by the way – Nikola Tesla never made an electric car that ran for months with no batteries. Somebody made up that story. Trust me.)
Other Ideas
I was all intrigued by the idea of wheel motors. What could be simpler? Mount a motor right in the wheels. Presto, no differential, no drive shafts, no CV joints, instant traction control and stability control. What kills the idea of wheel motors for road cars is unsprung weight.
Unsprung weight is the weight of the car that is below the springs – tires, wheels, brakes, hubs, etc. The higher the ratio of unsprung to sprung weight, the worse the car handles unevenness on the road. Tracking the road is so important that even inexpensive cars now commonly are sold with aluminum alloy wheels, alloy suspension components, etc. that knock a few pounds of unsprung weight off each corner of the car. Adding 30 unsprung pounds to each corner would mean abysmal handling.
We thought a lot about putting solar panels on the upward-facing portions of the body. The trouble is that the square footage available for solar panels would only increase your daily driving range by about 5 miles. While there is a sensible argument for such solar panels topping off your battery when you leave your car in the airport's long-term parking for a week, it just did not seem worth the complexity, weight, and cost.
Solar panels make so much more sense on the roof of your house, or out in the desert someplace where there is enough acreage available to power all your driving. This becomes obvious when you think of the electric grid as storage: solar panels produce energy during peak consumption times, and your electric car consumes energy at night while charging – when energy is the least expensive.
Solar panels on an electric car seem a bit like fins on a '59 Caddy.
Balanced Innovation
Our strategy here at Tesla Motors is to use the very best technology that is actually available. We innovate where we can and where it matters, but we avoid frivolous innovation and getting mired in unsolvable problems. I think we've done a pretty good job with the Tesla Roadster. We will continue to evaluate new technologies, innovate where sensible, and make the best choices for every future Tesla Motors model.
I have to disagree with a couple things here. First, motorcycles are not incredibly efficient. Many of them get worse fuel mileage than automobiles. Many of them have larger engines than automobiles-- The current crop of Harley Davidsons have as standard a 96 cubic inch motor. I think my 1987 Integra doesn't have that much displacement. شركة تنظيف شقق بالرياض شركة تنظيف مجالس بالرياض شركة تنظيف خزانات بالرياض My daily commute vehicle, a 2003 Harley Sportster 883cc, is EPA-rated 60 mpg hwy. The new 2007 HD big twins are 33 mpg. Not exactly incredibly efficient, given that they weigh less than half as much as cars that get the same mpg.
dan heath-caldwell
why dont you just put solar pannels in the body work? that way you dont have anything that looks like its out of back to the future, and it can constantly be charging your batteries, if you drive to work, leave your car in an open air car park, the batteries will be charged for your drive home, they may be expensive, but will make the car more practical and give it a longer range if you cant get to a power source
Well I hope someone sees my post....
I'm glad Martin addressed the solar panel idea. I ended up hear by looking for a way to send the idea of making the exterior of the car out of solar panel-type materials. The solar nanotechnology they're developing in Idaho sounds promising. I hope someone will keep a tab on their progress, here's a ridiculously long link....
I'd also really like to see Tesla Motors come out with an SUV or something along those lines. With their performance capabilities, they could develop some excellent off-road vehicles. It's been a dream of mine to see an vehicle that can take us to the boonies without polluting them at the same time.


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Valley soccer experiencing resurgence
TribLIVE Sports Videos
Valley boys soccer coach Andy DeAntonio refers to his team's resurgence as part of "the process."
For the Vikings, winning is important, but it isn't everything. Losing isn't a measure of ineptitude, just the technical result of a single game.
"Wins and losses won't ever validate what the team does," DeAntonio said. "I think it does to everyone else, but especially with this group of guys, the wins and losses are the outcome of the process. It's a whole lot nicer to win, but we understand if we played a nice game with or without a winning result."
The Vikings haven't had to spend much time measuring losses this year.
On Tuesday, they suffered their first loss of the season to Deer Lakes. The 2-0 loss lowered Valley's record to 5-1, a single victory shy of the team's combined wins over the past two seasons. It's also the most games the Vikings have won in a single season since 2004, when they finished 5-9-3. Last year, they were 2-15.
"This year, talent and skill has met hard work more than it has in my other years. The kids understand that those two things go together," DeAntonio said. "It's not about the end result; it's not about the win. It's about following the process to get the win."
Senior midfielder Steven Colagrande, a three-year starter and co-captain, is among those who have bought into DeAntonio's philosophy.
Colagrande said the primary function of "the process" is to foster team cohesion and allow players to look at the bigger picture. While the Vikings thrive on possession soccer, they are neither an attacking nor a defensive team but rather a balanced unit — one that is willing to pass until a flaw can be found to exploit.
"I like to say, 'Have a purpose with the ball. Play it into space. Don't play kickball,' " Colagrande said. "It's been hard for the team to grasp. But that's what the greatness of our team is, the balance of offense and defense."
Colagrande shared that everyone grasping "the process" required a lot of time and patience in practice, film sessions and individual instruction. But over time, players began to believe in the system, the results of which have been noticeable on the field this season.
"It's definitely a change," Colagrande said. "Coach says you should hold your chest out proud in school. It's hard coming from losing, going to that winning feeling. It's hard to get used to it and keep your composure about it."
DeAntonio echoed that sentiment.
"Without a doubt, they've built some belief," he said. "Yes, we're concerned with getting the win, but the clear message now is that they understand to refer back to the process. It has shown to be successful, so I think that the confidence is coming along, not only with that process but with themselves."
Stephen Catanese is a freelance writer.
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This week, All In the Mind takes you to the movies to explore the psychology of collective terror. The dawning of the Atomic Age brought with it a whole new pop culture genre - Nuclear Movies. From Mickey Rooney's Atomic Kid to the giant ants in Them! through to the seductive charms of 007, nuclear flicks were about much more than bombs, monsters, deformed villians and virulent plagues. They represented the shocking realisation that we were capable of our own mass destruction. Scary but ever timely contemplations on the cinematic psyche...
This [series episode segment] has
Marsat Ketchell joins Natasha Mitchell in conversation. Born and bred on Thursday Island in the Torres Strait, Marsat left home at the tender age of 15 to join the Royal Australian Navy. After 26 years of service he returned to his island home to become the first ever mental health worker, indigenous or non-indigenous, to be permanently based in the Torres Strait and Northern Cape York Peninsular region. Now he's part of a team keeping islanders healthy in body and mind.
This week, growing up with a parent who has mental illness. Small children frequently take on a carer role well before they're ready for it and their needs remain invisible to adult mental health workers and even to other family members. Many parents are frightened to disclose that they have children for fear that they'll be taken away. Natasha Mitchell reports on the critical issues, and efforts in Australia to put children's experiences at the fore.
The cyborg, that posthuman hybrid of flesh and machine, has long been fodder for futuristic Hollywood flicks like Terminator. Cyborgs make most of us nervous about what sort of future we're facing. But acclaimed philosopher and cognitive scientist Andy Clark reckons all of us are already Natural Born Cyborgs, with minds made to merge with the material world - your watch, paper, computer. Our mind, he argues, extends well beyond our brain, beyond our ancient skinbag and into the world at large. The cyborgian future is here...and it always been.
Suspend reality and join the world's philosophers in their contemplation of a very tricky problem indeed. Open your eyes and it feels as if our experience of vision and consciousness are continuous, complex, rich and colourful - thanks to some hard work put in by the brain. Actually, perhaps not. There are all sorts of gaps in our conscious experience which has prompted some to argue that we don't actually see the world as it really is. Yes, seriously, could it all be a grand illusion? The conundrum of human consciousness strikes again on All in the Mind.
The ticking time bomb that is an aneurysm. They can burst with very little warning and having one can be like living on a precipice. Surgery has its risks and life after a ruptured aneurysm brings all sorts of challenges. Only 1 in 10,000 of us are likely to experience one of these little beasts, usually in our brain. But in some extraordinary cases they run in the family across generations - mum, daughter, aunt, son - and an international study is trying to find out why this is. Sue Clark reports.
Once 'animal spirits' were fluid beasts, distilled from blood, that roamed our nervous system and rummaged the pores of our brain. The patterns of their flow carried the contents of our thoughts and were the going explanation for vision, memory, imagination, belief, passion and desire. When animal spirits misbehaved or became polluted they could make us ill, in both body and soul. For most of this millennium Westerners firmly believed in these lively and impish carriers of our identity and they only disappeared after the nervous system was found to be electrical in nature. This week, philosopher John Sutton sets a few animal spirits free to explore the mind in history.
Feared and reviled, physical pain may not be popular but it's an everyday experience for so many people. Patrick Wall, who died in 2002 after a long battle with prostate cancer, was the pin-up boy of pain researchers worldwide. They dubbed him the "pain man of the century". In this posthumous broadcast of an interview with All in the Mind's Natasha Mitchell, given in his home before his death, Patrick Wall discusses his influential work, and offers an insight into his own personal experience of pain from terminal cancer.
Is your memory failing? Considered popping in a memory chip? This week,
This week, All in the Mind whips out a handkerchief and has a good cry. Much of the science about sobbing has been speculative to date. Two longtime collaborators on a theory of weeping join Natasha Mitchell to contemplate whether adult crying is indeed cathartic, whether women cry more than men and why it is that only humans cry emotional tears? Weeping and wailing - it might be just what's needed at times like these.
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Sunday 11 October 2015
At the age of 24, acclaimed novelist Matt Haig's world suddenly fell apart—he could no longer see a reason to live. Haig's memoir is an honest and inspiring account of how he emerged from severe depression and anxiety, and learned to live again.


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In an analyst insight article from Euromonitor International analyst Robert Walker, Walker explores the new global market frontiers and their desire for beauty products.
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Growing Demand for Natural Beauty Products in Asia-Pacific Urges on Natural Ingredients
As more consumers in Asia-Pacific countries learn about and seek out more natural beauty and personal care products, there is a continually greater push for beauty ingredient suppliers to debut new innovations to provide solutions for beauty products and replace synthetic ingredients.
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Nu Skln Lays Out Serious Growth Plan for China
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Seven Scent Predicts 2013/14 Autumn-Winter Fragrance Trends
Tea and wood accords, leathers, spices, amber and red berries will be among the popular fragrance influences in the cooler months next year.


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Foreign workers the latest irritant for Kimberley gas supporters
It's been a rocky road at times for Woodside, the WA Premier and traditional owners of the James Price Point gas precinct site. Now the issue of foreign workers has highlighted the ongoing tensions.
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The political storm continues to rage over 1700 skilled worker, 457-visas being approved for Gina Rinehart's Roy Hill iron ore mine in the Pilbara. Now moves towards foreign workers in the neighbouring Kimberley region are starting to inflame competing interests between the main players behind gas processing north of Broome.
In response to questions about 457-visa workers for the proposed Browse gas plant north of Broome, a Woodside spokesman said "We aim to give skilled Australians priority employment on our projects, but the reality is that use of skilled migration is necessary in our industry to supplement the recruitment, training and development of local Australian workers within the oil and gas industry."
Woodside used the 457-visa program during the construction of the Pluto LNG project and says they may need to source some skilled foreign labour under the Commonwealth Government's new Enterprise Migration Agreement program for their Browse project in the Kimberley.
Former Executive Director of the Kimberley Land Council (KLC), Wayne Bergmann, moved to his current position as the CEO of KRED Enterprises, which is a non-profit company set up by the KLC to create economic opportunities and employment for Kimberley Aboriginal people. He is concerned about the use of foreign workers in a region which he describes as having "... a massive Aboriginal unemployment rate."
"It would be a sad day for projects like this to go ahead yet opportunities for employing Aboriginal people were missed by employing workers from overseas" says Mr Bergmann.
Woodside points out that the company intends on employing 300 Indigenous people during construction of a James Price Point LNG facility, should the project proceed. This would make up around three-and-a-half percent of the total construction workforce estimated to peak at around 8,500 workers.
This is cold comfort for Mr Bergmann who says that foreign workers shouldn't be considered until "...the over 4000 Aboriginal people out of work in the Kimberley are employed on these projects."
This tension over foreign workers is just the latest beef between the main parties behind the gas precinct proposed for the controversial site north of Broome at James Price Point. The State Government, Woodside and the KLC face stiff opposition from environmentalists and some traditional owners, but that doesn't stop them playing hardball with each other.
This week the Department of State Development has said the KLC's latest changes to the Native Title claim over the James Price Point area is "...in breach of the Browse Agreements."
It is these agreements on which employment targets are based as well as a compensation package for traditional owners worth over a billion dollars.
The KLC retorted that "The state government is walking away from the Browse LNG agreement."
In the past Mr Bergmann has called on the Federal Government to not approve the Browse project because of his concerns about State Government plans to manage the social impacts. And he once accused Premier Barnett of "...pomping around saying there's a failure to negotiate" in response to the Premier's moves to compulsorily acquire land for the gas precinct.
Woodside haven't escaped the wrath of either the Premier or Mr Bergmann. In May 2012, Premier Barnett made it clear that Woodside wouldn't be piping Browse gas to the North West Shelf without his say so.
"The North West shelf project is under a state agreement...and gas can only be brought down there if the State Government agrees" the Premier said.
And in September 2010 Mr Bergmann said "The State and Woodside refuse to engage with our senior negotiator. That's not good faith."
Of course, these have been the lowlights amidst many agreement signing ceremonies and statements that the Browse LNG project will make the difference for disadvantaged Kimberley Aboriginal people and boost the state and national economies. But it shows that not only does a Kimberley gas precinct need to overcome its opponents, but possibly more significantly it needs to survive the competing interests of industry, government and Aboriginal supporters of the project.
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A Chester police officer shot and killed a 21-year-old man during a pursuit Friday night, according to authorities and a witness.
The officer was in a patrol car chasing the man on Crosby Street around 8 p.m. when he got out and ordered the man to stop in the first block of East 23d Street, said Jamil Torres, 20, a neighbor who said he was outside when the shooting occurred.
Torres said the officer alleged that the man, whom he knew only as "Pooh," was carrying a gun.


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BionX electric bike motor conversion kits are available from NYCeWheels in a wide range of sizes, styles and price points to suit your particular needs. These bike motors range in voltage from 24 volts to 48 volts and in weight from 16 to 19 pounds. Once mounted, they can push your bike along at speeds of up to 20 miles per hour. Ranges run from 25 to 35 miles per charge.
Using a BionX electric bike motor conversion kit to convert your bike to electric power will make for faster commutes , let you tackle hills with ease and will greatly increase the fun factor in your bike riding. If you have any questions about BionX electric bike motor conversion kits, just contact NYCeWheels for advice you can trust.
The slimmed-down BionX PL250 Light system uses a smaller battery with the same amount of torque as the standard PL250. It's the lightest electric bike kit in our store, weighing in at only 12 pounds for the battery and motor together!
$1,495.00View Details
The 48 volt BionX SL350 motor kit is an upgrade in power and range from the PL350. It is the classic BionX motor kit offering excellent power and proven technology. This is a great kit for heavy riders, riders with lots of steep hills, or folks who need to pull a trailer.
$1,999.00View Details
The BionX PL500 offers the highest speed you can get from a BionX kit. This system will top out at 25 miles per hour. Only install this electric racing motor on bicycles designed for high speed electric bike riding.
$1,945.00View Details
The Bionx P250 is the standard in conversion kits with a 250W motor and a 24V NiMH battery and is the most affordable Bionx set-up. Suited best for the average rider with basic needs.
$1,195.00View Details


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Examining the algorithms of the diff utility
Diff finds the differences between two versions of a file. We'll show you how diff finds changes and matches in files without affecting a system's resources.
For a user at the command line, discovering the differences between two text files is easy: a simple command, such as diff Version_1.txt Version_2.txt, is all it takes. On closer inspection, however, it turns out that diff needs a large amount of memory and some ingenious algorithms to compare files. This article investigates how diff manages to find changes and matches in multiple megabyte files without affecting a system's resources.
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Bugzilla Bug
Bug database has a bug of its own that could allow an intruder to create an unauthorized account.
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Report focuses federal resources on achieving universal Internet access.
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Game on! Our Super Bowl Party Menu
Whether your guests are die-hard fans or just in it for the commercials, they'll want to dig into this Super Bowl spread. Extra napkins strongly advised.
Chocolate-Stout Brownies
A rich dose of stout adds big flavor to these fudgy, chocolate-glazed brownies
Our Favorite Brownie Recipes
Who doesn't love a good brownie? From cake-like to dense and gooey, these are the brownies of our dreams.
Blueberry Crumb Bars
The oat mixture makes a delicious crust for the bottom—and a tasty crumble on top. Lemon perks up the blueberry filling.


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The following problem affects a Certification authority running on the 64-bit edition of Windows Server 2008 and Windows Server 2008 R2. The problem does not occur on x86 (32-bit) platform of both operating systems.
When installing a subordinate enterprise CA using basicconstraintsextension section in a CAPolicy.inf file, the installation fails with a crashing management console. In this case, the following information is logged in the event log:
Log Name: Application Source: Application Error Date: 21.07.2009 12:40:27 Event ID: 1000 Task Category: (100) Level: Error Keywords: Classic User: N/A Computer: DMW2K8R201.forest3.test Description: Faulting application name: mmc.exe, version: 6.1.7100.0, time stamp: 0x49ee94f3 Faulting module name: ntdll.dll, version: 6.1.7100.0, time stamp: 0x49eeab11 Exception code: 0xc0000374 Fault offset: 0x00000000000c2a42 Faulting process id: 0xb08 Faulting application start time: 0x01ca09ef63d735ed Faulting application path: C:\Windows\system32\mmc.exe Faulting module path: C:\Windows\SYSTEM32\ntdll.dll Report Id: e6add21f-75e2-11de-80e5-00155d500124
The problem occurs when the CA certificate request is created and the basic constraint extension is evaluated in the CAPolicy.inf file. The following sample illustrates a CAPolicy.inf file causing the error:
[Version] Signature= "$Windows NT$" [Certsrv_Server] RenewalKeyLength = 4096 RenewalValidityPeriodUnits = 12 RenewalValidityPeriod = years CRLPeriod = weeks CRLPeriodUnits = 1 CRLDeltaPeriod = days CRLDeltaPeriodUnits = 0 [basicconstraintsextension] pathlength = 0 critical=true [RequestAttributes] CertificateTemplate = MySubordinateCA
The problem has been classified as a Windows bug. Until the bug is fixed, you have to remove basicconstraintsextension section from the CAPolicy.inf file and set the basic constraints extension at the certificate template that is used to enroll for the CA certificate.
Once the new certificate template is applied to the server where the CA is to be installed, the CA setup will pick up the certificate template MySubordinateCA and generate the certificate request for the CA. The basic constraints extension is applied to the certificate request from the template information.
Therefore, it doesn't matter if the subordinate CA is requesting a certificate from a 3rd Party CA or Windows based Standalone CA (which has no idea about the templates). The problem occurs during the certificate request creation and before sending the request to the parent CA.
If the server is a first one to be installed in the AD ( racine is in a work group) how can I managed templates ?
Is there a fix now ?
Is there a fix? I am encountering the same error. Also, the Enterprise Subordinate CA I am attempting to install is the 1st Enterprise CA in the hierarcy (my root CA is standalone offline). So how would I set the basicConstraints to CA=True, Critical since I don't have certificate templates yet?


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Baseball Prospectus
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December 24, 2014 6:00 am
The staff picks artifacts and symbols representative of the past year in baseball.
December 17, 2014 6:00 am
(Major-league players and teams are ineligible.)
December 10, 2014 6:00 am
The staff suggests some items you might consider putting under the tree this holiday season.
December 3, 2014 6:00 am
The staff looks back at some jaw-dropping deals.
November 26, 2014 6:00 am
If you invite a BP staffer over for movie night, don't screen any of these flicks.
November 19, 2014 2:42 pm
Systems Architect/Developer position avaialbe with Kansas City Royals
November 19, 2014 6:00 am
Because Stanton long balls are the best long balls.
November 12, 2014 6:00 am
The staff disagrees with the early odds of these clubs winning next year's World Series.
November 10, 2014 6:00 am
Presenting the staff results from the Internet Baseball Awards, along with abbreviated individual ballots.
Today we reveal the Baseball Prospectus staff choices for the Internet Baseball Awards. First, the cumulative results. Individual staff ballots may be found below below the group tallies..
AL Player of the Year
November 6, 2014 6:00 am
These front-office employees did good work or saw past efforts pay off in 2014.
October 29, 2014 6:00 am
These bullpen arms once proved pivotal in October.
October 23, 2014 6:00 am
Few expected these players to steal the show in October, but that didn't stop them from doing so.


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Heavy Duty Farm Sheds from YourShed
High quality Bluescope Steel Farm Sheds for long lasting reliable security and protection.
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In 2013, SPC slashed contracts, Simplot almost shut down a processing plant & Spring Gully briefly entered administration. Will 2014 be better for business?


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We show you what others paid so you have the confidence that you are getting a fair price from TrueCar Certified Dealers.
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We are staying for several days at an inn that has a kitchenette and were wondering if there is a store where we might buy snacks, fruit, cheese/crackers and wine to enjoy at this inn. Any suggestions/information would be greatly appreciated!
Definitely a big grocery store in the middle of town a couple of blocks west of the traffic circle. There are some shops out front so the parking and entrance are in the rear.
Thanks so much! I wasn't thrilled about the alternative which would have been stopping on our way to there from San Diego.
I don't know that I would call that particular grocery store "big". To me it's more of a small neighborhood grocery store with a limited number of items so if you just looking for basics, you should be okay. If you want more gourmet type items you may be out of luck.
It's not big ... and not big on "gourmet."
There are two shopping "centers" in Borrego. One is called the "Mall" and the other is the "Center." The grocery store in the "Center" which is on the north side of Palm Canyon Drive. It's the same complex which houses Pablito's (there's a large sign) and the Bottle Shop (visible from Palm Canyon).
However if you are driving over from San Diego, head through Santa Ysabel and stop at Don's Market. They have a fab meat market (make their own sausages and have venison) and good fresh veggies. Also pick up some of Dudley's bread. If the Dudley's store is closed, Don's carries a lot of their offerings.
Via the Borrego "Sun" (the local newspaper) I've been told that a grocery is supposed to be going in on Christmas Circle where there once was a liquor store, which has been empty for ages. However, like most things in Borrego, it's taking its time to open.
Besides the Bottle Shop there is another liquor/wine store across Palm Canyon Drive near Jilberto's.
Believe me, you can't miss any of this ... "downtown" Borrego is very small.
Enjoy the desert.
This is why I love Trip Advisor. Always get such great information! Thank you all!
Sal the next time we are in San Diego we will go and visit this area. The Anza park sounds interesting :-).
Do go belo ... just not in summer!
Our favorite place to stock up on wonderful eatables is the Friday morning Farmers' Market which is held in Christmas Circle. It is incredible and we always plan our visits to Borrego to arrive in time for it.
There is more than just fresh produce - there are also vendors who sell marvelous tamales, meats, fresh pita bread, dips and sauces . . . . enough for us to stock up for nearly a week of good eating.
Center Market which has already been mentioned does have a small supply of some good California wines, some crackers, a not great supply of cheese, basic things that you'd find in any small town grocery.
Also - don't forget the ice cream (and fudge) shop right at the front of the Center Mall group of stores.
Thank you.
The grocery store is now stocked with gourmet items and has wonderful offerings. We were there last winter and were so glad to have choices. Hope they have been able to keep it up because if folks don'tt purchase these items, they will have to stop carrying anything but basic groceries.


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11/2/11: Junior midfielder talks about the win over Putnam Valley in the Class B quaterfinals.
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Gregg Sarra and Carl Reuter break down the Long Island Championship games and discuss the Nassau County Awards Dinner.
Gregg Sarra and Carl Reuter discuss the fall's top student athletes, while Andy Slawson stops in to discuss Long Island wrestling.
Join Carl Reuter and Nick Garone as they discuss the Eastern National Championships, wrestlers to watch for in 2012-13 and talk to Rocky Point head coach Darren Goldstein.


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November 1, 2012
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can open anything on you site ?
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