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Copyright © 2003 Cell Press All rights reserved.
Structure, Volume 11, Issue 6, 627-636, 1 June 2003
Catalytically Active MAP KAP Kinase 2 Structures in Complex with Staurosporine and ADP Reveal Differences with the Autoinhibited Enzyme
Kathryn W. Underwood*, 1, , Kevin D. Parris*, 1, , Elizabeth Federico, Lidia Mosyak, Robert M. Czerwinski, Tania Shane, Meggin Taylor, Kristine Svenson, Yan Liu, Chu-Lai Hsiao, Scott Wolfrom, Michelle Maguire, Karl Malakian, Jean-Baptiste Telliez, Lih-Ling Lin, Ronald W. Kriz, Jasbir Seehra, William S. Somers and Mark L. StahlCorrespondence: Kathryn W. Underwood, (617) 665-7926 (phone), (617) 665-8993 (fax); and Kevin D. Parris, (617) 665-8916 (phone), (617) 665-8993 (fax)
1 These authors contributed equally to this work.
- MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41–364, in complex with staurosporine at 2.7 Å and with ADP at 3.2 Å, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the Km for ATP is very similar for MK2 41–364 and p38-activated MK2 41–400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41–364. However, phosphorylation of MK2 41–364 by p38 restores the Vmax and Km for peptide substrate to values comparable to those seen in p38-activated MK2 41–400, suggesting a mechanism for regulation of enzyme activity.
Mitogen-activated protein (MAP) kinases are a large and diverse group of Ser/Thr kinases involved in intracellular signaling pathways. The MAP kinase family can be separated into three major subgroups, including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs), and p38/reactivating kinase (RK). The ERKs are activated by mitogens and growth factors, whereas the JNKs/SAPKs and p38/RK are activated by bacterial lipopolysaccharide (LPS), interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and cellular stresses such as heat shock, osmotic shock, and UV damage. Exposure of cells to these factors results in the increased production of proinflammatory cytokines. Analysis of a specific inhibitor of p38 MAP kinase, SB203580, reveals that it inhibits LPS-induced cytokine synthesis in human monocytes, thus indicating that p38 is the MAP kinase responsible for stress-induced cytokine production Lee et al. 1994. SB203580 also prevents the activation of MAP kinase-activated protein kinase 2 (MK2), suggesting that this kinase is activated by p38 Cuenda et al. 1995.
Mice engineered to be homozygously deficient in MK2 show a reduction in TNF-α, interferon-γ, IL-1β, and IL-6 production and an increased rate of survival upon challenge with LPS, suggesting that this enzyme is a key component in the inflammatory process and a potential target for anti-inflammatory therapy Kotlyarov et al. 1999. Activation of MK2 results in the production of cytokines by regulating the translation and or stability of the encoding mRNAs through the AU-rich elements of the 3′-untranslated regions of the gene Neininger et al. 2002. MK2 also phosphorylates the transcription factor CREB, as well as leukocyte-specific protein-1 and heat shock protein 25/27, which are involved in the regulation of actin polymerization Tan et al. 1996,Lavoie et al. 1993,Stokoe et al. 1992b,Ben-Levy et al. 1995 and cell migration Hedges et al. 1999,Kotlyarov et al. 2002.
MK2 is a multidomain protein consisting of an N-terminal proline-rich domain, a catalytic kinase domain, an autoinhibitory domain, and, at the C terminus, a nuclear export signal (NES) and nuclear localization signal (NLS) Stokoe et al. 1993,Engel et al. 1998,Ben-Levy et al. 1998,Engel et al. 1993,Veron et al. 1993. Two isoforms of human MK2 have been characterized. One isoform consists of 400 amino acids; the other isoform comprises 370 residues and is thought to be a splice variant missing the C-terminal NLS Stokoe et al. 1993,Zu et al. 1994,Engel et al. 1998. MK2 is located in the nucleus of the cell, and, upon binding and phosphorylation by p38, the MK2 NES becomes functional and both kinases are cotransported out of the nucleus to the cytoplasm Ben-Levy et al. 1995,Ben-Levy et al. 1998,Engel et al. 1998,Stokoe et al. 1992a. Interestingly, transport of the MK2/p38 complex does not require catalytically active MK2, as the active site mutant, D207A, is still transported to the cytoplasm Ben-Levy et al. 1998. Phosphorylation of human MK2 by p38 on residues T222, S272, and T334 is thought to activate the enzyme by inducing a conformational change of the autoinhibitory domain, thus exposing the active site for substrate binding Ben-Levy et al. 1995,Engel et al. 1995. Mutations of two autoinhibitory domain residues, W332A and K326E, in murine MK2 demonstrate an increase in basal activity, and a C-terminal deletion of the autoinhibitory domain renders the enzyme constitutively active, providing additional evidence for the role of this domain in the inhibition of MK2 activity Engel et al. 1995.
In this study we have solved two crystal structures of a constitutively active C-terminal deletion version of human MK2 (residues 41–364), one in complex with ADP and the other with the broad-spectrum kinase inhibitor staurosporine. The overall fold of this structure of MK2 is very similar to the structures of other protein kinases. It is bilobal, consisting of an N-terminal domain with a five-stranded β sheet and a conserved α helix and a larger C-terminal domain containing largely α helices. cAMP-dependent protein kinase (PKA) Zheng et al. 1993, Ca2+/calmodulin-dependent protein kinase (cAMK) Goldberg et al. 1996, and Titin Mayans et al. 1998 are protein kinases that are structurally related to MK2. However, MK2 is distinct from these other kinases in that the structure of the inactive form of MK2 reveals that the nucleotide binding site is not sterically blocked Meng et al. 2002. Analysis of these structures will aid in the understanding of the regulatory mechanism of MK2.
Results and Discussion
Alignment of Homologs
MAP KAP kinase 2 (MK2) is an enzyme that belongs to a family of MAP kinase-activated protein kinases. Human members of this family include MK2, MK3, and MK5 McLaughlin et al. 1996,Ni et al. 1998. These proteins are highly homologous (Figure 1), and, in addition, all have been shown to be activated by the MAP kinase p38, although, to date, the only well-studied enzyme is MK2 Clifton et al. 1996,Ni et al. 1998. MK2 is phosphorylated on T222, S272, and T334 by p38 and has a putative autophosphorylation site at T338 Ben-Levy et al. 1995. All four of the phosphorylation sites are conserved in MK3, but only T222 is present in MK5. All of the isozymes have the ATP binding site motif GXGXXG (residues 71–76 in MK2), but only MK2 and MK3 have the bipartite nuclear localization signal KKIEDDASNPLLLKRRKK (residues 373–389 in MK2). The putative activation segment is highly conserved in all three isozymes (residues 207–233 in MK2), including the conserved motifs found to flank the activation segments of many kinases, DFG, and APE, with the latter being APQ in MK5 Johnson et al. 1996. Interestingly, the conserved p38 phosphorylation site, T222, is in the activation segment. MK2 and MK3 contain an N-terminal proline-rich domain, which is absent in MK5, and the C-terminal extension thought to contain the autoinhibitory domain in MK2 is elongated in MK5. Overall, MK2 shares 75% identity with MK3 and 42% identity with MK5.
Analysis of Protein Constructs
MK2 is a 400-amino acid protein consisting of five domains, an N-terminal proline-rich domain, a kinase catalytic domain, a C-terminal kinase autoinhibitory domain, a nuclear export signal, and a nuclear localization signal, which is also the postulated site for p38 binding Tanoue et al. 2000. After analyzing a large number of constructs for expression of MK2 in E. coli, we found that deleting the first 40 residues of the proline-rich domain greatly enhanced the expression levels and solubility of the enzyme. Additional analysis of various C-terminal deletions yielded evidence that the best crystals were obtained from protein ending with residue 364. MK2 41–364, a constitutively active form of the enzyme in which a portion of the C-terminal autoinhibitory domain was removed, was purified and produced crystals that diffracted to 2.7 Å.
As shown in Table 1, the Km for ATP is very similar for MK2 41–364 and p38-activated MK2 41–400, 7 μM versus 15 μM, respectively. However, binding of the peptide substrate, leukocyte-specific protein 1, to MK2 41–364 is more than 40-fold weaker than to the p38-activated MK2 41–400, 584 μM versus13 μM Km. In addition, the Vmax for MK2 41–364 is also much lower than that for the p38-activated MK2 41–400 (0.076 versus 8.9 μmol/min/mg, respectively). These data indicate that MK2 41–364, which lacks a portion of the autoinhibitory domain, binds ATP normally, but peptide substrate binding has been affected dramatically. However, when MK2 41–364 is phosphorylated by p38, the Vmax of the enzyme is greatly increased, to 11 μmol/min/mg, which is comparable to the Vmax of p38-activated MK2 41–400, 8.9 μmol/min/mg. Phosphorylation of MK2 41–364 also alters the Km for peptide substrate, such that the Km decreases from 584 μM to 20 μM, similar to the Km observed in activated MK2 41–400. These data suggest that, for optimal substrate binding and activity, MK2 must be phosphorylated. Clearly, phosphorylation of key residues within the autoinhibitory C-terminal α helix and T222 in the activation segment induce conformational changes in the enzyme that allow highly efficient binding of peptide substrate.
|Construct||p38a||Apparent Km ATPb (μM)||Apparent Km LSP-1c (μM)||Vmaxd (μmol/min/mg)|
|41–400||+||7.0 ± 0.7||13 ± 1||8.9 ± 0.1|
|41–364||−||15 ± 1.5||584 ± 50||0.076 ± 0.004|
|41–364||+||ND||20 ± 2||11 ± 14|
|a For activation, p38 (1.25 μg/ml), MK2 (200 μg/mL), and ATP (500 μM) were incubated for 1–2 hr at 25oC.|
b See Experimental Procedures.
c See Experimental Procedures.
d See Experimental Procedures.
The crystal structure of SeMet-labeled MK2 inhibited by staurosporine (Figure 2) was solved by multiple-wavelength anomalous dispersion (MAD) Hendrickson 1991. Experimental phases were determined at 3.1 Å, modified by solvent flattening and noncrystallographic symmetry averaging, and extended to 2.7 Å. The experimental map for staurosporine is shown in Figure 3A. The final model was refined to a crystallographic R factor of 23.9%, with an Rfree of 27.4% Table 2,Table 3. With this structure of MK2, the structure of ADP bound to MK2 was solved by molecular replacement. The final model for this costructure was refined to a crystallographic R factor of 25.9% and an Rfree of 29.2%. The electron density for ADP is shown in Figure 3B.
|Wavelength (Å)||λ3 = 0.95666||λ1 = 0.97926||λ2 = 0.97955||1.1||1.0|
|Resolution range (Å)||50–3.1||50–3.1||50–3.1||50–2.7||25–3.1|
|Rmergea (%)||6.7 (49.2)||6.6 (37.7)||6.0 (37.9)||4.7 (48.1)||5.5 (63.1)|
|Completeness (%)||99.6 (99.4)||100 (100)||100 (100)||100 (100)||99.3 (100.0)|
|I/σ(I)||10.8 (1.7)||16.2 (3.4)||18.0 (3.6)||20.2 (1.6)||34.9 (3.3)|
|MAD Phasing Statistics for MK2-Staurosporine|
|Resolution limits (A)||6.92||5.24||4.62||4.00||3.70||3.46||3.27||3.10||Overall|
|a Rmerge = Σ|Ih − <Ih>|/ΣIh, where <Ih> is the average intensity over symmetry equivalents. Numbers in parentheses reflect statistics for the last shell.|
b f′ and f″ reported values were refined by SHARP.
c Phasing power = Σ|Fh|/Σ|||FPHobs| − |FPHcalc||, where Fh is the calculated heavy atom structure-factor amplitude.
d Figure of merit = <ΣP(α)eia/Σ|P(α)|>, where α is the phase and P(α) is the phase probability distribution.
|Maximum resolution (Å)||2.7||3.2|
|<B value> (Å3)||29.5||86.4|
|Rms deviations from ideal geometry|
|a Rwork = Σ||Fobs| − |Fcalc|/Σ|Fobs|. Rfree is equivalent to Rwork, except that it is calculated for a randomly chosen 5% of reflections omitted from the refinement process.|
Analysis of MK2 Structures and Comparison with Other Kinases
The MK2 kinase core domain contains an overall fold that is very similar to the structures of other protein kinases (Figure 4). It is bilobal, consisting of a smaller N-terminal domain that is largely β sheet and a larger C-terminal domain dominated by α helices. MK2 shares two structural features within the N-terminal lobe that have been shown to be important in the regulation of many protein kinases Huse and Kuriyan 2002. These structural elements are the highly conserved αC helix (residues 99–113) and the β1-β2 loop (residues 13–32), which has been termed the phosphate binding loop, or “P loop.” This P loop contains the highly conserved ATP binding motif GXGXXG (Figure 2).
Although the overall structure of the catalytic domain is highly conserved in many protein kinases, the mechanisms of regulation are quite diverse and, in some cases, require dramatic conformational changes Huse and Kuriyan 2002. The N-terminal and C-terminal lobes of many protein kinases are connected by a flexible hinge (residues 142–145; DGGE in MK2); thus, the relative positions of the domains determine whether the kinase is in the “open,” or inactive state, versus the “closed,” or active conformation Huse and Kuriyan 2002. MK2 can be classified as an “RD” kinase, as the catalytic aspartate, D186, is immediately preceded by an arginine, R185. Activation of a number of RD kinases requires phosphorylation on one or more threonine, serine, or tyrosine residues within the activation segment. Activation of MK2 requires phosphorylation, by p38, of the activation segment residue T222.
Three protein kinases closely related to MK2, both by structure and shared regulatory mechanisms, are cAMP-dependent protein kinase (PKA) Zheng et al. 1993, Ca2+/calmodulin-dependent protein kinase (cAMK) Goldberg et al. 1996, and Titin Mayans et al. 1998(Figure 4). All are Ser/Thr kinases and all require phosphorylation of a residue within the activation segment for activation of the kinase. Like MK2, all share an additional level of negative regulation by either a C-terminal autoinhibitory domain or a regulatory subunit. Although PKA lacks an autoinhibitory domain, a bound regulatory subunit maintains the kinase in an inactive conformation. Activation of PKA requires binding of an allosteric regulator, cAMP, to the regulatory subunit, resulting in a conformation change and release of the catalytically active kinase subunit. cAMK is maintained in an inactive conformation by a C-terminal autoinhibitory domain that blocks both substrate and ATP binding. Activation of cAMK requires Ca2+/calmodulin binding, which is thought to induce a conformation change resulting in the displacement of the autoinhibitory domain from the peptide and ATP binding sites. Titin also contains a C-terminal autoinhibitory domain that sterically blocks both substrate and ATP binding. Titin activation also requires Ca2+/calmodulin binding, which is also thought to induce a conformation change resulting in the displacement of the autoinhibitory domain from the peptide and ATP binding sites. The recently published structure of the autoinhibited, inactive form of MK2 47–400 reveals a unique mechanism of kinase regulation Meng et al. 2002. The C-terminal autoinhibitory α helix extends along the entire surface of one face of the C-terminal lobe adjacent to the active site and binds, hypothetically, as a pseudosubstrate (Figure 4B). D366 acts as a phosphothreonine mimetic by coordinating with the basic residues R185 and K212 within the active site of the enzyme. This “pseudosubstrate” region is thought to be positioned in a manner that would effectively block binding of both protein and peptide substrates.
The N-terminal lobe of the kinase domain in the inactive MK2 structure has significant structural differences compared to the catalytically active MK2 structures presented here (Figure 4A and 4B). First, the αC helix is one full turn shorter in the autoinhibited structure than in the structures we present here. Additionally, the β1 strand, which is present in our structures as well as in the majority of other kinases, is replaced by an α helix in the autoinhibited MK2 stucture. It is interesting to speculate that this significant conformational change in the structure of the N-terminal lobe may be part of the MK2 activation process, but another possibility as to the difference in the structures may be due to the conditions used for crystallization and heavy-atom derivatization. Meng et al. generated crystals with P1 symmetry containing six molecules per asymmetric unit. Upon soaking a single crystal in a mercury compound overnight, the crystal symmetry shifted to R3, containing two molecules per asymmetric unit. The authors solved the structure using a data set collected from the mercury-modified crystal. Since there are four cysteines in the N-terminal lobe (98, 114, 133, and 140) and each has been derivatized by the addition of mercury, it is conceivable that the binding of these mercury compounds has led to a disruption of the five-stranded β sheet and a conformational shift of the β1 strand to an α helix. However, the answer to this question requires further structural studies.
The C-terminal lobe contains another important regulatory feature, the MK2 activation segment (residues 207–233), a fragment of which extends outward from the surface of the catalytic domain into solvent as part of a loop terminating at residue 239. In the structure of the autoinhibited form of MK2, this portion of the activation segment that extends away from the core is completely disordered Meng et al. 2002. In our structures, the conformation of this loop is partially stabilized by interactions with the C-terminal domain of a symmetry-related molecule, as electron density was observed for all residues except 216–226. Included in this unobserved section is T222, which is one of the p38 phosphorylation sites. These structural data suggest that the activation segment in unphosphorylated MK2 is highly dynamic and solvent accessible.
As discussed previously, MK2 41–364 is a constitutively active truncated version of the enzyme that does not contain the complete C-terminal autoinhibitory sequence. Of the four MK2 structures in the asymmetric unit in the complex with staurosporine, C-terminal residues 345–364 are disordered in three, but only residues 358–364 are disordered in the fourth. The additional residues ordered in the latter structure are in contact with a symmetry-related molecule. The structure of ADP bound to MK2 is most like the former copies of MK2 with staurosporine. The symmetry interactions that appear to stabilize residues 346–357 are not present, and, thus, the entire autoinhibitory domain is disordered in the structure with ADP. By comparison with the autoinhibited structure, MK2 41–364 contains all of the residues that comprise the autoinhibitory domain α helix. However, in our structures, this autoinhibitory helix does not occupy the same location in the C-terminal lobe (Figure 4A and 4B). Instead, it is displaced to one side and is disordered after two turns. The location from which the autoinhibitory helix was displaced is now occupied by the loop containing the activation segment from a symmetry-related molecule. The significance of the movement of the autoinhibitory helix and the location of the activation loop cannot be readily deduced from the structures we report here and that of the full-length autoinhibited enzyme.
The MK2 structures presented here are of unphosphorylated enzyme. MK2 41–364, in the unphosphorylated state, is catalytically active and shows normal binding of ATP. However, the Km for LSP-1 peptide substrate is 45-fold higher and the Vmax is 100-fold lower than those for p38 phosphorylated MK2 41–400 (see Table 1). Yet, when MK2 41–364 is phosphorylated by p38, it exhibits virtually identical peptide substrate binding and catalytic rates to phosphorylated MK2 41-400. These data suggest that, when p38 phosphorylates MK2 41–400, key residues on the autoinhibitory C-terminal helix and residue T222 of the activation segment shift the equilibrium of the pseudosubstrate region from a bound to an unbound state. The activation segment equilibrium is shifted from a highly dynamic state, as observed in the MK2 structures presented here and elsewhere Meng et al. 2002, to a more stable bound state, required for efficient binding of peptide substrate and a high catalytic rate. Constitutively active MK2 41–364, however, lacks key residues within the pseudosubstrate region, and, as observed in the structures presented here, most of the autoinhibitory C-terminal helix is disordered and probably highly mobile, thus allowing a lower level of peptide substrate binding and catalysis. Phosphorylation of MK2 41–364 by p38 restores full enzymatic activity, presumably, as described above, by shifting the equilibrium of the phosphorylated activation segment to the bound state, allowing efficient binding of peptide substrate. These data also clearly show that residues 365–400 are not required for full catalytic activity in vitro.
Structural Analysis of the ADP and Staurosporine Costructures
The ATP binding site of protein kinases is located in a deep cleft between the N-terminal and C-terminal lobes of the catalytic domain. The β1 and β2 strands in the N-terminal lobe constitute the P loop, which contains a glycine-rich motif, GXGXXG, that is highly conserved in all protein kinases. The conserved glycines confer two important structural properties to the P loop, the lack of side chains, which allow loop backbone amides to interact with ATP phosphates without steric hindrance, and backbone flexibility, which allows the P loop to adopt multiple conformations. Conformational flexibility of the P loop is an important factor in the regulation of many protein kinases. The structure of the MK2 P loop, within the context of a five-stranded β sheet, and the spatial relationship of the αC helix are consistent with the structures of many protein kinases and also constitute an important part of the kinase active site.
A number of catalytic residues conserved in all protein kinases are spatially oriented to allow efficient transfer of phosphate from ATP to a protein substrate. As one of the products of the reaction, adenosine-5′-diphosphate is an efficient probe for these interactions. The interactions between ADP and MK2 in the structure we have determined are very similar to those observed in other kinases in complex with ADP or ATP mimics. Specifically, the adenine ring is buried in the protein, with hydrophilic interactions with the L141 main chain nitrogen and the E139 main chain oxygen. The phosphate groups are positioned adjacent to the P loop and have ionic interactions with N191, D207, K93, and I74. Although magnesium was present in the crystallization media, no discernible electron density was found that could be identified as the magnesium ion.
The binary complex structure of MK2 41–364 and staurosporine is very similar to the ternary complex structure of PKA, PKI inhibitor peptide, and staurosporine Prade et al. 1997 and thus forms the basis for a comparative analysis of the MK2 and PKA active sites. The analysis revealed that all active site residues discussed below are spatially conserved. R185 and D186 (R165 and D166 in PKA) are catalytic loop residues that are invariant in all RD kinases. The catalytic aspartate, D186, is thought to act as a base to remove a proton from the protein substrate hydroxyl group. The phosphates of ATP are positioned for hydrolysis by interactions with backbone amide protons in the P loop and by ionic interactions with K93 (K72 in PKA), which is positioned and stabilized properly by E104 (E91 from PKA), a residue from the αC helix. This ion pair interaction between K93 and E104 is one of the criteria used to judge whether a kinase is in the active or inactive conformation Huse and Kuriyan 2002. This interaction as well as the interaction of K93 with the α and β phosphates of ADP indicates that the structures we report here are of MK2 in its active conformation (Figure 5).
As observed in many other RD kinases, including PKA, that require phosphorylation of a threonine residue within the activation segment for activation and efficient catalysis, two basic residues, R165 and K189 in PKA, are required for coordination and charge neutralization of the phosphoryl threonine. These residues are also spatially conserved in the MK2 structures (R185 and K212).
Comparison of the MK2 structures cocrystallized with ADP and staurosporine show that the most significant difference lies in the β sheet containing the GKGING (71–76) loop (Figure 5). This loop, between β strands 1 and 2, and the loop on the other side of β strand 2 that connects to β strand 3 (residues 83–88) move extensively as a result of staurosporine binding deeper into the ATP binding pocket. The residue at the center of the loop, I74, moves 4.2 Å closer to the activation segment when bound with staurosporine than when bound with ADP. Conversely, in the structure with ADP, T86, found in the loop between β strand 2 and 3, moves closer to the protein core by 2.5 Å than in the structure with staurosporine. This coupled movement of the loops connecting strands 1, 2, and 3 allows the structural integrity of the β sheet to remain while moving to accommodate different molecules in the active site.
Staurosporine binds to the ATP binding pocket of MK2 in a manner similar to that observed in other kinases (Protein Data Bank codes 1AQ1 [CDK2], 1BYG [SRC], 1QPD and 1QPJ [LCK], and 1STC [cAMP]). The pyrrolidinone moiety makes two potential hydrogen bonds, one to the main chain carbonyl oxygen of E139 and the other to the main chain nitrogen of L141. The amino group of staurosporine interacts with the carbonyl oxygen of E190 and makes a potential hydrogen bond to glutamate. The remaining interactions inside the active site are van der Waal-type interactions. The bulk of the amino acids that make up the lining of the ATP binding site between these structures differ, leading to cavities and protrusions in the active site that differ among these proteins, suggesting that the design of ATP binding site inhibitors is a possibility. Similar analyses of the ADP/MK2 costructure compared to 1B38 and 1HCK (CDK2), 1CSN (Casein), 1GOL (ERK-2), and 1PHK (phosphonylase) show a similar pattern of depressions and extrusions in the lining of the ATP binding pocket. The conservation of several of these features supports the hypothesis that ATP binding site-specific compounds can be developed. Specifically, the pocket leading to H108 in MK2 has no homolog in any of the structures listed above.
Cells exposed to heat shock, cytokines (TNF and IL-1β), or ultraviolet light display an increase in p38 MAP kinase activity due to phosphorylation by the upstream kinases MKK3 and MKK6. p38 in turn phosphorylates a variety of substrates, including the transcription factors ATF-2 and CHOP-1, and other kinases, such as MK2 and MK3. The p38/MK2 signal transduction cascade plays a pivotal role in the production of proinflammatory cytokines. Mice homozygously deficient in MK2 show a reduction in TNF, IL-1β, IL-6, and IFN-γ synthesis and an increased rate of survival upon exposure to LPS compared to that in wild-type mice Kotlyarov et al. 1999. MK2 controls the synthesis of cytokines by regulating the translation and/or stability of the encoding mRNAs through the AU-rich elements of the 3′-untranslated regions of the gene Neininger et al. 2002. These data indicate that MK2 is a vitally important enzyme in inflammatory-based diseases and is a target for anti-inflammatory drug design.
In this study we present the structures of a catalytically active MK2 41–364 in complex with ADP and staurosporine. From these structures, as well as the previous structure of the inactive enzyme 47–400 Meng et al. 2002, we observe that MK2 is regulated quite differently from other kinases that have a similar fold. Specifically, the autoinhibitory domain of MK2 does not block the nucleotide binding site and phosphorylation of the residues in the activation segment and C-terminal autoinhibitory domain is necessary for optimal binding of the substrate and activity of the enzyme. From this work we will have an important tool for designing inhibitors to MK2.
Cloning and Expression of MK2
The MAP KAP kinase 2 gene was PCR cloned into the NcoI and XhoI sites of pET16b (Novagen) with Hot tub polymerase (Amersham Pharmacia Biotech). The expressed protein contains residues 41–364, excluding the N-terminal proline-rich sequence. To produce selenomethionine-labeled MK2, we expressed the protein in BL21(DE3) (Novagen) E. coli at 25°C. Precultures were grown in shake flasks in LeMaster media supplemented with L-methionine, and expression cultures were grown in LeMaster media supplemented with L-selenomethionine that was replenished upon culture induction. Cultures were induced with 0.5 mM IPTG for four hours. Unlabeled MK2 was also expressed in E. coli BL21 (DE3). The culture was induced with 0.5 mM IPTG and cells were harvested 4 hr postinduction.
Purification of MK2 41–364
The purification was performed at 4°C. Five grams of bacterial cells was homogenized in 200 ml of Buffer A (50 mM Tris [pH 7.5], 10 mM DTT, and 0.24 mg/ml AEBSF) plus 90 μg/ml TPCK, 2.5 mM aminobenzamidine, and 500 μl protease inhibitor cocktail for use in purification of poly-(histidine)-tagged proteins (Sigma), RNase, and DNase. Cells were lysed by four passages through a Microfluidics microfluidizer. The lysate was collected and centrifuged at 20,000 × g for 30 min. The supernatant was applied to a Poros HQ column (Applied Biosystems). The flow-through was loaded onto a Poros HS column (Applied Biosystems), and the bound protein was eluted with a gradient of Buffer A plus 1 M NaCl. Ammonium sulfate to 0.8 M was added to the peak fraction, and the protein was loaded onto a polypropyl aspartamide column (Nest Group) equilibrated with Buffer B (50 mM HEPES [pH 7.5], 10 mM DTT, and 0.8 M ammonium sulfate). The protein was eluted with a gradient, and the peak fraction was concentrated in a Millipore Ultrafree concentrator. The protein was applied to a Superdex 200 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES (pH 7.5), 200 mM NaCl, and 10 mM DTT. Protein purity was >95%.
ATP, ADP, phosphoenolpyruvate (PEP), NADH, and pyruvate kinase/lactate dehydrogenase enzymes were purchased from Sigma Chemical (St. Louis). Activated p38 MAP kinase was purchased from Upstate Biotech (Lake Placid, NY). LSP-1 peptide (RTPKLARQASIELPSM) was purchased from AnaSpec (San Jose, CA).
Activation of MK2 Constructs
The MK2 constructs 41–400 and 41–364 were activated by the phosphorylation of the constructs by p38 MAP kinase. The activation was done in 20 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM DTT, 0.50 mM ATP, 0.2 mg/ml MK2, and 0.125 μg activated p38. The reaction was incubated at 25°C for 1–2 hr and then placed on ice to be used for kinetic analysis.
The rate of MK2 was characterized in 20 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM DTT, and 100 mM NaCl. The kinetics was followed by linking the turnover of ATP to the turnover of NADH to NAD. This was followed spectrophotometrically at 340 nm. The continuous assay contained 20 units pyruvate kinase, 30 units lactate dehydrogenase, 0.25 mM NADH, 2 mM PEP, and 1.6–8.0 μg/ml MK2. For determining the apparent Km for ATP, the ATP concentration was varied from 0.005 to 0.25 mM, while the peptide was held constant at 0.2 mM for activated MK2 or at 1.0 mM for the constitutively active form of MK2 (41–364). For determining the apparent Km for LSP-1, the LSP-1 concentration was varied from 0.01 to 0.5 mM, while ATP was held constant at 2 mM. The kinetic analysis was carried out in a 96-well plate at 25°C, on a Molecular Devices spectrophotometer.
Peptide LSP, based on the protein substrate of MK2 lymphocyte/leukocyte-specific protein, was used for the investigation of the kinetic mechanism for the enzyme MK2. Data was fit to 1 for normal Michaelis-Menten kinetics.
The selenomethionine-labeled protein was concentrated to ∼5 mg/ml according to the Bradford method Bradford 1976 in a solution containing 20 mM HEPES (pH 7.5), 200 mM NaCl, 10 mM DTT, and 5 mM MgCl2. Prior to crystallization, staurosporine (0.375 mM) was added from a DMSO stock. Diffraction-quality conical crystals were obtained at 18°C from 2 M ammonium sulfate, 100 mM HEPES (pH 7.5), and 2% PEG 400. These crystals, which appeared in 7–10 days, belong to space group P63, with cell dimensions of a = b = 160.20 Å and c = 133.48Å, and contain four molecules of MK2 in the asymmetric unit. Native MK2 was crystallized in the presence of ADP, and the protein solution was prepared as above, except that 5 mM ADP was added, instead of the staurosporine. Diffraction quality native MK2/ADP cocrystals were obtained at 18°C from 2.0 M ammonium sulfate. These bipyramidal crystals belong to space group F4132, with cell dimensions of a = b = c = 253.05 Å, and contain one molecule of MK2 and one molecule of ADP in the asymmetric unit.
Data Collection, Phasing, and Refinement
A three-wavelength MAD phasing experiment was carried out on beamline 5.0.2 at the ALS, Berkeley, using an ADSC Quantum-4 CCD detector from a single crystal of SeMet-MK2 cooled to −180°C. All data were integrated and scaled with HKL2000 Otwinowski and Minor 1997. The positions of the selenium atoms were determined in Shake and Bake Weeks and Miller 1999 and refined in SHARP De La Fortelle and Bricogne 1997 at 3.1 Å Table 2. The structure of the MK2 was built into the original 3.1 Å-resolution solvent-flattened symmetry-averaged MAD map with the X-AUTOFIT features within QUANTA (Molecular Simulations, San Diego, CA). The phases were then extended from 3.1 Å to 2.7 Å with symmetry averaging in DM CCP 4 1994. This model was then used as the initial model for refinement with the program CNX Brünger et al. 1998 against the 2.7 Å data. Prior to refinement, 5% of the data were randomly selected and designated as an Rfree test set to monitor the progress of the refinement. After seven cycles of refinement with REFMAC Murshudov et al. 1999 and rebuilding with QUANTA, refinement converged with a model that contained 4 molecules of MK2, 4 staurosporine molecules, 43 water molecules, and 2 sulfate ions at an Rcryst of 23.9% (Rfree of 27.4%). Of the four molecules of MK2 in the asymmetric unit, molecule “C” was the most complete, with residues 41–153 and 227–357 visible in the electron density. The refinement statistics are given in Table 3.
Single-wavelength (1.0 Å) data for the MK2/ADP cocrystals were collected on beamline 5.0.1 at the ALS, Berkeley with an ADSC Quantum-4 CCD detector from a single crystal cooled to −180°C. The data were processed with HKL2000 Otwinowski and Minor 1997, and the statistics are given in Table 2. The structure of the MK2/ADP complex was solved by molecular replacement with a composite consisting of the overlapped MK2 monomers from the staurosporine structure as the molecular replacement probe with AMORE CCP 4 1994. The molecular replacement solution was then rebuilt into a 3.2 Å-resolution solvent-flattened map. After the initial placement of the protein chain into density, the model was rebuilt with omit maps calculated with BUSTER Bricogne 1993 in order to eliminate any bias from the molecular replacement solution. The structure was refined in CNX by the methods described above. Refinement converged after six rebuilding cycles, with an Rcryst of 25.9% and an Rfree of 29.2%. The final model consisted of residues 46–152, 159–217, 227–265, and 274–345 and the ADP moiety. The refinement statistics are given in Table 3.
We thank Sang Hsu and Mei Tai Wang for preparation of DNA constructs, James Wilhelm for mass spectrometry, and Thomas Ernst and his staff at beamline 5.0.2 at the ALS for assistance in diffraction data collection.
The coordinates for the two structures described in this paper have been deposited in the Protein Data Bank under ID codes 1NXK for the MK2/Staurosporine structure and 1NY3 for the MK2/ADP structure.
Ben-Levy et al. 1998 (1998). Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr. Biol 8, 1049–1057. Abstract | Full Text | PDF (531 kb) | CrossRef | PubMed
Clifton et al. 1996 (1996). A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress. FEBS Lett 392, 209–214. CrossRef | PubMed
Engel et al. 1995 (1995). Constitutive activation of mitogen-activated protein kinase-activated protein kinase 2 by mutation of phosphorylation sites and an A-helix motif. J. Biol. Chem 270, 27213–27221. CrossRef | PubMed
McLaughlin et al. 1996 (1996). Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. J. Biol. Chem 271, 8488–8492. CrossRef | PubMed
Neininger et al. 2002 (2002). MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels. J. Biol. Chem 277, 3065–3068. CrossRef | PubMed
Ni et al. 1998 (1998). MAPKAPK5, a novel mitogen-activated protein kinase (MAPK)-activated protein kinase, is a substrate of the extracellular-regulated kinase (ERK) and p38 kinase. Biochem. Biophys. Res. Commun 243, 492–496. CrossRef | PubMed
Prade et al. 1997 (1997). Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential. Structure 5, 1627–1637. Abstract | Full Text | PDF (1159 kb) | CrossRef | PubMed
Stokoe et al. 1992b (1992). Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313, 307–313, b. CrossRef | PubMed
Received: December 13, 2002
Revised: March 6, 2003
Accepted: March 14, 2003
Published: June 3, 2003


text:
Hillstream Loach (Beaufortia kweichowensis)
The Butterfly Hillstream Loach is a great looking, peaceful, small and very unusual fish that deserves more attention than they currently get.
They are also great for ridding a tank of any algae.
Their body, shape and pattern make them very unique, along with their ease of care, make them the perfect candidate for the community tank.
Its body is flat, narrowed from below, and has fan-shaped pectoral and ventral fins edged with dark colouring. The caudal fins are small and hard to see.
The stomach is translucent, and you can see the internal organs, and their mouths are located below. They are brown with dark spots.
However, this fish is not recommended for the beginner aquarist as they are susceptible to illness and medication because they have no scales and will require proper water purity and temperatures.
|Scientific Name||Beaufortia kweichowensis|
|Other Names||Chinese Hillstream Loach, Hong Kong Pleco, Butterfly Hillstream loach, Chinese Sucker Fish|
|Best kept as||Trios|
|Lifespan||8 to 10 year|
|PH||6.5 - 8.0|
|GH||3 - 10|
|68 - 75℉|
20 - 23.9℃
Photos of the Hillstream Loach
Chinese butterfly loach inhabits deep rivers with a fast flow and upstreams highly saturated with oxygen, small tributaries of streams and rivers; zones restricted with crests and sometimes with waterfalls. in Jiangxi river system, the South of China, and the main branch of Zhu Jiang River.
This species was described in the upper part of Guizhou province, and itâ€™s widely spread in the independent region Guangxi and Guangdong province.
In their natural environment, a Hillstream Loach will spend their time either working their way across the substrate looking for food or stationed out on a rock.
Their substrate is mainly sand, small rocks, round stones and gravel. In some instances, there is not even streamside vegetation. The water is crystal clear, with a small amount of dissolved oxygen.
Such sunlight and water conditions are right for biofilm development which consists of different types of algae and microorganisms. This biofilm covers all the rocks and stones surfaces like a blanket.
What to feed the Hillstream Loach
The Hillstream Loach is not a fussy eater, but you will need to bear it in mind that they have tiny mouths.
They will happily accept good quality small flakes, sinking pellets, Algae wafers, thawed frozen or live Bloodworm, little Cyclops, Artemia, Blanched Spinach, Mysis shrimp and Kale and natural Algae is an advantage for this fish.
How to breed the Hillstream Loach
Breeding hillstream loaches, although challenging, is possible. There have been no recorded successes in the home aquaria though.
You will need your water parameters and conditions to be perfect for this species to reproduce, and even then it could be unsuccessful.
You will know when your loaches are ready to reproduce because you will see the males displaying a mating dance to the females. If she is interested she will stay by his side instead of going about her usual business.
When the male has been given the green light, he will then build a nest by sorting through and digging the substrate, here the female will place her eggs, and the male will fertilise them.
The eggs will start hatching a couple of weeks after fertilisation, and there will be no need to remove the parents as they will not eat them.

domain: hobby


Are Smartphones Making Us Any Less Smarter?
The smartphones have reduced our recall skills as now we don't store data in our memory but store it in our phones or computers
Photo Credit :
So you have bought or are planning to buy the new iPhone 8. And you are aware that the rapid increase in the usage of smartphones has guided you to a whole new world of connectivity. Smartphone users worldwide are now in constant touch with their distant friends and relatives, have access to seamless entertainment and virtually infinite information sources.
When you have a smartphone in your hand, you get unlimited access to news, views, and opinions from all around the world. No matter where you are you can share your status, photographs, do online purchases and can even have a video call with someone you know. Remember the times when you would want to meet a person and the medium to do so was slow and expensive. Whereas, now you are constantly connected to anyone and everyone on a press of a button.
Do you know that 10 years back Steve Ballmer, CEO, Microsoft predicted that the iPhone will not gain significant market share and will always remain a niche product. Ballmer went on record saying that Microsoft software will be present in 80% of 1.3 billion sold mobile phones. But the result has been the other way round - in 2016 Apple share stands at 11.5% as compared to Window phone share of a mere 0.4%. Nokia at launch of iPhone was the market leader and welcomed iPhone but in a complacent tone said that it won't change Nokia's thinking. A year later Nokia CEO penned a blistering note to staff about how the company had failed to adapt. Blackberry also ignored the writing on the wall and considered iPhone (read smartphones) as just another competitor in a red ocean.
Some Internet reactions questioned the future of smartphones in general, and considered touch screen as a bad idea that will never work. Some balked at the idea of a short 5 hour battery and unwieldy large screen. Another one said that "I mean it looks pretty but it's not something you foresee being the next ipod for the phone industry".
But no one knew that how smart the smart phone really was going to be. We've known for a while now that mobile phones can have an adverse effect on our ability to concentrate, sleep and even socialise. But now new research has uncovered even more damning evidence for smartphone addicts, showing that the devices are actually reducing our intelligence.
Actually, today your brain is becoming dependent on smartphones and your intellect is weakening day by day. Research suggests that an average iPhone user unlocks his phone more than 80 times a day. This includes immediately upon waking up, just before going to sleep, and oftentimes in the middle of the night.
The smartphones are distracting you from attending to difficult problems in hand or complex situations. This is evident from the fact that you get distracted as soon as your phone rings, vibrates or a message is beeped. Adrian Ward, a cognitive psychologist and marketing professor at the University of Texas at Austin has researched and concluded that, "The division of attention impedes reasoning and performance". During this study, the UT Austin researchers found that someone's ability to hold and process data significantly improved if his or her smartphone was in another room while taking a test to gauge attentional control and cognitive processes. Participants who kept their phones in a pocket or bag also outperformed those who kept their phones on the desk while taking the same test. Again, even if the phone was turned off and face down on the desk, the mere sight of one's own smartphone seemed to induce "brain drain" by depleting finite cognitive resources.
Smartphones were invented with good intention by providing some useful functions to make us smarter, but it has increased our anxiety levels too. Researchers have observed that the closer you are to your phone, your brainpower decreases. Researchers call it a phenomenon of "brain drain" that can reduce your learning capabilities, logical reasoning, abstract thought, problem solving, and creativity.
The combination of a letterbox, newspaper, video player, music player, a camera, a library and your personal secretary, compressed in your smartphone is lethal for your brain functioning. This all is what smartphones do to us, no wonder we cannot live without them.
Starting of the 21st century, when we first started surfing on the world-wide-web we thought it would make us smarter. Strange but it's true, our knowledge and understanding may have diminished gradually as smartphone grant us easy access to the information sources without taxing our brain. Research from McGill University in Canada found that drivers who depend on GPS-style navigation to get around, as opposed to those who rely on their own spatial abilities, had less activity and gray matter volume in the hippocampus region of their brain-an area important for memory consolidation.
The smartphones have reduced our recall skills as now we don't store data in our memory but store it in our phones or computers. We access the electronically stored information and hardly push our brain to do the mental effort to remember and recall it. The researchers named this phenomenon the "Google effect" which implies that our learning is dependent on search engines therefore we believe that there is no need to encode and decode the information by using our brain. It means that whenever we need an information or search for a solution, we can immediately pick up our phone and look for it. Even a sibling's birthday is not remembered now - we are just training in how to find out the birthday. Contrast this with 30 years ago when birthdays, anniversary dates, and important phone numbers used to be on tips - leaving a few husbands out at that.
The advent of computers and smartphones has created a generation of people believing that they are more knowledgeable and smarter than the earlier generation but actually they are not. Man is a social animal but the smartphones have changed everything. There was a time when we would call our friend to check for information but with the unlimited flow of information through smartphones, many people may first pick up their smartphone and perform a Google search rather than calling a friend.
Researchers have proved that the quality of social interactions in the presence of smartphones has made us less empathetic towards people and situations. Further, they found evidence that have negative effects on closeness, connection, and conversation quality. The findings demonstrate that the presence of mobile phones can interfere with human relationships, an effect that is most clear when individuals are discussing personally meaningful topics.
Psychologist and philosopher William James said in an 1892 lecture, "the art of remembering is the art of thinking." Only by encoding information in our biological memory can we weave the rich intellectual associations that form the essence of personal knowledge and give rise to critical and conceptual thinking. The lesser the information we record in our memory the lesser we think. But when we look for information through our smartphones, we often end up thinking that this information is generated through our smart intelligence and not through the smartphone.
Imagine you were a librarian in a large library of year 1980. That role gave you access to lots of books and encyclopedias. But did it make you the smartest human being? Or you were just a clerk having lots of knowledge chests in your custody. How many librarians have won Nobel Prizes or Booker Prizes or filed patents?
Do we have a history behind our memory? Can we allow smartphones to be smarter than us? Till the time we are dependent on smartphones and use it as our memory card, we will sacrifice our ability to think. Therefore we need to reduce our proximity to smartphones and give our minds more space to think. James Barrat has said about Artificial Intelligence being man's last invention, as post that, all inventions would come from the superior artificial intelligence (as per Turing's test). Is smartphone a precursor to that artificial intelligence?
Disclaimer: The views expressed in the article above are those of the authors' and do not necessarily represent or reflect the views of this publishing house. Unless otherwise noted, the author is writing in his/her personal capacity. They are not intended and should not be thought to represent official ideas, attitudes, or policies of any agency or institution.


Kaizer Chiefs defender Ramahlwe Mphahlele wants MTN8 statement
Posted: 12 July 2017 Time: 11:16
Ramahlwe Mphahlele says Kaizer Chiefs are targeting success in the MTN8 to bring back that winning feeling at Naturena and also send out a strong message to their title rivals.
After winning the league and cup double with Mamelodi Sundowns in the 2015/16 PSL season, Mphahlele finished his debut campaign with no trophies as Amakhosi flattered to deceive yet again under coach Steve Komphela.
As they prepare for the new campaign, Amakhosi players do not need to be told of the wrath they’d have to face if they go a third consecutive season without a trophy, so Mphahlele has called on the team to do their best to win the MTN8 early in the campaign.
“You know when you win something, you start believing you can win and that’s what we need at the moment. We need to win one cup, so the MTN8 will be very important,” Mphahlele tells KickOff.com.
“We have to concentrate on that and try to bring it home. We owe it to the fans. They have been there for us and sometimes we even feel ashamed. We owe it to the chairman [Kaizer Motaung]. We owe them a trophy or two,” he adds.
Defensively, Amakhosi need to improve after losing concentration in key moments of the game. It’s what let them down in matches against the likes of Baroka FC, Golden Arrows and Cape Town City.
“That’s what we want to work on. I saw the interview of the chairman, saying the defence [can be better]. We made some crucial mistakes, but we have learnt. Next season we will do better. We will take games seriously and we will concentrate more. I think we owe it to everyone to do better.”


Genie Bouchard and Mason Rudolph could be getting serious ... the tennis star showed up to the Steelers game Sunday with friends to cheer on her new beau!!!
We broke the story ... Genie and the Steelers backup QB kicked off a romantic fling recently -- and clearly, the fire is still burning hot.
'Cause just weeks after 26-year-old Genie was spotted out and about near Rudolph's Pittsburgh-area home ... she headed to 25-year-old Mason's game against the Titans in Tennessee.
And, while Mason didn't play in the win (he's backing up a healthy Ben Roethlisberger this season) ... didn't seem Genie minded too much -- check out her smile!!!
Yeah, looks smitten to us!!
Neither Genie nor Mason has gone public with the relationship yet ... but posting pics from games means we could be getting close to IG offish any day now.
Congrats on the young love!


text:
MEMA Weighs in on NHTSA’s State Model Policy
WASHINGTON, D.C. – The Motor & Equipment Manufacturers Association (MEMA), the voice of a mobility industry on the forefront of innovation and new safety technologies, presented oral comments before the National Highway Traffic Safety Administration (NHTSA) during a Dec. 12 public meeting regarding a section of the Federal Automated Vehicle Policy (FAVP) dedicated to model state policy. This is the second public meeting the agency has held on the FAVP; the first was on Nov. 17.
To create a national framework, “Federal and state governments must work together to establish policies that accelerate deployment of these technologies while also balancing public safety and building trust,” said Leigh Merino, MEMA senior director of regulatory affairs, during the hearing. “Federal leadership working closely with the states is essential to avoid a potential patchwork of varying state laws and requirements.”
In its statement, MEMA once again urged for broad-based clarity and requested that NHTSA ensure that its policy clearly treat test vehicles separately from production vehicles. As it relates to the State model policy, NHTSA’s FAVP proposed a framework of requirements for the states among which is the assertion that each test vehicle follows the Performance Guidance “set forth by NHTSA” and “meets all applicable” federal motor vehicle safety standards.
MEMA strongly believes that a state should not codify the NHTSA vehicle performance guidance by way of its own laws, policies and/or application requirements for automated vehicle testing. “Doing so will avoid the impact of unintended consequences that may hinder supplier development and innovation of automated systems and prevent opportunities for real-world testing experiences in various states,” Merino said.
MEMA’s testimony represents just one step in a multi-pronged effort to communicate concerns about the policy to regulators and legislators to advance members’ business interests. On Nov. 22, MEMA submitted comments to NHTSA regarding its FAVP, and MEMA also testified on Nov. 15 at the House Energy and Commerce Subcommittee on Commerce, Manufacturing and Trade during a hearing on self-driving vehicles.
MEMA and its four specialized divisions comprise the leading international trade association in the fast-changing mobility industry, and legislators and regulators rely on MEMA’s input for a wide range of policies that affect MEMA membership. Testifying before Congress is one way MEMA represents the industry in Washington, D.C. For more information on NHTSA’s AV Policy, contact Leigh Merino.
The Motor & Equipment Manufacturers Association (MEMA) and its four specialized divisions comprise the leading international trade association in the fast-changing mobility industry. Representing vehicle suppliers that manufacture and remanufacture components, technologies, and systems for use in passenger cars and heavy trucks, MEMA works to ensure that the marketplace and legislative and regulatory environment support the development and implementation of new technical capabilities transforming the automotive industry, including autonomous vehicles and vehicle connectivity. By directly employing more than 871,000 Americans and generating a total employment impact of 4.26 million jobs, MEMA’s member companies are the largest sector of manufacturing jobs in the U.S. MEMA’s members are represented through four divisions: Automotive Aftermarket Suppliers Association (AASA), Heavy Duty Manufacturers Association (HDMA), Motor & Equipment Remanufacturers Association (MERA) and Original Equipment Suppliers Association (OESA). For more information on how MEMA is leading transformation in the mobility industry, visit www.mema.org.
MEDIA CONTACT: Cindy Sebrell: 202-312-9250 or firstname.lastname@example.org

domain: law


April 20, 2022
The April Trauma-Informed Primary Care (TIPC) Implementation Advisory Committee meeting took place on 4/20/22 from 10am to 12pm PDT. Members of the committee advised on promising models, best practices, evolving science and clinical expertise for the implementation of trauma-informed care systems in California. This meeting was also open to the public.
For more information, contact email@example.com


text:
Other obligations prevent me from writing until later today -- and I intend to focus on Rahm Emanuel's war-crimes-protecting proclamation that Obama's desire for immunity extends beyond CIA officers perpetrating torture to the "policy makers" who ordered it (watch today as the hardest-core Obama loyalists start explaining how the UN doesn't matter, international treaties are irrelevant, and war criminals need not be held accountable) -- but, until then, I wanted to highlight this extremely important and well-reported story from CQ's Jeff Stein, which involves allegations of major corruption and serious criminal activity on the part of Democratic Rep. Jane Harman. Here's one crucial prong of the story:
Rep. Jane Harman , the California Democrat with a longtime involvement in intelligence issues, was overheard on an NSA wiretap telling a suspected Israeli agent that she would lobby the Justice Department reduce espionage-related charges against two officials of the American Israeli Public Affairs Committee, the most powerful pro-Israel organization in Washington.
Harman was recorded saying she would "waddle into" the AIPAC case "if you think it'll make a difference," according to two former senior national security officials familiar with the NSA transcript.
In exchange for Harman's help, the sources said, the suspected Israeli agent pledged to help lobby Nancy Pelosi, D-Calif., then-House minority leader, to appoint Harman chair of the Intelligence Committee after the 2006 elections, which the Democrats were heavily favored to win.
Seemingly wary of what she had just agreed to, according to an official who read the NSA transcript, Harman hung up after saying, "This conversation doesn't exist."
That's not even the most significant part. Back in October, 2006, Time reported that the DOJ and FBI were investigating whether Harman and AIPAC "violated the law in a scheme to get Harman reappointed as the top Democrat on the House intelligence committee" and "the probe also involves whether, in exchange for the help from AIPAC, Harman agreed to help try to persuade the Administration to go lighter on the AIPAC officials caught up in the ongoing investigation." So that part has been known since 2006.
Stein adds today that Harman was captured on an NSA wiretap conspiring with an Israeli agent to apply pressure on DOJ officials to scale back the AIPAC prosecution. But the real the crux of Stein's scoop is that then-Attorney General Alberto Gonazles intervened to kill the criminal investigation into Harman -- even though DOJ lawyers had concluded that she committed crimes -- because top Bush officials wanted Harman's credibility to be preserved so that she could publicly defend the Bush administration's illegal warrantless eavesdropping program:
[C]ontrary to reports that the Harman investigation was dropped for "lack of evidence," it was Alberto R. Gonzales, President Bush's top counsel and then attorney general, who intervened to stop the Harman probe.
Why? Because, according to three top former national security officials, Gonzales wanted Harman to be able to help defend the administration's warrantless wiretapping program, which was about break in The New York Times and engulf the White House. . . .
Justice Department attorneys in the intelligence and public corruption units who read the transcripts decided that Harman had committed a "completed crime," a legal term meaning that there was evidence that she had attempted to complete it, three former officials said. . . .
Then-CIA Director Porter J. Goss reviewed the Harman transcript and signed off on the Justice Department's FISA application. . . . Goss, a former chairman of the House Intelligence Committee, deemed the matter particularly urgent because of Harman's rank as the panel's top Democrat.
But that's when, according to knowledgeable officials, Attorney General Gonzales intervened.
According to two officials privy to the events, Gonzales said he "needed Jane" to help support the administration's warrantless wiretapping program, which was about to be exposed by the New York Times.
Harman, he told Goss, had helped persuade the newspaper to hold the wiretap story before, on the eve of the 2004 elections. And although it was too late to stop the Times from publishing now, she could be counted on again to help defend the program
He was right.
On Dec. 21, 2005, in the midst of a firestorm of criticism about the wiretaps, Harman issued a statement defending the operation and slamming the Times, saying, "I believe it essential to U.S. national security, and that its disclosure has damaged critical intelligence capabilities."
And thanks to grateful Bush administration officials, the investigation of Harman was effectively dead.
Indeed, as I've noted many times, Jane Harman, in the wake of the NSA scandal, became probably the most crucial defender of the Bush warrantless eavesdropping program, using her status as "the ranking Democratic on the House intelligence committee" to repeatedly praise the NSA program as "essential to U.S. national security" and "both necessary and legal." She even went on Meet the Press to defend the program along with GOP Sen. Pat Roberts and Rep. Pete Hoekstra, and she even strongly suggested that the whistleblowers who exposed the lawbreaking and perhaps even the New York Times (but not Bush officials) should be criminally investigated, saying she "deplored the leak," that "it is tragic that a lot of our capability is now across the pages of the newspapers," and that the whistleblowers were "despicable." And Eric Lichtblau himself described how Harman, in 2004, attempted very aggressively to convince him not to write about the NSA program.
Stein's entire story should be read. It's a model of excellent reporting, as it relies on numerous sources with first-hand knowledge of the NSA transcripts (and what sweet justice it would be if Harman's guilt were established by government eavesdropping). It should be noted that Harman has issued a general denial of wrongdoing (but does not appear to deny that she had the discussion Stein reports), and the sources in Stein's story are anonymous (though because they're disclosing classified information and exposing government wrongdoing, it's a classic case of when anonymity is justifiable; and note Stein's efforts to provide as much information as possible about his sources and why they are anonymous).
There are many questions that the story raises -- Josh Marshall notes just some of those vital questions here -- and Harman's guilt therefore shouldn't be assumed. But obviously, given all the very serious issues this story raises -- involving what seem to be credible allegations of very serious wrongdoing by a key member of Congress, the former Attorney General and one of the most powerful lobbying organizations in the country -- full-scale investigations are needed, to put it mildly.
* * * * *
Two other notes:
(1) I was interviewed by ReasonTV regarding my report on drug deciminalization in Portugal and Obama's record thus far on civil liberties (the interview was two weeks ago before release of the OLC memos):
(2) Kudos to The New York Times and Scott Shane not merely for amplifying a key fact about the OLC memos -- that 2 of the detainees were waterboarded a total of 266 times -- but, even more notably, for crediting Marcy Wheeler as the person who discovered that fact and first broke the story. Ironically, establishment journalists often complain that bloggers are "parasitic" on their work, yet many feel no compunction about simply copying what they read on blogs without any credit whatsoever. Some reporters are conscientious about crediting blogs --Shane is one of them -- but it's far more common for them to use the work of bloggers with no credit. Marcy is one of the smartest, most knowledgeable and most diligent writers around, and it's excellent to see her get just some of the credit her work deserves.

domain: news


text:
Consumer Alert: Did airlines forget their pandemic promises?
From refunds to vouchers, passengers are getting shorted. Here's what to do.
Have airlines conveniently forgotten the promises they made to you during the pandemic? Travelers like Manuele Wasserman think so.
Keep reading with a 7-day free trial
Elliott Confidentialto keep reading this post and get 7 days of free access to the full post archives.

domain: travel


text:
Director of Health Promotion at the Ghana Health Service, Dr. Aboagye DaCosta believes Frontiers Health Services are providing one of the most important mechanisms in the fight against the Covid-19 at the Airport in spite of the bastardization by many Ghanaians.
According to him, their introduction at the Kotoka International Airport is a great innovation to check the Covid-19 cases coming into the country, but it received a lot of criticisms when it was first introduced.
Speaking on Okay FM's 'Ade Akye Abia' Morning Show, Dr. Aboagye DaCosta said it is refreshing to see that Ghanaians are recognizing that the operations of the Frontiers Health Services are important in ensuring that the Covid-19 cases do not overwhelm the country like it is happening in Europe and other countries.
"The Covid-19 cases would have overwhelmed us like it is happening in Europe and other countries . . . since we had two cases last year March 12, 2020, we saw that the cases were imported and so if we don't do anything about our Airport, the situation would overwhelm us," he noted.
"We have come up with a lot of interventions to curtail the Covid-19 cases in Ghana until we introduced the testing kits at the Airport which people kicked against, but today, we all can attest to the benefit of the testing kits at the Airport," he stressed.
Frontiers Health Services, managers of Ghana’s COVID-19 airport testing program have issued an alert over the rising number of positive COVID-19 cases coming into the country.
The company in a letter to the Managing Director of Ghana Airports Company Limited, Yaw Kwakwa said it recorded the highest daily number of positive COVID-19 cases at the airport on Saturday, April 24, 2021.
Reacting to the caution letter, Dr. Aboagye DaCosta has assured Ghanaians that the government together with the Ghana Health Service and the Ministry of Health will not relent on their efforts in ensuring that all Covid-19 cases are detected from other countries through the protocols which will be enrolled soon at the Airport.
"We see what is happening at the Airport and we had discussions about the situation and certain protocols will be rolled out soon. I don't want to be the one to break the new protocols at the Airport but very soon the government will announce it to the general public," he hinted.
Source: Daniel Adu Darko/Peacefmonline.com
|Disclaimer: Opinions expressed here are those of the writers and do not reflect those of Peacefmonline.com. Peacefmonline.com accepts no responsibility legal or otherwise for their accuracy of content. Please report any inappropriate content to us, and we will evaluate it as a matter of priority.|


An overturned taco truck forced the temporary closure of north and southbound lanes on the 101 Freeway through Hollywood Hills Monday morning.
The crash was reported just after 3:00 a.m. after the truck crashed and overturned into the northbound lanes of the freeway near North Cahuenga Boulevard, according to the California Highway Patrol’s traffic incident log.
The incident forced officials to stop traffic on both sides of the freeway and issue a SigAlert at about 3:35 a.m.
No injuries were reported in the crash, the Los Angeles Times reported.
The SigAlert was canceled and all lanes were reopened just before 6 a.m., the CHP tweeted.


Manson Given Life Term In Hinman-Shea Murders
Tuesday, November 30th, 1971
LOS ANGELES, Nov. 30 — A penalty verdict of life imprisonment was returned against Charles Manson yesterday for the Gary Hinman-Donald (Shorty) Shea murders in 1969.
A Superior Court jury of eight men and four women had deliberated five hours and 20 minutes since receiving the case last Friday.
Judge Raymond Choate set Dec. 20 at 9 a.m. for formal pronouncement of sentencing.
After the verdicts were read, Manson yelled at jurors and spectators: “You’re in prison…you’re all in prison.”
Jurors recommended three life imprisonment terms for Hinman’s murder, Shea’s murder and a charge of conspiracy to murder and to rob Hinman.
The 37-year-old defendant looked haggard when he entered the courtroom and did not react during the actual reading of the verdicts.
Judge Choate thanked the jurors for their “sacrifice,” which he called “unusual and demanding.” The case has been in trial since last July.
After the judge thanked jurors, Manson muttered, “shake their hands.”
The same jury last Nov 2 convicted Manson of two counts of first-degree murder and the conspiracy charge after a week of deliberation.
Hinman, a musician, was murdered in his Topanga Canyon home in late July 1969, about two weeks before the Tate-LaBianca murders.
Manson faces eight death sentences in connection with the seven Tate-LaBianca murders, including one for a conspiracy to murder conviction.
Shea assertedly was murdered about two weeks after the Tate-LaBianca killings, but a body has never been found.
The victim was a stuntman who lived on and off at the Spahn Ranch in Chatsworth, where Manson and his cult of nomadic hippies lived during part of 1968 and 1969.
One of Manson’s followers, Robert Beausoleil 24, was convicted of Hinman’s murder last year in a retrial. He is on death row.
Another follower, Susan Atkins 23, pleaded guilty to Hinman’s murder earlier this year and was sentenced to life in prison.
Miss Atkins, along with three other Manson cultists, also face death sentences in connection with the Tate-LaBianca murders.
The others are Patricia Krenwinkel 24, Leslie Van Houten 22 and Charles (Tex) Watson 25. Steve Grogan 20, was convicted and sentenced to death for Shea’s murder.
His death verdict by another Superior Court jury came earlier this month.
In addition, Bruce Davis 26, another Manson follower, is scheduled to go on trial in Judge Choate’s courtroom in a matter of days in connection with the Hinman-Shea killings.
Still another cultist, Mary Brunner 26, has been indicted for Hinman’s murder, but a trial has not yet been scheduled.
Newsmen asked defense attorney Irving A. Kanarek to comment on the verdict.
“I believe the evidence shows Charles Manson is innocent,” Kanarek said. “There is a reasonable doubt in the minds of some of the jurors.”
One reporter asked if this were the case, why did the jury convict Manson of two counts of first-degree murder in the first place.
“Because of the Manson mania,” he replied.
Kanarek declared that everyone on the jury knew that Manson has already been sentenced to death for the Tate-LaBianca murders.


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Tecmo Koei announced Yaiba: Ninja Gaiden Z will be released for PC as well as consoles. The PC version will be available as a digital download on Steam in early 2014, which sounds like it won’t be too far away from the PS3 or Xbox 360 version.
Yaiba: Ninja Gaiden Z is a collaborative project with Keiji Inafune from comcept, Team Ninja, and Lost Planet 3 developer Spark Unlimited. You play as Yaiba, a cybernetic ninja out for revenge after Ryu Hayabusa cut off his limbs.
Siliconera spoke with Inafune about Yaiba: Ninja Gaiden Z and he told us he wanted to make Yaiba a completely different kind of protagonist from Ryu and explained why they went with a comic book art style.

domain: game


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For the second time in four seasons, the Cowboys will open up the NFL Preseason game in Canton, OH. Dallas has been selected to be a participant in this year's Hall of Fame Game, against the Miami Dolphins. Normally, teams are chosen based on the class being inducted and this year is no different. Super Guard Larry Allen will have his bust enshrined as will coaching legend Bill Parcells.
Allen spent the first 12 of his 14 seasons with the Cowboys and was a part of the squad to win Super Bowl XXX. He was an 11 time Pro Bowler, 7 time All Pro and was a member of the NFL's Team of the Decade for both the '90s and the '00s.
Allen was selected to the Cowboys Ring Of Honor on November 6th, 2011 along with Drew Pearson and Charles Haley. Haley was not selected for the Hall of Fame this last go around.
Bill Parcells has won two Super Bowls as head coach, and performed several ressurection efforts of other franchises, including changing the Dallas Cowboys from three consecutive 5-11 seasons to making the playoffs in two of four seasons. He is widely credited for building the team that Wade Phillips took over and continued success with.
Important for the Cowboys is the fact that Dallas will get to start training camp a week earlier than most of the league. Teams are allowed to start training camp 15 days prior to their first game. Last year Dallas was at a decided disadvantage. They played the first Monday game of the preseason, meaning they started later than everyone except their opponents. That was confounded by the fact that they were chosen for the regular season kickoff game against the World Champion Giants. That game was of course on the road, so the team lost a few days for travel and that was even made worse by the league scheduling the game for Wednesday to accomodate the DNC.
In 2010, Cowboys fans flooded the Pro Football Hall of Fame for Emmitt Smith's induction. Dallas won the inaugural game over the Cincinnati Bengals, 16-7. It was their first victory in four tries in the contest.
If you're interested in heading out for either the ceremony or the game, check out the Hall of Fames Official Website, here. They have tickets available and information for the fan experience.


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How Far Would You Agree That Pitt's Success As A Reformer To 1793 Was Dependent On Royal Support?
William Pitt the Younger's political success revolved around an effective working relationship with King George III, without which Pitt never could have maintained the support needed for his economically revolutionary reform policies such as Income Tax. Lacking the selfish eloquence to thrive among Lords, Pitt's support depended upon those who were tryiing to please the king. The more feble minded were content with Pitt's success at the progess he made towards financial recovery kept the public from questioning the rise of the aloof lifestyles of the Lords and furthermore the king. Pitt's dependency on the king should not be cause to question his strength as rime minister but highights a house without intention to prioritise a discipline on organisations amongst the cabinet.
The Regency Crisis of 1788 is a pefect example of his reliance on the king, moreover the concept of George having confidence in Pitt's abilities can be lessened by the King's despisition of Charles James Fox, nd his selfish social nature. The French Revolution had shown that this typica aristocratic behaviour could have catastrophic results, and so caused fear for the monarchy despite the heavy loyalism in Britain. The crisis showed the weakness of Pitt's position, as with the King no longer in the political equation, Pitt's grasp on his position rapidly declined. It was essential for Pitt that he held the kings support. Had the Kings sudden incapability been permanent then a regency, headed by the Prince of Wales would have gone ahead and either Fox himself, or a Foxite would have been instated in Pitt's position. It was Fox's intention to restrict the monarchys influence, as this was the easiest solution to overcome Pitt. Pitt was unable to tsand y himself as an 'independent whig,' without the general support given to Pitt for having the Kings backing, the few supporters Pitt could...
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domain: history


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Declining dementia: Role of education, stroke prevalence
The prevalence of dementia, particularly vascular dementia and mild dementia, declined substantially between 1986 to 1987 and 2008 to 2010, a new study has found.
“The decline was mainly explained by the higher proportion with more than basic education and the lower odds ratio for dementia in those with stroke in the later born birth cohort,” said researchers.
The study involved two cohorts of 85-year-olds: those born in 1901 to 1902 and were examined in 1986 to 1987 (n=494; mean age 85.55±0.12 years; 71.1 percent female) and those born in 1923 to 1924 and were examined in 2008 to 2010 (n=571; mean age 85.91±0.17 years; 62.9 percent female).
Dementia diagnoses were classified as Alzheimer’s disease, vascular dementia or mixed dementia based on aetiology. Logistic regression models were used to determine relationship between birth cohort, sex, education and stroke on the prevalence of dementia.
The prevalence of dementia was significantly lower in the 2008 to 2010 cohort than in the 1986 to 1887 cohort (21.7 vs 29.8 percent; odds ratio [OR], 0.66; 95 percent CI, 0.50 to 0.86; p=0.003) regardless of method of diagnosis. [Sci Rep 2017;7:6136]
The trends were similar when dementia was diagnosed using either MMSE scores (OR, 0.72; 0.54 to 0.96) or examinations only (OR, 0.69; 0.52 to 0.91). Mild dementia decreased significantly from the 1986 to 1987 cohort (8.3 percent) to the 2008 to 2010 cohort (2.3 percent; p<0.01).
Logistic regression showed that education (OR, 0.70; 0.51 to 0.96), prevalent stroke (OR, 3.78; 2.28 to 6.29; p<0.001) and the interaction of stroke and birth cohort (OR, 0.50; 0.26 to 0.97; p=0.04) were significantly associated with the prevalence of dementia after adjusting for sex, education, stroke and other potential confounders.
In contrast, birth cohort alone (OR, 0.98; 068 to 1.41; p=0.920) and marital status (OR, 1.09; 0.76 to 1.57; p=0.628) were not.
According to the researchers, the current findings of decreasing dementia prevalence corroborate what is also increasingly being reported by other research groups.
“Recent epidemiological studies in several countries (UK, USA and Sweden included) have revealed a decrease in the incidence of age-standardized rates of dementia (i.e. the expected tsunami of dementia in ageing populations may be less problematic than predicted),” said Dr Barry Halliwell, chairman of the Biomedical Research Council at the Agency for Science Technology and Research (A*STAR) in Singapore.
“Note that the decrease is observed in the West. We don’t know if this is true for Asian countries such as Singapore and China,” noted Dr Feng Lei, a research assistant professor at the Department of Physiological Medicine in the Yong Loo Lin School of Medicine in Singapore.
While both doctors agreed to the roles that stroke and education play in dementia, they also highlighted the importance of taking other factors into consideration. “[M]any other factors play important roles, for example: diet, social engagement, physical activities, etc,” said Dr Lei.
“Education is indeed a powerful protective factor, as beautifully illustrated in the Nun study and many subsequent ones, but I would be surprised if changes in other risk factors did not contribute to the decrease observed [in the present study],” added Dr Halliwell. [Oncotarget 2015; 6:14082-14091; Curr Alzheimer Res 2012:9:6]


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Cary Ballet Tickets
Want to get your hands on some Cary Ballet tickets? The search is over as TonsOfTickets has a huge inventory of Cary Ballet tickets for the full schedule ready to be delivered to you. This is where you can find a massive selection of Cary Ballet tickets for any of the dates available. Call us anytime and our excellent customer service will bring you all the information you need to get to your event.Cary Ballet Schedule
Sorry, Cary Ballet does not have any events :(Purchasing and Shipping your Cary Ballet Tickets
To purchase Cary Ballet tickets from TonsOfTickets, simply click the “Buy” button and proceed to find your desired tickets. You'll then be brought to our secure check out system where you can make your purchase. We are not affiliated with any box offices, venues, performers or team. Cary Ballet tickets may be sold on this site for more than face value. All of our Cary Ballet tickets are 100% guaranteed. Your Cary Ballet tickets will be shipped through FedEx (unless otherwise noted) and are typically shipped within 1 business day of the time you placed your order. In many cases Cary Ballet tickets are available through E-Tickets which can be downloaded and printed through your email.

domain: entertainment


text:
- Latest available (Revised)
- Original (As made)
This is the original version (as it was originally made). This item of legislation is currently only available in its original format.
2.—(1) This article modifies the effect of section 165 of the Copyright, Designs and Patents Act 1988 (parliamentary copyright) in its application to works made by or under the direction or control of the National Assembly for Wales.
(2) The section has effect as if for subsection (1) there were substituted—
“(1) Where a work is made by or under the direction or control of the National Assembly for Wales—
(a)the work qualifies for copyright protection notwithstanding section 153(1) (ordinary requirement as to qualification for copyright protection), and
(b)the National Assembly for Wales Commission(1) is the first owner of any copyright in the work.”.
(3) The section has effect as if for subsection (4) there were substituted(2)—
“(4) For the purposes of this section, works made by or under the direction or control of the National Assembly for Wales include—
(a)any work made by a relevant person in the course of his duties, and
(b)any sound recording, film or live broadcast of the proceedings of the Assembly (including proceedings of a committee or sub-committee of the Assembly),
but a work shall not be regarded as made by or under the direction or control of the Assembly by reason only of its being commissioned by or on behalf of the Assembly.
(4A) The following are relevant persons for the purposes of subsection (4)—
(a)the presiding officer of the National Assembly for Wales elected under section 25 of the Government of Wales Act 2006(3),
(b)the deputy presiding officer of the Assembly elected under that section,
(c)the members of the Assembly Commission appointed under the standing orders of the Assembly by virtue of section 27(3) of that Act, and
(d)the members of the staff of the Assembly, within the meaning of that Act (see paragraph 3(2) of Schedule 2 to that Act).”.
(4) The section has effect as if in subsection (5) for “the House of Commons or the House of Lords” there were substituted “the National Assembly for Wales”.
Latest Available (revised):The latest available updated version of the legislation incorporating changes made by subsequent legislation and applied by our editorial team. Changes we have not yet applied to the text, can be found in the ‘Changes to Legislation’ area.
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Pizza, much like the earth, is circular, flat, and supports the existence of Karl Malone. Enjoy something that always delivers with this Groupon.
- $10 for $20 worth of wood-fired pizza and Italian food
M & T's Pizzeria
As a small, family-oriented eatery, M & T's Pizzeria is proud of its gorgeous wood-fired brick pizza oven. Guests can try the primary offering in sizes ranging from 8 to 14 inches, sprinkling on toppings such as beef, ham, and grilled chicken. However, the kitchen team also prepares alternate fare, ranging from subs to burgers and pasta.

domain: food


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Justice brings services closer for Gauteng and North West
The days of travelling long distances and incurring costs to access justice services for the community of Diepsloot have been drastically cut as of 1 December 2014, due to the alignment of courts into new magisterial districts in the Gauteng and North West province.
Diepsloot community travelled as far as Pretoria and Atteridgeville and paid about R70 taxi fare to access courts for maintenance and family law services.
The project of rationalisation of the magisterial districts to municipal boundaries was officially launched at the Diepsloot Youth Centre on 28 November 2014 and now Diepsloot residents and surrounding areas will no longer have to travel to Pretoria, but would go to the Randburg Magistrate’s Court, where the distance will only cost them R13.
Justice and Correctional Services Minister Michael Masutha, MP, expressed that communities are losing confidence in the ability of the justice system to deal effectively and appropriately with crime as courts are too far from where they live.
“In an attempt to bring back communities to the centre of the justice system, we have moved courts closer to them which will, in turn, also cut travelling costs,” said the minister.
The department’s Chief Operations Officer, Dr Khotso De Wee highlighted that it is through the partnership of the Departments of Justice and Correctional Services, Public Works, the Judiciary, National Prosecuting Authority (NPA), South African Police Services (SAPS) and municipalities that the realignment of new magisterial boundaries has been a success.
“The rationalisation process has commenced only in Gauteng and the North West province, the rest of the provinces will undergo the same process in 2015,” said Dr De Wee.
The launch was followed by an Imbizo where members of the community got the opportunity to address issues of crime, domestic violence and rape in Diepsloot.
Ms Lizzy Chauke, whose sister was shot dead recently and whose niece is also in a critical condition in hospital from a gun wound, pleaded with the minister and the police to find the perpetrator.
“I have lost a sister and I am uncertain about whether my niece will be able to see again as she was shot in the eye. I only ask that the police bring back the Tactical Response Team (TRT) in Diepsloot as they did a good job in reducing the high crime rate in our community previously,” she appealed.
Police Major General, Mr Norman Taioe reported that Diepsloot has a high rate of domestic violence, rape and murder cases and assured that the community’s cry for a strong police presence in the area will be highly considered.
“I am confident that the realignment of magisterial districts is the best solution in improving service delivery. We as the police are committed to this project,” he concluded.
By Mosima Mashao
Published: 05 Dec 2014

domain: sociology


text:
Applied Filter's LOADSAVE
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Don, a wanted criminal, dies in a police chase. DSP D'Silva is the only one who knows about his death, and to get hold of the gang he trains Don-lookalike Vijay. But Vijay faces danger from the police and from within the gang.
Nab, a story of destiny and fate, begins with a lottery ticket. A drunk who cannot pay his tab trades his 'winning' ticket to the waiter...
Inspector Arjun Singh (Vinod Khanna) of Bombay Police has attained a sordid reputation of being of the most ruthless policemen in India...
A cabaret dancer and singer, Sheela (Zeenat Aman) has two admirers: a small-time thief Rajesh (Feroz Khan)...
Vikram has prospered in a life of crime and lives a hedonistic, self-indulgent lifestyle. When Durga...
While returning from Army Base, Captain Ajit Kapoor stops over at a rest-house, where there is a party on, and he meets a charming, vivacious young lady. When he reaches home, his guardian, Father Joseph, wants him to get married. When Joseph accompan
Tony's daughter Mary gets intimate with a college friend. When he refuses to marry Mary even on Tony's insistence...
Sitamgar is a 1985 Bollywood action film starring Dharmendra, Rishi Kapoor, Parveen Babi, Poonam Dhillon.
Vishal is a poet and lives a middle-class lifestyle with his widowed and wheelchair-bound mom. He is in love with Aruna..
Wealthy, powerful and influential Seth Dharamdas leads a financially secure life in a palatial mansion...
On a visit to scenic Agra, Pooja meets with a reknown artist, Kabir. Kabir is instantly attracted to Pooja...
Dharma's boxing career fades when he receives an injury due to which he lose his job at Kashmir Silk Mills. This then raised his tension prompting him to take to alcohol in a big way leaving his family in a destitute way of living consisting of his pr
Dharamdas is killed by Dhanraj and his man later one by one Dharamdas killers are killed but it puts question to everyone as whose behind all this killings?
Suman (Raakhee) and Amit (Amitabh Bachchan) love each other and plan to marry soon. Amit lives with his younger brother...
Bhavani starts living a honest life, not knowing that his estranged sons are leading a life of crime, and that his daughter is now living with the family, whose son he killed.
Jhutha Sach () is a 1984 Bollywood film directed by Esmayeel Shroff and starring Dharmendra and Rekha. The film was remade in Telugu as Kodetrachu. It was the second movie for child actor Jugal Hansraj, after Masoom.
The plot centers on a wealthy but uneducated father who goes to college to show solidarity with his discouraged son Jason and learns that he cannot buy an education or happiness.
While on a tiger-hunt, two aristocrats, Thakur and Sehgal get into a dispute about who which one of them shot the tiger. This dispute escalates into fisticuffs and a gunfight, with Sehgal getting killed, and Thakur getting seriously wounded. Sehgal's
Suraj lives a wealthy lifestyle with his Police Inspector dad, Sangram, mom, and sister, Chutki. He joins the Police Force...
Captain Chawla has been employed for years with a cargo shipping company run by Ranbir. While celebrating the new year...

domain: artistic


text:
Written by Evie
Do you know the famous cartomancer, Miss Lenormand? Marie-Anne Lenormand (1772-1843) was a bookseller, the daughter of a cloth merchant, and she received an excellent education. But, from an early age, this famous fortune-teller began to draw cards. It was around 1780 that she moved to Paris, where she became famous, and was known as "the Sibyl of the Faubourg Saint-Germain," significant political figures including Napoleon himself consulted her. She became the confidant of the future Empress Josephine. Marie-Anne Lenormand used all the techniques to seduce and convince: the lines of the hand, rock crystal, cards... All of Paris came to consult her!
You've drawn one of the six cards of Lenormand's Conquest Read more
Discover the battles you will have to fight Read more
Discover the symbolism behind this tarot Read more
This draw means facing new situations! Read more
Meaning of the twelve cards of the Order of Time Read more
We propose that you discover the game of Tarot here: an original card game, feminine and poetic composed of 54 cards which fall under five categories: the cards of the Golden Fleece, the cards of the War of Troy, the Great Work, unforeseen events, and the order of time. This game of Tarot links the real world to the spiritual realm with reason and intuition. It's up to you to discover it...
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text:
All-New Ford Focus RS High Performance Hatch with Innovative All-Wheel Drive, Set for U.S. Debut!
- Third-generation Ford Focus RS will be available in major markets around the world and for the first time in the United States; Focus RS follows the reveal last month of the new Ford GT supercar – a new era of Ford performance that will deliver 12 new vehicles by 2020
- All-new Ford Performance All-Wheel Drive with Dynamic Torque Vectoring Control paired with a powerful 2.3-liter EcoBoost® four-cylinder engine will provide impressive performance
- Aggressive, functional design inside and out makes Focus RS the most powerful production Focus ever, with highly capable performance on both road and track
- Professional rally driver and star of Gymkhana films Ken Block teamed with Ford as a consultant on development of Focus RS
“The all-new Focus RS is a serious machine with high-performance technology and innovative engineering that sets a new benchmark for driving exhilaration on the road and track,” said Raj Nair, group vice president, Global Product Development, Ford Motor Company. “The RS line has a proud history of technical breakthroughs that have migrated to mainstream Ford vehicles to benefit all of our customers, and the new Focus RS is no exception. It’s a great example of our passion for innovation through performance, and creating vehicles that make people’s hearts pound.”
Developed by a small team of global Ford Performance engineers, the new Focus RS is the third generation in the line, following models launched in 2002 and 2009. It will be the 30th car to wear the legendary RS badge, following such technology trendsetters as the 1970 Escort RS1600 with 16 valves, 1985 Sierra RS Cosworth with turbocharging and radical aerodynamics and 1992 Escort RS Cosworth with four-wheel drive.
Sporting a dramatic exterior design that delivers enhanced aerodynamics and cooling, the new Focus RS offers technologies new to the RS line including Ford SYNC® connectivity system.
The all-new Focus RS is the first-ever RS model that will be sold around the world, including the United States, and produced for all markets at Ford’s Saarlouis, Germany, manufacturing plant beginning late this year.
Interested? Stay in the Loop! Submit your information below and we’ll contact you as more information becomes available.

domain: design


This is music video for Skafandr (russian music group)
music created by Skafandr
design, animation, etc created by Alexander "Satim" Timofeev
This work was designed in minimalism 2d-animation style (like logotypes
or road sings). In this work I did'nt want to do cool 3d and video effects, smooth animation, etc... This work
simple and elegant, as Skafandr music.


Our old buddies Howth (rhymes with growth) perform their song, “Tourist Town,” as a stripped down 4-piece rock band at Muchmore’s in Williamsburg, Brooklyn, in this episode of BTR Hear & There that we originally presented in March of 2015.
“Tourist Town” appeared on Howth’s 2015 album, Trashy Milky Nothing Town.
Meet Nikkiesha McLeod. Charlie Kaufman’s ‘I’m Thinking of Ending Things.’ Plus music by Photo Ops. | listen
Several organizations have put together awesome musical lineups, but only for voters. | read


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Cunningham on Obama: "What we have here is this little boy who grew up in Jakarta, Indonesia, at the age of 6 to 10, rejected by his own father, rejected by his own mother, rejected by his stepfather, raised by his grandparents"
In addition to reviewing print and online media, Media Matters monitors at least 150 hours of television and radio each week. This section features highlights (or low-lights) from our monitoring efforts, other noteworthy clips as well as original videos.
Family Research Council (FRC) president Tony Perkins has appeared significantly less frequently on CNN and MSNBC in the wake of petitions calling on the networks to stop hosting him. Perkins, whose organization has been labeled an anti-gay "hate group," continues to appear frequently on Fox News.


Machete used in video store robbery
A pregnant woman and teenage girl were threatened by a pair of robbers with a machete during a video store hold-up overnight.
Two men jumped the counter of the Mount Annan shop, in Sydney's south-west, about 9.30pm, police say.
The pair threatened a 14-year-old girl with the machete and demanded money from the till, before going to a back office and confronting the pregnant store manager, 41.
They made off with a sum of cash, police say.
Witnesses reported the men driving off in a red Ford Laser, and five people were arrested about 2.20am.
The four men and one woman, aged between 25 and 45, are assisting Campbelltown Police with their inquiries.