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In the yeast Saccharomyces cerevisiae, transcription of a secreted acid phosphatase, PHO5, is repressed in response to high concentrations of extracellular inorganic phosphate. To investigate the signal transduction pathway leading to transcriptional regulation of PHO5, we carried out a genetic selection for mutants that express PHO5 constitutively. We then screened for mutants whose phenotypes are also dependent on the function of PHO81, which encodes an inhibitor of the Pho80p-Pho85p cyclin/cyclin-dependent kinase complex. These mutations are therefore likely to impair upstream functions in the signaling pathway, and they define five complementation groups. Mutations were found in a gene encoding a plasma membrane ATPase (PMA1), in genes required for the in vivo function of the phosphate transport system (PHO84 and PHO86), in a gene involved in the fatty acid synthesis pathway (ACC1), and in a novel, nonessential gene (PHO23). These mutants can be classified into two groups: pho84, pho86, and pma1 are defective in high-affinity phosphate uptake, whereas acc1 and pho23 are not, indicating that the two groups of mutations cause constitutive expression of PHO5 by distinct mechanisms. Our observations suggest that these gene products affect different aspects of the signal transduction pathway for PHO5 repression.
ALL cells must respond appropriately to changes in their environment. When microorganisms are limited for nutrients, they respond by regulating expression of genes important for survival. In the yeast Saccharomyces cerevisiae, transcription of a gene for a secreted acid phosphatase, PHO5, is regulated in response to changes in the extracellular concentration of inorganic phosphate. PHO5 transcription is repressed in high-phosphate medium and derepressed in low-phosphate medium.
A genetic pathway for PHO5 regulation has been established by Oshima and colleagues (Oshima 1982). PHO2, PHO4, and PHO81 are positive regulators of PHO5; deletion of these genes results in an inability to induce PHO5 upon phosphate starvation (Toh-eet al. 1973). Another set of genes, including PHO80, PHO85, and PHO84, are required for PHO5 repression, and loss-of-function mutations in these genes result in constitutive expression of PHO5, even in high-phosphate medium (Phoc phenotype; Uedaet al. 1975).
In recent years, progress has been made in elucidating the molecular mechanism of a signal transduction pathway leading to the transcriptional regulation of PHO5 (Lenburg and O'Shea 1996). PHO4 encodes a transcription factor that is required for PHO5 expression (Toh-eet al. 1973). When yeast cells are grown in high-phosphate medium, Pho4p is phosphorylated by the Pho80p-Pho85p cyclin/cyclin-dependent kinase (CDK) complex (Kaffmanet al. 1994). Phosphorylated Pho4p is localized predominantly to the cytoplasm (O'Neillet al. 1996), and PHO5 transcription is repressed. When yeast cells are starved for phosphate, the activity of the Pho80p-Pho85p cyclin-CDK complex is inhibited by the CDK inhibitor (CKI) Pho81p (Schneideret al. 1994; Ogawaet al. 1995). Under these conditions, Pho4p is unphosphorylated and localized to the nucleus, where it activates PHO5 transcription.
In high-phosphate medium, repression of PHO5 transcription requires inactivation of the CKI activity of Pho81p. However, it is still not known where the signal to repress PHO5 transcription is generated, nor how the signal results in activation of the kinase activity of the Pho80p-Pho85p cyclin-CDK complex. Loss-of-function mutations in PHO81 are epistatic to mutations in PHO84, suggesting that PHO84 functions upstream of PHO81. Biochemical data are consistent with this model—Pho84p is a transmembrane protein required in vivo for high-affinity phosphate uptake (Tamaiet al. 1985; Bun-Yaet al. 1991), and recombinant Pho84p expressed in Escherichia coli is capable of transporting phosphate in vitro when assembled into synthetic phospholipid vesicles (Berheet al. 1995). Whether Pho84p is directly involved in the signaling that leads to PHO5 repression is currently unclear.
In an effort to understand the signaling process for PHO5 repression, we designed a genetic selection using a PHO5-HIS3 reporter to identify genes that function upstream of PHO81 and are required for PHO5 repression. Our strategy was to isolate mutants that exhibit a Phoc phenotype that is also PHO81 dependent. Our goal was to identify components of the signal transduction pathway required to prevent inhibition of the Pho80p-Pho85p complex by Pho81p in high-phosphate medium. Loss-of-function mutations in these genes should cause accumulation of unphosphorylated Pho4p in the nucleus in high-phosphate medium as a result of inhibition of Pho80p-Pho85p kinase activity by Pho81p. We have determined that mutations in five genes (PHO84, PHO86, PMA1, ACC1, and PHO23) result in constitutive PHO5 expression in a PHO81-dependent manner. Mutations in PMA1, ACC1, and PHO23 have not been reported to confer a Phoc phenotype. Analysis of these mutants indicates that these genes are likely to affect different aspects of the signaling pathway. Possible mechanisms for the action of these gene products in the phosphate repression signaling pathway are discussed.
Yeast strains, plasmids, media, and genetic methods: Yeast strains used in this study are described in Table 1. Our yeast strains are derived from K699 or have been crossed into the K699 genetic background (Schwob and Nasmyth 1993). Plasmids are listed in Table 2. Standard yeast media are as described (Ausubelet al. 1993). Low-phosphate medium is phosphate-depleted medium (O'Connell and Baker 1992). No-phosphate medium is synthetic medium consisting of yeast nitrogen base lacking inorganic phosphate. Yeast nitrogen base lacking inorganic phosphate was made with components described in the Difco manual, except that potassium phosphate was substituted with the same amount of potassium chloride. Uptake medium consists of no-phosphate medium containing 10 mm potassium acetate to buffer it to pH 4.2 or 10 mm potassium citrate to buffer it to pH 3.0. Media supplemented with fatty acids were prepared by adding 1% Tween 40 (v/v) with 0.05% (w/v) palmitic acid. Crossing, sporulation, and tetrad analysis were done by standard genetic methods (Shermanet al. 1978). Yeast cells were transformed by the lithium acetate method (Itoet al. 1983) or by electroporation (Becker and Guarente 1991).
Mutagenesis and isolation of novel, PHO81-dependent Phoc mutants: The PHO5-HIS3 reporter fusion was constructed as follows. The PHO5 promoter was amplified using the polymerase chain reaction (PCR) from plasmid pMH313 (Han and Grunstein 1988) with the following two primers: 5′-GC GGAATTCGATCCGAAAGTTGCA-3′ and 5′-TTAAGCTCTA ATGGTTACCCTAGGGCG-3′. The amplified product contains nucleotides -550 to -1 of the PHO5 promoter, which includes the Pho4p- and Pho2p-binding sites, as well as sites for three of four positioned nucleosomes that undergo transitions in low-phosphate medium (Almer and Horz 1986). This PCR fragment was cut with EcoRI and BamHI and subcloned into pRS314 (Sikorski and Hieter 1989) to create pRS314-PHO5. The HIS3 open reading frame and 3′-untranslated region, a total of 1100 bp, were amplified using PCR from plasmid pJJ217 (Jones and Prakash 1990) with the primers 5′-GCGGGATCCACAGAGCAGAAAGCC-3′ and 5′-TCCCCGCG GATCACCACAACTAAC-3′. This amplified product was cut with BamHI and SacII and cloned into pRS314-PHO5 to create plasmid pPHO5-HIS3. Finally, nucleotides -460 to -110 of the 5′-untranslated region of HIS3 were amplified from plasmid pJJ217 by PCR with the following primers: 5′-GCGGGGCC CCTGCACGGTCCTGTT-3′ and 5′-GCGGAATTCGAGTCAT CCGCTAGG-3′. This amplified product was cut with ApaI and EcoRI and cloned into plasmid pPHO5-HIS3 to create plasmid pPHO5-HIS3int. To integrate this fusion construct into the yeast genome, plasmid pPHO5-HIS3int was cut with ApaI and SacII, and the liberated 1.9-kb piece was gel purified and transformed into a pho80Δ strain (EY0486). The net result of the targeted integration at the HIS3 locus was to delete most of the HIS3 promoter and replace it with 525 bp of the PHO5 promoter. After selection for stable transformants on SD -His, individual colonies were tested for the integration of the construct at the HIS3 locus by colony PCR using primers 5′-TTAAGCTCTAATGGTTACCCTAGGGCG-3′ and 5′-TTTAG CTTCTCGACGTGGGCCTTT-3′. A 950-bp product was produced from cells that had undergone the integration event, but not from cells that contained random insertions. The resulting strain was crossed to a wild-type strain (EY0091). The heterozygous diploid was sporulated to generate the starting strain for the selection, EY0100.
A logarithmically growing culture of strain EY0100 in YPD was washed and resuspended in distilled water, mutagenized with UV to 33% survival, and plated onto SD -His with the addition of 2 mm 3-amino-1,2,4-triazole. These plates were incubated for 4 days at 25° in the dark. Colonies were picked and patched onto YPD plates. The Phoc mutants were identified by assaying the histidine prototrophs for constitutive PHO5 expression using acid phosphatase plate assays (Toh-eet al. 1973) on rich medium. Recessive mutants were isolated by mating the Phoc mutants to a wild-type strain (EY0183) and assaying the resulting diploid strains for constitutive PHO5 expression. Mutations in genes that were previously known to be required for PHO5 repression (i.e., PHO80, PHO85, or PHO84) were identified by crossing the recessive mutants to a pho80Δ pho85Δ double mutant (EY0179) or a pho84Δ mutant (EY0211) and analyzing the resulting diploids for the Phoc phenotype using acid phosphatase plate assays. Complementation tests among the remaining non-pho80, non-pho85, and non-pho84 recessive Phoc mutants were performed by mating them to each other after backcrossing. PHO5 expression was examined in these diploids by acid phosphatase plate assays.
To test whether these novel recessive mutants are PHO81 dependent, we crossed one mutant from each group to EY0150 (pho81Δ::TRP1). The resulting diploid cells were sporulated, and the double-mutant haploid cells were identified from the nonparental ditype tetrads by examining PHO5 expression in low-phosphate medium.
Plasmid cloning: Each novel recessive PHO81-dependent Phoc mutant was backcrossed at least three times to a wild-type strain. The Phoc phenotype segregated 2:2 for each class, suggesting that the mutation resides in a single gene. The wild-type versions of the genes containing the novel PHO81-dependent Phoc mutations were cloned by screening a centromere-based (YCp50) yeast genomic library (a gift from A. Murray and K. Hardwick) for plasmids that were able to complement the Phoc mutant phenotype in acid phosphatase plate assays. Because we found that the lithium acetate transformation method was somewhat mutagenic and that our PHO81-dependent Phoc mutants picked up suppressors or reverted at a fairly high rate, we transformed the DNA library primarily by electroporation. Transformants were streaked to isolate single colonies, which were patched and tested for plasmid dependence on 5-FOA plates. Plasmids were then isolated from these transformants and retransformed into the original mutant strains to test complementation.
Phosphate uptake assays: Phosphate uptake assays were performed with a modification of a previously described procedure (Tamaiet al. 1985). Phosphate uptake was measured on whole cells. Yeast strains grown to log phase in synthetic high-phosphate medium were inoculated into no-phosphate medium at an optical density at 600 nm (OD600) of 0.1-0.2 and grown for 4-6 hr at 30°. Cells were washed and resuspended in uptake medium at pH 4.5 or pH 3.0 to a final OD600 of ∼0.5-0.8, and they were incubated at 30° for 20 min before uptake experiments were performed. The final external inorganic phosphate concentration was adjusted to 10 μm, and an appropriate amount of H 323PO4 (Dupont NEN Research Products) was added as a radioactive label. The labeled substrate was added to prewarmed cells, and samples were withdrawn at different time intervals. Samples were filtered immediately through 0.8-μm nitrocellulose membrane filters (Millipore, Bedford, MA) and washed twice with 5 ml of 100 mm KH2PO4. The amount of phosphate taken up by the yeast cells was determined by scintillation counting.
Quantitative studies of PHO5-GFP expression: Log-phase yeast strains harboring pPHO5-GFP (EB0180) were inoculated into SD -Ura or SD low-phosphate -Ura medium. The pPHO5-GFP plasmid was made by fusing the PHO5 promoter (nucleotides -550 to -1 upstream of the ATG) in-frame to the N terminus of green fluorescent protein (GFP; Chalfieet al. 1994) cloned into the URA3-marked vector pRS316 (Sikorski and Hieter 1989). Emission at 510 nm was measured after excitation at 395 nm of whole cells using a fluorometer (Photon Technology International, Inc., Monmouth Junction, NJ). The final emission was normalized to the cell density as measured by OD600. Background fluorescence was determined by using wild-type cells transformed with pRS316.
Pho4-GFP localization: pACPHO4-GFP (EB0347) consists of the PHO4 promoter (nucleotides -323 to -1 upstream of the ATG) and the entire PHO4 coding region fused in frame to the N terminus of a GFP mutant (S65T; Heimet al. 1995) cloned into the URA3-marked vector pRS316. pACPHO4-GFP was used to transform different yeast strains. Using a BX60 microscope (Olympus Corp., Lake Success, NY), localization of the fusion protein was examined by direct fluorescence of live yeast grown in SD -Ura.
A genetic selection to isolate mutants that express PHO5 constitutively: To identify novel factors that signal PHO5 repression, we designed a selection to isolate mutants that express PHO5 constitutively (Figure 1). Our starting strain for this selection was his3::PHO5-HIS3 (EY0100), in which the promoter of the HIS3 gene was replaced by the PHO5 promoter (see materials and methods). We demonstrated that HIS3 gene expression is subject to the same regulatory control as the PHO5 gene in three ways. First, in high-phosphate medium, this strain was a histidine auxotroph, whereas in low-phosphate medium, it was a histidine prototroph. Second, in high-phosphate medium, a his3::PHO5-HIS3 pho80Δ strain was a histidine prototroph. Third, a his3::PHO5-HIS3 pho2Δ strain was a histidine auxotroph (data not shown). Thus, the ability to grow in the absence of histidine reflects PHO5 expression in the strain his3::PHO5-HIS3.
To carry out the selection for Phoc mutants, we plated approximately one million live, mutagenized yeast cells on SD -His plates containing aminotriazole. A total of 420 colonies of heterogenous sizes grew, and 167 of 420 conferred unambiguously the constitutive PHO5 expression phenotype. The Phoc phenotype was scored by the acid phosphatase assay in which a chromagenic phosphatase substrate, α-naphthylphosphate, was applied in an agar overlay; yeast strains expressing PHO5 turn red by these assays (Toh-eet al. 1973). We determined that 81 mutants contained dominant Phoc mutations. Among the remaining 86 recessive mutants, we recovered 24 alleles of either PHO80 or PHO85, 44 alleles of PHO84, and 18 alleles of mutations in genes not previously identified in the screen of Ueda et al. (1975). These 18 novel Phoc mutants fell into eight complementation groups. Mutants in four of these complementation groups also had a slow growth phenotype at 16°. Genetic analysis has revealed that the mutation that causes the cold-sensitive (cs) phenotype is tightly linked to the Phoc mutation in each case.
Four novel recessive Phoc mutant groups are PHO81 dependent: To distinguish genes involved in the signaling process from those that affect other aspects of PHO5 regulation (e.g., transcriptional repression), we analyzed the epistatic relationship between pho81 and the novel Phoc mutant classes. We reasoned that a pho81 mutant should be epistatic to mutants defective in the signaling process (see also discussion); in these mutant strains, deletion of the PHO81 gene should result in Pho4p being localized to the cytoplasm and an uninducible PHO5 expression phenotype. Double mutants of pho81 and novel Phoc mutants were generated (see materials and methods) and examined for PHO5 expression and Pho4-GFP localization. Four complementation groups showed a PHO81 dependence for PHO5 expression and Pho4-GFP localization. Each of the four groups contains only one mutant allele, namely wk29, wk153, wk196, and wk383. Mutants wk29, wk153, and wk196 also have a slow growth phenotype at 16°.
We measured PHO5 expression in these four PHO81-dependent mutants in high-phosphate medium in two ways. We first used acid phosphatase plate assays (Tohet al. 1973) and the color intensity of different Phoc mutant strains as indicators of the level of PHO5 activity (Figure 2, A and C). Then, to better quantitate PHO5 expression, we measured PHO5 promoter activity by using a pPHO5-GFP construct in which the PHO5 promoter was fused to the GFP (EB0180; Figure 2, B and D). The strength of the PHO5 promoter was measured by GFP fluorescence at 510 nm (see materials and methods).
Results from these two methodologies show strong correlation; mutants with a stronger Phoc phenotype (darker color) have a higher fluorescent emission at 510 nm. Figure 2, A and B, demonstrates that the four novel PHO81-dependent mutants express PHO5 constitutively, but to different degrees. Among the four PHO81-dependent complementation groups harboring pPHO5-GFP, wk153, which confers the strongest Phoc phenotype, emits a GFP fluorescence at 510 nm that is approximately five- to eightfold above that of a wild-type strain. wk196 and wk29 are of an intermediate Phoc phenotype and emit GFP fluorescence approximately threefold above wild type, whereas wk383, which confers the weakest Phoc phenotype, emits GFP fluorescence approximately twofold above wild type (Figure 2B).
To examine PHO5 expression in double mutants of pho81Δ and the novel recessive Phoc mutants, we performed acid phosphatase plate assays and GFP fluorescence assays in low-phosphate medium, as shown in Figure 2, C and D, respectively. Only the results for the PHO81-dependent mutants are shown. A pho80Δ pho81Δ double-mutant strain was included as a control for PHO5 expression in low-phosphate medium because PHO80 is a gene known to function downstream of PHO81. Similarly, a pho84Δ pho81Δ double-mutant strain was also included as a control because pho81 is epistatic to pho84. The four mutant groups show a phenotype similar to that of pho84Δ pho81Δ, indicating that pho81 is epistatic to these novel Phoc mutants, and that they are likely to define genes that function upstream of PHO81. The four complementation groups whose Phoc phenotype is independent of PHO81 were not analyzed further in this study.
We next used the localization of Pho4-GFP in high-phosphate medium to monitor the PHO81 dependence of our Phoc mutants. The localization of Pho4p has been characterized previously (O'Neillet al. 1996). In both wild-type and pho81Δ strains, Pho4-GFP is localized to the cytoplasm in high-phosphate medium (Figure 3). In mutants that express PHO5 constitutively, such as pho84Δ, Pho4-GFP is localized exclusively to the nucleus. However, in the double mutant pho84Δ pho81Δ, Pho4-GFP is again localized to the cytoplasm because pho81 is epistatic to pho84. In contrast, in a pho80Δ pho81Δ strain, Pho4-GFP is localized to the nucleus because pho80 is epistatic to pho81. Thus, the epistatic relationship between the Phoc mutants and pho81 can be studied easily using this assay. In the mutant wk153, Pho4-GFP is localized exclusively to the nucleus, while in wk29, Pho4-GFP is localized both to the nucleus and to the cytoplasm. It should be noted that this result correlates with the observation that wk153 expresses more GFP when harboring the pPHO5-GFP construct and has higher acid phosphatase activity in high-phosphate medium than wk29; this is consistent with the model in which the concentration of nuclear Pho4p modulates the level of PHO5 expression. In contrast, in the double mutants wk153 pho81Δ and wk29 pho81Δ, Pho4-GFP is predominantly cytoplasmic, indicating that pho81 is epistatic to wk153 and wk29. Thus, the Pho4-GFP localization is consistent with both the results from acid phosphatase plate assays for PHO5 expression and the PHO5-GFP fluorescence assays, and it confirms that pho81 is epistatic to wk153 and wk29. The Pho4-GFP localization analysis in wk196 and wk383 is very similar to that of wk29 (data not shown).
The novel PHO81-dependent Phoc mutations represent alleles of PHO86, PMA1, ACC1, and PHO23: During our mutant characterization, Yompakdee et al. (1996) reported the isolation of a novel mutant that expresses PHO5 constitutively, pho86-1, on the basis of a screen for arsenate-resistant mutants. Pho86p is a membrane protein with two putative transmembrane domains (Yompakdeeet al. 1996) and has no homology to other proteins. On the basis of the following observations, we conclude that wk153 is an allele of PHO86. First, wk153 failed to complement the Phoc phenotype of a pho86Δ::TRP1 strain (EY0404). Second, after the diploid resulting from the cross of wk153 and EY0404 was sporulated, the mutation that caused the 16° slow growth phenotype segregated away from Trp+ spores, indicating that this mutation in wk153 was tightly linked to the PHO86 locus. Third, a low-copy-plasmid expressing PHO86 (a gift from Nobuo Ogawa) was able to complement both the PHO5 constitutive phenotype and the 16° slow growth phenotype of wk153. We therefore referred to wk153 as pho86-153.
To clone the remaining PHO81-dependent genes, we determined which genes could complement the PHO5 constitutive expression phenotype of each mutant by screening a low-copy yeast genomic library (see materials and methods). Both the Phoc and the slow growth phenotype at 16° of the wk196 mutant were complemented by two plasmids. The overlapping region of the insert fragments in these two plasmids contains two intact ORFs, PMA1 and YGL007w. Subsequent subcloning localized the complementing region to a HindIII-XhoI genomic fragment (EB0658), that contains only one complete ORF, PMA1, which encodes a p-type plasma membrane ATPase (Serranoet al. 1986).
To confirm that the mutation responsible for the Phoc phenotype in wk196 was tightly linked to the PMA1 locus, we subcloned the PstI/XhoI fragment of the PMA1 ORF into a LEU2-marked integrating vector, pRS305 (Sikorski and Hieter 1989). This plasmid (EB0660) was linearized with HindIII, a unique site within the PMA1 open reading frame, and was targeted to the genomic site via homologous recombination in a wild-type strain (EY0183). The integration of this vector at the PMA1 locus was confirmed by Southern blotting (data not shown). The resulting strain (EY0469) was crossed to a wk196 strain, and the diploid was sporulated. All 31 tetrads examined were of the parental ditype (i.e., the mutation that caused the Phoc phenotype segregated away from the Leu+ spores), indicating that this mutation in wk196 was tightly linked to the PMA1 locus. On the basis of our complementation and linkage analyses, wk196 was named pma1-196.
One plasmid (EB0657) complementing the Phoc phenotype of the wk29 mutant strain was isolated, and it contained only one intact ORF, ACC1, which encodes acetyl-CoA carboxylase. This plasmid also rescues the slow-growth phenotype of wk29 at 16°. To demonstrate that ACC1 corresponded to the wk29 locus, a HindIII fragment that contained part of the ACC1 gene was subcloned into pRS306, a URA3-marked integrating vector. The plasmid (EB0661) was linearized with NarI and targeted to the genomic site by homologous recombination. Southern blotting confirmed the integration of the vector at the ACC1 locus (data not shown). The resulting strain (EY0468) was crossed to the wk29 strain, and the diploid was sporulated. All 28 tetrads examined were of the parental ditype, demonstrating linkage of ACC1 to wk29. We then referred to wk29 as acc1-29.
The mutant phenotype of wk383 was complemented by three identical plasmids isolated from independent clones. Further subcloning localized the complementing region to a PstI-SpeI genomic fragment, which was subcloned into the PstI and SpeI sites of pRS314. The resulting plasmid (EB0659) contains only one ORF, YNL097c, which we named PHO23, and wk383 was named pho23-1. The wk383 mutation was shown to be linked to the RAS2 gene that is adjacent to YNL097c by mating wk383 to a ras2Δ::LEU2 strain (a gift from J. Whistler and J. Rine) and analyzing the resulting tetrads.
To confirm that the YNL097c ORF corresponds to the wk383 locus, we constructed a disruption vector. An EcoRI-SphI fragment containing the HIS3 gene (from pJJ215, EB0098) was inserted into the NcoI-SphI fragment of EB659, with the EcoRI and NcoI ends blunted by treatment with Klenow. The TATA box and approximately half of the YNL097c ORF, including the ATG start codon, were deleted in the resulting plasmid (EB0662). EB0662 was then cut with EcoRI and NotI and transformed into a diploid wild-type cell so that one of the endogenous YNL097c copies in the yeast genome was replaced by one-step gene replacement (Rothstein 1983). His+ transformants were selected and sporulated. All His+ haploid cells also exhibited a Phoc phenotype in >20 tetrads analyzed. The disruption of YNL097c was confirmed by Southern blotting (data not shown). The resulting strain has no growth defect on either YPD or synthetic media with high or low phosphate at a variety of temperatures, indicating that PHO23 is nonessential for cell viability. The Phoc phenotype of the pho23Δ:: HIS3 strain is similar to that of pho23-1.
PHO23 encodes a putative 330-amino-acid polypeptide, and the hydropathy profile of this protein suggests that it has no transmembrane domains. It has weak homology with two hypothetical yeast proteins, Yhr090p and Ymr075p, according to a search of the yeast genome database using the BLAST program (Altschulet al. 1990). Moreover, Pho23p is similar to a human protein p33ING1, a tumor suppressor functioning in the p53-signaling pathway (Garkavtsevet al. 1998). Interestingly, Pho23p shares significant similarity with p33ING1 at the C terminus, with 61% identity over 52 residues using the BLAST program.
Enhanced Phoc phenotype in double mutants between acc1, pma1, and pho23: To test whether the ACC1, PMA1, and PHO23 genes function in a common pathway in PHO5 regulation, we examined the constitutive PHO5 expression phenotype in the double mutants acc1-29 pma1-196 (EY0464), acc1-29 pho23Δ (EY0465), and pma1-196 pho23Δ (EY0466). We examined the expression of PHO5 in high-phosphate medium using acid phosphatase plate assays. Each of the double mutants displayed a stronger Phoc phenotype than either parent strain (data not shown). Normally, the synergy in phenotypes in a double mutant resulting from two total loss-of-function mutations implies that the two genes do not function in a linear pathway. However, in our case, the interpretation is complicated because ACC1 and PMA1 are essential genes, and we isolated only partial loss-of-function alleles of these two genes. Thus, the enhanced Phoc phenotype in these double mutants is either because these novel Phoc genes do not function in a linear pathway signaling PHO5 repression, or because acc1-29 or pma1-196 are only partial loss-of-function alleles.
Phosphate uptake analysis defines two classes of mutants: Because PHO84 encodes a high-affinity phosphate transporter and PHO86 is implicated to function in the phosphate transport system (Yompakdeeet al. 1996), we performed phosphate uptake assays to test whether the remaining genes that we identified are also involved in the high-affinity phosphate transport system. Figure 4 documents the results of phosphate uptake experiments. The pho84Δ, pho86Δ, and pma1 strains have a clear defect in phosphate uptake. This result is consistent with the function of Pma1p as a proton pump, as well as the fact that pH is able to affect phosphate uptake kinetics in yeast (Nieuwenhuis and Borst-Pauwels 1984; Tamaiet al. 1985). In contrast, acc1-29 and pho23-1 are able to take up phosphate as well as a wild-type strain. Moreover, the acc1-29 pho23Δ (EY0465) double mutant does not have a phosphate uptake defect (data not shown), confirming that neither ACC1 nor PHO23 are involved in high-affinity phosphate transport.
The phosphate uptake defect of pma1-196 can be rescued by lowering the pH: To determine which aspects of Pma1p function are required for constitutive PHO5 expression, we obtained several pma1 mutant strains from other labs and assayed them for PHO5 expression. The strains we obtained have mutations in residues required for ATPase activity [A608T (van Dycket al. 1990), H285Q (Wachet al. 1996), and A135V (Naet al. 1993)]; for membrane targeting [P434A and G789S (Chang and Fink 1995)], and for proton pumping activity [I183A (Wanget al. 1996) and S368F (Perlinet al. 1989)]. Only one of the mutants expressed PHO5 constitutively; I183A conferred a weak Phoc phenotype. This mutant has decreased proton pumping ability, although its ATPase activity is comparable to that of the wild type (Wanget al. 1996), raising the possibility that an impaired proton pump causes poor phosphate uptake, which in turn results in constitutive PHO5 expression by an unknown mechanism.
To determine if the proton pumping defect in pma1-196 is indeed the reason for poor phosphate uptake, we examined if the phosphate uptake defect of pma1-196 could be rescued by providing a more acidic extracellular environment to restore the proton gradient across the plasma membrane. We measured phosphate uptake in a wild-type strain and pma1-196 at pH 4.5 and pH 3.0, as shown in Figure 5. At pH 3.0, pma1-196 could take up phosphate at about the same rate as a wild-type strain, whereas at pH 4.5, it had a phosphate uptake defect (Figures 4 and 5). We noted that the wild-type strain could take up phosphate at a higher rate at pH 4.5 than at pH 3.0, consistent with the observation that the optimum pH for the high-affinity phosphate transport system is between 4 and 5 (Nieuwenhuis and Borst-Pauwels 1984; Tamaiet al. 1985). We were not able to investigate whether lowering the pH would rescue the Phoc phenotype in pma1-196 because pma1-196 would not grow in low pH medium. These data suggest that the proton pumping defect in pma1-196 is the reason for its poor uptake of phosphate and the resulting Phoc phenotype.
The PHO5 constitutive expression phenotype of acc1-29 can be rescued by supplementation of fatty acids: The Phoc phenotype of acc1 indicates a possible connection between fatty acid synthesis and PHO5 regulation. Recently, several acc1 alleles that confer novel phenotypes have been isolated, leading to the proposal that Acc1p performs a function in addition to its role as an acetyl-CoA carboxylase (Schneiter and Kohlwein 1997). To determine if the effect of the acc1 mutation on PHO5 regulation was allele specific, we examined PHO5 expression in various acc1 mutants in high-phosphate medium, including acc1-2150 [a temperature-sensitive fatty acid auxotroph (Mishinaet al. 1980)], acc1-7-1 [defective in mRNA export (Schneiteret al. 1996)], and acc1cs [synthetic lethal with hpr1 (Guerra and Klein 1995)]. Constitutive PHO5 expression was observed in all acc1 alleles tested using acid phosphatase plate assays, indicating that the Phoc phenotype is not allele specific, but that it can be attributed to a general defect in acc1 mutants. To investigate if this general defect is caused by the inefficient synthesis of long chain fatty acids, we supplemented the medium with palmitic acid and examined PHO5 expression in acc1-29. The Phoc phenotype of acc1-29 is rescued by the supplementation of palmitic acid (Figure 6), suggesting that inefficient fatty acid synthesis causes constitutive PHO5 expression in acc1 mutants.
Isolation of mutants defective in signaling PHO5 repression: In this study, we have described a genetic selection for mutants that express PHO5 constitutively in high-phosphate medium. Because we are interested in how the high-phosphate signal is generated and transduced, we have focused on mutants whose phenotype is dependent on the function of the CDK inhibitor Pho81p. Repression of the phosphate response pathway by a high concentration of extracellular phosphate is a complex process, consisting of successful detection and generation of the phosphate signal, proper activation of the kinase activity of the Pho80p-Pho85p cyclin-CDK complex, appropriate localization of Pho4p to the cytoplasm, and correct establishment of transcriptional repression (e.g., repressive chromatin structure). Many mutations could interfere with these processes and cause inappropriate expression of PHO5 in high-phosphate medium. Molecules that are involved in transducing a high-phosphate signal should be specifically required to prevent inhibition of the Pho80p-Pho85p complex by Pho81p; the pho81 mutant will be epistatic to mutations in these signaling molecules. We isolated five mutant classes whose phenotypes are PHO81 dependent.
Two of the five mutant classes were previously known to confer a Phoc phenotype: PHO84 encodes a high-affinity phosphate transporter, and PHO86 is required in vivo for high-affinity phosphate uptake (Yompakdeeet al. 1996). It is unclear if PHO86 is a positive regulator of PHO84 or if it is directly involved in signal transduction.
We isolated additional mutations in genes previously unknown to be involved in PHO5 regulation. PMA1 encodes a plasma membrane ATPase, which is required for the in vivo function of several transporters, including the high-affinity phosphate transport system (Vallejo and Serrano 1989; Kotyk 1994). PMA1 is likely to be a positive regulator of PHO84 because Pma1p functions as a proton pump and Pho84p is a proton-phosphate symporter (Borst-Pauwels 1981).
Two other classes of PHO81-dependent Phoc mutants include ACC1 and a new gene, PHO23. Mutations in these two genes do not cause high-affinity phosphate uptake defects, suggesting that they cause the Phoc phenotype by mechanisms distinct from those of PHO86 and PMA1. ACC1 encodes an acetyl-CoA carboxylase, which catalyzes the rate-limiting step in the de novo synthesis of fatty acids. Mutant alleles of ACC1 were initially isolated as fatty acid auxotrophs. The ACC1 gene is essential for cell viability and is evolutionarily conserved. Its role in the phosphate metabolism pathway is unclear (see next section). Null alleles of PHO23 result in only a partial Phoc phenotype, which argues against Pho23p being a direct inhibitor of Pho81p function because hyperactive alleles of PHO81 confer a much more severe Phoc phenotype (data not shown). Alternatively, Pho23p may play a role in inhibiting Pho81p if there exist additional genes, possibly the two homologues, with redundant function. In summary, these genes are likely to affect different aspects of the transduction of the phosphate signal.
Constitutive PHO5 expression in acc1 mutants: In our screen for constitutive PHO5 expression mutants, we isolated an allele of ACC1. We have shown that the Phoc phenotype is not allele specific. Moreover, the Phoc defect can be rescued by supplementing exogenous palmitic acid (Figure 6). These results suggest that the defect in PHO5 repression in an acc1 mutant is caused by the inefficient synthesis of fatty acids, and that there may exist crosstalk between fatty acid biosynthesis and phosphate metabolism pathways. What is the mechanism of action of ACC1 in the signal transduction pathway for PHO5 repression? If ACC1 functions upstream of PHO81, it is possible that some metabolite(s) of fatty acids may serve as a second messenger to signal PHO5 repression. In acc1 mutants, this metabolite would be absent, resulting in constitutive expression of PHO5. Another possibility is that a protein involved in PHO5 regulation requires specific fatty acylation for its in vivo function. The intracellular level of the required fatty acid in acc1 mutants would be so low that fatty acylation is impaired, leading to the constitutive expression of PHO5.
Recently, some acc1 mutants that have defects in nuclear morphology have been isolated (Schneiter and Kohlwein 1997), leading to the speculation that ACC1 is directly involved in maintaining membrane structure. acc1-7-1 was isolated as a mutant defective in mRNA export, and further analysis revealed a defect in the nuclear envelope of the mutant yeast strain (Schneiteret al. 1996). Whether the membrane structure of organelles in acc1-29 is defective has not been investigated. The fact that the high-affinity phosphate transport system is intact in the acc1-29 mutant suggests that there is no general defect in plasma membrane structure in this mutant strain.
Is the high-affinity transport system directly involved in signaling? Because PHO84, PHO86, and PMA1 are required for PHO5 repression, one intriguing possibility is that the high-affinity phosphate transport system is directly involved in signaling repression of PHO5 transcription. There are two models that could explain the constitutive PHO5 expression in pho84 or pho86 mutants. It is possible that in addition to their phosphate transport functions, Pho84p and Pho86p may also act as sensor proteins for the levels of extracellular phosphate and transduce a repression signal when phosphate levels are high. Alternatively, the phosphate transport function of the high-affinity phosphate uptake system may be only indirectly involved in the signaling process. In pho84 or pho86 mutants, defects in phosphate uptake might result in a low level of intracellular phosphate (or some metabolite) that serves as a messenger; a lack of this messenger may cause a defect in the production of a repression signal.
The first model, in which the phosphate transporter is also the phosphate sensor, has a precedent in bacteria. The Pho regulon of E. coli is also regulated by extracellular phosphate levels; its transcription is repressed when phosphate levels are high and is induced when phosphate levels are low (Wanner 1996). In the E. coli system, the phosphate signal is transduced by a two-component regulator complex consisting of phoR and phoB. Repression of the Pho regulon also requires an intact Pst phosphate transport system and a protein called PhoU. PhoR senses phosphate starvation signals and activates phoB, which is a transcription factor required for transcription of the Pho regulon (Wanner 1996). Mutations in the Pst transport complex that separate the transport and repression functions have been isolated, suggesting that the phosphate transporter is able to directly sense changes in the extracellular concentration of phosphate (Coxet al. 1988).
In yeast, there are also examples of transporters serving as sensors. It has been proposed that some glucose transporters act as receptors for sensing glucose levels. Dominant gain-of-function mutations in two yeast genes, SNF3 and RGT2, which encode glucose transporters, have been isolated (Ozcanet al. 1996). These mutants cause induction of the expression of the glucose-regulated gene HXT2 in the absence of glucose, suggesting that the glucose signal is transmitted into the cell by glucose transporters that also act as glucose receptors that sense extracellular glucose levels (Ozcanet al. 1996). Interestingly, both Snf3p and Rgt2p belong to the same family of 12-transmembrane domain transporters as Pho84p (Bun-Yaet al. 1991). Recently, an ammonium permease, Mep2p, was also proposed to be involved in a signal transduction pathway that regulates pseudohyphal growth in response to ammonium starvation (Lorenz and Heitman 1998).
There are also examples of intracellular metabolites that serve as signals to control gene expression. For example, intracellular concentrations of glutamine are able to regulate gene expression. In yeast, the expression of GLN1, which encodes a glutamine synthetase, is inactivated by an increase in the intracellular glutamine concentration (Magasanik 1991). Similarly, the expression of glnA, the structural gene for bacterial glutamine synthetase, is also inactivated by high levels of intracellular glutamine. This process depends on the regulation of another two-component complex with NRII as the sensor and NRI as the effector (Parkinson 1993).
Previous studies on the yeast phosphate transport system suggest that in addition to the high-affinity uptake system, there exists a constitutive low-affinity phosphate transport system with a proposed Km of 1 mm (Nieuwenhuis and Borst-Pauwels 1984; Tamaiet al. 1985). We supplied the medium with external phosphate ≤100 mm to saturate the low-affinity transport system, and we examined PHO5 expression in pho84Δ and pho86Δ strains. The Phoc phenotype of pho84Δ and pho86Δ strains cannot be suppressed at phosphate levels 100-fold above the Km of the low-affinity phosphate uptake system, suggesting that these mutants may be defective in transducing a repression signal downstream rather than simply in phosphate uptake. However, whether the high-affinity phosphate uptake system can directly sense changes in extracellular phosphate levels remains to be determined.
We thank D. Perlin, A. Chang, A. Goffeau, R. Schneiter, S. Kohlwein, A. Tartakoff, E. Schweizer, H. Klein, N. Ogawa, J. Whistler, and J. Rine for their generous gifts of yeast strains. We thank A. Murray and K. Hardwick for the YCp50 genomic library, N. Ogawa for the PHO86 low-copy plasmid, and J. Li for the LYS2 disruption vector. We also thank A. Yi for making the pPHO5-GFP construct. We thank the members of the O'Shea lab for their technical assistance, advice, and encouragement. We are especially grateful to M. Lenburg, L. Huang, F. Banuett, and M. Christman for their critical comments on the manuscript. This work was supported by grant GM-51377 from the National Institutes of Health (E.K.O.) and a Howard Hughes Medical Institute predoctoral fellowship (W.-T.W.L.).
Communicating editor: M. Carlson
- Received May 1, 1998.
- Accepted August 7, 1998.
- Copyright © 1998 by the Genetics Society of America


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2011 Smart electric drive - first drive
2011 Smart electric drive - first drive
Smart plans to bring some 1,500 Fortwo EVs into the U.S. Of those, about 80 percent will head directly to municipalities and institutions. The rest? Well, in true Smart fashion they have mostly been spoken for by early adopters who can't live without the small electric car. Those who have chosen to plop their money down are looking at a $2,400 down payment and $599 a month for 48 months. Of course, there is tax, title and license fees that bring the final payout some where above the $31K mark.
Bottom line—the 2011 Smart Fortwo Electric Drive is functional and will be a great city runabout. Now we just have to see if the price will be a barrier to its success.
[Source: Leftlane]

subset: commoncrawl


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As a provider of a Software-as-a-Service (SaaS) digital asset management solution, we get plenty of security-related questions from people who are a bit apprehensive about trusting their DAM software needs to the cloud.
That was the topic of a recent Google Hangout between Widen leaders and RCR Wireless. As CEO Matthew Gonnering and CTO Gary Norris point out, the Amazon Web Service cloud is not only secure, but also enables Widen to offer its DAM customer more innovation at a lower cost. A true win-win.
Here's a video of Matthew and Gary recapping the interview and offering a few extra thoughts. The full interview is below. If you want to chat with us in more detail about the security of cloud-based DAM, just get in touch!
Matthew Gonnering and Gary Norris after their RCR Wireless News interview
RCR Wireless News interview


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Can al-Qaeda and like-minded terrorist groups be deterred? The Bush and Obama administrations both eventually concluded that they can be, according to an important new book by New York Times' National Security Correspondents Eric Schmitt and Thom Shanker. In Counterstrike: The Untold Story of America's Secret Campaign Against Al Qaeda, Schmitt and Shanker use extensive interviews with high-ranking U.S. officials to trace the evolution of U.S. counterterrorism (COTERROR) strategy over the last decade since 9/11. Their use of first-hand accounts by participants in the debates makes the book Bob Woodwardesqe and differentiates it from other histories of the Global War on Terror (GWoT); most notably, Peter Bergen's The Longest War: The Enduring Conflict Between America and Al-Qaeda.
Page numbers referring to the text appear in parenthesis.
I. The new deterrence
The most important takeaway from Counterstrike is the story of how the United States gradually came to adopt a deterrence strategy against al-Qaeda. This "new deterrence" strategy both drew from and expanded upon the successful deterrence strategy the United States adopted vis-à-vis the Soviet Union.[i] Much like "nuclear deterrence," Washington's deterrence strategy towards al-Qaeda has been mostly improvised and adopted in an ad hoc fashion. A constant obstacle to implementing this strategy has been the tension between the different government bureaucracies with a stake in COTERROR. Although the authors point to many areas where the U.S. government has made significant strides in integrating the different "stovepipes" or bureaucracies, the search for a "whole-of-government" approach in the COTERROR realm remains elusive.
In the immediate aftermath of the 9/11 attacks, the Bush administration rejected the notion that a deterrence strategy could be applied to transnational terrorist organizations like al-Qaeda. In its 2002 National Security Strategy of the United States, for instance, the administration argued, "traditional concepts of deterrence will not work against a terrorist enemy whose avowed tactics are wanton destruction and the targeting of innocents; whose so-called soldiers seek martyrdom in death and whose most potent protection is statelessness." This was not an unreasonable assumption; after all, as the national strategy pointed out, al-Qaeda operatives do not value territory or even their own lives, both of which had been crucial to deterring the Soviets.
A few years after the 9/11 attacks, however, a viable long-term strategy for prosecuting the GWoT had still not materialized. Frustrated, senior Defense officials began searching for such a strategy in the lower echelons of the Pentagon. Douglas Feith, undersecretary of defense for policy, eventually found one in an ambitious new project being undertaken by Barry Pavel, a mid-ranking official who the authors describe as a "veteran Cold Warrior," and Matthew Kroenig, then a summer graduate intern at the Pentagon who was pursuing his doctorate in nuclear proliferation issues at University of California at Berkeley.[ii]
Pavel and Kroenig acknowledged that al-Qaeda didn't value territory or their own lives in the way that the Soviet Union had. Nonetheless, they argued that the terrorists did value other things that the United States could threaten. As Kroenig explains, "Terrorists are deterrable. While they may have a preference structure that's different from ours, they do value things- things we could hold at risk-and we can, therefore, influence their decisions (51)."
In their presentation-which made its way to then-Secretary of Defense Donald Rumsfeld who presented it to President Bush at Camp David in 2005- Pavel and Kroenig listed twelve different things that al-Qaeda leaders and operatives value:
- Calculus of chances for success of their attacks
- Personal glory
- Personal reputation
- Support among Muslim populations
- Publicity
- Network cohesion and dependability
- Trust in fellow cell members
- Well-being of their family
- Enhancement of the Muslim community
- Material assets
- Growing membership for the movement
- Strategic success (53)
II. Implementing the new Deterrence
Although Washington has remained vigilant in carrying out "kill and capture" missions against the terrorists, from 2006 on the United States has begun supplementing these kinetic operations with ones designed to threaten these twelve things. Counterstrike's best moments are when the authors explain the (often creative) ways in which Washington has tried to operationalize this new deterrence strategy.
A. Financial Disruption
Some of this is relatively straightforward. For example, to target al-Qaeda's material assets the United States has tried to crackdown on the terrorist group's financial operations. Thus, one of the primary characters in the book is Juan Zarate, a former Justice Department COTERROR official who transferred a few weeks before 9/11 to the Treasury Department to handle this same topic for that department. Working with other governments-notably those in the Persian Gulf that are home to most of al-Qaeda's high-level donors and financial markets- they have tried to shut down the financial hubs through which al-Qaeda leaders fund their operations.
But targeting the group's finances also brings up another point the United States' national security apparatus eventually came to appreciate. Namely, that although hardcore terrorists like bin Laden and Ayman al-Zawahiri might not be deterrable, other crucial members of the al-Qaeda network are not so ideologically committed to the Jihadist cause.[iii] The group's financers are a good example.[iv] These financers help al-Qaeda leaders transfer money through modern global financial networks undetected, which is crucial to al-Qaeda's ability to recruit members and carry out attacks. However, the financers perform their services for the group because of the personal monetary benefits they receive from doing so, not because of any ideological convictions for Jihadism. By targeting these individuals, then, the United States can deter talented financers from working with al-Qaeda. This, in turn, hinders the non-deterrable terrorist leaders' ability to act upon their vicious intentions.
B. Discrediting al-Qaeda
Another interesting part of the book is the authors' discussion of how the Bush and Obama administrations differed in their approaches to discrediting al-Qaeda in the eyes of the Muslim world. The Bush administration carried out a "war of ideas" that pitted Western values of liberal democracy and free-market capitalism against al-Qaeda's violent ideology in a sort of zero-sum competition for the hearts and minds of the Arab Muslim world.
While certainly desirable, the Obama administration concluded that promoting American values was not required to discredit al-Qaeda. Rather, all discrediting al-Qaeda required was to aggressively attack its message, ideology and tactics. Given that these were usually incompatible with the beliefs of the vast majority of humanity in general, and Muslims in particular, this task was much more achievable. As one Defense official explains, "We have come to a realization of what is in the art of the possible and what is not and what's our role, and more importantly, what is not our role. Now it is learning how to better execute those parts that we think we actually have a role in (177-178).[v]
Counterstrike reveals many of the creative tactics the Obama administration has devised for the parts they decided they had a role in. One example is a YouTube video the administration created last Spring that spliced together a video of al-Qaeda leader Ayman al-Zawahiri from 2008 saying "there is no hope to remove the corrupt regimes in Muslim countries except by force" with pictures of the Egyptian masses celebrating the resignation of Hosni Mubarak last February (175).
Another strategy the Obama administration has relentlessly pursued is highlighting al-Qaeda attacks that kill Muslims, particularly vulnerable targets such as women and children. To do this, however, the administration has learned that any message from the United States is likely to be viewed skeptically, at best, by most of the Middle East. Therefore, the administration has sought to enable more credible sources to relay the facts. For example, Schmitt and Shanker note that the military often gives background facts of an attack to Arab and Muslim journalists who then chose whether and how to write stories based upon them.[vi]
A more familiar example is the particular pieces of intelligence that the Obama administration chose to release from the trove collected from the raid on bin Laden's compound last May. Thus, the administration released videos of an elderly, defeated-looking bin Laden huddled under blankets watching press accounts of himself. Administration officials also tried to emphasize the fact that the terrorist leader had been living in comfortable quarters far from the violent battlegrounds of Western Pakistan, which many of his ardent followers inhabited. All of this was an attempt to demystify bin Laden, who had painted himself as a courageous underdog fighting the U.S. superpower on behalf of the Muslim world (Ummah).
In all these examples the point isn't to make the Arab world love the United States, rather just the much simpler task of making Muslims hate al-Qaeda.
C. Cyberwarfare COTERROR tactics
But perhaps the most interesting and creative COTERROR tactics were the ones designed to sow mistrust between the members of al-Qaeda and make them doubt the reliability of their primary modes of communication. To do this the administration has increasingly relied on cyberattacks; the authors in fact dedicate an entire chapter to discussing the Obama administration's decision-making regarding cybersecurity in general.
That the West has taken to using cyberattacks to thwart al-Qaeda operations was not unbeknown before this book. A prominent example took place last June when MI6 hacked into al-Qaeda in the Arabian Peninsula's English-language publication, Inspire, and replaced bomb-making instructions with Ellen DeGeneres recipe for "The Best Cupcakes in America."
Counterstrike, however, devolves much deeper into the West's cyberwarfare tactics against al-Qaeda. Schmitt and Shanker also report that cyberwarfare triggered some of the most intense interagency debates among those dealing with terrorism. As the authors note, "It is an ironic but important footnote to history that these debates over taking down terror Web sites resulted in the largest interagency meetings held since the 9/11 attacks (139)."
In fact, it is in the arena of cyberwarfare that the U.S. government seems to have struggled most with achieving a "whole-of-government" approach to COTERROR. The issue of shutting down terrorist websites, for instance, presents an irreconcilable clash of interests between the various agencies' charged with prosecuting the GWoT. To cite just one example: whereas the military favors shutting the sites down because terrorist operations that kill U.S. troops are often planned on Jihad forums, intelligence agencies advocates allowing them to continue because monitoring them yields valuable intelligence.[vii]
Despite these types of difficulties, Counterstrike documents a number of successful ways in which the United States has used cyberwarfare to disrupt terrorist operations. For example, Schmitt and Shanker report that the administration has hacked into terrorists' cell phones and sent text messages to other terrorists that sow confusion and distrust between the operatives. A senior officer from CyberCom explains one instance of this:
We had the ability to hack into their phones and we would text message guys. It was primarily telling them 'Hey, brother, another guy is cheating you out of money.' The bad guys would get this text message and be like, 'Oh Shit!'… We texted a few of his guys and basically some of his guys gave him up. It was really freaking cool. (147-148).[viii]
The authors report other interesting cyberwarfare tactics the U.S. has relied upon. For example, Schmitt and Shanker write, "There is at least one case confirmed by American officials in which a jihadist Web site was hacked by cyberwarriors to lure a high-value al-Qaeda leader to a surreptitious meeting with extremist counterparts only to find a U.S. military team in waiting (147)."
Although al-Qaeda is likely to catch onto these ploys quickly, that is partially the point. As U.S. drones push terrorists further and further underground, they become increasingly dependent upon cell phones and the Internet to communicate with each other and outsiders. By making al-Qaeda operatives distrust these modes of communications, Washington is significantly increasing the difficulty terrorists encounter in trying to carry out their violent activities. And since, as Pavel and Kroenig noted, terrorists value successful attacks, their failure to conduct successful operations just might ultimately deter them from trying altogether.
Zach Keck is an Associate Editor of e-IR.
[i] Although the authors make the point that the new deterrence strategy drew from those used against the Soviet Union during the Cold War, they do not elaborate on this fact in great detail. For example, they do not say whether certain Cold War administration's deterrence strategies were more influential or not. Rather it seems that what they have in mind is just the general principle of deterrence; convincing an adversary to not take an action. As we shall see, linking the Cold War nuclear deterrence to the new deterrence is somewhat inappropriate since the latter does not rely on the promise of overwhelming retaliation, which was at the core of the United States' and Soviet Union's deterrence strategy towards each other during the Cold War. The literature on deterrence is vast. For a useful recent overview of this literature see, Patrick Morgan, Deterrence Now (New York, Cambridge University Press, 2003). For an analysis of the variations in U.S. strategy towards the Soviet Union across different Cold War administrations see, John Lewis Gaddis, Strategies of Containment: A Critical Appraisal of American National Security Policy During the Cold War (New York: Oxford University Press, 2005). For an insightful analysis of how the Soviet Union perceived U.S. strategy and the international balance of power during the Cold War, see, William Wohlforth, The Elusive Balance: Power and Perceptions during the Cold War (Ithaca, NY: Cornell University Press, 1989).
[ii] Kroenig is now a recognized nuclear proliferation scholar who's written at least two books and numerous scholarly articles on various aspects of this phenomenon. He is currently an assistant professor at Georgetown University in Washington, D.C. Before his current stint at Georgetown he also returned to the Pentagon for a year under the Obama administration where he worked on Iran's nuclear program and other Middle East defense issues.
[iii] As Barry Pavel, the co-author of the new deterrence, explains, "The stereotype and monolithic image of the guy with the suicide bomb on his back isn't the only type of actor in a terrorist network. It is far-flung; it is multifaceted and multifunctional…. This guy is a banker; he is deterrable. This guy is a financier, this guy is a logistician (181)."
[iv] Another example is the profit-seeking criminal groups that often sell al-Qaeda and like-minded groups weapons purely because of the monetary reward (188).
[v] Similarly, with regards to working with different groups to counter al-Qaeda's message, another senior defense official says, "This is not about getting them to love us. It is about getting them not to challenge our interests (178)."
[vi] This strikes me as an excellent example of how the Obama administration sometimes prefers to "lead from behind," as an anonymous senior administration official put it to Ryan Lizza, a reporter for The New Yorker. In this instance, the anonymous source was referring to the Obama administration's response to the Arab Spring but the comment seemed to imply that the principle held in other areas as well.
[vii] The authors also report that intelligence agencies also insert agents into these Jihadist forums who spend weeks building up their credibility and then begin posting comments that are aimed to create confusion and dissent among the users of these sites.
[viii] At the beginning of this month, Wired magazine's Danger Room blog reported a similar tactic only applied to a Jihadist forum instead of cell phones. In this case, (presumably) Western intelligence agents hacked into a prominent Jihadist forum writer's account and sent his fellow forum members messages and perhaps spyware.


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Cottage 03
Sullivan County Real Estate.
The definition of a weekend hideout. Super-efficient, super low energy costs, super stylish living on 6+ private acres. 1 mile from the Delaware River.
Sullivan County NY real estate offers wide open spaces, peacefulness and proximity to NYC you will not experience anywhere else. Prices and values can seem refreshingly attainable here. Weekend life in Sullivan County revolves around coffee on the front porch, small dinner parties, county fairs and one horse towns like Callicoon, Narrowsburg and Barryville.
The Homes of Catskill Farms are engineered to be as energy efficient as a home can be. Enhanced insulation paves the way for superior performance from our high efficiency furnaces and on-demand hot water heaters. Many of our homes now have heat pumps for their heating source, eliminating the need for gas or oil as a fuel source.
For over 18 years, Catskill Farms has been adding to the architecture and real estate of the Catskills. Our Modern New Homes with style fill the need of a broad cross-section of city dwellers looking for that Catskills getaway.
Click here to read more about Sullivan County Real Estate. Catskills Real Estate at its finest.


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Hello Trulovers! For those UK readers, I hope that you had a lovely warm and fun filled weekend. Today's inspiration board is brought to you by Julie's readers request for ideas for her upcoming wedding in Provence. Julie from Arizona USA, is renting out a large private farmhouse and for the main wedding day is looking to create an informal wedding style and is seeking backyard BBQ wedding ideas.
Of course I was only too happy to help and given the lovely weather currently, this board left me counting day the moments until summer is here!
A rustic backgarden wedding really does open up the world of possibilities not only for decor but of course also food. I will turn to the food and refreshments first! With French cuisine being renowned the world over, if you are planning a DIY BBQ wedding reception, the supermarkets, butchers and local shops can provide you with all of the fare that you will need. In order to combine the American take on BBQ with the French, which not add in some speciality items for your more daring wedding guests.
Whilst the wedding look is rustic, a great deal of planning and decorations go into a achieving a beautiful backyard style wedding. Do take into account the time of year of your wedding reception and be sure to tie in the season colours of the countryside. Wild flowers can also be a wonderful addition to decoration, but a great florist can help you create the rustic ambience with style and those unique wedding decorations.
Signage: Gina Cristine Weddings, Outdoor Reception: Anne-Claire Brun via 100LayerCake, Bouquet: Trendy Bride, Couple: Brent Van Auken Photography via Snixy Kitchen, Ceremony focal point: The Nichols via Style Me Pretty, Hanging Drinks: Capitol Romance, ,Family Tree: GreenAutumn Photography via Style Me Pretty, Cake: Naomi Kenton Photography via Style Me Pretty, Petanque: Xavier Navarro via Knot and Pop,
That is it for another week of wedding inspiration, but don't forget if you want me to create a style and board for you email info@frenchweddingstyle with Inspire Me in the subject line.


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The Stainless Steel Modern Curved Bighorn Door Pull features mitered ends for a clean, contemporary look. Choose from 12 inch, 24 inch, or 36 inch lengths in satin stainless, polished stainless, or matte black finishes. The Modern Curved Stainless Steel Handle can be mounted on wood, fiberglass, metal, and even glass. Choose from surface mounted, thru-bolt mounted, or back-to-back mounted.
Available in 3 Sizes:
Note: This door pull is handmade in the USA when you order it—allowing it to be custom designed to fit your specific door application, including Other Lengths. Unfortunately, because of the made-to-order nature of the door handle, it is Not Returnable. Please Contact Us with your requirements or if you have any questions.
When Selecting a Mounting Hardware:
Sold individually (1 door pull handle). If you order Back-to-Back Mounting please Order 2 Pulls.


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Best iPhone Case Brands – #1 For Quality & Style
Why Quality Counts With Cases
Simple, really: if you use a cheap case that lacks the necessary protections, when you drop your iPhone, as we all invariably do, the case won't do much to protect it – you'll have a bust iPhone. For this reason, you should ALWAYS use good-quality iPhone cases.
Cheap iPhone cases, the kind you can find pretty much everywhere, are designed, predominantly, to stop your iPhone from getting scratched. And that's about it. Any drops or fumbles will result in shattered back panels and displays, bumps, scuffs, and dents.
MORE: The Best iPhone Guide: Find The Best iPhone For You…
And when a lot of iPhones cost in and around $1000 these days, paying a little extra for a case that delivers proper protection, both for drops and scratches, is a wise investment. Look after your iPhone and you can sell it for very good money, even after a couple of years.
Increase Resale Value of Your iPhone
Apple's iPhones cost A LOT of money. Starting with the iPhone X, the average price of an iPhone flagship model increased dramatically overnight. This is bad for anyone looking to buy a new one, but good news for those that have and want to sell the phone.
You will experience depreciation, obviously, but 12-month-old iPhone X models are selling for in and around the $600 mark. If it's a higher storage model, you could get more. And that's only $300 depreciation in 12 months. If only your car held onto its value like an iPhone!
DC Wonder Woman Collection by Casetify – View All Cases
For this reason, it really does pay to ensure your iPhone is properly protected. If you want to get the MAX resale value for your iPhone, it needs to look as good as new – that means no scuffs, no scratches, and it works perfectly. If you've got a 12-month-old, mint-condition iPhone, you could sell it right at the top of the market; and this means you'd only have to pay, say, $300 for a brand new model like the iPhone 11 Pro Max, for instance.
The older the iPhone, the less you'll get for it on the resale market. But you'll still get good resale value on iPhones right down to the iPhone 7 and iPhone 8. It won't be iPhone X money. But it'll still be a few hundred bucks, providing the phone is in mint condition.
Case in point: retailers expect to sell an iPhone at around 60-70% of its retail price six months after its release. The iPhone 6 to iPhone 8 all got around 65%, according to market data.
A Good iPhone Case Will Make You Money
Honestly, a few years ago, I'd never even really thought about any of this kind of stuff. I basically got a phone and then used it until it broke or I lost it. But when phones started costing $1000 a pop, I changed my ways – I started getting more interested in resale values and keeping my handsets in mint condition.
Nowadays, whenever I buy a new phone – be it an iPhone or a Samsung Galaxy S handset – I ALWAYS get a decent case from one of the case makers listed above.
Virtual Super Land Range by Casetify – View All Cases
I then do my damnedest to keep the phone safe and away from anything that will mess with it; that includes water and dust and everything else in between.
Earlier this year, I sold my iPhone X and got $650 for it. I then used that money to put towards a top-of-the-range iPhone XS. In the end, I spent about $450 on the new phone, saving myself almost $600 in the process. I say, SAVE, but really I just used the value of my old phone to finance the new one. And you can ONLY do this if your phone is in mint condition.
OK, So What's The Best iPhone Case Then?
Personally, I HATE "best iPhone case" posts. Why? Because taste is subjective and what I like you may dislike intensely. Therefore, it is far more productive to simply highlight the companies that make the best quality cases and then let you pick out the one you like. I tend to go for hardly-there cases that don't hide the design of the phone too much.
But I know for a fact that plenty of my buddies love full-on cases that completely hide the back of the phone entirely. As I said: tastes are subjective. All you need to be concerned about is going with a quality manufacturer (our #1 picks are listed in the table below). Once you've figured this out, you can then pick out a case you like.
Another option? Want something completely unique for your iPhone? Well, you can design your own iPhone cases at Casetify; you have full control over the color, the text on the case, and pretty much everything else in between. It's not really my kind of thing, but it is super-cool if you're the kind of person that likes bespoke stuff – even on your iPhone!


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Sniper Elite 4 – "Italy 1943" Story Trailer | Xbox One
Italy, Second World War. Explore the Allied invasion plans and discover the covert operations behind Sniper Elite 4's breathtaking campaign. Featuring beautiful concept art and new in-game factions and environments, ""Italy 1943″" is the first in a series of scene-setting story trailers for Sniper Elite 4.
Launching 14 Feb 2017.
PRE-ORDER NOW and get the Target: Fuhrer campaign mission and Camo Rifle Skins Pack FREE!


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Evolv launches outsourcing tool for auditors
SMSF auditing firm Evolv has developed a white-labelled outsourcing tool that performs the majority of SMS audit procedures on behalf of SMSF auditors.
Evolv executive director Ron Phipps-Ellis says the new offering, Evolv Grey, produces a financial and compliance audit though Evolv's software and resources.
"Auditors then review our work and as their clients' trusted adviser, continue to sign the audit report and charge the audit fee they are comfortable with," Mr Phipps-Ellis said.
He said the SMSF audit industry is large and disparate, and mergers and acquisitions in this space are uncommon.
"[This] new offerings provide an alternate way to collaborate and do our jobs more efficiently – together."


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The Japanese Nuru Erotic Massage is widely accepted as the most erotic massage in the world. It has become one of the most sought after massages in Japan, where it originated from, and those fortunate enough to have experienced this amazing massage will understand exactly why this is so. Often described as the ultimate pleasure in life and considered by many to be even better than actual sex, a Nuru massage is totally unlike any other erotic massage you may have experienced before.
Using a specially designed slippery mattress, both client and masseuse are covered in a unique Nori seaweed based clear gel and the masseuse massages the client solely by slipping, sliding and grinding his body with her own. This creates the most erotic sensations enjoyed by both client and masseuse.
And now Katrina offers the authentic Japanese Nuru Erotic Massage for her clients, the only professional masseuse in Devon to do so.
What to expect during an Erotic Nuru Massage with Katrina
Your massage begins and ends with a shared shower with Katrina, giving you the chance to connect sensually and familiarise yourself with her body and touch. After showering you will receive a brief full body massage to relax you further and then, laying on the mattress Katrina will pour warm Nuru Gel all over your body, massaging it into every inch of your body. She will then pour the gel over her body from head to toe and join you on the slippery Nuru mattress where she will begin to massage you by sliding her entire body over you, sideways, up and down and across. Instead of using hands and fingers, however, she will use her breasts, buttocks, groin and torso to slide continually over you, creating pleasures you didn't know existed, unique to the Nuru massage.
Half way through the massage Katrina will ask you to turn over on your back and the whole process is repeated but this time obviously more intimate as her body slides continually over yours as you lay face to face. You should savour every moment as only a Nuru massage can create the amazingly intense erotic sensations you will now be experiencing. Nothing is more erotic and sensual than having a beautiful woman sliding her gel-soaked naked body over your similarly gelled body. After you have soaked up the euphoria of your Nuru massage you shower again one last time together and your Japanese Nuru Massage experience is complete.
Couples Nuru Erotic Massage
Just imagine watching your partner laying on a gel-soaked special mattress, body covered with slippery Nuru Gel, while I slide my gel covered body up and down, side to side and across, sitting astride their waist and massaging their body with my breasts, buttocks, groin and torso, stimulating levels of eroticism in both of you that you didn't even know existed. And then it is your turn to lay down and be pleasured while your partner watches you. Absolutely nothing can beat the sensations a couple will experience in this highly erotic massage together.
Welcome to the world of Japanese Nuru Erotic Massage............
For the ultimate in erotic massage for a couple I offer both of you the chance to enjoy a gel-soaked erotic body to body massage with me with both of you covered in sensual Nuru Gel laying on the Nuru mattress and being massaged by me, also soaked in the Gel. This truly is the ultimate in Couples massage. Don't just take my word for it. Try it!
Health Benefits
As well as the amazing sensual benefits from having a Nuru massage, there are also considerable health benefits as well. The Nori seaweed content of the Gel is an essential ingredient in many of the skin moisturizing products currently on the market and the benefits to a person's skin are obvious. It is also important in improving blood circulation, removing harmful toxins and improving the function of vital organs. The combination of Nori seaweed and the massage can stimulate recovery and healing of aching and tired muscles while the massage itself can remove stress and anxiety due to the nature of the massage. The massage is also without doubt one of the best ways of promoting genuine total relaxation.
A Nuru massage has been clinically proven to improve sexual functions and ailments such as Erectile Dysfunction and Premature and Delayed Ejaculation. One of the aims of a Nuru massage is to prolong and enjoy pleasure for as long as possible.
Here at Devon Tantric Massage for Men Katrina only uses genuine Nuru Gel Products containing Nori seaweed specially imported from Japan for her Nuru massages. Please note a Nuru Erotic massage is not a sexual service.
One person
One hour Japanese Nuru Massage £150
One and a half hour Japanese Nuru massage £175
Two hours Japanese Nuru massage £225
Two persons (Couple) massaged together at the same time
One and a half hour Japanese Nuru Massage £175
Two hours Japanese Nuru Massage £225
In order to protect the identity of our cliients we only accept cash as payment.
Nuru Massage in Devon * Nuru Massage in Cornwall * Nuru Massage in Exeter * Nuru Massage in Plymouth
Nuru Massage in Torquay * Nuru Massage in Newton Abbot * Nuru Massage in Totnes


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Following on from yesterday, (or was it the day before yesterday?), and the delicatessen in our local market and his wife's potato prints, I have just remembered this piece from that hugely clever writer AA Gill on markets:
"My weakness, my pleasure, is markets. Whenever they say, what would you like to see? The museum, the opera house, the red light district, the bridge over the river? I always say the market. I want to see where the women buy their vegetables. I want to see the fish, the butchers, the quarter of cobblers and tailors. You can't fake a market. You can't make it what it's not. It is as true a reflection of the people it serves as anything; what they have, don't have, what they make and import, and what their pretensions and weaknesses are."
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Red Wing Shoes MOC TOE 8146 Brown Laced Boot - Brown!
Classic ladies & men work boots. Inspired by the "Work Boots", the construction worker - Iron Workers the high-rise builder.
The "Original" working boots Made in USA!
The complete shaft and heel area has no extra lining. The foot area was finished with cowhide leather. The special "Poron" insole made of leather is shock absorbing and antibacterial.
As upper material for the welted (Goodyear world) an extremely thick "Oil Slick" cowhide leather was used. The vegetable tanned leather was strongly oiled so that a very pleasant grip was created. In addition, the leather is water-repellent and easy to pluck. The legendary "MOC LUG" decorative seam, which has become one of the trademarks of the Red Wing Shoes over the years, is unmistakable. In order to achieve a colour contrast, the decorative seams on the MOC LUG or on the leather outer frame were sewn in a white shade.
The Red Wings 8146 Briar Oil Slick Moc Lug have a one-piece "Vibram" outsole.
The "8" last of the Red Wings 8146 Brown Work Boots boot has a normal foot width, a so-called "D" width. The height can be individually adjusted by the 7-hole lacing.
The laced boot 8146 Briar Oil Slick Moc Lug of the brand Red Wing Shoes has a shaft height of 6 inch, approx. 15 cm.
The Vibram outsole has a thickness of approx. 1.5 cm.
The heel has a height of approx. 3 cm.
Important notes:
Due to the handcrafted production method of the Red Wing Shoes products, differences in colour or design may occur compared to the illustrated models!
Each pair of Red Wings is unique!
Waxed and oiled leathers (OIL SLICK) also require special care products.
With the welted production method (GOODYEAR WELT) a small running-in phase must always be observed.


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New Delhi, Jun 29 The American India Foundation (AIF) today felicitated 27 young leaders from the United States and India on the completion of their 10-month service under AIF's William J Clinton Fellowship for Service in India.
"These young fellows contributed their skills and resources to NGOs in the field of livelihoods, education, and public health across 15 states in India," Mark A White, Mission Director of USAID-India, said at the event.
The event brought together philanthropists, CSR practitioners, leading businessmen, NGO founders and fellows, who shared testimonials and glimpses into their journeys of service in India, along with an art exhibition portraying the impact of their work on the ground.
"The William J Clinton Fellowship for Service in India helps shape the next generation of leaders committed to impactful change while also strengthening civil society to be more efficient and effective," White added.
The fellowship pairs a select number of highly skilled young professionals with leading NGOs and social enterprises in India in order to accelerate impact and create effective projects that are replicable, scalable, and sustainable.
While congratulating the Clinton Fellows, Mathew Joseph, Country Director-AIF, said, "Through values like teamwork, perseverance, and humility they address the challenges they face in their service and ultimately succeed in making positive and lasting impacts in their host organisations."
Since the first batch of fellows who assisted in the post-earthquake rehabilitation of Gujarat in 2001, the AIF Clinton Fellowship Program has successfully paired 452 fellows in service with 202 NGOs and social enterprises across 23 states, thus representing a collective force and organised network dedicated to social change in India.
AIF was founded in 2001 at the initiative of President Clinton following a suggestion from then Indian Prime Minister Atal Bihari Vajpayee.
Working closely with local communities, AIF partners with NGOs to develop and test innovative solutions and with governments to create and scale sustainable impact.
This article was initially published in Outlook on June 29, 2018. Read the original article here.


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Novice Squad
This squad continues the refinement and development of all competitive strokes and marks the change from a learn to swim class to a squad environment. The swimmers will generally do 25m at a time with a major focus on good technique. This squad will also introduce new skills such as streamlining, tumble turns and racing dives.
Junior Squad
This squad is a continuation of the skill and technique acquisition from Novice squad and starts to introduce more focus on the sport of competitive swimming along with the introduction of "squad skills" like using the pace clock and reading training sets from the board to carry out specific sets on a time cycle.
Intermediate Squad
This squad is for swimmers with squad experience who are endeavouring to improve their fitness, skills and technique to a competitive level. This squad also prepares swimmers to be able to step up to the senior squad.
Senior Squad
The Senior Squad is aimed at high school aged swimmers who wish to step back from the commitment required to compete at external meets and concentrate on fitness and continued involvement in club competition.
Competition Squad
This squad is for competitive swimmers seeking to achieve at a high level and are committed to swimming as their chosen sport. Entry into this squad is at the discretion of the Head Coach, with a minimum number of sessions required to ensure consistent levels of training and performance are achieved.


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Central Oregon Insights
8:00am - 9:00am
Listen Live
2018-04-13 05:56:00
Metro Source Media Networks, Inc.
(Seattle, WA) -- New details are coming to light regarding allegations of abuse in the Hart family, whose vehicle crashed off a California cliff and into the ocean late last month. KATU-TV reports that an intake report from Washington state's Child Protective Services details claims from the family's neighbors that two of the children asked them for help several times in the days leading up to the crash. The documents filed March 23rd state that 15-year-old Devonte Hart asked his neighbor in Woodland for food multiple times a day for about a week in mid-March. Devonte allegedly told the neighbor that his parents punished him and his siblings by denying them food for entire days at a time. And last year, 16-year-old Hannah Hart allegedly jumped from her second-story window and ran to her neighbors' home, claiming that both her moms were racist and were abusing her and whipping her with belts. The teen was also reportedly missing her two front teeth.
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The Shroud of Turin is a piece of cloth that some people claim was the burial cloth used to bury Jesus. Is it authentic? How important is it that it's real or not? Today on Disclosure, Shawn and Jean look at some of the arguments made for and against this mystery.
Episode Chapters
Arguments Made Against the ShroudStarting at 31 min45 sec
What are some of the arguments that say the shroud isn't real?
Arguments Made for the ShroudStarting at 35 min03 sec
What are some of the arguments in favor of it being authentic?
If It Wasn't Painted, Then How Was It Made?Starting at 40 min00 sec
To date, no one has been able to duplicate the shroud. How do some people believe it was made?
Does It Matter?Starting at 44 min53 sec
Is it ultimately something we should base our faith on?


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With her singles creating a buzz locally and internationally, Jamaican/Canadian artist Kreesha Turner released Part 1 of her "#TheBody" EP. This sexy and energetic project features 7 tracks with production from Yonni, Dunlap Exclusive, and Big Duke, and contributions from T.O.K., Rock City, 2 Chainz, Kardinal Offishall, Mya, Tank, King Chip and Eric Bellinger. With top acts giving her cosigns, it's only right to get this in your daily playlist. Also included is her popular "MJ" single. Get yourself some Kreesha by checking out the EP below and share your thoughts in the comments section.
Continue reading "Mixtape Movements: @KreeshaTurner – The Body Pt. 1"


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Live from the Crown Plaza – just getting on the Internet now so we join the event in progress.
Live video from your iPhone using Ustream
Also, DaTechGuy just came in and he is tweeting away as well. Presently, Jack Kimball (who has already endorsed Ovide) is introducing Ovide with another one of his stemwinder speeches. Jeff Chidester is the MC for the evening.


text: Warning: The following module is missing from the file system: views_data_export. For information about how to fix this, see the documentation page. in /home/clmeimsgcfi/public_html/includes/bootstrap.inc on line 1128 Spiny Lobster Management | CLME IMS Jump to navigation English Español Spiny Lobster Management Home > Spiny Lobster Management Choose a subject to see records related to that subject, or search all subjects using the search box above. Taxonomy Ecosystems Statistics Trade Biological Monitoring Legislation Publications Key Actors Footer Links Home My Account Contact


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text: Valentine's Day is nearly here. A day to show that special someone in your life how much you love them. Or, if you are cynical like me, a day to eye-roll at the print-a-sentiment cards, overpriced chocolates and booked-up restaurants. Despite my cold-heartedness, the day does remind me how important it is to now and… Continue reading Between Love and Loathing- a story of self love


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Don't be that person!
Don't be that person!
Okay I totally get it and agree…..however this picture makes me happy as it's from our old 'hood! Looks like something you'd see in illinois rather than cali. 🙂
I am sorry, but I have studied the picture and cannot find anything wrong. Possibly the building designer should have eliminated the middle steps. Obvisiously not needed and a waste of the owners money.


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Book your group at Umaya Resort & Adventures in advance and we'll guarantee the best rate available (subject to availability).Room Rate Includes: Complimentary transfer between the Placencia Airstrip and the Resort.Complimentary refreshment upon arrival.One (1) complimentary room for every 10 confirmed. Complimentary on-site activities include: kayaks, paddle boards, bicycles, volleyball, bocce court, croquet court, water aerobics, and yoga. Post conference food and beverage 20% discount.
Blackout dates apply. Subject to availability. Room rate does not include 9% hotel tax and 15% Resort fee.Single occupancy in Lagoon Bay View Suite.Cannot be combined with other promotions.Promotion is eligible for all meeting and conference bookings.
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@ Super8: That's pretty cool, I guess to some Japanese people, it may be mind blowing to see an American license plate on a Japanese car, but we love them over here, Nissan Z, Toyota Celica, Honda S2000, Honda Prelude, Honda Civic SI/Type R, NSX, Toyota MR2, Subaru Impreza WRX/WRX STI, Mitsubishi Lancer Evo, Mitsubishi Eclipse, Mazda MX5 Miata, Mazda RX7, Mazda RX8. Someone at work drives a Subaru Imprexa WRX. When it comes to hot Japanese cars, I mostly see Mitsubishi Eclipses. Toyota Celicas, and Mazda MX5 Miatas around where I live.
Dodge Challenger R/T 383 Magnum specs
|Car type
|Curb weight
|1595 kg (3516 lbs)
|4.86 m (191 in) long, 1.93 m (76 in) wide, 1.29 m (51 in) high
|2.79 m (110 in)
|Origin country
|United States
|Submitted by
Powertrain specs
|Engine type
|Chrysler B-series V-8 383 (FirePower)
|6.3 l (383 ci / 6276 cc)
|340 ps (335 bhp / 250 kw) @ 5200 rpm
|576 Nm (425 lb-ft) @ 3400 rpm
|Power / liter
|54 ps (53 hp)
|Power / weight
|213 ps (210 bhp) / t
|Torque / weight
|361 Nm (266 lb-ft) / t
|4 Speed Manual (3.23 ratio)
|front engine, rear wheel drive
Challenger R/T 383 Magnum competition
Super8 8y ago
@ Shaggy: If I can remember correctly, there some Japanese people that own classic Muscle Cars including me. Here the list of the cars that I remember ever see.
1965 Shelby GT350
1967 Shelby GT500 (I heard there's only one in here)
1970 Chevrolet Chevelle SS (My father's car)
1968 Ford Mustang GT
1959 Chevrolet Impala
1964 Chevrolet Impala SS (My brother's car)
1966 Ford Galaxie Fastback (My car)
1970 Dodge Challenger R/T (383 and 440 Magnum)
Shaggy 8y ago
The 383 Magnum is a good engine, 335 horsepower and very durable. I believe my dad had a 383 in his '71 Charger, but the engine was detuned to 300 horsepower for 1971. It's kind of crazy to see a Japanese license plate on an American muscle car. Is it true that in Japan the license plate you get on your car is the license plate that stays on the car its whole life no matter how many times the car changes hands from owner to owner and registration doesn't need to be renewed?
Shaggy 8y ago
@ Super8: 440 Magnum, 375 horsepower if I'm correct.
BR2' 8y ago
Id rather have the SixPack, But not that i would complain with this car either way.
But, Id rather have the girl instead of the car..
Does sh know her undergarments are showing?..