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Shakeup in Nigerian Oil Sector Not a Simple Matter
LONDON Sep 4, 2007 (Dow Jones Newswires)
Nigerian President Umaru Yar'Adua's plans to break up the state-owned Nigerian National Petroleum Corp. and restructure the state energy sector has prompted largely positive reactions, but it's also left analysts with more questions than answers.
"This is a serious proposal, to allow it (NNPC) to become a serious player along the Saudi Arabia Oil Company model, but my only critique is that there is no policy platform with details and analysis," said Sebastian Spio-Garbrah, Africa and Middle East analyst at Eurasia Group.
Yar'Adua took office in April following a general election condemned by both domestic and international observers. He has pledged to overhaul the oil industry in Nigeria, Africa's largest crude producer, and his latest move to reform bureaucracy and improve the stability of the energy sector is viewed as a step in the right direction.
"The most significant aspect of the forthcoming changes seems to be that President Yar'Adua has decided to listen to his advisers and the petroleum experts," said Global Insight analyst Thomas Pearmain.
Yar'Adua's predecessor, Olusegun Obasanjo, rejected recommendations from the Gas Reform Committee and the National Council on Privatization to restructure the industry.
"Obasanjo said all the right things eight years ago but (there's) not much to show for it," said a London-based trader of West African crude. "It's early days, but, yes, Yar'Adua appears to mean business and wants to be seen as his own man."
"Yar'Adua has staked his presidency on turning around the country's energy sector by taking on responsibility for the Energy Ministry," Pearmain said.
Global Insight's analysts expect Yar'Adua to push for the development and export of the country's gas supplies and to call a state of emergency in the power sector, he added.
The restructuring of the NNPC would further decentralize the industry, which is such a crucial part of the economy and hopefully will make the sector more efficient and stamp out corruption, Spio-Garbrah of Eurasia Group said. Global Insight's latest figures show Nigeria's energy sector provides over 85% of foreign export earnings and about 60% of government revenues.
A reorganized and restructured NNPC would be a much better joint-venture partner for the major oil companies working in Nigeria, but in the longer term there may be disadvantages for them.
"If the new National Oil Company of Nigeria becomes a strong player, which the NNPC never has been, the oil majors may face stiff competition," he said.
"If the new NOC begins to want to bid on oil blocks and other exploration and production projects against a Royal Dutch Shell PLC (RDSB.LN) or an Exxon Mobil Corp. (XOM), clearly they are going to win out," Eurasia Group's Spio-Garbrah said.
"The worse-case scenario could be like what happened with Saudi Arabia Oil Company, where in the long run the government says we want to buy you out, or reduce your stake in the joint ventures," he added.
The government plans to replace the NNPC with five independent entities to separately handle exploration, production, distribution, domestic fuel pricing and the awarding of oil licenses.
The NNPC is the biggest company controlled wholly or partly by the Nigerian government, owning 57% of joint-venture business in upstream operations.
In a bid to emphasize the seriousness of the government's intentions, the president, vice president and other senior ministers will sit on a newly formed National Energy Council, set up to implement the new policy within six months.
Under the new structure, the NNPC's core roles of exploration and production will fall to the NOC, which is to be empowered to conduct itself like any other oil and gas company, Minister of State for Petroleum Odein Ajumogobia said Wednesday.
The old Department of Petroleum Resources - formerly a part of the Energy Ministry - which in the past oversaw the often controversial awarding of oil licenses and blocks, will now be an independent entity known as the Petroleum Inspectorate Commission.
The Energy Ministry itself will now become the National Petroleum Directorate, which alongside the NOC will be overseen by the newly created National Oil and Gas Assets Holding and Management Committee.
On the domestic front, the responsibilities of the Petroleum Products Prices Regulatory Agency, which currently fixes domestic fuel prices, will fall to the Petroleum Products Distribution Agency.
However, the actual implementation of these plans may not be as straightforward as stated, Spio-Garbrah said.
"On paper they can separate the companies, but whether they will become financially viable as stand-alone entities at the moment is doubtful," he said.
What's more, with all the other problems in the country at the moment, having the president, vice president and other senior ministers overseeing the new framework via the National Energy Council may not be practical.
"The finer details of this plan means that you actually need people who can focus their attention on this for the next few months, and I don't think that they will have the time to dedicate to it while at the same time dealing with the economy and the Niger Delta situation," Spio-Garbrah said, referring to unrest that has hit all the oil companies operating in the area.
"I think that in six months the legal details can be rearranged, but it will probably take a year or two at the very best to really get these new entities functional, with budgets and asset inventories, and an assessment of current NNPC liabilities."
Also, the Niger Delta States have seen increased oil revenues, but unrest there has slowed the development of the infrastructure and caused frequent refinery outages.
"It's a laudable plan. I think it will shake up the energy sector there eventually, but I think the details are too hazy to know how successful this will be," Spio-Garbrah said.
Copyright (c) 2007 Dow Jones & Company, Inc.
- Nigeria's State Oil Company Signs Deals With Chevron, Shell (Aug 03)
- Nigeria To Continue Oil Exploration In Lake Chad Basin Despite Kidnapping (Jul 31)
- Nigeria Says Has Rescued Oil Firm Staff Kidnapped By Suspected Boko Haram (Jul 26)
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subset: commoncrawl


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December 11, 2009 - A brand new telescope called the Visible and Infrared Survey Telescope for Astronomy (VISTA) has started observing the cosmos with astonishing results. Located at the ESO's Paranal Observatory, Chile, VISTA has become the world's largest telescope dedicated to mapping the southern hemisphere's sky in visible and infrared wavelengths.
The 3-ton camera inside VISTA houses 16 special detectors with a combined photographic power of 67 million pixels (for reference, high-quality cameras in cellphones can take 3 million pixel images). This enables very wide angle images to be captured, while maintaining unparalleled definition when zooming into features of special interest such as this star-forming region.
"Observing at wavelengths longer than those visible with the human eye allows VISTA to study objects that are otherwise impossible to see in visible light because they are either too cool, obscured by dust clouds or because they are so far away that their light has been stretched beyond the visible range by the expansion of the Universe." -- ESO Press Release.
VISTA's scope doesn't stop at observing stars and nebulae in the Milky Way. Looking into intergalactic space, many types of galaxies - containing billions of stars -- are resolved. One example is this cluster of galaxies in the constellation of Fornax where the beautiful barreled spiral of NGC 1365 can be seen (lower right).
A portrait of VISTA, a system developed by a consortium of 18 universities in the United Kingdom led by Queen Mary, University of London. This is the biggest survey telescope of its kind, revolutionizing ESO's capability of observing the southern skies, combining outstanding photographic capabilities with a huge light-collecting mirror.


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VIDEO: American Sniper will open nation-wide on Friday January 16th 2015
January 13, 2015
My websites and blogs are Pro-US Military, Pro-Law Enforcement and Pro-US Government dedicated to exposing radical Muslim terrorists and their recruiting websites hosted in the USA. My rating is 5 out of 5 GOLD STARS my only complaint is that US Navy SEAL Chris Kyle did not kill more Muslim Savages in real life. Peter Travers of Rolling Stone gave the film American Sniper three and a half stars out of four, saying "Bradley Cooper, as Navy SEAL Chris Kyle, and director Eastwood salute Kyle's patriotism best by not denying its toll. American Sniper received positive reviews from critics. Rotten Tomatoes gives the film a rating of 75%, based on 76 reviews, with an average rating of 7.1/10. The site's consensus states, "Powered by Clint Eastwood's sure-handed direction and a gripping central performance from Bradley Cooper, American Sniper delivers a tense, vivid tribute to its real-life subject." The film received a limited release in New York City, Los Angeles and Dallas on December 25, 2014, and will open nation-wide on January 16, 2015.
"Lone Wolf' Terrorists Radicalized By Jihadist Websites As Cyber Vigilante PI Bill Warner Takes On Terror Sites. FOX NEWS VIDEO TAMPA…Tracking Terrorists Hidden In The Web Is Private Investigator Bill Warner. For terrorists, the internet is a virtual town square. It's used by Al Qaeda for recruiting and training. The FBI knows this, and so does Bill Warner, a private eye in Sarasota, Florida who spends his time online tracking terrorist websites. Super Spy Cyber Vigilante Private Investigator Bill Warner takes on Al-Qaeda website and it's members death threats. "They got this Jihad Jane, Jihad Jamie, these people are popping out of the woodwork now," Warner said, explaining why he tries to get these terror sites shut down and stop communication, the life blood of terrorists.
Bill Warner exposes the real danger of radical islamic websites and terrorists like Faisal Shahzad and the Bridgeport CT Terror Cell that Plotted the Times Square Car bomb. As Faisal Shahzad was tracked, investigators have also started looking at a Connecticut-based Web site, the Tehreek-e-Taliban Pakistan News Channel on YouTube, purporting to represent a faction of the Taliban in Pakistan, sources said. The site went up last week, and within 24 hours of the failed bombing was laying claim to an attack on American soil, law enforcement sources said.
Bill Warner Private Investigator Sarasota Fl at www.wbipi.com


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RNA transcripts in which all guanosine residues are replaced by inosine are degraded at a highly accelerated rate when incubated in extracts from HeLa cells, sheep uterus or pig brain. We report here the partial purification and characterization of a novel ribonuclease, referred to as I‐RNase, that is responsible for the degradation of inosine‐containing RNA (I‐RNA). I‐RNase is Mg2+ dependent and specifically degrades single‐stranded I‐RNA. Comparison of the Km of the enzyme for I‐RNA with the Ki for inhibition by normal RNA suggests a ∼300‐fold preferential binding to I‐RNA, which can account for the specificity of degradation. The site of cleavage by I‐RNase is non‐specific; I‐RNase acts as a 3′→5′ exonuclease generating 5′‐NMPs as products. The presence of alternative unconventional nucleotides in RNA does not result in degradation unless inosine residues are also present. We show that I‐RNase is able to degrade RNAs that previously have been modified by the RED‐1 double‐stranded RNA adenosine deaminase (dsRAD). dsRADs destabilize dsRNA by converting adenosine to inosine, and some of these enzymes are interferon inducible. We therefore speculate that I‐RNase in concert with dsRAD may form part of a novel cellular antiviral defence mechanism that acts to degrade dsRNA.
The processing and turnover of various classes of RNA in prokaryotic and eukaryotic cells involves multiple steps in which ribonucleases (RNases) play a role. Consequently, it is not surprising that there are a large number of distinct RNases to fulfil these roles (reviewed in Deutscher, 1993; Beelman and Parker, 1995). Among the criteria by which RNases can be distinguished are their action as endo‐ or exonucleases, whether they cleave only adjacent to specific bases, whether they are specific for a particular sub‐class of RNA such as rRNA, whether they act upon double‐ or single‐stranded RNA and whether they cleave phosphodiester bonds to leave 5′ or 3′ nucleotide monophosphates (NMPs). In addition, some RNases, such as RNase L (see below), are not constitutively active, but exhibit inducible activity. As well as the more well known roles of RNases in turnover of mRNAs and processing of tRNA and rRNAs, they are also used in various circumstances by bacterial, plant and mammalian cells as cytotoxic agents to kill cells selectively. Some such cytotoxic nucleases have even been tested for use in anti‐cancer therapy (Wu et al. 1993).
In the course of investigating a proposed non‐Watson–Crick base pairing interaction between the terminal guanosine residues in pre‐mRNA introns, we tested the splicing activity of RNA transcripts in which all of the internal guanosine residues had been replaced by inosine (Scadden and Smith, 1995). Inosine is identical to guanosine except for the lack of the exocyclic amine group at position 2 of the purine ring. Unexpectedly, when incubated in HeLa cell nuclear extracts, the inosine‐containing RNAs (I‐RNAs) appeared to be considerably less stable than the guanosine‐containing transcripts (G‐RNAs). This observation was repeated subsequently with several other unrelated RNAs, many of which were not splicing substrates. These observations indicated that the instability of the I‐RNAs was not attributable to increased accessibility to degradation by general RNases due to failure of the I‐RNA to be incorporated into spliceosomes. In addition, the various substrates were not predicted to contain strong secondary structure which may have been weakened by inosine substitution leading to preferential degadation. These data therefore suggested that HeLa nuclear extracts contain a nuclease activity that is specific for I‐RNA.
Inosine occurs naturally in several places within cellular RNA. One well‐characterized site is in the 'wobble' position of some tRNA anticodons (Elliot and Trewyn, 1984). Inosine has also been detected in vitro as the result of hydrolytic deamination of adenosine residues in double‐stranded RNA (dsRNA) by dsRNA adenosine deaminases (dsRAD; Bass and Weintraub, 1988; Wagner et al., 1989). The conversion of A–U to I–U base pairs by dsRAD results in destabilization of the substrate dsRNA. Such A→I changes appear to have a role in at least some cases of site‐specific RNA editing. For example, the editing in the brain of mRNA encoding the glutamate receptor subunit B (GluRB) involves the formation of double‐stranded structures around the editing site (Higuchi et al., 1993; Maas et al., 1996) and can be catalysed in vitro by either purified dsRAD (Hurst et al., 1995; Dabiri et al., 1996; Maas et al., 1996) or a related enzyme, RED‐1 (Melcher et al., 1996). Inosine is recognized as G by the translation machinery so what was originally a glutamine codon is recognized as arginine after editing. Pure Xenopus dsRAD has also been shown to edit hepatitis delta virus RNA efficiently in vitro (Polson et al., 1996).
Despite the accumulation of evidence for a role in RNA editing, expression of dsRAD activity is much more widespread than the pre‐mRNAs that it is known to edit (Wagner et al., 1990; Higuchi et al., 1993), suggesting the likelihood of a more general role for the enzyme. A clue to a less specialized function is that levels of dsRAD increase in response to interferon (Patterson and Samuel, 1995; Patterson et al., 1995). Interferon induction and activation by dsRNA are hallmarks of enzymes that are involved principally in cellular antiviral defence (Samuel, 1991). The significance of activation by dsRNA is that viral infection often involves the production of high levels of dsRNA as a consequence either of viral replication or of transcription from opposite strands of DNA viruses. For instance, protein kinase R (PKR) is induced by interferon and activated by dsRNA in an autophosphorylation reaction (Clemens, 1996). Activated PKR phosphorylates the translation initiation factor eIF2α resulting in a shut‐down of protein synthesis in virally infected cells. Likewise, oligo 2′‐5′ A synthetase is interferon inducible and activated by dsRNA (Witt et al., 1993). Its product, 2′‐5′ oligo A, is an activator of RNase L, which degrades single‐stranded RNA (ssRNA) in virally infected cells. Therefore, dsRAD, as an enzyme that is interferon inducible and that acts upon dsRNA, probably plays a role in cellular antiviral defence. Multiple A→I conversions would cause miscoding of viral messages. Nevertheless, the viral RNAs potentially would still clog up the cellular translation machinery. An RNase specific for I‐RNA potentially could play an important role in disposing of the products of dsRAD, producing a more effective and streamlined antiviral defence pathway. Based upon this speculation, we have sought to characterize the properties of the I‐RNase activity that we detected initially in HeLa nuclear extracts (Scadden and Smith, 1995).
Here we report the partial purification and characterization of I‐RNase activity from pig brain. We show that I‐RNase is an Mg2+‐dependent 3′→5′ exoribonuclease which has specificity for single‐stranded I‐RNA. The presence of alternative unconventional nucleotides in RNA does not result in degradation unless inosine residues are also present. The specificity of I‐RNase is not determined by the site of cleavage since the products of I‐RNase cleavage are predominantly 5′‐NMPs. Rather, the specificity appears to arise at the level of binding to RNA with an ∼300‐fold higher affinity for I‐RNA than for G‐RNA. Finally, we show that I‐RNase is able to specifically degrade RNA that has been modified by the enzyme RED‐1. I‐RNase, therefore, appears to be a novel RNase whose properties are consistent with a role in cellular viral defence.
Our original observation of enhanced degradation of I‐RNA in HeLa nuclear extracts (Scadden and Smith, 1995) has been repeated consistently with a wide variety of transcripts (see Figures 1, 2, 3, 4, 5, 6 for use of various RNAs). We subsequently have detected I‐RNase activity in crude extracts made from sheep uterine smooth muscle and pig brain (Figure 1A), which suggests that I‐RNase is a widespread activity at least in mammalian tissues. All the subsequent experiments described here used I‐RNase which has been partially purified from a pig brain extract. Partial purification of I‐RNase involved three chromatographic steps, and has yielded a fraction which has high I‐RNase activity and is free of other general nucleases (see Materials and methods).
The basic assay for I‐RNase activity is illustrated in Figure 1B. The transcript ΔKP contains exons 2 and 3 of the rat α‐tropomyosin gene with no intron between (Smith and Nadal‐Ginard, 1989). The ΔKP template was linearized with either BamHI or BglII, which enabled synthesis of transcripts of two different lengths which contained either guanosine or inosine (G‐RNA or I‐RNA). The G‐RNA transcript was ∼50 nucleotides longer than the I‐RNA transcript and served as an internal control for assessing degradation of I‐RNAs in the same tube. Figure 1B shows a time course of degradation of I‐RNA and G‐RNA ΔKP transcripts. Quantitation by phosphorimaging indicated that I‐RNA transcripts were degraded at a rate of 469 ± 23 ng RNA/min/mg protein (n = 3), while the G‐RNA transcripts were degraded at a rate of 18.8 ± 0.6 ng RNA/min/mg protein (n = 3). The rate of degradation of I‐RNA transcripts was thus at least 25‐fold greater than that of the G‐RNAs. After several hours of incubation with an excess of I‐RNase, G‐RNAs were also substantially degraded by I‐RNase (data not shown). This could indicate that I‐RNase does not have an absolute specificity for I‐RNA. Alternatively, the degradation of G‐RNA could reflect low levels of other nucleases in the I‐RNase preparation. The degradation of I‐RNA transcripts was seen to be dependent on the presence of MgCl2 and did not require ATP (Figure 1C).
We carried out a number of experiments to investigate the substrate specificity of the I‐RNase. Firstly, we wanted to establish whether the novel activity was specific for I‐RNAs or if it recognized and degraded RNAs containing any unconventional nucleotides. To this end, ΔKP transcripts were synthesized in which UTP was substituted by 5‐bromo UTP (5‐BrU) in addition to containing either G or I. As I and 5‐BrU were being substituted for G and U residues in the ΔKP transcripts, respectively, the number of modified nucleotides incorporated was different in each case. While the ΔKP transcripts contained 100 I or 112 G residues, only 41 or 57 5‐BrU residues were included in the I‐ and G‐RNAs, respectively. Therefore, a direct comparison of the effect of an equivalent number of modified residues in an RNA transcript could not be made. Nevertheless, a sufficient number of 5‐BrU residues were present in the modified RNA to make a qualitative assessment of its effect on degradation. RNA transcripts which contain <41 I residues (e.g. pBluescript II SK I‐RNA) were still degraded preferentially by I‐RNase (see Figure 4, lanes 1 and 2). Degradation of ΔKP transcripts containing either U or 5‐BrU in addition to I or G were thus analysed over a time course from 0 to 60 min (Figure 1D). Quantitation by phosphorimaging showed that there was no consistent difference in the rate of degradation of 5‐BrU‐ and U‐containing RNA. In the experiment of Figure 1D, the rates were indistinguishable (462 ng RNA/min/mg for I‐RNA and 19 ng RNA/min/mg for G‐RNA). This observation demonstrated that I‐RNase is specific for inosine rather than for any unconventional base. Moreover, this demonstrates that I‐RNase is not inhibited by the presence of other anomalous nucleotides.
I‐RNase does not cleave at specific sites
The specificity of I‐RNase could arise either from preferential binding of the enzyme to I‐RNA with subsequent non‐specific degradation, or from a high specificity for cleavage adjacent to I residues. The latter case would be analogous to RNase T1 which specifically cleaves to the 3′ side of G residues leaving a 3′ phosphate group. To address the question of whether I‐RNase cleaves only adjacent to I residues, we digested the transcript B3P3 with I‐RNase (Figure 2A). B3P3 contains two blocks of 24 pyrimidine residues separated by a single G (or I) residue (Mullen et al., 1991). If I‐RNase cleaved only at I residues, two major cleavage products of 25 and 29 nucleotides would be detected, corresponding to the two pyrimidine blocks. As a control, cleavage was also carried out with RNase T1 which cleaves after G residues and with somewhat reduced kinetics at I residues (Steyaert et al., 1991). Cleavage of B3P3 RNA by RNase T1 yielded two major bands of 25 and 29, as expected (Figure 2A). Note that RNase T1 digestion was almost complete within the time that it took to quench the 'zero' time reactions (Figure 6A, lanes 7 and 11). In contrast, cleavage of B3P3 I‐RNA by I‐RNase yielded no specific cleavage products at any time, but instead gave rise to a faint ladder of degradation products. Similar results were obtained when a different pyrimidine‐rich transcript was used for analysis. These data demonstrate that I‐RNase does not cleave specifically at I residues, but rather suggest that it may cleave at every phosphodiester bond.
We next analysed the products of complete digestion by I‐RNase. Analysis of I‐RNase‐digested ΔKP I‐RNA was carried out by thin layer chromatography (TLC). ΔKP I‐RNA transcripts were synthesized using either [α‐32P]UTP or [α‐32P]CTP. If cleavage by I‐RNase produces 3′‐NMPs, the label would be transferred to all nucleotides adjacent to U or C residues, resulting in a mixture of the four labelled 3′‐NMPs. In contrast, cleavage yielding 5′‐NMPs would result in the label remaining associated only with UMP or CMP respectively. As shown in Figure 2B, a single [32P]NMP spot was detected upon digestion of each I‐RNA substrate. The major spot resulting from digestion of either [32P]UTP‐ or [32P]CTP‐labelled I‐RNA corresponds to the generation of 5′‐UMP or 5′‐CMP respectively, as determined by the position of unlabelled standards. The spot detected at the origin in both lanes probably represents a small proportion of incompletely digested oligoribonucleotides since its relative intensity diminished when increasing amounts of I‐RNase were used to digest the transcripts (data not shown). Therefore, I‐RNase appears to digest I‐RNA completely, yielding 5′‐NMPs.
Preferential binding to I‐RNA
The preceding data demonstrate that the specificity of I‐RNase does not result from cleavage only adjacent to I residues. An alternative explanation for I‐RNase specificity is that the enzyme binds preferentially to I‐RNA. To investigate this possibility, competitor RNAs were mixed with the ΔKP I‐ and G‐RNA substrates prior to incubation with I‐RNase. If the competitor RNA is able to bind to I‐RNase it should cause a decrease in the rate of degradation of the test I‐RNA. Both I‐RNA and G‐RNA competitor RNAs were tested over a range of molar excesses (Figure 3A, lanes 5–11 and 13–19). Both RNAs competed the I‐RNase degradation of the target ΔKP I‐RNA transcript and the I‐RNA competitor appeared to be a more effective competitor (compare lanes 5–7 with 13–15). However, this analysis underestimates the competition by I‐RNA since the competitor itself is digested. This can be seen by comparing the amount of competitor at 0 h in lanes 4 and 12 with the amount remaining after 2 h incubation in lanes 5 and 13. While the G‐RNA competitor remains intact, the I‐RNA competitor is largely degraded. Note that the competitor RNAs were labelled to much lower specific activities. Thus, these data demonstrate that I‐RNase binds both G‐RNA and I‐RNA and suggest that I‐RNA is bound with higher affinity. Interestingly, we found that poly(I), poly(G) and yeast tRNA (seven species of which contain inosine) were all much less effective at competition than the mixed composition RNAs shown in Figure 3A (data not shown).
To obtain a more rigorous estimate of the difference in the ability of I‐ or G‐RNA to bind to I‐RNase, we performed simple Michaelis–Menten kinetic analysis and determined the Km of I‐RNase for I‐RNA and the Ki for G‐RNA. The initial rate of degradation of ΔKP I‐RNA at various concentrations was determined for a constant concentration of I‐RNase by measuring the decrease in acid‐precipitable radioactivity. Since I‐RNase is an exonuclease (see below), the loss of precipitable counts should be proportional to the breakage of phosphodiester bonds; this condition would not necessarily hold for endonucleases because initial cleavage events might yield fragments that are still fully precipitable. The Km for ΔKP I‐RNA was determined subsequently by Lineweaver–Burke plots (Figure 3B) to be 9.0 ± 4.4×10−9 M (n = 4). The rate of degradation was then assessed under the same conditions, but in the presence of a fixed concentration of one of two different G‐RNA competitors (ΔSL or ΔKP). Analysis of these data indicated that G‐RNA acted as a purely competitive inhibitor, with no change in the Vmax of the enzyme but a decrease in the apparent Km (Figure 3B). In duplicate assays, the Ki of I‐RNase for ΔSL G‐RNA was determined to be 1.5×10−6 M and 1.2× 10−6M (Km is decreased by a factor of 1+[I]/Ki where [I] is the concentration of inhibitor). The Ki for the ΔKP G‐RNA competitor was similarly determined to be 1.2×10−6 M. Assuming that Km and Ki provide a reasonable estimate of the dissociation constants for I‐RNA and G‐RNA, these data suggest that the binding of G‐RNA to I‐RNase is ∼300‐fold weaker than the binding of I‐RNA. It is worth recalling that Km is only a good estimate of the dissociation constant, KD, if the rate of subsequent catalytic steps are slow compared with the dissociation rate for the enzyme–substrate complex (see Fersht, 1985). Given that it is unlikely that the rate constant of these steps for G‐RNA is greater than for I‐RNA, the apparent 300‐fold difference in affinities is likely to be a conservative estimate. Thus, preferential binding of I‐RNA to I‐RNase is probably sufficient to explain the high specificity for degradation of I‐RNA.
I‐RNase does not degrade dsRNA
We next investigated the specificity of I‐RNase for dsRNA or ssRNA. Complementary RNA transcripts were synthesized using the polylinker of pBluescript SK+ (Stratagene) as a template. One strand was labelled with [32P]UTP and contained guanosine or inosine, while the complementary transcripts contained guanosine and were unlabelled. The labelled pBluescript SKI‐RNA, but not the G‐RNA, was degraded by I‐RNase, as expected (Figure 4A, lanes 1–4). However, if it was first annealed with the complementary RNA strand (see Materials and methods for annealing conditions), I‐RNA degradation was largely inhibited (Figure 4A, lanes 5–7). Addition of a 1‐ to 5‐fold excess of complementary RNA led to protection from degradation (compare lanes 5–7 with lane 4). That this inhibition of degradation was due to base pairing rather than competitive inhibition by binding of the complementary RNA to I‐RNase was confirmed by the much higher concentrations of an unrelated non‐complementary RNA competitor that were required to achieve similar levels of inhibition (lanes 8 and 9; 25‐ and 100‐fold excess respectively). These observations demonstrate that I‐RNase does not degrade fully base‐paired I‐RNA.
We next tested whether I‐RNase is able to degrade I‐RNA which is largely double‐stranded, but which contains unpaired single‐stranded regions of 8, 26 or 38 nucleotides containing two, nine or 10 I residues respectively. These bulged RNA duplexes were created by annealing a full‐length pBluescript SK I‐RNA transcript with a complementary pBluescript SK G‐RNA transcript containing an internal deletion in the polylinker. No degradation of any of the test substrates by I‐RNase was detectable (Figure 4B). Since we know that I‐RNase can degrade short single‐stranded I‐RNAs of ∼30 nucleotides (data not shown), but not fully base‐paired RNA (Figure 4A), this observation suggests that I‐RNase is an exonuclease and that the inosine‐containing single‐stranded regions of these substrates are protected from degradation by the flanking double‐stranded regions.
I‐RNase is a 3′→5′ exonuclease
Exonucleases degrade nucleic acids in either a 3′→5′ or a 5′→3′ direction. To determine the polarity of I‐RNase, we have created transcripts containing a stable hairpin at either the 5′ or 3′ end. Based upon the preceding observations, if I‐RNase is a 5′→3′ exonuclease, degradation would be blocked by the presence of a hairpin at the 5′ end of the I‐RNA substrate but not when the stem–loop is at the 3′ end. Conversely, if I‐RNase functions as a 3′→5′ exonuclease, its activity would be blocked by a stem–loop located at the 3′ but not the 5′ end of the substrate. Uncapped I‐ and G‐RNA transcripts were synthesized from the template pSLA using either the SP6 or the T7 promoter (transcripts were initiated with 5′‐GMP, refer to Materials and methods). This generated complementary transcripts which had a stem–loop at either the 5′ or 3′ end, respectively. Upon incubation with I‐RNase, the I‐RNA transcript with the stem–loop at the 5′ end was degraded (Figure 5A, lane 4) while the transcript with a 3′ hairpin remained intact (lane 8). This observation, which was made consistently in four identical assays, suggests that I‐RNase is a 3′→5′ exonuclease. To confirm this conclusion, degradation of a second pair of transcripts was tested (in triplicate). I‐ and G‐RNA transcripts were synthesized from the constructs pΔSL and pSLB. The SLB and ΔSL transcripts were identical except that SLB transcripts had a stable hairpin at the extreme 3′ end. Only the I‐RNA without a 3′ hairpin was degraded significantly (Figure 5B, compare lanes 5 and 9). The data shown were obtained with uncapped I‐ and G‐RNA; identical data were obtained when the RNAs were capped. Taken together, these data confirm that I‐RNase is a 3′→5′ exonuclease.
I‐RNase degrades the products of RED‐1 deamination
One instance in which inosine occurs naturally in cells is as the result of deamination of dsRNAs by enzymes such as dsRAD (Hurst et al., 1995; Dabiri et al., 1996; Maas et al., 1996) or RED‐1 (Melcher et al., 1996). The inosine‐containing products of deamination may be substrates for degradation by I‐RNase. The previous experiments were carried out on RNAs in which I had been incorporated in place of G by bacteriophage RNA polymerases (Figures 1,2,3,4,5). We next tested whether I‐RNase could specifically degrade I‐RNAs in which inosine was introduced by the more physiologically relevant mechanism of RED‐1 modification.
Complementary RNA transcripts labelled with [α‐32P]ATP were synthesized using the template pBluescript SK, from either the T3 or T7 promoters. The RNAs were then annealed, and the dsRNA treated with recombinant RED‐1. In order to determine what percentage of the A residues were converted to I, a sample of RED‐1‐treated RNA was digested with RNase P1 and then analysed by TLC under conditions where the spots corresponding to 5′‐AMP and 5′‐IMP were separated. Inosine was only detected in dsRNA which had been treated with RED‐1 (Figure 6A, lane 1). As expected, untreated dsRNA and ssRNA incubated in the presence or absence of RED‐1 remained unmodified (lanes 2, 3 and 4, respectively). Quantitation of the double‐stranded pBluescript RNA deamination by phosphorimaging revealed that 13.6 ± 0.5% (n = 3) of A residues were converted to I. Allowing for the adenine content of the double‐ and single‐stranded regions, this corresponds to 17.6% conversion of adenosines in dsRNA, which is about a third of the maximum obtainable value with dsRAD (Polson and Bass, 1994). This in turn corresponds to the introduction of an average of 3.2 I residues into the T3‐synthesized RNA strand and 2.8 into the T7 strand if all adenines in dsRNA are equally susceptible to deamination (which strictly is not the case, Polson and Bass, 1994). Following deamination of the double‐stranded pBluescript RNA by RED‐1, the modified RNA was treated with I‐RNase under standard conditions. Prior to incubation of the modified double‐stranded pBluescript RNA with I‐RNase, the duplex was disrupted by heat denaturation. The results showed that the RNAs which contain inosine as the result of deamination by RED‐1 were degraded selectively by I‐RNase (Figure 6B, compare lanes 1 and 2). Quantitation of the data of Figure 6B by phosphorimaging showed that ∼50% of the initial amount of RNA remained at time 2 h when the RNA had been treated with RED‐1 followed by I‐RNase (compare lanes 1 and 2). In contrast, dsRNA incubated under the same conditions in the absence of RED‐1 and then with I‐RNase was undegraded at 2 h (compare lanes 3 and 4). ssRNA (T3 strand) pre‐incubated in the presence or absence of RED‐1 was not degraded by I‐RNase (lanes 5–8). In the absence of denaturation of the RNAs prior to incubation with I‐RNase, no degradation was observed under the assay conditions used (data not shown). Neither was any degradation of ΔKP G‐RNA observed following heat denaturation and I‐RNase treatment under identical conditions. These results have been reproduced qualitatively three times, although the data has not been quantitated on each occasion.
These data therefore demonstrate that RNAs with as few as 3–4 I residues introduced by RED‐1 become specific substrates for I‐RNase upon denaturation. Moreover, they demonstrate that the preferential degradation of I‐RNA is due to the presence of inosine rather than to the absence of guanosine; this distinction could not be made formally from the previous data. The data are consistent with I‐RNase playing a role in degradation of deaminated RNA in the cell, although modification to the levels observed here does not destabilize the dsRNA sufficiently to allow direct access of I‐RNase without prior denaturation (see Discussion).
In this study we have characterized a novel ribonuclease, I‐RNase, which is specific for inosine‐containing ssRNA, which is divalent cation dependent, and which acts as a 3′→5′ exonuclease yielding 5′‐NMPs. The most interesting of these properties is the novel specificity for I‐RNA. I‐RNase preferentially degraded RNA that contained inosine as a result of incorporation by bacteriophage polymerases during in vitro transcription (Figures 1,2,3,4,5), and also by the more physiologically relevant route of modification by the RED‐1 deaminase (Figure 6). The basis of I‐RNase specificity is not due to exclusive or preferential cleavage adjacent to I residues (Figure 2). Rather, competition experiments (Figure 3) indicated that the specificity arises at the level of binding to RNA. This was not immediately obvious from the standard analysis involving gel electrophoresis and autoradiography of samples (Figure 3A). However, a more rigorous kinetic analysis indicated that G‐RNA behaved as a purely competitive inhibitor of I‐RNA degradation by I‐RNase. The determined Ki for G‐RNA (∼1.3×10−6 M) was some ∼300‐fold higher than the Km of the enzyme for I‐RNA. Therefore, assuming that these constants provide a reasonable estimate of the binding affinity (see Results section above), preferential binding to I‐RNA appears to provide the basis for the specificity of the enzyme. The specificty of I‐RNase is for RNA that contains inosine rather than for RNA that lacks guanosine, as demonstrated by its ability to specifically degrade RED‐1‐modified RNA (Figure 6). Finally, the specificity of the enzyme appears to be for inosine, rather than for any non‐conventional bases, as indicated by the stability of G‐RNA containing 5‐BrU (Figure 1D). It might be expected that naturally occurring uridine analogues such as pseudouridine and dihydrouridine would also not be targeted by I‐RNase. It is worth noting that although I‐RNase activity is novel, it is possible that the enzyme is a previously characterized nuclease that hitherto has not been tested with I‐RNA substrates.
Biological substrates of I‐RNase?
Given the novel specificity of I‐RNase, what might be its natural substrates? The properties of I‐RNase that we have characterized allow some informed speculation between possible substrates. Inosine commonly occurs in cellular RNAs as a result of three processes. The first is in the wobble position of the anticodon loop of some tRNAs. This was thought originally to occur via insertion of the base hypoxanthine (Elliot and Trewyn, 1984), but has been shown more recently to involve hydrolytic deamination of adenine (Auxilien et al., 1996). We have shown that single‐stranded regions containing up to 10 inosines flanked by dsRNA stems are not degraded (Figure 4B). Therefore, the single inosine within the anticodon loop of tRNAs is unlikely to be a target. Consistent with this, we found that yeast tRNAs were particularly weak competitors of I‐RNase (data not shown). The second process which introduces inosine into RNA is specific editing of some cellular RNAs by deamination of A to I. The A to I conversions at specific positions of the GluRB mRNA (and probably other cellular RNAs) lead to changes in the coding capacity of the mRNA because the I residues in the mRNA are decoded as G rather than A. This editing is most likely carried out by the RED‐1 enzyme (Melcher et al., 1996) and/or dsRAD (Hurst et al., 1995; Dabiri et al., 1996; Maas et al., 1996) and relies upon localized secondary structure within the exons which is probably sufficiently destabilized by the specific editing event to prevent subsequent modifications (Higuchi et al., 1993; Maas et al., 1996). Specifically edited mRNAs with a limited number of inosines also seem unlikely to be I‐RNase targets for two reasons. In GluRB mRNAs the edited positions are distant from the 3′ end; the 3′‐untranslated region alone is 2.7 kb (Koehler et al., 1994). Given that I‐RNase is a 3′→5′ exonuclease, it is likely that specific degradation requires I residues to lie within proximity to the 3′ end. It is possible that the poly(A) tail may in itself be sufficient to protect many edited mRNAs either because it is a region devoid of inosines and/or because poly(A)‐binding proteins would block entry of 3′→5′ exonucleases. We are planning to investigate this issue. Second, compartmentation could conceivably protect edited mRNAs. We have only detected I‐RNase in nuclear fractions, but our inability to detect I‐RNase in the cytoplasmic S‐100 fractions was due to the high levels of general nuclease activity. Therefore, while we cannot rule out cytoplasmic I‐RNase activity, the possibility remains that it could be restricted to the nucleus. In this case, edited mRNAs that are transported to the cytoplasm efficiently would no longer be vulnerable to I‐RNase attack. Thus, tRNAs and edited mRNAs, which have specific functions in the cell, are not probable targets for I‐RNase.
The third source of I in RNA, related to specific editing, is the wholesale conversion of A to I within extended regions of dsRNA as a result of dsRAD activity (Bass and Weintraub, 1988; Wagner et al., 1989). The resultant I–U base pairs are less stable than the original A–U base pairs, causing the RNA to become less double‐stranded in character. dsRAD was originally characterized in Xenopus laevis oocytes as an activity that caused irreversible unwinding of dsRNA, and subsequently has been identified in a number of organisms throughout the animal kingdom (Wagner et al., 1990; reviewed in Bass, 1993). The binding of dsRAD to dsRNA does not appear to be sequence specific, although in short RNA duplexes there is some selectivity in the choice of modification sites (Polson and Bass, 1994). In long dsRNA duplexes (>100 bp) modification is more efficient, and up to 50% of adenosines can be modified in either strand of the duplex (Nishikura et al., 1991; Polson and Bass, 1994).
While dsRAD and its related isoform RED‐1 have been implicated in specific RNA editing of cellular GluRB mRNA (Hurst et al., 1995; Dabiri et al., 1996; Maas et al., 1996; Melcher et al., 1996) and hepatitis delta virus RNA (Polson et al., 1996), it is likely that one or both of these enzymes may also have other roles. One of the earliest suggestions, due to the targeting of dsRNA, was a role in antiviral defence (Bass and Weintraub, 1988). The demonstration of induction of dsRAD expression by interferon is highly supportive of such a role (Patterson and Samuel, 1995; Patterson et al., 1995). In addition, the presence of A→G and U→C hypermutations of viral RNAs during persistent infection observed with some negative‐stranded RNA viruses is consistent with dsRAD acting upon viral dsRNA intermediates (Bass et al., 1989; Cattaneo and Billeter, 1992). The I‐RNase activity, like dsRAD, appears to be widespread (readily detectable in HeLa cells, smooth muscle and brain) consistent with a general role rather than a cell type‐specific function. Moreover, the enzyme appears to be more active on larger RNAs with multiple I residues (compare Figure 6B with previous figures). We therefore speculate that I‐RNase may have a general role in cellular antiviral defence by degrading RNAs that have been 'tagged' by wholesale A→I modifications. Although dsRAD is capable of destroying the integrity of viral open reading frames, such RNAs potentially could still compete with cellular RNAs for the translation machinery. Efficient degradation of such RNAs would give the cell a better chance of combating the viral attack.
Consistent with this speculation, we showed that I‐RNase was able to degrade RNA that had first been modified by RED‐1 (Figure 6). Nevertheless, we found that under the conditions of our experiment, the modified dsRNA had to be denatured first to allow subsequent degradation by I‐RNase. Taken at face value, this suggests that the immediate products of deamination of dsRNA are not substrates for I‐RNase. One possible explanation is that I‐RNase degrades heavily deaminated RNA inside the cell, but that some other enzymatic activity such as an RNA helicase is necessary to present the modified RNA in recognizable form. The coupled action of an RNA helicase with a 3′→5′ exonuclease has been demonstrated in the Escherichia coli degradosome (Py et al., 1996). Moreover, the conditions of the RED‐1/I‐RNase experiment in Figure 6 were not ideal. First, the 17.6% A→I conversion was well below the maximal obtainable, and so the double‐stranded region was not fully destabilized. Probably, this could be improved by using longer RNA duplexes which are more efficient deaminase substrates. It is possible that by increasing the level of A→I modification the helix may be sufficiently destabilized that heat denaturation of the RNA is unnecessary prior to degradation by I‐RNase. Moreover, other specific features of the Bluescript polylinker duplex made it a suboptimal substrate for this experiment. The RNA duplex at the 3′ end of the T3 strand was closed by a G–C base pair followed by two A–U base pairs and then 13 G–C base pairs. Given that adenines at the 3′ end of the duplex are poor substrates for dsRAD (Polson and Bass, 1994) and so are probably not modified, the 3′ end of this duplex would be expected to stay fully double stranded and so resistant to I‐RNase unless first denatured. Likewise, a 9 bp GC stretch towards the other end of the duplex probably protected the 3′ end of the T7 strand from I‐RNase attack. Nevertheless, even under less than optimal conditions, the modified Bluescript RNA was a substrate for I‐RNase. We are planning to test whether other dsRNAs can be degraded directly by I‐RNase after modification by RED‐1 or other members of the dsRAD family.
Enzymes that are involved in antiviral pathways are often inducible by interferon and/or virus infection. We have attempted to detect changes in the levels of I‐RNase activity in interferon‐treated cells. However, we have found that in unfractionated cell extracts the background general nuclease activities are too high to allow changes in I‐RNase activity to be discerned. However, it is possible that the enzyme could participate in an inducible pathway without being induced itself. One further speculation that can be made is that the proposed dsRAD/I‐RNase pathway could also mediate antisense‐mediated inhibition of gene expression (Nellen and Lichtenstein, 1993).
In conclusion, we have characterized an RNase whose novel specificity suggests a role in cellular antiviral defence. The elucidation of the precise biological substrate specificity of I‐RNase and its role and localization in the cell will be greatly facilitated by its purification to homogeneity and molecular cloning. We are currently pursuing this goal.
Materials and methods
Constructs and transcripts
Constructs used as templates for in vitro transcription or for transfection were all prepared by standard cloning procedures (Sambrook et al., 1989). The construct ΔKP comprises spliced exons 2 and 3 of α‐TM, generated by deletion between the KpnI and PvuII sites at the ends of the intron in construct pGC+DX (Smith et al., 1993). The construct B3P3 contains the branch point and polypyrimidine tract of α‐TM exon 3 between the SmaI and AccI sites cloned into the SmaI site of pGEM 4 (Mullen et al., 1991). The construct pBluescript SK was used to synthesize complementary RNAs to investigate the ability of I‐RNase to degrade dsRNAs. The T3 promoter was used for the synthesis of transcripts containing I or G when pBluescript SK was linearized with Asp718. The T7 promoter was used to synthesize complementary RNAs which contained G when pBluescript SK was linearized with XbaI. To make dsRNAs, the complementary RNAs were mixed together in TE buffer and then heated at 70°C for 5 min. The RNAs were cooled slowly to 30°C (60–90 min in a heating block). The constructs pBluescript SK Δ8, pBluescript SK Δ26 and pBluescript SK Δ38 were made by deleting the polylinker sequence of pBluescript SK between the XbaI site and the BamHI, EcoRI and HindIII restriction sites respectively. Each of these plasmids were linearized with NotI, and the transcripts were synthesized using the T7 promoter. The construct pSLA was made by inserting the hairpin‐forming sequence 5′ GGG(CGG)6 GAATTC(CCG)6 CCC 3′ into the PvuII site of pSP70 (this is the construct referred to as HP in Liu et al., 1996). The complementary sequences which anneal to form the stem are underlined. pSLA was linearized with BglII, and the SP6 promoter was used to synthesize the transcript 5′‐SLA. To synthesize the transcript 3′‐SLA, pSLA was linearized with XhoI, and the T7 promoter was used for transcription. Both of these transcripts were synthesized using 5′‐GMP to prime transcription. The construct ΔSL was used to transcribe linear RNAs from the T7 promoter with the sequence: GpppGGGAGACAAGCUUGAGCUGGAUGCCGCC UCUGCUGCUGCGCACAUUUCAUUUAUA UUCUGUCCUUUCCCCUUUUU CUCCUCUUCUUUACCUCCUCCCCUUUGGUU GGAGGUGGGUGGGUGAGAAUU. The construct pSLB was used to synthesize RNAs, using the T7 promoter, which contained a sequence at the 3′ end which forms a stable stem–loop, as follows: GpppGGGAGACAAGCUUGAGCU GGAUGCCGCCUCUGCUGCUGCGCACAUUUCAUUUAUAUUCUGUCCUUU CCCCUUUUUCUCCUCUUCUUUACC UCCUCCCCUUUGGUUGGAGG UGGGUGGGUGAGAAUUGCUUG CAAUUCUCACCCACCCAGAAUU. The sequence which anneals to form the stem is underlined; the loop has the sequence of a stable tetraloop. The identity of all constructs was confirmed by sequencing.
Extract preparations and partial purification of I‐RNase
HeLa cell nuclear extracts were prepared using the modifications of Abmayr et al. (1988). Pig brain extract was prepared as follows: 100 g of pig brain was homogenized in 200 ml of a low salt buffer [20 mM HEPES‐KOH, pH 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.5 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg/ml aprotinin, 2 μg/ml chymostatin, 2 μg/ml leupeptin, 5 mM NaF, and 5 mM β‐glycerophosphate], and the salt concentration was then raised to 0.3 M in a total volume of 400 ml. The homogenate was stirred at 4°C for 30 min, centrifuged at 8000 g for 20 min, and the supernatant subsequently was centrifuged at 100 000 g for 2 h at 4°C. Finally the extract was dialysed against 20 mM HEPES‐KOH, pH 7.9.
I‐RNase was partially purified from the pig brain extract by three successive chromatography columns: DEAE–Sepharose, CM–cibacron blue and heparin–Sepharose. Each of these columns were run using 20 mM HEPES‐KOH, pH 7.9, 50 mM KCl buffer, and the proteins eluted using salt gradients up to 3 M KCl. The column fractions were dialysed against a buffer containing 20 mM HEPES‐KOH, pH 7.9, 50 mM KCl for subsequent identification of the fractions containing I‐RNase. Activity was assessed using the standard degradation assay.
Sheep uterine smooth muscle tissue extract was prepared by essentially the same method as was used for the pig brain extract; the only exception being that the smooth muscle tissue was ground in liquid nitrogen using a mortar and pestle rather than broken down by homogenization. I‐RNase was partially purified from the sheep uterine smooth muscle extract using a heparin–Sepharose column. This column was run using 20 mM HEPES–KOH, pH 7.9, 50 mM KCl buffer, and the proteins were eluted using a salt gradient from 0 to 1 M KCl
In vitro transcription
Standard 32P‐labelled RNAs were transcribed from cloning vectors with SP6, T3 or T7 RNA polymerase (Promega) as described previously (Smith and Nadal‐Ginard, 1989; Mullen et al., 1991). Unless otherwise stated, transcription was initiated using a m7G(5′)ppp(5′)G dinucleotide primer. For uncapped transcripts (SLA, SLB, ΔSL, Figure 5), 5′‐GMP was added to the transcription reaction at a final concentration of 2 mM. For synthesis of inosine‐containing transcripts, ITP was added in place of GTP at an equivalent concentration. TLC was used to verify the composition of the I‐RNA transcripts (Scadden and Smith, 1995). In 50 random transcription reactions, the efficiency of I‐RNA transcription ranged from 40 to 80% of the G‐RNA transcription efficiency. In the absence of CAP analogue or 5′‐GMP, the I‐RNA transcription efficiency was reduced further to <10% of the G‐RNA transcription efficency, consistent with the previous report that T7 RNA polymerase can use ITP for transcriptional elongation but that a primer is also required (Milligan and Uhlenbeck, 1989).
Labelled mixed composition competitor RNAs were trace labelled with [α‐32P]UTP at a specific activity at least 10‐fold lower than substrate RNAs.
Degradation assays
The standard assay for degradation used the same conditions that we use for in vitro pre‐mRNA splicing. Each 10 μl reaction contained 20–50 fmol of [32P]RNA transcript, 2.5 mM MgCl2, 500 μM ATP, 20 mM creatine phosphate, 2.5 U/μl RNasin, buffer E [12 mM Tris (pH 7.9), 12% (v/v) glycerol, 60 mM KCl, 0.12 mM EDTA, 0.3 mM DTT], and 40–60% extract/fraction. Reactions generally were incubated at 30°C for 0–2 h. Degradation assays were then subjected to proteinase K digestion, phenol/chloroform extraction and ethanol precipitation in the presence of 10 μg of carrier tRNA. Reaction products were analysed by electrophoresis in 8 M urea, 5–10% polyacrylamide gels, followed by autoradiography or phosphorimaging.
For assaying I‐RNase activity by loss of acid‐precipitable counts, 10 μl reactions (Figure 3B) were assembled according to the standard protocol, using the ΔKP I‐RNA. When the incubation at 30°C was complete, the reaction was added to 1 ml of 10% trichloroacetic acid, which additionally contained 10 μg of tRNA. Following incubation on ice for 10 min, the samples were filtered onto glass fibre discs, and the counts then determined by Cerenkov counting. I‐RNase assays were carried out using various amounts (10–250 fmol) of ΔKP I‐RNA, assayed over various time courses (0–90 min). This enabled the initial rates of degradation of I‐RNA to be determined at each substrate concentration. A Lineweaver–Burke plot was then constructed and the Km for I‐RNA determined. Equivalent assays were also performed in the presence of 12 pmol of ΔSL or ΔKP G‐RNA competitor. This enabled determination of the apparent Michaelis constant Kapp, for I‐RNA in the presence of the G‐RNA competitor, thus allowing the Ki for the competitor (G‐RNA) to be calculated using the equation Kapp = Km(1+ [I]/Ki), where [I] is the concentration of G‐RNA inhibitor.
Composition analysis of I‐RNA transcripts
[32P]UTP‐ or [32P]CTP‐labelled RNA transcripts were digested with an excess of I‐RNase for 2 h, and the digestion products were fractionated by TLC on PEI plates using 0.65 M sodium formate, pH 3.4 as the solvent (Scadden and Smith, 1995).
Deamination reaction
Equimolar amounts of pBluescript T3 Asp718 and T7 XbaI RNAs were annealed as described above in 'Constructs and transcripts'. The resultant dsRNA, if correctly annealed, would contain an 82 bp duplex with 5′ overhangs of 39 and 11 nucleotides on the T3 and T7 sides respectively. dsRNA (200 fmol) was deaminated in a 25 μl reaction containing the following components: 0.5 μg of poly(A), 5 mM EDTA, 0.2 μg of bovine serum albumin, 25 mM Tris–HCl, pH 7.9, 100 mM KCl, 5% glycerol, and 6.25 μl of His‐tagged recombinant RED‐1 (a gift from Mary O'Connell, University of Basel). The reaction was incubated at 30°C for 1 h. The RNA was then extracted with phenol/chloroform, recovered by ethanol precipitation and resuspended in 10 μl of double‐distilled water. The dsRNA was denatured prior to subsequent degradation assays by heating at 90°C for 5 min followed by snap cooling on ice. For analysis of the efficiency of the deamination reactions, the RNA was digested with RNase P1 for 1 h at 50°C, and the digestion products fractionated by TLC on PEI plates. The solvent used comprised saturated (NH4)2SO4, 0.1 M Na acetate, pH 6.0, and isopropanol (mixed in the ratio 79:19:2). The identity of the spots corresponding to 5′‐AMP and 5′‐IMP was verified by the migration of unlabelled standards.
We thank Clare Gooding and Liu Zhi‐Ren for supplying some of the constructs, John Le Quesne for assistance in the initial stages of this investigation, and Mary O'Connell and Walter Keller for the gift of RED‐1. We also thank Richard Jackson, Gavin Roberts and Justine Southby for critically reading the manuscript. This work was funded by a grant from the Wellcome Trust to C.W.J.S. A.D.J.S. was supported by scholarships from the Cambridge Commonwealth Trust and the New Zealand Federation of University Women, and the Mary Caldwell Research Fellowship (Newnham College, Cambridge).
- Copyright © 1997 European Molecular Biology Organization


text: Well, I sent my 200q to the mechanic because I just couldn't find out what was causing the annoying vibration. Turns out it was a blown valve cover, allowing oil to seep into (mainly) #5 cylinder and caused it to not spark very well. I tell him "great" and all is well. Until I get a call today, the timing belt was NEVER CHANGED!! I bought the car at 101k under the assumption that all service was up to date, but now at 122k, he calls to tell me that the timing belt has more cracks than a nudist convention!! He said that he can guarantee that it is the original timing belt and it was 32k over due for a change. I went to take a look and I can't believe it held together! I feel like buying a lotto ticket or something. Joseph Vanzeipel 200q20v 122k, original timing belt


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Product or Service
catalytic deoxidizer
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1. Grab your favorite chili recipes, or one of ours:
- Chicken Chili
- Low Carb Chicken Chili
- Bison Chili
- Southwest Style Bison Chili with Quinoa
2. Bake sweet potatoes in the oven (they are sweetest this way, but the microwave will work if you are in a pinch!). Here's a tutorial on the Restaurant Style Baked Sweet Potato.
3. Top your sweet potato with warm chili and add your favorite toppings. Some suggestions:
- Organic Shredded Cheese
- Chopped Green Onions or Chives
- Dollop of Greek Yogurt (healthier alternative to Sour Cream)
- Sliced Jalapenos
4. Enjoy!


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Festive, plentyful, and so delicious, paella is a party all-star. Paella is a wonderful versatile dish that can be adapted to your own personal style. This version can be made quickly with ingresients that are easy to find.
- Yield
- 4-6
- 1 tablespoon vegetable oil
- 14 ounces chorizo, cut into 1/2-inch slices on the diagonal
- 1 small onion, chopped
- 1 red bell pepper, chopped
- 2 cloves garlic, chopped
- 1/4 teaspoon pimentón or paprika
- 2 cups short- or medium-grain rice
- 1 pinch saffron threads
- 2 bay leaves
- 4 cups chicken stock
- 1 1/4 pounds roast chicken (meat from 3-pound chicken skinned, deboned, and torn into chunks)
- 1/2 cup frozen peas
- 1 tomato, chopped
- Coarse salt and freshly ground black pepper to taste
- Heat oven to 400°F. Heat oil in a large paella pan or casserole over high heat. Add chorizo and cook until browned and fragrant, about 1 minute. Add onion, red bell pepper, and garlic. Cook, stirring occasionally, until vegetables are tender, about 3 minutes. Add rice (medium- or short-grain rice), pimentón, saffron, and bay leaves. Stir to combine and coat the rice, about 1 minute. Add chicken stock and bring to a boil. Add roast chicken, frozen peas, and tomato; stir to combine. Transfer skillet to oven. Cook uncovered until rice is tender and no liquid remains, about 20 minutes. Remove from the oven. Fluff with a fork and serve immediately.
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Nutritional Info
- Calories1132
- Carbohydrates97 g(32%)
- Fat50 g(76%)
- Protein69 g(137%)
- Saturated Fat17 g(83%)
- Sodium1701 mg(71%)
- Polyunsaturated Fat6 g
- Fiber3 g(11%)
- Monounsaturated Fat24 g
- Cholesterol194 mg(65%)


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Finally a pre-war one bedroom that will suit your needs with a generous layout, great closet space, open windowed kitchen, separate dining area, windowed bath, hardwood floors, and charming pre-war detail. This pin-drop quiet home is located in a full service building on Riverside Drive and 75th Street in the heart of the Upper West Side.
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It's always with great excitement that I approach a new Oztorical* romance, and Téa Cooper is one of my absolute favourites from this sub-genre. So, of course, when I got the chance to read her latest, The Horse Thief, I grabbed it with both hands. The fact that it is… Read more »
Author: Téa Cooper
Forgotten Fragrance by Téa Cooper: Review
I don't need to say how much I love Australian-set historical romance, because I've said it many times before. BUT…….I'm going to anyway! I shall even express it in GIF form. There's not nearly as much of it as I'd like, but I can always count on Téa Cooper to… Read more »
Lily's Leap by Téa Cooper: Review
Readers of this blog will know I am bang up for some good historical romance of late. One period of time we rarely get to see in romantic fiction is Colonial-era Australia, and thanks to Téa Cooper, her soon-to-be-released Lily's Leap takes us straight back to this wild and fascinating… Read more »


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BRAMPTON, ON, April 14 /CNW/ - Bramalea-Gore-Malton MPP Kuldip Kular will launch Ontario's annual Spring Seatbelt Campaign at Peel Children's Safety Village on behalf of Transportation Minister Jim Bradley. Dr. Kular will join community road safety partners to highlight how to properly use booster seats to protect kids and to highlight the importance of buckling up. Date: Wednesday, April 15, 2009 Time: 12:00 p.m. Location: Peel Children's Safety Village Powerade Sports Park 7605 Kennedy Road South, Brampton Directions: From Highway 401 (travelling east or west): - Exit at Highway 410 and travel north to Derry Road, - Travel west on Derry Road to Kennedy Road, - Travel north on Kennedy Road and follow signage to Powerade Sports Park. Editor's/Producer's Note: Photo opportunity. ------------------------------------------------------------------------- ontario.ca/transportation-news Disponible en français
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For further information: Nicole Lippa-Gasparro, Minister's Office, (416) 327-1815; Bob Nichols, Communications Branch, (416) 327-1158
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Join the El Cerrito Arts & Culture Commission at its booth on Pomona Ave (right next to the El Cerrito Art Association booth). This year we're featuring a variety of hands-on make-and-take projects using fabric, ephemera, un-usable books, and other fun stuff gathered from the El Cerrito Recycling Center Exchange Zone.
Stop by to make stickers, magnets, bookmarks, collage masterpieces, and unique mail art. Learn how easy it is to make upcycled envelopes at home using old magazines, calendars, maps, and discarded books. Declare your independence from buying new art supplies – learn how to give new life to materials that would otherwise be sent to landfill or recycled! And beside saving money, creative reuse is great for the environment. All projects are appropriate for children and adults.
Any questions? Email me at email@example.com And click here to see more details at my blog post.


text: And so it begins.... I just came back from the San Francisco Jimmy Choo. And the Cruise Party is here...REMEMBER IT'S A PRESALE for the December Sale too!!!!!!! Thursday, November 29 5-8 pm Cocktails to be Served San Francisco Boutique in Bloomingdales RSVP 415-856-5431 All purchases will include complimentary shipping and gift wrap Hope all you Bay Area Choo Girls can make it. I know I'll be there with my Choos on!


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Panneaux solaires
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We develop, design, manufacture and sell variety of mono-crystalline modules ranging from 10W to 185W in power output(We can also produce other specifications according to customers' requirement), built to the general specifications for use in a wide range of on-grid and off-grid residential, commercial, industrial and other solar power generation systems.
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Hill media strike a new tone with Conservatives over karaoke at Stornoway
The Hill Times' Rachel Aiello and Global News' Monique Muse.
Conservative MP Erin O'Toole.
National PR's Chris Froggatt and The Huffington Post's Catherine Lévesque.
Conservative Party Director of Communications Cory Hann.
Earnscliffe's Yaroslav Baran with Mike Storeshaw.
CBC's Julie Van Dusen, Reuters reporter Randall Palmer, and Conservative MP Maxime Bernier.
iPolitics' Janice Dickson and Kelsey Johnson.
Conservative MP Pierre Paul-Hus and Global's Laura Stone.
Hill Times photographer Jake Wright and Conservative MP Kellie Leitch.
Richard Madan and Rona Ambrose's chief of staff Garry Keller.
CTV's Richard Madan, Conservative interim leader Rona Ambrose and Conservative MP Tony Clement.
TVO's Steve Paikin and Tony Clement, probably giving him an Instagram selfie lesson.


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Tuesday, September 6, 2011
Sept 6: A GOOD STORY IS A GOOD STORY with Host Marsha Casper Cook
Please join Marsha Cook every Tuesday at 9 PM-EST. for an entertaining and informative discussion on writing screenplays and books.
What a great show - We will be having several of the authors of WORLD CASTLE PUBLISHERS on every couple of weeks to introduce some very good writers. On this show the special guests are Charlotte Blackwell, Kathi Barton and Joann Hamann Buchanan, who has her own show on Shark Radio. This is going to be a fun show with lots of information about writing, blogging and publishing, and lets not forget marketing. Denise spooner will be there to add to the discussion, open up the chat room and the telephone lines for callers.
Direct link to show page http://www.blogtalkradio.com/worldofinknetwork/2011/09/07/a-good-story-is-a-good-story-with-host-marsha-casper-cook
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Archive for the 'Scams For Fun And Profit…' Category
It Just So Happens….
This came to me on the web, the best piece I've seen written on the subject of Barack Hussein Obama:
by Don Fredrick, ©2012, blogging at The Obama Timeline
(Oct. 1, 2012) As I noted in the introduction to my book, The Obama Timeline, a jury at a murder trial will often find the accumulated circumstantial evidence so overwhelming that a guilty verdict is obviouseven though there may be no witness to the crime. "The jurors in the Scott Peterson trial believed the collection of evidence more than they believed Scott Peterson. Among other things, the jury thought that being arrested with $15,000 in cash, recently-dyed hair, a newly-grown goatee, four cell phones, camping equipment, a map to a new girlfriend's house, a gun, and his brother's driver's license certainly did not paint a picture of a grieving husband who had nothing to do with his pregnant wife's disappearance and murder."
In the four years I have been gathering information aboutand evidence against Barack Hussein Obama, I have encountered hundreds of coincidences that strike me as amazing. None of those coincidences, by themselves, may mean much. But taken as a whole it is almost impossible to believe they were all the result of chance. Consider the Obama-related coincidences:
Obama just happened to know 60s far-left radical revolutionary William Ayers, whose father just happened to be Thomas Ayers, who just happened to be a close friend of Obama's communist mentor Frank Marshall Davis, who just happened to work at the communist-sympathizing Chicago Defender with Vernon Jarrett, who just happened to later become the father-in-law of Iranian-born leftist Valerie Jarrett, who Obama just happened to choose as his closest White House advisor, and who just happened to have been CEO of Habitat Company, which just happened to manage public housing in Chicago, which just happened to get millions of dollars from the Illinois state legislature, and which just happened not to properly maintain the housingwhich eventually just happened to require demolition.
Valerie Jarrett also just happened to work for the city of Chicago, and just happened to hire Michelle LaVaughan Robinson (later Obama), who just happened to have worked at the Sidley Austin law firm, where former fugitive from the FBI Bernardine Dohrn also just happened to work, and where Barack Obama just happened to get a summer job.
Bernardine Dohrn just happened to be married to William Ayers, with whom she just happened to have hidden from the FBI at a San Francisco marina, along with Donald Warden, who just happened to change his name to Khalid al-Mansour, and Warden/al-Mansour just happened to be a mentor of Black Panther Party founders Huey Newton and Bobby Seale and a close associate of Nation of Islam leader Louis Farrakhan, and al-Mansour just happened to be financial adviser to a Saudi Prince, who just happened to donate cash to Harvard, for which Obama just happened to get a critical letter of recommendation from Percy Sutton, who just happened to have been the attorney for Malcolm X, who just happened to know Kenyan politician Tom Mboya, who just happened to be a close friend of Barack Hussein Obama, Sr., who just happened to meet Malcolm X when he traveled to Kenya.
Obama, Sr. just happened to have his education at the University of Hawaii paid for by the Laubach Literacy Institute, which just happened to have been supported by Elizabeth Mooney Kirk, who just happened to be a friend of Malcolm X, who just happened to have been associated with the Nation of Islam, which was later headed by Louis Farrakhan, who just happens to live very close to Obama's Chicago mansion, which also just happens to be located very close to the residence of William Ayers and Bernardine Dohrn, who just happen to have been occasional baby-sitters for Malia and Sasha Obama, whose parents just happen not to mind exposing their daughters to bomb-making communists.
After attending Occidental College and Columbia University, where he just happened to have foreign Muslim roommates, Obama moved to Chicago to work for the Industrial Areas Foundation, an organization that just happened to have been founded by Marxist and radical agitator Saul "the Red" Alinsky, author of Rules for Radicals, who just happened to be the topic of Hillary Rodham Clinton's thesis at Wellesley College, and Obama's $25,000 salary at IAF just happened to be funded by a grant from the Woods Fund, which was founded by the Woods family, whose Sahara Coal company just happened to provide coal to Commonwealth Edison, whose CEO just happened to be Thomas Ayers, whose son William Ayers just happened to serve on the board of the Woods Fund, along with Obama.
Obama also worked on voter registration drives in Chicago in the 1980s and just happened to work with leftist political groups like the Democratic Socialists of America (DSA) and Socialist International (SI), through which Obama met Carl Davidson, who just happened to travel to Cuba during the Vietnam War to sabotage the U.S. war effort, and who just happened to be a former member of the SDS and a member of the Committees of Correspondence for Democracy and Socialism, which just happened to sponsor a 2002 anti-war rally at which Obama spoke, and which just happened to have been organized by Marilyn Katz, a former SDS activist and later public relations consultant who just happened to be a long-time friend of Obama's political hatchet man, David Axelrod.
Obama joined Trinity United Church of Christ (TUCC), whose pastor was Reverend Jeremiah Wright, a fiery orator who just happened to preach Marxism and Black Liberation Theology and who delivered anti-white, anti-Jew, and anti-American sermons, which Obama just happened never to hear because he just happened to miss church only on the days when Wright was at his "most enthusiastic," and Obama just happened never to notice that Oprah Winfrey left the church because it was too radical, and just happened never to notice that the church gave the vile anti-Semitic Nation of Islam leader Louis Farrakhan a lifetime achievement award.
Although no one had ever heard of him at the time, Obama just happened to receive an impossible-to-believe $125,000 advance to write a book about race relations, which he just happened to fail to write while using the cash to vacation in Bali with his wife Michelle, and despite his record of non-writing he just happened to receive a second advance, for $40,000, from another publisher, and he eventually completed a manuscript called Dreams From My Father, which just happened to strongly reflect the writing style of William Ayers, who just happened to trample on an American flag for the cover photograph of the popular Chicago magazine, which Obama just happened never to see even though it appeared on news-stands throughout the city.
Obama was hired by the law firm Miner, Banhill and Galland, which just happened to specialize in negotiating state government contracts to develop low-income housing, and which just happened to deal with now-imprisoned Tony Rezko and his firm Rezar, and with slumlord Valerie Jarrett, and the law firm's Judson Miner just happened to have been a classmate of Bernardine Dohrn, wife of William Ayers.
In 1994 Obama represented ACORN and another plaintiff in a lawsuit against Citibank for denying mortgages to blacks (Buycks-Roberson v. Citibank Federal Savings Bank), and the lawsuit just happened to result in banks being blackmailed into approving subprime loans for poor credit risks, a trend which just happened to spread nationwide, and which just happened to lead to the collapse of the housing bubble, which just happened to help Obama defeat John McCain in the 2008 presidential election.
In 1996 Obama ran for the Illinois State Senate and joined the "New Party," which just happened to promote Marxism, and Obama was supported by Dr. Quentin Yong, a socialist who just happened to support a government takeover of the health care system.
In late 1999 Obama purportedly engaged in homosexual activities and cocaine-snorting in the back of a limousine with a man named Larry Sinclair, who claims he was contacted in late 2007 by Donald Young, who just happened to be the gay choir director of Obama's Chicago church and who shared information with Sinclair about Obama, and Young just happened to be murdered on December 23, 2007, just weeks after Larry Bland, another gay member of the church, just happened to be murdered, and both murders just happened to have never been solved. In 2008 Sinclair held a press conference to discuss his claims, and just happened to be arrested immediately after the event, based on a warrant issued by Delaware Attorney General Beau Biden, who just happens to be the son of Joe Biden.
In 2003 Obama and his wife attended a dinner in honor of Rashid Khalidi, who just happened to be a former PLO operative, harsh critic of Israel, and advocate of Palestinian rights, and who Obama claims he does not know, even though the Obamas just happened to have dined more than once at the home of Khalidi and his wife, Mona, and just happened to have used them as occasional baby-sitters. Obama reportedly praised Khalidi at the decidedly anti-Semitic event, which William Ayers just happened to also attend, and the event Obama pretends he never attended was sponsored by the Arab American Action Network, to which Obama just happened to have funneled cash while serving on the board of the Woods Fund with William Ayers, and one speaker at the dinner remarked that if Palestinians cannot secure a return of their land, Israel "will never see a day of peace," and entertainment at the dinner included a Muslim children's dance whose performances just happened to include simulated beheadings with fake swords, and stomping on American, Israeli, and British flags, and Obama allegedly told the audience that "Israel has no God-given right to occupy Palestine" and there has been "genocide against the Palestinian people by (the) Israelis," and the Los Angeles Times has a videotape of the event but just happens to refuse to make it public.
In the 2004 Illinois Democrat primary race for the U.S. Senate, front-runner Blair Hull just happened to be forced out of the race after David Axelrod just happened to manage to get Hull's sealed divorce records unsealed, which just happened to enable Obama to win the primary, so he could face popular Republican Jack Ryan, whose sealed child custody records from his divorce just happened to become unsealed, forcing Ryan to withdraw from the race, which just happened to enable the unqualified Obama to waltz into the U.S. Senate, where, after a mere 143 days of work, he just happened to decide he was qualified to run for President of the United States
Obama just happened to save $300,000 on the purchase of a $1.65 million Chicago mansion for which he deposited only $1,000 in earnest money, while the seller's adjacent empty lot which was appraised at no more than $500,000 just happened to be sold at the inflated price of $625,000 to Rita Rezko, who just happened to earn only $37,000 per year working for Cook County government, and who just happened to be married to Tony Rezko, who just happened to be Obama's main money man for his political campaigns, and who only days before the Obama mansion purchase just happened to obtain a $3.5 million loan from wealthy Iraqi Nadhmi Auchi, who just happened to have been kicked out of Iraq, and who just happened to have been convicted of corruption charges in France, and who just happened to ask Rezko to ask then-U.S. Senator Obama to help him obtain a visa to travel to the United States.
Rita Rezko just happened to borrow the money for the $625,000 empty lot from the Mutual Bank of Harvey, which just happened to be run by Tony Rezko's pal Amrish Mahajan, whose wife Anita just happened to have been charged with fraudulently receiving $2 million in Illinois taxpayer dollars for drug tests never performed by her company, K. K. Bio-Science, which just happened to have a no-bid contract with the state, and whose computers just happened to disappear right before investigators arrived to take them away for evidence.
Obama just happened to obtain a $1.32 million mortgage for his mansion even though the payments of $8,000 per month (plus at least $1,500 per month in property taxes) exceeded 50 percent of his $162,100 U.S Senate salary income, and even though Michelle Obama was claiming that she and her husband were still paying off substantial student loans and were struggling to pay for piano lessons for their daughters, one of whom just happens to look remarkably like one of the daughters of Malcolm X.
Obama just happened to obtain his mansion mortgage from Northern Trust Bank, whose Board of Directors just happened to include Susan Crown, who just happened to be part of the wealthy Crown family, which just happened to donate to Obama's campaigns, and which just happened to have ownership in defense contractor General Dynamics Corporation, and the Crown family just happened to sit on the board of energy company Exelon, formerly known as Commonwealth Edison, which just happened to have had Thomas Ayers as its CEO, and the Crown family also owned the Maytag appliance company, which just happened to move its operations to Mexico, after its employees just happened to donate to Obama's campaign, after he just happened to pledge that he would keep their jobs in Galesburg, Illinois.
In June 2005, just months after Obama became a U.S. Senator, Michelle Obama just happened to be named a "non-executive director" of the board of TreeHouse Foods, a supplier of Wal-Mart, for a salary of $51,200 in 2005 and $101,083 in 2006, and she just happened to be given 7,500 TreeHouse stock options, worth approximately $72,375, even though she just happened to know nothing about the private sector or running a business.
In 2006 Obama pushed for a $1 million earmark for the University of Chicago, and his wife Michelle just happened to be promoted to Vice-President of Community and External Affairs for the hospitals with a salary increase from $121,900 to $316,962, and she just happened to receive public relations help from Obama's political strategist David Axelrod, whose mother just happened to write for a communist newspaper.
In 2006 Sarah P. Herlihy, an associate of the Chicago law firm of Kirkland and Ellis, whose employees later contributed $87,722 to Obama's presidential campaign, and whose partner Bruce I. Ettleson just happened to be a member of Obama's campaign finance committee, just happened to write a paper calling for the elimination of the "natural born citizen" requirement in the U.S. Constitution.
Obama just happened to visit Kenya in 2006 to support his cousin, Raila Odinga, a Muslim socialist candidate for president, who just happened to have ties to both al-Qaeda and Libya's Muammar Qaddafi, and who just happened to have been educated in communist East Germany, and who just happened to name his son Fidel, and who just happened to plan on establishing Shari'ah Muslim law in Kenya, and whose activities prompted the Kenyan government to lodge an official protest of Obama's passport abuse and misconduct, and Obama's actions just happened to have been denounced by the U.S. State Department as being in direct opposition to U.S. National Security, and after Odinga, for whom Obama just happened to have raised $950,000, lost the election, his Muslim followers just happened to burn Christian women and children alive in a church where they had sought refuge.
In 2006 Obama endorsed Alexi Giannoulias in his race for Illinois State Treasurer and stated that he is "…one of the most outstanding young men I could ever hope to meet" even though Giannoulias just happened to be only 29 years old and even though his family's Broadway Bank just happened to finance Chicago crime figures like Michael "Jaws" Giorango, a Chicago thug with convictions for bookmaking and promoting prostitution, and even though virtually all of Chicago's Democrat politicians were keeping their distance from Giannoulias, whose reputation was so questionable he even failed to get the endorsement of the Chicago Democrat Party which just happens to almost never be concerned about questionable reputations.
Obama's mother, Stanley Ann Dunham, once worked for the Ford Foundation's Asia program, which just happened to be run by Peter Geithner, who just happened to be the father of Timothy Geithner, who just happened to neglect to pay Social Security taxes on much of his income, which just happened to somehow qualify him to be Obama's Treasury Secretary.
During the 2008 campaign Obama's passport records just happened to have been illegally searched by an employee of a firm headed by John O. Brennan, and Lt. Quarles Harris, Jr., who was cooperating with federal investigators in connection with the incident, just happened to be found with a bullet in his head, and the murder just happened never to be solved, and Obama later just happened to make Brennan his terrorism and intelligence advisor.
On election night in 2008 in Chicago's Grant Park, Obama just happened to wear a black suit and a red tie, and his older daughter just happened to wear a red dress, and his younger daughter just happened to wear a stark black dress, and his wife Michelle just happened to wear an arguably unattractive black dress that appeared to have a giant red X design, which just happened to prompt some to wonder if their clothing just happened to denote black power, communism, and Malcolm X, and at the very least prompted others to wonder why anyone would have his daughter wear a jet-black dress for a celebratory occasionor where one could even just happen to find a store that sells black dresses for little girls.
From election night forward there are hundreds of other "just happeneds," not the least of which is the long-form birth certificate released by Obama in April 2011 which just happened to consist of multiple image layers, including various objects which can be separated and rotated with computer software which just happens to be impossible if a birth certificate is merely scanned and not computer-constructed by a forger.
Oh, and Obama just happened to have used more than one Social Security number over the years, and one of them is associated with 713 Hart Senate Office Building and starts with the digits 282, which signifies issuance in Ohio, a state in which Obama just happened to have never lived or worked, and another Social Security number used by Obama starts with 042, which signifies Connecticut, another state in which Obama just happened to have never lived or worked, and it just happened that no one in the mainstream media has ever bothered to ask Obama why he has used multiple Social Security numbers or why the 042 number comes up as invalid in the E-Verify system used by employers to confirm whether immigrant job applicants have valid numbers.
I could go on… but you get the idea.
P.S. If Obama just happens to win reelection on November 6, remember that hyperinflation just happens to be the inescapable consequence of printing trillions of dollars to cover massive government deficits.
From Hot Air – Demos Don't Really Care Where The Money Comes From
From Hotair.com:
If the term "Doodad Pro" doesn't mean anything to you, get up to speed so that you have the background needed to appreciate this new story if/when it drops. Start with Patrick Ruffini's post from the end of the 2008 campaign explaining how lax security on Obama's campaign donation website created an opportunity for fraudulent donations. The Washington Post picked up the story a few days later, reporting that the campaign not only was accepting money from untraceable prepaid credit cards but that it "had chosen not to use basic security measures to prevent potentially illegal or anonymous contributions from flowing into its accounts, aides acknowledged." Team O's defense at the time was that they preferred to manually eliminate any bogus donations that flowed in by scrutinizing their books afterward and rejecting the ones that looked suspicious. Why they'd prefer a time-consuming process like that when they could screen donations before they were accepted with online security measures, as many other campaigns do, wasn't clear.
Fast-forward four years, when the campaign once again had to decide which measures to take to prevent online donation fraud. Time to learn a lesson and institute front-end screening? Apparently not: A video posted to YouTube in April suggested that they'ddisabled their donation security measures again. (The measures may since have been reinstated. I'm not going to donate to find out.) No way to tell yet if this sort of thing is what the forthcoming scandal story is about, but the description in the excerpt sure sounds familiar. And if it is the same thing, then it's essential to understand that people have been complaining about it for years. Team O is fully aware of the objections by now; if it's still happening, it's because they didn't care enough to stop it. And if I'm wrong about all this and the story has to do with some entirely different donation scam, well, that's even more interesting. To raise $150 million in a month, you need an awful lot of donors. And we know that when it comes to donors, Obama's not picky.
How'd Harry Get Filthy (Rich)?
Please, please, don't miss this National Review article! Click on the link to view.
Maybe Harry didn't get filthy rich from being a Nevada senator; just rich, he was already filthy, and just applied those tendencies when he had the position to mis-use his power. What's that old adage: Power corrupts. Of course, he got rich while destroying the rest of the country along with his sweetheart-in-crime, Barney Frank. This current financial mess is the result of Barney and Harry engineering the collapse of the real estate market by pushing bad loans on financial institutions, all so they could buy votes. All while Harry TOOK, not earned, profits from real estate ventures as an inside trader. I wish they both had much longer terms…life in a federal pen would be about right. While they hold hands and skip into the sunset, we all suffer. One of these days I'll tell you what I really think.
1,828 Convicted of Corruption!
Barack Hussein Obama's Training Ground
From THE AMATUER by NYT Correspondent Edward Klein
Since 1972, four Illinois governors (out of seven) have been convicted of corruption. …Chicago…has the dubious distinction of being the federal with the most convictions since 1976. Since then 1,828 elected officials, appointees, government employees and a few private individuals have been convicted of corruption in Illinois, and 84% of those were in the Northerns District—a judicial zone which contains the entire Chicago metropolitan area. During this time around 1/3 of the city's aldermen have been convicted or indicted…
Chicago is where Obama cut his political teeth. His wife Michelle worked for Mayor Daley's political machine, as did his senior advisor, Valerie Jarrett.
…"Obama has the political sense but he lacks an executive sense," says Walter Anderson, a leadership expert whose lectures at the New School for Social Research in New York City were turned into a bestselling book, The Confidence Course."
Underprivileged or Under-Proud?
Underprivileged or Under-proud
by L. J. Martin
The next time you're tempted to give money to a panhandler (bum) then remember this exchange between a cop and an Oklahoma City hand-out, palm-up scumbag, as just reported on Fox News:
Cop: "Why don't you get a job?"
Bum: "Number one, I'm lazy; Number two I make $60,000 a year."
That would be the end of story if it convinced you not to perpetuate this behavior on American streets. I was semi-cured when I was approached by a Native American in a shopping center in Missoula, Montana, who was with his wife (babe in arms) and his elderly mother. He asked, "Man, can you help me out? I need gas to get back home to the rez." I, feeling guilty that my work ethic had paid off, gave him five bucks. A week later, same guy, same sidewalk in front of the shopping center. I walked in the pack-and-ship I frequent and asked the clerk. "What's the story with the guy bumming money out front." The answer, "Oh, he's here every summer working the sidewalk. The grandma and baby thing works really well."
I should have been cured.
Two years later I was leaving a Subway where I'd just bought a sandwich, and had it cut in half as my wife might have enjoyed it and I was on my way home. Driving out of the parking lot, wa la, a panhandler with his dog and sign "Hungry. Will work for food." Again, feeling guilty about my success and forgetting to attribute it to work ethic, I stopped and pushed the down button on my truck window. "Hey, I just bought this sandwich at Subway and was taking half of it home to my wife, but you're welcome to it." He jumps up and takes it with, "God bless you man, you may have saved my life." I drive away and in my large truck side mirror see him unrolling the sandwich…for his dog.
Now, I love dogs and in retrospect am not the least unhappy about the dog getting fed, but I'm perturbed about the lie, and the fact I'm a sucker…again.
Giving anything to panhandlers just makes them convinced that they are smarter than you and I, and can live off the guilt that drives liberal thinking in this country where so many of us have so much (including those so-called poverty level folks, who, with cell phones and flat screen T.V.'s and warm roofs over their heads are ultra wealthy in the eyes of so much of the rest of the world). By the way, that bum who makes sixty thousand a year takes money from those poverty level folks who think him underprivileged. And, yes, he is very, very underprivileged: he's not privileged to have any pride.
Tell the bums where the nearest shelter is, which they won't go to because most shelters have rules, and their only rule is being able to con you
L. J. Martin is the author of 30 book length works and the conservative blog, http://fromthepeapatch.com. He lives in Montana with his wife, who's a best-selling author of romantic suspense. They winter in California, where both were born. For more see www.ljmartin.com and www.wolfpackranch.com.


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J. Pekka Mäkelä: 391
Published January, 2004
paperback, 207 pages.
Like Scifi
"Mäkelä adds diversity to the Finnish SF and takes it towards the historical novels by Mika Waltari."
– Suonna Kononen, Karjalainen
"Extremely well written, well constructed story, a pleasure to read. Like Philip K. Dick, Mäkelä has eye for the everyday problems, like the protagonist's clumsiness in relationships and his efforts to find sense in his time-distorted life."
– Juha K. Tapio, Kaleva
If you could travel in time, where? To do what? How about rescuing something mindless fanatics are about to demolish? Like... the manuscripts in the legendary Library of Alexandria?
Or, if your workmate would tell, during a quiet night shift, that he had been accidentally taken to such a trip, would you believe him? Would you follow him to a place where he says they hid the treasures?
"391's time trip to ancient Alexandria is a fascinating read, a time trip to humaneness, to how little we have changed during the times, and how little we may change in the future."
– Anne Leinonen, Kosmoskynä