text:
- Car Alarms[10]
- Doors[10]
- Automatic Door Operators[1]
- Garage Door Openers[10]
- Other Door & Window Accessories[10]
- Shutters[7]
- Gates[10]
- Curtain[6]
- Contact Person : Ms. Zhang Arabella
- Company Name : Changsha J-King Electronic Science & Technology Co., Ltd.
- Tel : 86-574-88593231
- Fax : 86-574-88593231
- Address : Zhejiang,NingBo,130 NANSHAN ROAD,FENGHUA , NINGBO, CHINA
- Country/Region : China
Related Product Searches:Aluminum Rolling Door,High Quality,aluminum rolling door, aluminum garage doors,JR11602
1) Slat option: 55,60,77,90,100mm2) Rail option: 80,100mm guide rail 3) Motor: 40~300Nm tubular motor
4) ISO9001:2008 International Quality System; CE; ROHS;
Aluminum rolling door profiles are made of 6063-T5 double-layer aluminum alloy with strength powder coating or aluminium slat with polyurethane inside or galvanized steel slat with polyurethane inside, and specified color could be customed as customer's request. Specified size aluminum roller door could be custom made. Roll door could be installed outside and operated manually or electrically from the inside. With opening ways controlled by motor control,switch or manual mode, electric, remote controlled operation. Common colors: White, cream, grey(All kind of styles and colors are adjustable to users' requirements. ) The manufacturing process is based on EU standard. It is safe operation and free-noise. It is one kind of shutter with much function such as energy saving ,keep safety
rainproof , heat / cold insulation. noiseproof , sunproof ,wind & sand proof ,cold proof ,peek proof ,rustless,etc .
It is widely used for garage door, industrial door, commercial door,residential security door.
Aluminum Rolling Door


text: I am looking to by a android tablet but I need a secure way to mount it on the wall so it does not get stolen. I have yet to purchase the tablet as I want to make sure the tablet I buy will work with the mounting bracket. Everything I am finding search the web is for the iPad but I do see some that will work for any tablet, but it looks like they can be taken off easy. Does anyone know of a mounting bracket where I don't have to worry about someone walking up and stealing it?

subset: commoncrawl


text:
Bomberman vs Digger combines three classic oldies, spiced up with original game elements, studio-quality sound effects, and engaging graphics. The application is entertaining, sometimes puzzling and overwhelming, but always thrilling. As you might have guessed from the name, this game is loosely based on the oldies any die-hard gaming fan loves tenderly and unconditionally: Boulder Dash, Digger, and Bomberman. However, this reincarnation of the holy trinity is not a dumb copy of the old classics; the game DJs from DivoGames have created a new kind of remake, with explosions, shrewd robotic monsters, and groovy bonuses. You start out with a digging machine and burrow your way to the diamonds. Having collected every piece, don't forget to destroy all the metal fiends you meet on your way to proceed to the next level.
Version 2.15 features some improvements in the game balance.
Windows 95/98/Me/NT/2000/XP, DirectX 7.0 drivers
60-minute trial


text:
It looks like you're using an Ad Blocker.
Please white-list or disable AboveTopSecret.com in your ad-blocking tool.
Thank you.
Some features of ATS will be disabled while you continue to use an ad-blocker.
Your facts don't work well with history, and most importantly don't approve violence today. ps. In the future would be nice if you can state resources for your facts. This will improve conversation and you might decide not to post some of fabrications you did in that post.
Originally posted by Lazarus Short
Originally posted by rootbranch2012
On that day, God made a covenant with Abram, saying: "To your descendants I have given this land, from the river of Egypt as far as the great river the Euphrates. The land of the Kenites, Kenizites, Kadmonites; the Chitties, Perizites, Refaim; the Emorites, Canaanites, Giga#es and Yevusites." (Genesis 15:18-21)
"And I will give to you and to your descendants after you, the land of your temporary residence, all the land of Canaan as an eternal possession and I will be a God to them." (Genesis 17:8)
Originally posted by SuperFrog
The problem with quoting Scriptural "pieces/parts" to support your theo-illogical stance, is that you may have missed the bigger picture. I have have read on in the story, and know beyond the shadow of a doubt that these promises were made as YHWH was courting His future Bride, Israel. The land was a sort of dowry, but in time, after they eloped at Sinai, Israel sinned and went whoring after other gods/husbands. YHWH properly divorced her and kicked her out of the house (land of Canaan). Neither Israel nor Judah nor the Jews have any claim to the land from the standpoint of the land covenant. The covenant will not be renewed until Jesus takes the bride as His again, and that is yet future. The Jews have cleverly set themselves up as the fulfillers of prophecy, and subverted theologians, Bible teachers, Sunday school lesson-writers, and many others. It strikes me as more than coincidence that Christianity has been on the decline since 1948, and it seems the USA has been too, even though Israel-Firsters insist that if we support Zionist-Occupied Palestine, we will be blessed. Come again? Clearly, we are cursed.
14 "Therefore, son of man, prophesy and say to Gog: 'This is what the Sovereign Lord says: In that day, when my people Israel are living in safety, will you not take notice of it? 15 You will come from your place in the far north, you and many nations with you, all of them riding on horses, a great horde, a mighty army. 16 You will advance against my people Israel like a cloud that covers the land. In days to come, Gog, I will bring you against my land, so that the nations may know me when I am proved holy through you before their eyes.
17 "'This is what the Sovereign Lord says: You are the one I spoke of in former days by my servants the prophets of Israel. At that time they prophesied for years that I would bring you against them. 18 This is what will happen in that day: When Gog attacks the land of Israel, my hot anger will be aroused, declares the Sovereign Lord. 19 In my zeal and fiery wrath I declare that at that time there shall be a great earthquake in the land of Israel. 20 The fish in the sea, the birds in the sky, the beasts of the field, every creature that moves along the ground, and all the people on the face of the earth will tremble at my presence. The mountains will be overturned, the cliffs will crumble and every wall will fall to the ground. 21 I will summon a sword against Gog on all my mountains, declares the Sovereign Lord. Every man's sword will be against his brother. 22 I will execute judgment on him with plague and bloodshed; I will pour down torrents of rain, hailstones and burning sulfur on him and on his troops and on the many nations with him. 23 And so I will show my greatness and my holiness, and I will make myself known in the sight of many nations. Then they will know that I am the Lord. '
Originally posted by ATSGrunt
The Indians went through an extermination type holocaust too and you don't see them causing problems the way the Israeli's constantly do! I'm 5th Mohawk Indian, so I do have a deep cited hate for the constant # treatment i got my whole life, along with same for the tribes of this country that once were. At least you Israelites got a country, the Indians just got a reservation, bitch! Guess that is what happens when you let people who say they are fighting for God, yet murder and kill the whole #ing way, proves that they are actually doing Satan's bidding. God job to the Satanists...
Originally posted by rootbranch2012
Wars involving Israel
List of Palestinian rocket attacks on Israel, 2012
Chronology of Terrorist Attacks in Israel
Summary of Terrorist Attacks in Israel
reply to post by SuperFrog
Your facts don't work well with history, and most importantly don't approve violence today. ps. In the future would be nice if you can state resources for your facts. This will improve conversation and you might decide not to post some of fabrications you did in that post.
Originally posted by rootbranch2012
You're doing what the authors of the NT did. They took some basic facts and then added, subtracted, twisted and fabricated as they saw fit.
Fact - God, the Creator made a covenant with Abraham and his seed, Isaac and Jacob ever after to give the Land of Israel to them. That's a fact. It's called the Promised Land for a reason. Why do you think it was called the Promised Land? Because someone felt like using that name because it just sounded interesting?
Read. Understand. Learn.
The Promised Land - Israel
Originally posted by rootbranch2012
Right. They just went to a slow grave, eating themselves up inside, drinking and drugging themselves to death to numb the pain of having been forced to renounce their heritage, become owners of houses of gambling and live on nuclear waste sites. It is a damning history and even more compounded when it is celebrated as Thanksgiving aka The Genocide of the American Natives. That's just consistently rubbing it in. So - to the final solution, eh? Till nothing is left of them. Tuck them away in the desert so nobody ever has to deal with them and let them self-destruct. A grand scheme all around.
And why aren't you speaking out against the atrocities? Maybe because most Americans would much rather not deal with it at all. It's done, over and let's watch TV and get high. That's not the way history with the Jews goes. It is specifically been historically documented lest we forget. Everything was recorded. Everything was written down. And when it was burned we wrote it exactly as it was at Sinai - word for word. Because we also have an oral tradition to back everything up in case catastrophe hits, so we REMEMBER. We are commanded to REMEMBER. Never forget. Never ever. Because we are our roots. We are our ancestors. And just as we were redeemed once from Egypt, so it will be again. That is our heritage, strength and what we pass on to our children and our children's children.
Originally posted by g146541
I have always known that the terrorist state of israel was a nation and not a religion.
They get away with alot as they fly under the guise of many banners, state, religion, ethnicity, Etc.
Screw the terrorist state of israel, I say stop all aid, financial and military goods and see just how "Gods chosen people" do.
There will still be enough jews and semites to go around, they won't go extinct.
but it's right to defend itself with any means possible to protect and ensure it's survival.
Originally posted by DOLCOTT
reply to post by g146541
Obviously you are an atheist, if you do not believe in the Bible (Old Testament), you most obviously do not understand the connection between the land of Israel which was given to them (JEWS)by GOD. As Christians we are instructed to help the JEWS and teach the message of Christ although they the Jews turned their backs and forced his execution.
I do not see why this is so complex a subject for mankind to understand, the Muslims claim this same piece of land as Holy for themselves. Is the USA any worse for protecting Israel, than Iran protecting the Palestines?????????????????????????????????????///
The site claims to deny ignorance, it appears ignorance is displayed more than denied.


text: Save This Event For Later 5TH Annual Ride for the Waffles Jacksonville, NC, Sun Oct 23, 2016 Contact Event | Event Website | Google Calendar Start line address: 711 New Bridge St., Jacksonville, NC 28540 Presented by Bicycle Gallery This is a past event. If you wish to see the details for this event click, "Continue" below. If you are looking for a current event, please search the event calendar .


text:
- freely available
- re-usable
Genes 2013, 4(2), 134-151; doi:10.3390/genes4020134
Abstract: In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp loaders contain conserved Walker A and Walker B sequence motifs, which participate in ATP binding and hydrolysis, respectively. Mutation of the glutamate residue in Walker B motifs (or DExx-boxes) in AAA+ proteins typically reduces ATP hydrolysis by as much as a couple orders of magnitude, but has no effect on ATP binding. Here, the Walker B Glu in each of the four active ATP sites of the eukaryotic clamp loader, RFC, was mutated to Gln and Ala separately, and ATP binding- and hydrolysis-dependent activities of the quadruple mutant clamp loaders were characterized. Fluorescence-based assays were used to measure individual reaction steps required for clamp loading including clamp binding, clamp opening, DNA binding and ATP hydrolysis. Our results show that the Walker B mutations affect ATP-binding-dependent interactions of RFC with the clamp and DNA in addition to reducing ligand-dependent ATP hydrolysis activity. Here, we show that the Walker B glutamate is required for ATP-dependent ligand binding activity, a previously unknown function for this conserved Glu residue in RFC.
1. Introduction
DNA replication requires the coordinated activity of many enzymes and proteins. Members of the AAA+ (ATPases associated with a variety of cellular activities) family of ATPases contribute to many steps in DNA replication including recognizing and unwinding origins where DNA synthesis will be initiated, unwinding duplex DNA to give DNA polymerases access to the template, and loading sliding clamps onto DNA (reviewed in [1,2]). Sliding clamps are ring-shaped proteins that encircle DNA and at the same time bind to DNA polymerases to increase the processivity of DNA synthesis by decreasing polymerase dissociation from DNA [3,4]. Without sliding clamps, synthesis by DNA polymerases would be too inefficient, due to frequent dissociation from the template, to support genome duplication.
Sliding clamps are assembled onto DNA by the activity of clamp loaders, which are AAA+ family members. This clamp loading reaction requires clamp loaders to bind clamps, open clamps, place open clamps around DNA, close clamps, and release the clamps to allow binding of DNA polymerases. This mechanical reaction is driven by ligand-induced conformational changes in the clamp loader. ATP binding promotes binding of the clamp loader to the clamp and DNA [5,6,7,8,9]. DNA binding triggers hydrolysis of ATP, and ATP hydrolysis stimulates release of the clamp on DNA [5,10,11].
As typical of AAA+ family members, clamp loaders are oligomeric assemblies of subunits that contain a conserved protein fold, the AAA+ module (reviewed in [12]). In addition to the AAA+ module, clamp loader subunits contain unique C-terminal domains that interact with one another to form a pentameric ring. The AAA+ modules are attached to the C-terminal domains to form a more open structure such that the clamp loader resembles a cap. Each AAA+ module contains two domains, and ATP binds at a site on the first domain and is sandwiched between the two domains (Figure 1A). Conserved nucleotide interacting sequence motifs including the Walker A and B motifs and a polar sensor 1 residue are located in the first domain and the basic sensor 2 residue is located in the second [13,14,15,16,17]. In AAA+ oligomers, ATP sites are also located at the interface of two subunits such that residues from the adjacent subunit including a conserved Arg finger form an integral part of the ATP active site. Given the location of ATP binding sites between two domains in a single subunit and at the interface of domains in a complex [14,15,18,19], mechanisms by which ATP binding and hydrolysis could promote conformational changes within individual subunits and within the complex as a whole are easy to envision.
The clamp loading reaction can be divided into two stages based on the ATP requirements (Figure 1C). In the first stage, ATP binding promotes clamp binding and opening as well as DNA binding. In the second stage, DNA binding promotes ATP hydrolysis that in turn stimulates clamp and DNA release. Key residues in the Walker A and B motifs are believed to be important for nucleotide binding and hydrolysis, respectively [20,21,22]. The Walker B motif contains an acidic amino acid residue, usually Asp, that is believed to coordinate a Mg2+ ion to promote ATP hydrolysis. AAA+ proteins contain a second conserved acidic residue that is usually Glu, and the consensus sequence is hhhhDExx in which h is a hydrophobic residue and xx residues are not well conserved but often AD [16]. This DExx-box was first recognized in helicases [23,24,25]. In P-loop NTPases that lack this second acidic residue in the Walker B motif, a Glu residue is usually found in the same spatial location relative to ATP [26,27]. These Glu residues have been proposed to activate a water molecule to hydrolyze ATP [22,25,27]. Thus, mutation of the Walker B Glu residue would be predicted to decrease the ATP hydrolysis activity of clamp loaders and hydrolysis-dependent steps such as release of the clamp on DNA, and this has been shown for an archaeal clamp loader [28]. However, recent work with another ATPase, bacterial SecA, has shown that mutation of the conserved glutamate to glutamine affects ATP-dependent conformational transitions and dynamics [29]. Similarly, in this work, we show that the Walker B Glu residue in replication factor C (RFC) contributes to ATP-dependent binding to PCNA and DNA.
2. Results and Discussion
2.1. Generation of RFC Walker B Mutants
Clamp loaders are well-characterized members of the AAA+ family proteins. RFC contains five subunits and each subunit contains sequences that are conserved among AAA+ proteins including the Walker B motif or DExx-box. To determine whether the Walker B Glu residues in RFC are important to ATP hydrolysis only, or whether they also make other contributions to enzyme activity, the activities of RFC Walker B (WB) mutants were measured for different steps of the clamp loading reaction. The conserved glutamate (E) residue in the WB motif was mutated to glutamine (Q) or alanine (A) to generate two types of WB mutants, referred to as WB-EQ or WB-EA mutants, respectively. Mutations were made in the four active ATPase subunits, Rfc1 (Glu-425), Rfc2 (Glu-141), Rfc3 (Glu-118), and Rfc4 (Glu-115) to generate a quadruple RFC mutant. Rfc5 naturally contains a mutation in the WB motif (D141N) and lacks a trans-acting arginine finger both of which reduce its ATPase activity. The mutant complexes were expressed and purified in the same way as WT RFC. Effects of the WB-EQ and WB-EA mutations on individual steps in the clamp loading reaction were evaluated.
The N-terminal region, the first 283 amino acids, of Rfc1 has DNA binding activity [32,33]. Deletion of these residues does not adversely affect clamp loading activity, and in some assays, actually simulates activity possibly by reducing nonspecific DNA binding [34,35,36]. Nor does deletion of this region affect cell viability or DNA replication [34]. In all RFC complexes used in this study, this N-terminal region was replaced with a 6X His-tag and kinase recognition motif [37,38], and the RFC complex lacking mutations in the ATP site is referred to as wild-type RFC.
2.2. Measurement of PCNA Binding and Opening
ATP binding to RFC promotes PCNA binding and opening, and ATP hydrolysis is not required for either of these interactions [5,39,40,41]. The nonhydrolyzable ATP analog, ATPγS, supports both PCNA binding and opening by RFC [5,41]. Therefore, if the only function of WB Glu residues is in catalyzing ATP hydrolysis, then mutation of these glutamates should not affect either PCNA binding or opening.
2.2.1. PCNA Binding
A fluorescence intensity-based binding assay [42] was used to measure equilibrium binding of RFC to PCNA. The fluorescence of 10 nM MDCC labeled PCNA (PCNA-MDCC) was measured in presence of 0.5 mM ATP and increasing concentrations of RFC. Binding of RFC to PCNA-MDCC increases the fluorescence of MDCC in a concentration-dependent manner (Figure 2). A dissociation constant, Kd, of 7.1 ± 2.1 nM was calculated for WT RFC in presence of ATP, which is comparable to the Kd value of 1.3 nM measured previously using surface plasmon resonance [5]. Kd values were higher for both WB mutants binding PCNA-MDCC. Calculated Kd values were 25 ± 1 nM and 27 ± 12 nM, for the WB-EQ and WB-EA mutants, respectively.
Given that PCNA binding should not require ATP hydrolysis, weaker PCNA binding by the WB mutants should not be due to a defect in ATP hydrolysis. For comparison, we measured the effect of the "non-hydrolyzable" analog of ATP, ATPγS (5'-O-(3-thio)triphosphate) on PCNA binding activity of WT RFC [6]. A Kd value of 13 ± 2 nM was calculated from PCNA-MDCC binding data in assays with 0.5 mM ATPγS. Hence, ATPγS increased the Kd value by about a factor of 2.
2.2.2. PCNA Opening
As with PCNA binding, PCNA opening requires ATP binding but not hydrolysis [40,41], and therefore, if the WB Glu residues contribute only to ATP hydrolysis, PCNA opening should not be affected. PCNA opening was measured using a fluorescence "unquenching" assay [43]. In this assay, PCNA is labeled with Alexa Fluor 488 (AF488), one molecule on each side of the monomer interfaces. When PCNA is closed, the two fluorophores interact and are quenched. When PCNA is opened at one interface, two fluorophores are separated and the quenching is relieved. The fluorescence of 10 nM AF488-labeled PCNA (PCNA-AF4882) was measured in presence of 0.5 mM ATP and increasing concentrations of RFC (Figure 3). Two important types of information can be obtained from this assay. The RFC-concentration-dependence of the increase in fluorescence provides a measure of equilibrium binding constants, and the relative values of AF488 intensity at saturating RFC concentrations reflect the fraction of PCNA molecules in an open conformation. When PCNA-AF4882 was titrated with WT RFC, the fluorescence intensity increased by a factor of about 2 at saturating concentrations of RFC. In contrast, the titration of PCNA-AF4882 with the WB mutants gave only about a 1.2-fold increase in AF488 fluorescence. These data indicate that at equilibrium WT RFC forms about 5 times more open RFC•PCNA complexes than are formed with either of the WB mutant RFC complexes. The PCNA opening assay also shows that WB mutants have a lower affinity for PCNA than does WT RFC as observed in the PCNA-MDCC binding assay (Figure 2). A dissociation constant, Kd, of 11 ± 4 nM was calculated for the WT RFC in presence of ATP. The Kd values observed for the mutants were 40 ± 27 nM (WB-EQ) and 40 ± 13 nM (WB-EA). The error in the Kd value calculated from PCNA opening data is greater than from binding data because the overall change in fluorescence was relatively small for the mutants.
To determine whether the decreased opening activity for the mutants is due to decreased ATP hydrolysis, PCNA opening by WT RFC was measured in assays with ATPγS. In these experiments, 0.5 mM ATPγS was substituted for ATP. When PCNA-AF4882 was titrated with WT RFC in assays with ATPγS, a smaller increase in AF488 fluorescence (1.5-fold) was observed at saturating RFC concentrations than in assays with ATP, but the Kd value (5.8 ± 2.6 nM) was the same. The effect of ATPγS on PCNA opening differs from the effects of the WB mutations. The WB mutations affected both the fraction of PCNA molecules opened and the Kd value, whereas ATPγS only affects the fraction of PCNA molecules opened, and the opening defect is smaller than for the WB mutants.
ATP binding to RFC induces conformational changes that increase the affinity of RFC for PCNA and stabilizes PCNA in an open conformation. Given that the affinity of the WB mutants for PCNA and the fraction of PCNA molecules that are opened by the WB mutants are both decreased relative to WT RFC, this would suggest that the WB mutations affect ATP-dependent conformational changes required for these interactions. One possibility is that interactions between the conserved Glu and ATP are important for ATP-binding induced conformational changes in RFC. A second possibility is that mutations to the Glu residue reduce ATP binding to RFC and as a result affect ATP-dependent interactions with PCNA.
2.3. Measurement of ATP Binding
To test the possibility that decreased PCNA opening (and binding) by the WB mutants is due to weaker ATP binding, opening of PCNA-AF4882 was measured as a function of ATP concentration. The rationale being that if ATP binding to the WB mutants is weaker, increased ATP concentrations will increase ATP binding. The fluorescence of 10 nM PCNA-AF488 was measured in presence of 250 nM RFC and 0–4 mM ATP (Figure 4a). The fraction of clamps in an open conformation does not change for WT RFC over a concentration range of 0.25 to 4 mM ATP as indicated by intensity values that are the same, 1.8 to 2, within experimental error. For WT RFC, this result is consistent with reported Kd values of less than 2 µM for ATP binding [6,39] because 0.25 mM ATP is already a saturating concentration. Increasing the ATP concentration to 4 mM does not increase the fraction of open clamps in assays with either of the Walker B mutants; and at all concentrations the relative intensity is lower than for WT RFC, about 1.2 to 1.3. If the weaker PCNA opening activity of the Walker B mutants were due to weaker ATP binding, the fraction of open clamps would be expected to increase with increasing ATP concentrations unless ATP concentrations of 4 mM are still not high enough for a significant fraction of RFC to bind ATP.
To determine whether WB mutants are defective in ATP binding, ATP binding was measured more directly by using a fluorescent ATP analog, TNP-ATP. TNP-ATP is weakly fluorescent when free in solution, and the fluorescence increases when bound to a protein in a less polar environment [44,45]. Because the fluorophore is attached to ATP, the TNP-ATP concentration was held constant at 50 µM and the concentration of RFC was varied from 0.1 to 0.8 µM. For both WT RFC and WB mutants, TNP-ATP fluorescence increased linearly with increasing concentrations of RFC demonstrating that the WB mutants bind TNP-ATP as does WT RFC. Note that saturation in TNP-ATP binding was not observed due to the requirement of high concentrations of RFC (at least 10 µM) that were experimentally inaccessible. The small differences in TNP-ATP fluorescence between WT RFC and the WB mutants are not enough to account for the weak PCNA opening activity of the mutants given that the TNP-ATP concentration is much lower than the ATP concentrations used in Figure 4a.
Together, results in Figure 4 show that the RFC WB mutants bind ATP and that a lack of ATP binding cannot explain the weaker PCNA binding and opening activity of the WB mutants. Interestingly, in assays with no ATP (Figure 4a, 0 mM ATP), the same fraction of PCNA molecules is opened by the mutants as opened in assays with ATP suggesting that ATP does not stimulate PCNA opening by the WB mutants. Thus, mutation of the Walker B Glu residues negatively affects high affinity ATP-dependent PCNA binding and opening. A similar finding has been made for the conserved Arg finger residue that acts in trans to help catalyze ATP hydrolysis in the adjacent subunit. Mutation of the Arg finger motif in both the E. coli and S. cerevisiae clamp loaders does not affect ATP binding by the clamp loaders, but leads to defects in ATP-dependent DNA and clamp binding [46,47,48]. Taken together, these studies suggest that one simple function cannot be attributed to individual amino acid residues in the ATP active site; the contribution that these residues make to clamp loader function is more complex.
2.4. Measurement of DNA Binding
DNA binding by RFC requires ATP binding, but not hydrolysis [5]. But, because the WB Glu mutations affect ATP-dependent interactions with PCNA, effects of these mutations on DNA binding were also measured. The binding of RFC to primed template (p/t) DNA was measured in an anisotropy-based assay. A 60-nucleotide template was labeled with X-rhodamine isothiocyanate (RhX) on the 5' end via an amino linker and annealed to a 26-nucleotide primer to create a p/t substrate with a 34-nucleotide 5'-template overhang. Assays contained 20 nM p/t DNA-RhX, 0.5 mM ATPγS and varied concentrations of RFC. ATPγS was used instead of ATP to inhibit DNA-triggered ATP hydrolysis and release of PCNA on DNA. Briefly, DNA and RFC were added sequentially to a cuvette containing buffer and ATPγS, and polarized intensities were measured after each addition (Figure 5a). The Kd values calculated from RhX-aniostropy data for the clamp loaders binding DNA were 91 ± 28 nM (WT RFC), 690 ± 400 nM (WB-EQ) and 1,060 ± 200 nM (WB-EA). These values show that the WB mutations also decrease ATP-dependent DNA binding activities of the RFC mutants.
The DNA binding by RFC has been shown to be stimulated by PCNA binding [5]. Because our data shows that WB mutations decrease PCNA opening, we analyzed the effect of PCNA on DNA binding to determine whether PCNA would no longer stimulate DNA binding for the WB mutants. For these experiments, DNA binding was measured in RhX-anisotropy assays as above except that 2 µM PCNA was added to the cuvette after adding DNA and RFC, and the results are plotted for each RFC in Figure 5b. The Kd values calculated for DNA binding in the presence of PCNA were 37 ± 13 nM (WT), 460 ± 200 nM (WB-EQ) and 1,800 ± 700 nM (WB-EA). Other studies showed that PCNA reduced the Kd for RFC-DNA binding by about a factor of 3 [5], and this binding assay gave similar results for WT RFC. PCNA binding had no effect on DNA binding by the RFC WB mutants; binding was weak in both the absence and presence of PCNA. These data are consistent with expectations based on weaker PCNA binding and opening activities of the RFC WB mutants in that PCNA did not stimulate DNA binding by the mutants.
2.5. Measurement of ATP Hydrolysis
The Walker B glutamate is predicted to activate a water molecule for catalysis of ATP hydrolysis [25,26,27]; therefore, weaker ATP hydrolysis activities are expected for the RFC WB mutants. ATPase activity was measured in a steady-state reaction that uses the coupled pyruvate kinase/lactate dehydrogenase assay to report on the ADP product produced. Briefly, pyruvate kinase uses the ADP product to convert phosphoenol pyruvate to pyruvate, and lactate dehydrogenase uses NADH to reduce pyruvate to lactate. Thus, one mole of NADH is converted to NAD+ per mole of ADP produced by hydrolysis. The fluorescence of NADH was monitored to measure ATP hydrolysis. To convert fluorescence units to concentration of ADP, a standard curve was generated by adding known amounts of ADP to the coupled enzyme system. Because RFC is a DNA-dependent ATPase, the concentration of DNA was varied in ATPase reactions (Figure 6a). Assays were done at Vmax conditions with respect to ATP concentration and contained 500 µM ATP which is 50-fold greater than the reported Km value of 9.7 ± 3 µM [49]. PCNA was not included in these assays. In the absence of DNA, steady-state hydrolysis rates were 17.4 ± 0.4 nMs−1 (WT), 15.3 ± 2.1 nMs−1 (WB-EQ) and 9.3 ± 3.9 (WB-EA) nMs−1. Hence, the basal rate of ATP hydrolysis was not affected by the WB mutations. On the other hand, the effects of the WB mutations on DNA-stimulated ATPase activity were large. At the highest concentration of DNA, 2 µM, ATP hydrolysis rates were 125 ± 20 nMs−1 (WT), 16 ± 1 nMs−1 (WB-EQ) and 11 ± 1 (WB-EA) nMs−1. While adding DNA stimulates ATPase activity of WT RFC by a factor of 7, DNA has no effect on ATPase activity of the RFC WB mutants. Given that the WB mutants bind DNA more weakly than WT RFC, decreased rates of ATP hydrolysis are expected at DNA concentrations where DNA binding to the WB mutants is low. However, based on the fractions of RFC bound at 2 µM DNA calculated from Kd values obtained in Figure 5a, about 95% WT RFC should be bound to DNA, and about 75% of the RFC WB mutants should be bound to DNA. Therefore, the weaker DNA binding activity of RFC WB mutants (Figure 5a) cannot account for the lack of DNA-stimulation of ATPase activity at this high DNA concentration.
The ATPase activity of WT RFC is stimulated by PCNA to a lesser degree than by DNA, and stimulated to the greatest extent by the presence of both PCNA and DNA [50,51]. To determine whether PCNA would affect the ATPase activity of the Walker B mutants, ATPase activity was measured in assays containing 1 µM PCNA and 1 µM PCNA with 1 µM p/t DNA (Figure 6b). While PCNA alone increased the ATPase activity of WT RFC by a factor of about 2 and PCNA with DNA increased the ATPase activity of WT RFC by more than 10-fold, PCNA had no effect on the ATPase activities of the Walker B mutants. Under all conditions tested, the ATPase activity of the RFC WB mutants was about the same as the basal level of ATPase activity of WT RFC in the absence of PCNA and DNA.
3. Experimental Section
3.1. Buffers
Assay buffer contains 30 mM HEPES-NaOH, pH 7.5, 150 mM sodium chloride, 10 mM magnesium chloride, 0.5 mM EDTA, 2 mM DTT and 2% glycerol. Storage buffer for RFC contains 30 mM HEPES-NaOH, pH 7.5, 300 mM sodium chloride, 0.5 mM EDTA, 2 mM DTT and 10% glycerol. Storage buffer for PCNA is the same as RFC storage buffer except for the inclusion of 150 mM sodium chloride. RFC purification buffer A contains 30 mM HEPES-NaOH pH 7.5, 10% (v/v) glycerol, 0.5 mM EDTA, 1 mM DTT and 0.04% Bio-Lyte 3/10 ampholyte (Bio-Rad). RFC purification buffer B contains 30 mM HEPES-NaOH pH 7.5, 500 mM NaCl and 30 mM imidazole.
3.2. Construction of RFC Expression Vectors
RFC expression vectors generated previously include pLANT/RIL expressing Rfc1 and Rfc5 [37], pET-11 expressing Rfc2, Rfc3, and Rfc4 [37], and pET-11 expressing Rfc3 and Rfc4 [52]. The coding sequence for Rfc2 was PCR amplified from S. cerevisiae genomic DNA and subcloned into a pCDFDuet vector (Novagen) between NcoI and BamHI restriction sites in the first T7 expression cassette. In all RFC1 constructs, the sequence encoding the first 283 amino acid residues was deleted and replaced with a sequence encoding a 6X His-tag and kinase recognition motif. Site-directed mutations were introduced into the coding sequences for individual RFC subunits using the QuikChange Mutagenesis kit (Strategene) as per the manufacturer's instructions. Site-directed mutagenesis was done on vectors that contained coding sequences for only one RFC subunit, and mutant coding sequences were subcloned into expression vectors that contained multiple coding sequences. Vectors were co-transformed into E. coli BL21 (DE3) cells in the combinations listed in Table 1 to produce WT and mutant RFC complexes.
|RFC Complex
|Expression Vector
|Coding Sequences
|Wild-type RFC
|RFC1 and RFC5
|RFC2, RFC3, and RFC4
|RFC1 (E425Q) and RFC5
|RFC3 (E118Q) and RFC4(E115Q)
|RFC1(E425A) and RFC5
|RFC3(E118A) and RFC4(E115A)
3.3. Proteins
WT and mutant RFC complexes were purified as described previously with modifications listed below [53]. Transformed E. coli BL21(DE3) cultures (2.4 L) were grown to an OD of 0.8 at 30 °C (Table 1). After cooling to 15 °C, IPTG was added to a final concentration of 1 mM and cells were incubated for an additional 18 h at 15 °C. Cells were harvested by centrifugation, resuspended in 30 mL Buffer A containing 1 M NaCl and lysed using a French press at 14,000 p.s.i. The lysate was clarified by centrifugation and diluted with buffer A to a final NaCl concentration of 150 mM. The diluted supernatant was applied to two 5 mL Hi-Trap SP columns (GE healthcare) attached in tandem and equilibrated with buffer A containing 150 mM NaCl. Protein was eluted in a 100-mL gradient of 150 to 600 mM NaCl. The five-subunit RFC complex eluted at approximately 450 mM NaCl. The fractions containing RFC complex were pooled and dialyzed overnight against buffer B. The dialyzed protein was applied to a 1-mL Hi-Trap chelating column (GE healthcare) charged with NiSO4 and equilibrated with buffer B. Protein was eluted using a 10-mL gradient of 30 to 500 mM imidazole. Fractions containing RFC eluted at approximately 450 mM imidazole. The fractions were pooled, dialyzed against RFC storage buffer and stored at −80 °C. Typical yields were 5–10 mg of RFC. PCNA was expressed, purified and labeled as previously described [42].
3.4. Oligonucleotides
A primed template consisting of a 26-nucleotide primer annealed to a 60-nucleotide template was used. The sequences are as follows: primer, 5'-ACA CGA CCA GTA ATA AAA GGG ACA TT; and template, 5'-TTC AGG TCA GAA GGG TTC TAT CTC TGT TGG CCA GAA TGT CCC TTT TAT TAC TGG TCG TGT. For anisotropy experiments, the 60-mer template was covalently labeled with X-rhodamine isothiocyanate (RhX) (Invitrogen) at by incorporation of a C6 amino linker on the 5'-end as described [54].
3.5. Equilibrium PCNA Binding and Opening Measurements
These fluorescence assays were done as described previously [43]. Data were fit to Equation 1 in which PCNAo and RFCo are the initial concentrations of PCNA and RFC, respectively, and Imax and Imin are the maximum and minimum fluorescence intensities, respectively, to calculate Kd values.
3.6. TNP-ATP Binding Measurements
The binding of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (Santa Cruz Biotechnology) to RFC was measured. TNP-ATP was excited at 408 nm and the emission spectra were recorded at 555 nm using a 3 nm bandpass. To generate each point, emission spectra were recorded after reagents were sequentially added to the cuvette. First, assay buffer was measured for a background signal, then 50 μM TNP-ATP for unbound ATP, and then RFC, from 0 to 0.8 μM was added to measure the signal for bound ATP. Storage buffer was added instead of RFC to generate the 0 nM RFC point. After correcting for buffer background, the intensity of bound ATP was divided by the intensity for free ATP for each point. All the values for 0 to 0.8 nM RFC were then divided by the value obtained for 0 nM RFC, setting this point to 1, to account for the change in fluorescence due to dilution.
3.7. DNA Binding Assay
The anisotropy of an RhX probe on the 26/60 mer p/t DNA was measured. RhX was excited at 585 nm and polarized emission was measured at 605 nm over 30 s using 8 nm bandpass as described [55]. The quadratic equation, (Equation 1) was used to calculate Kd values for DNA binding except that the initial DNA concentration was substituted for the PCNA concentration, and maximum and minimum anisotropy values were substituted for intensity values.
3.8. ATP Hydrolysis Assay
ATPase data was collected on a QuantaMaster QM1 spectrofluorometer (Photon Technology International) at room temperature. NADH was excited at 340 nm and emission was measured at 460 nm using a 2 nm bandpass. To the assay buffer, 1 mM phosphoenolpyruvate, 0.2 mg/mL NADH, 68 units/mL pyruvate kinase, 99 units/mL lactate dehydrogenase, 0.5 mM ATP and varying concentrations of p/t DNA were added and mixed to give the final concentrations indicated. Reactions were initiated by adding 0.45 μM RFC (final concentration). Time courses were measured for 600 s and reaction rates were calculated from a linear fit of the time course data. Storage buffer was added instead of RFC to obtain the dilution effect on NADH fluorescence and a no enzyme reaction. To convert change in fluorescence to the rate of ADP produced, a standard curve was generated by adding varying concentrations of ADP (0 to 200 µM) to the coupled assay system and measuring the fluorescence at each ADP concentration. The slope obtained from a linear plot of fluorescence vs. ADP concentration was used to convert reaction time courses to nM ATP hydrolyzed/s.
4. Conclusions
Clamp loaders undergo a series of ligand-induced conformational changes that modulate the interactions of the clamp loader with the clamp and with DNA to allow the clamp loader to catalyze the mechanical clamp loading reaction. Two different residues, a conserved Asn (corresponding to N79 in Rfc4 [56]) and a conserved Arg (corresponding to R84 in Rfc4 [57]), have been proposed to function as "glutamate switches" to link ligand binding to ATP hydrolysis. In both cases, interaction between the switch residue and the DExx glutamate residue are proposed to position the Glu residue in a catalytically inactive conformation prior to DNA binding. The interaction between the switch residue and Glu changes when DNA binds to allow the Glu residue to adopt a catalytically active conformation. The loss of DNA-dependent, but not DNA-independent, ATPase activity by RFC Walker B mutants is consistent with, but does not prove, this model. However, our data may add another layer of complexity to the model. Because the mutation of the Glu residues decreases ATP-dependent ligand binding, perhaps interaction of the Glu residue with ATP possibly via a bridging water molecule helps position other residues to interact with PCNA and DNA. Then, interactions of these or other residues with PCNA and DNA reposition Glu to activate the water molecule for catalysis of ATP hydrolysis. When the Glu residue is altered, both ligand binding and ligand-induced ATP hydrolysis activities are reduced. It is interesting to note that while the Glu residue is conserved in the E. coli clamp loader DnaX subunits, the switch residues are not. Perhaps another residue functions as the switch residue in the E. coli clamp loader. Both this work and another study of the bacterial SecA protein suggest that the contribution that the Walker B Glu residue makes to the activity of mechanoenzymes is more complex than simply activating a water molecule for ATP hydrolysis [29].
This work was supported by NIH GM082849 to LBB and GM38839 to MOD.
References and Notes
- Davey, M.J.; Jeruzalmi, D.; Kuriyan, J.; O'Donnell, M. Motors and switches: Aaa+ machines within the replisome. Nat. Rev. Mol. Cell. Biol. 2002, 3, 826–835. [Google Scholar] [CrossRef]
- Duderstadt, K.E.; Berger, J.M. Aaa+ atpases in the initiation of DNA replication. Critical Rev. Biochem. Mol. Biol. 2008, 43, 163–187. [Google Scholar] [CrossRef]
- Kong, X.-P.; Onrust, R.; O'Donnell, M.; Kuriyan, J. Three-dimensional structure of the β subunit of E. Coli DNA polymerase iii holoenzyme: A sliding DNA clamp. Cell 1992, 69, 425–437. [Google Scholar] [CrossRef]
- Stukenberg, P.T.; Studwell-Vaughan, P.S.; O'Donnell, M. Mechanism of the sliding b-clamp of DNA polymerase iii holoenzyme. J. Biol. Chem. 1991, 266, 11328–11334. [Google Scholar]
- Gomes, X.V.; Burgers, P.M. Atp utilization by yeast replication factor c. I. Atp-mediated interaction with DNA and with proliferating cell nuclear antigen. J. Biol. Chem. 2001, 276, 34768–34775. [Google Scholar] [CrossRef]
- Gomes, X.V.; Schmidt, S.L.; Burgers, P.M. Atp utilization by yeast replication factor c. II. Multiple stepwise atp binding events are required to load proliferating cell nuclear antigen onto primed DNA. J. Biol. Chem. 2001, 276, 34776–34783. [Google Scholar]
- Hingorani, M.M.; O'Donnell, M. Atp binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase iii holoenzyme. J. Biol. Chem. 1998, 273, 24550–24563. [Google Scholar] [CrossRef]
- Naktinis, V.; Onrust, R.; Fang, F.; O'Donnell, M. Assembly of a chromosomal replication machine: Two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particl. II. Intermediate complex between the clamp loader and its clamp. J. Biol. Chem. 1995, 270, 13358–13365. [Google Scholar] [CrossRef]
- Tsurimoto, T.; Stillman, B. Replication factors required for sv40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. J. Biol. Chem. 1991, 266, 1950–1960. [Google Scholar]
- Bertram, J.G.; Bloom, L.B.; Hingorani, M.M.; Beechem, J.M.; O'Donnell, M.; Goodman, M.F. Molecular mechanism and energetics of clamp assembly in Escherichia coli. The role of atp hydrolysis when γ complex loads β on DNA. J. Biol. Chem. 2000, 275, 28413–28420. [Google Scholar]
- Hingorani, M.M.; Bloom, L.B.; Goodman, M.F.; O'Donnell, M. Division of labor-sequential atp hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA. EMBO J. 1999, 18, 5131–5144. [Google Scholar] [CrossRef]
- Erzberger, J.P.; Berger, J.M. Evolutionary relationships and structural mechanisms of AAA+ proteins. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 93–114. [Google Scholar] [CrossRef]
- Cullmann, G.; Fien, K.; Kobayashi, R.; Stillman, B. Characterization of the five replication factor c genes of saccharomyces cerevisiae. Mol. Cell Biol. 1995, 15, 4661–4671. [Google Scholar]
- Guenther, B.; Onrust, R.; Sali, A.; O'Donnell, M.; Kuriyan, J. Crystal structure of the delta' subunit of the clamp-loader complex of E. Coli DNA polymerase III. Cell 1997, 91, 335–345. [Google Scholar] [CrossRef]
- Jeruzalmi, D.; O'Donnell, M.; Kuriyan, J. Crystal structure of the processivity clamp loader gamma (γ) complex of E. Coli DNA polymerase III. Cell 2001, 106, 429–441. [Google Scholar] [CrossRef]
- Neuwald, A.F.; Aravind, L.; Spouge, J.L.; Koonin, E.V. Aaa+: A class of chaperone-like atpases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 1999, 9, 27–43. [Google Scholar]
- Walker, J.R.; Hervas, C.; Ross, J.D.; Blinkova, A.; Walbridge, M.J.; Pumarega, E.J.; Park, M.O.; Neely, H.R. Escherichia coli DNA polymerase III tau- and gamma-subunit conserved residues required for activity in vivo and in vitro. J. Bacteriol. 2000, 182, 6106–6113. [Google Scholar] [CrossRef]
- Bowman, G.D.; O'Donnell, M.; Kuriyan, J. Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex. Nature 2004, 429, 724–730. [Google Scholar] [CrossRef]
- Kelch, B.A.; Makino, D.L.; O'Donnell, M.; Kuriyan, J. How a DNA polymerase clamp loader opens a sliding clamp. Science 2011, 334, 1675–1680. [Google Scholar] [CrossRef]
- Gorbalenya, A.E.; Koonin, E.V.; Donchenko, A.P.; Blinov, V.M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and rna genomes. Nucleic Acids Res. 1989, 17, 4713–4730. [Google Scholar] [CrossRef]
- Saraste, M.; Sibbald, P.R.; Wittinghofer, A. The p-loop—A common motif in atp- and gtp-binding proteins. Trends Biochem. Sci. 1990, 15, 430–434. [Google Scholar] [CrossRef]
- Walker, J.E.; Saraste, M.; Runswick, M.J.; Gay, N.J. Distantly related sequences in the alpha- and beta-subunits of atp synthase, myosin, kinases and other atp-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982, 1, 945–951. [Google Scholar]
- Hodgman, T.C. A new superfamily of replicative proteins. Nature 1988, 333, 22–23. [Google Scholar] [CrossRef]
- Linder, P.; Lasko, P.F.; Ashburner, M.; Leroy, P.; Nielsen, P.J.; Nishi, K.; Schnier, J.; Slonimski, P.P. Birth of the d-e-a-d box. Nature 1989, 337, 121–122. [Google Scholar]
- Subramanya, H.S.; Bird, L.E.; Brannigan, J.A.; Wigley, D.B. Crystal structure of a dexx box DNA helicase. Nature 1996, 384, 379–383. [Google Scholar]
- Abrahams, J.P.; Leslie, A.G.; Lutter, R.; Walker, J.E. Structure at 2.8 a resolution of f1-atpase from bovine heart mitochondria. Nature 1994, 370, 621–628. [Google Scholar]
- Story, R.M.; Steitz, T.A. Structure of the reca protein-adp complex. Nature 1992, 355, 374–376. [Google Scholar] [CrossRef]
- Seybert, A.; Wigley, D.B. Distinct roles for atp binding and hydrolysis at individual subunits of an archaeal clamp loader. EMBO J. 2004, 23, 1360–1371. [Google Scholar] [CrossRef]
- Kim, D.M.; Zheng, H.; Huang, Y.J.; Montelione, G.T.; Hunt, J.F. Atpase active-site electrostatic interactions control the global conformation of the 100 kda seca translocase. J. Am. Chem. Soc. 2012, 135, 2999–3010. [Google Scholar]
- Bowman, G.D.; Goedken, E.R.; Kazmirski, S.L.; O'Donnell, M.; Kuriyan, J. DNA polymerase clamp loaders and DNA recognition. FEBS Lett. 2005, 579, 863–867. [Google Scholar] [CrossRef]
- Simonetta, K.R.; Kazmirski, S.L.; Goedken, E.R.; Cantor, A.J.; Kelch, B.A.; McNally, R.; Seyedin, S.N.; Makino, D.L.; O'Donnell, M.; Kuriyan, J. The mechanism of atp-dependent primer-template recognition by a clamp loader complex. Cell 2009, 137, 659–671. [Google Scholar] [CrossRef]
- Fotedar, R.; Mossi, R.; Fitzgerald, P.; Rousselle, T.; Maga, G.; Brickner, H.; Messier, H.; Kasibhatla, S.; Hubscher, U.; Fotedar, A. A conserved domain of the large subunit of replication factor c binds pcna and acts like a dominant negative inhibitor of DNA replication in mammalian cells. EMBO J. 1996, 15, 4423–4433. [Google Scholar]
- Halligan, B.D.; Teng, M.; Guilliams, T.G.; Nauert, J.B.; Halligan, N.L. Cloning of the murine cdna encoding vdjp, a protein homologous to the large subunit of replication factor c and bacterial DNA ligases. Gene 1995, 161, 217–222. [Google Scholar] [CrossRef]
- Gomes, X.V.; Gary, S.L.; Burgers, P.M. Overproduction in escherichia coli and characterization of yeast replication factor c lacking the ligase homology domain. J. Biol. Chem. 2000, 275, 14541–14549. [Google Scholar] [CrossRef]
- Podust, V.N.; Tiwari, N.; Stephan, S.; Fanning, E. Replication factor c disengages from proliferating cell nuclear antigen (pcna) upon sliding clamp formation, and pcna itself tethers DNA polymerase delta to DNA. J. Biol. Chem. 1998, 273, 31992–31999. [Google Scholar]
- Uhlmann, F.; Cai, J.; Gibbs, E.; O'Donnell, M.; Hurwitz, J. Deletion analysis of the large subunit p140 in human replication factor c reveals regions required for complex formation and replication activities. J. Biol. Chem. 1997, 272, 10058–10064. [Google Scholar]
- Finkelstein, J.; Antony, E.; Hingorani, M.M.; O'Donnell, M. Overproduction and analysis of eukaryotic multiprotein complexes in escherichia coli using a dual-vector strategy. Anal. Biochem. 2003, 319, 78–87. [Google Scholar] [CrossRef]
- Kelman, Z.; Yao, N.; O'Donnell, M. Escherichia coli expression vectors containing a protein kinase recognition motif, his6-tag and hemagglutinin epitope. Gene 1995, 166, 177–178. [Google Scholar] [CrossRef]
- Chen, S.; Levin, M.K.; Sakato, M.; Zhou, Y.; Hingorani, M.M. Mechanism of atp-driven pcna clamp loading by s. Cerevisiae rfc. J. Mol. Biol. 2009, 388, 431–442. [Google Scholar] [CrossRef]
- Yao, N.Y.; Johnson, A.; Bowman, G.D.; Kuriyan, J.; O'Donnell, M. Mechanism of proliferating cell nuclear antigen clamp opening by replication factor c. J. Biol. Chem. 2006, 281, 17528–17539. [Google Scholar]
- Zhuang, Z.; Yoder, B.L.; Burgers, P.M.; Benkovic, S.J. The structure of a ring-opened proliferating cell nuclear antigen-replication factor c complex revealed by fluorescence energy transfer. Proc. Natl. Acad. Sci. USA 2006, 103, 2546–2551. [Google Scholar]
- Marzahn, M.R.; Bloom, L.B. Improved solubility of replication factor c (rfc) walker a mutants. Protein Expr. Purif. 2012, 83, 135–144. [Google Scholar] [CrossRef]
- Thompson, J.A.; Marzahn, M.R.; O'Donnell, M.; Bloom, L.B. Replication factor c is a more effective proliferating cell nuclear antigen (pcna) opener than the checkpoint clamp loader, rad24-rfc. J. Biol. Chem. 2012, 287, 2203–2209. [Google Scholar]
- Hiratsuka, T. Fluorescence properties of 2' (or 3')-o-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate and its use in the study of binding to heavy meromyosin atpase. Biochim. Biophys. Acta 1976, 453, 293–297. [Google Scholar] [CrossRef]
- Hiratsuka, T. New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. Biochim. Biophys. Acta 1983, 742, 496–508. [Google Scholar] [CrossRef]
- Sakato, M.; O'Donnell, M.; Hingorani, M.M. A central swivel point in the rfc clamp loader controls pcna opening and loading on DNA. J. Mol. Biol. 2012, 416, 163–175. [Google Scholar] [CrossRef]
- Snyder, A.K.; Williams, C.R.; Johnson, A.; O'Donnell, M.; Bloom, L.B. Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: II. Uncoupling the β and DNA binding activites of the γ complex. J. Biol. Chem. 2004, 279, 4386–4393. [Google Scholar]
- Paschall, C.O.; Thompson, J.A.; Marzahn, M.R.; Chiraniya, A.; Hayner, J.N.; O'Donnell, M.; Robbins, A.H.; McKenna, R.; Bloom, L.B. The Escherichia coli clamp loader can actively pry open the beta-sliding clamp. J. Biol. Chem. 2011, 286, 42704–42714. [Google Scholar] [CrossRef]
- Schmidt, S.L.; Gomes, X.V.; Burgers, P.M. Atp utilization by yeast replication factor c. III. The atp-binding domains of rfc2, rfc3, and rfc4 are essential for DNA recognition and clamp loading. J. Biol. Chem. 2001, 276, 34784–34791. [Google Scholar] [CrossRef]
- Tsurimoto, T.; Stillman, B. Functions of replication factor c and proliferating-cell nuclear antigen: Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage t4. Proc. Natl. Acad. Sci. USA 1990, 87, 1023–1027. [Google Scholar] [CrossRef]
- Yoder, B.L.; Burgers, P.M. Saccharomyces cerevisiae replication factor c. I. Purification and characterization of its atpase activity. J. Biol. Chem. 1991, 266, 22689–22697. [Google Scholar]
- Yao, N.; Coryell, L.; Zhang, D.; Georgescu, R.E.; Finkelstein, J.; Coman, M.M.; Hingorani, M.M.; O'Donnell, M. Replication factor C clamp loader subunit arrangement within the circular pentamer and its attachment points to proliferating cell nuclear antigen. J. Biol. Chem. 2003, 278, 50744–50753. [Google Scholar] [CrossRef]
- Johnson, A.; Yao, N.Y.; Bowman, G.D.; Kuriyan, J.; O'Donnell, M. The replication factor c clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading. J. Biol. Chem. 2006, 281, 35531–35543. [Google Scholar]
- Perez-Howard, G.M.; Weil, P.A.; Beechem, J.M. Yeast tata binding protein interaction with DNA: Fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics. Biochemistry 1995, 34, 8005–8017. [Google Scholar] [CrossRef]
- Ason, B.; Bertram, J.G.; Hingorani, M.M.; Beechem, J.M.; O'Donnell, M.; Goodman, M.F.; Bloom, L.B. A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends: DNA triggers a change in binding specificity of the γ complex clamp loader. J. Biol. Chem. 2000, 275, 3006–3015. [Google Scholar]
- Zhang, X.; Wigley, D.B. The "glutamate switch" provides a link between atpase activity and ligand binding in AAA+ proteins. Nat. Struct. Mol. Biol. 2008, 15, 1223–1227. [Google Scholar] [CrossRef]
- Neuwald, A.F. Bayesian shadows of molecular mechanisms cast in the light of evolution. Trends Biochem. Sci. 2006, 31, 374–382. [Google Scholar] [CrossRef]
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).


text:
Once more, carry-on requirements may vary based on the aircraft type being used on an individual flight—smaller regional jets will have more restrictive carry-on rules than wide-body airliners. These standards are also subject to change at any time, so it's always a good idea to check with the airlines prior to travel, so you don't get caught by any last-minute rules changes.
As consumers, it's important to be wary of products claimed to be "FAA-approved" or "Government-approved" carry-on bags — there's no such thing. Federal agencies issue guidelines (see FAA Carry-On Advisory), but it's up to the individual carriers to set the rules.
The over-arching guideline — with very few exceptions, like canes and baby strollers — is anything you take on board must fit in an overhead compartment or under the seat in front of you, and that varies depending on the type of plane you're on. Additionally, every airline states that carry-on items are subject to a space-available limitation — you may have to check your carry-on bag if there's no room available. Hedge your bets by ensuring your carry-ons will fit under the seat in front of you.
That said, there are some general guidelines that can help you to select bags that are carry-on kosher. Obviously, giving these limitations a wide berth increases the likelihood that your new luggage won't be made suddenly obsolete by new regulations.
The maximum size for a carry-on is determined by each airline and the size of the aircraft, so make sure to check with them before flying. In general, most airlines have adopted a maximum carry-on size of 45 linear inches, determined by adding up the length, width and depth of a bag (typically 22 x 14 x 9 inches). These dimensions are determined by taking the longest measurement possible, so if your prospective carry-on has protruding handles and wheels they should be included in your calculations.
Some "wide-body" roll-aboards currently on the market measure 20 x 16 x 9 inches, which do meet the commonplace 45 linear-inch rule, but won't fit into the 14 x 9 x 22-inch sizing boxes typically used to determine whether bags meet carry-on dimensional requirements—which means they may often have to be checked at the gate. If in doubt, contact your air carrier, or stick with the 20 x 16 x 9 standard.
Be aware that overfilling a bag — especially one with protruding outside pockets — can turn a dimensionally legal bag into an oversize one that will have to be checked.
While most airlines do not weigh carry-on items, most have a de facto 40-pound limit per piece.
On domestic flights, the typical allowance is one carry-on bag, and one personal item. For most air carriers, that personal item is taken to be a purse, briefcase, laptop bag or camera bag. In almost every instance this personal item must be smaller than your allotted carry-on (you may not, for example, have two 22 x 14 x 9-inch bags and count one as a personal item). Some airlines set size/weight limits for personal items, so check with your airline.
* It is always recommended to check with your carrier prior to departure.
Carry-on Guidelines By Carrier


text:
Edit: Also I have had this computer for 5-6 years so it seems like it was about time for something to go out on it. It's been a very good computer up untill now.
Edited by Rrwood87, 08 February 2012 - 02:31 PM.
Jump to content
Geeks To Go is a helpful hub, where thousands of volunteer geeks quickly serve friendly answers and support. Check out the forums and get free advice from the experts. Register now to gain access to all of our features, it's FREE and only takes one minute. Once registered and logged in, you will be able to create topics, post replies to existing threads, give reputation to your fellow members, get your own private messenger, post status updates, manage your profile and so much more.Create Account How it Works
Edited by Rrwood87, 08 February 2012 - 02:31 PM.
Not necessarily as it is a process of elimination, that is why I asked you to check the drivers!
Hmm, So it looks like to be sure im going to have to buy a new PSU and then if thats not it a new video card?
What is the answer please!
Boot into Safe Mode and check in the Device Manager to see if there is a yellow ! next to the video card.
Edited by Rrwood87, 10 February 2012 - 03:24 PM.
I explained earlier;
I am on it right now in Safemode with networking and everything seems to be working fine besides the video card not being enabled\
Booting in safe mode uses only the bare essentials including Windows generic drivers, this puts less stress on both the computer and the PSU, a failing PSU is one of the main causes of PC problems and especially display related ones as video chips require a substantial amount of power.
The PSU again may be the culprit if it cannot provide the power that the HDD needs to function correctly, as a precautionary measure if you have important data on the HDD you should back it up immediately, you can use Puppy Linux for this http://www.geekstogo...over-your-data/
Another failure has occurred.
Edit: I went through the f 12 boot to utility partition because those were the instructions to try and get to Dell Diagnostics but it would not let me get to the screen that has Express test extended test custom test or syptom tree just gave an error about no partition available or something like that
Edited by Rrwood87, 12 February 2012 - 01:20 AM.
0 members, 0 guests, 0 anonymous users
Community Forum Software by IP.Board
Licensed to: Geeks to Go, Inc.


text:
Funeral arrangements announced for Mullahoran man killed in New Zealand
The remains of the young Mullahoran man who died in New Zealand last week, will return to his native Loughduff at the weekend.
Martin Cadden, who had just turned 22, died after the car he was travelling in left the road and hit a tree outside the town of Leeston, Canterbury on Friday, January 2.
It has been confirmed that he will return to Ireland in the early hours of next Sunday morning and will be subsequently waked at the family home in Loughduff until Monday evening. The family have requesed House private procedures from 4pm on Monday.
Mr Cadden's reamains will then be removed to Loughduff Church at 7pm and he will be laid to rest following Funeral Mass at 12 noon on Tuesday.
The late Martin Cadden had celebrated his birthday at the end of December and was working as a farmer in New Zealand having completed an agricultural course at Ballyhaise College in Co Cavan.
He had been living in the Southbridge area, near Christchurch, since 2013 and worked for Ellesmere Agricultural Ltd.
Parish Priest in Mullahoran, Fr Owen Devaney said the community was both "shocked and devastated".
The late Martin Cadden is survived by his mother Ann, father Michael and sisters Roisín and Mary. Ar dheis Dé go raibh a anam dilis.


text: MacNAUGHTON, S. C. 'Chris' – Lyon's Brook. The family of Sarah Christine 'Chris' MacNaughton sadly annonce her passing on December 2, 2017, at age 105. Chris lived life to the fullest, engaging in many church, community and family events. She enjoyed her card parties, knitting, gardening, reading the newspapers and diligently completing most of their puzzles, before calling it a day. Chris was a lifelong member of the Lyon's Brook United Church and the United Church Women (UCW), as well as a member of the Lyon's Brook Women's Institute. She was also a member of the Lyon's Brook United Church choir for over 50 years, as well as organist and choir director when needed. Her real joy in life was her two boys, Peter and Ted. She welcomed with love their wives, Patricia 'Pat' and Eileen. Chris was very proud of her granddaughters, Sara and Paula; and her grandsons, Eddie and Patrick; great-granddaughter, Brittany; and great-grandson, Devin. She is also survived by sister-in-law, Bernice Hutchinson of Lindsay, Ontario. Chris was predeceased by her loving husband, Harold (1987); her parents, W.E.C. 'Cooke' and Christena 'Tena' MacCallum; six brothers, Edward, Earl, Bert, Ernie, Harry and Wilbur. Chris was the last surviving member of her immediate family. Also left to mourn are several nieces and nephews, cousins and her card playing buddies. All will miss her but grateful for a long life well lived. Chris was employed for several years at the Department of Fisheries Training School in Pictou, a position she thoroughly enjoyed. Visitation will be 6:30-9 p.m. Tuesday in McLaren Funeral Home, Pictou. Funeral will be held 2 p.m. Wednesday from Lyon's Brook United Church, with Rev. Jim Webber-Cook officiating. Burial will be in the Seaview Cemetery. Family flowers only please. Donations in her memory may be made to the Lyon's Brook United Church, Lyons Brook Hall or a charity of your choice. www.mclarenfuneral.ca


text:
Author Websites
Book Reviews
Coffee Thoughts
Cafe' Chronicles
Big Newsletter
Chat/Party Events
Featured Authors
Free Reads
CTRR Awards
Writing Classes
The Coffee Crew
Hey, Authors!
Contact Us
Book 1 : A Quick Bite
Book 2 : Love Bites
Book 3 : Single White Vampire
Book 4 : Tall Dark & Hungry
Book 5 : A Bite to Remember
Book 6 : Bite Me if You Can
Book 7 : The Accidental Vampire
Book 8 : Vampires are Forever
Book 9 : Vampire Interrupted
Book 10 : The Rogue Hunter
Book 11 : The Immortal Hunter
Book 12 : The Renegade Hunter
Bitten by Cupid Anthology
Book 13 : Born to Bite
Book 14 : Hungry for You
Book 15 : The Reluctant Vampire
The Bite Before Christmas (Anthology)
Book 16: Under a Vampire Moon
An Argeneau Novel
November 30, 2010
Harper Collins Publishers
384 Pages
Paranormal Romance
Rating: 5 cups
Alex is a culinary genius, but her luck lately has gone completely awry. From wrong paint colors, to fires, to the most recent and worst possible stroke of bad luck… her head chef walking out. When a chef is recommended from France who also happens to be desperately handsome and charming, she feels her luck may have finally taken a turn for the better.
Cale cannot stand the smell of food and cannot microwave a thing, but when there is a chance that Alex could be the life mate he has searched the world for two thousand years for, he is willing to fake it. When he finds her stirring up more trouble than he counted on and someone putting her in danger, he finds she has awakened more than one hunger.
Attraction strikes the moment they meet but Alex will not think of dating a man who is working for her, no matter how strongly attracted she is to him. And Cale, although more sure the longer he is around her that he has at long last found his perfect match, is not sure how to convince her otherwise. Will he manage to convince her to take the chance of a lifetime or will she find out what he is before he can show her how he feels for her?
Sands has cooked up a paranormal feast with this comedic addition to the Argeneau series. Cale is all business and Alex is an artist. The combination of their personalities makes for a brilliant and humorous trip into the intricately crafted world of immortals that this series is known for. With the suspenseful twist of the restaurants mysterious chain of disasters, and the passionate romance of the main characters, this story is a well rounded tale that has only one flaw… it ends.
Reviewer for Coffee Time Romance & More
Awards | Author Services | Contact Information | New Author Information


text:
UEFA chief Michel Platini has said he is in favour of increasing the number of teams taking part in the World Cup finals from the current 32 to 40.
The Frenchman has been behind several innovative ideas since taking over at the helm of European football's governin"g body in 2007, notably in encouraging the sport's global growth.
"I am in favour of there being more teams from Africa, Asia, Oceania and north America playing in the World Cup," he told AFP.
"So why not move from 32 teams to 40?. That would give us eight groups of five. And going by our research, it would only add three days to the World Cup schedule."
Platini's views came just two days after FIFA chief Sepp Blatter said he was in favour of increasing the number of World Cup finalists.
"We need to ensure that the African and Asian federations get what they deserve. The Europeans and South Americans between them have the majority of places at the World Cup although they have fewer member federations than Africa and Asia," Blatter said.
"It is not normal that Africa has just five places."
Source: AFP


text:
|Erik Mona Chief Creative Officer, Publisher
|1 person marked this as a favorite.
Nobody picked up on the real problem with this post:
A friend and I bought a bunch of singles from Heroes & Monsters on the after-market, and 5 of them showed up broken. We've both been buying DDM since Harbinger. Over the grand total of DDM purchased - for both of us combined - we had 3 broken ones. Over half the minis purchased (DDM & Pathfinder) came from the same vendor, so their shipping and handling practices aren't the issue.
I hope this isn't going to be an on-going problem with the PF minis. Aside from that, I love them. Just thought Paizo should be made aware.
The Pathfinder minis are less bendable than DDMs to be sure, but the fact that the Kaven paint master broke actually has nothing whatsoever to do with the final minis, as the early outputs we use to create the paint masters are made of a completely different (and much much much more brittle) material than the final production run miniatures.
We're keeping an eye on the plastic rigidity for the production runs, and it may be that we need to alter the balance to make them more durable, but please do not correlate the two, because they are completely different things.
If the actual minis were made the the stuff the paint masters are made of, every single miniature ever would arrive broken, and you guys would have burned down the Paizo offices a long time ago.
|Erik Mona Chief Creative Officer, Publisher
My turn. I understand about the situation with the broken sword from a paint master. Like others I too have concerns about the plastic. I sincerely hope the issue with brittle plastic in Heroes and Monsters are corrected with RotR. DDMs were too bendy. But I rarely worried about breakage. It would be revolutionary to the hobby to find a grade of plastic that was firm without being brittle. I would prefer a bendy sword to a broken one.
I'm on it. I'd like to see a little more "give" with the minis, provided we don't have to trade off too much in terms of the sculpt quality. I'm discussing this with our partners at WizKids, and we're working on getting the balance exactly right.
Please note that this may take us a set or two, but I am working diligently on it as we speak!
|Erik Mona Chief Creative Officer, Publisher
Of course this could just be older packaging (predating the cut of 2 huges from the set) so this could be one of the 2 mystery cut minis, yeah sure. Very tricksy Eric Mona, very tricksy. So looks like th e white was cut (costly? must be a good dragon) and I think we'll see that white dragon in a later release... Makes me wonder what the other cut hiuge was, there aren';t that many in the original pathway
The news is out, and I'm afraid this is my biggest personal disappointment of the set so far. The Huge White Dragon, one of the very first miniatures I spoiled as being part of this set, has unfortunately been cut from the set by WizKids due to cost issues.
The White Dragon WILL appear in a future release, and I have reason to believe that may be sooner rather than later, but when WizKids told us we needed to cut from seven to four Huges, we had to make some grim sacrifices, and the Huge White Dragon was the most difficult of all.
Basically, we had to look at the sculpts we'd approved and decide which ones would work as part of future sets or independent releases, and which ones wouldn't. The White Dragon, obviously, isn't tied directly to the Rise of the Runelords Adventure Path in the same was as the Lamia Harridan or the Karzoug Statue, so we're holding it for later release.
I promise that the Karzoug Statue (really a Huge Stone Golem) is a _fantastic_ miniature both in terms of sculpt and paint job. It's not a dragon, but I think people will really like it, and it's definitely one of the key encounters of the entire Adventure Path.
So yeah, the bad news is that the Huge White Dragon will NOT be a part of the Rise of the Runelords set. But the sculpt looks amazing, and we WILL be releasing it soon in another format.
I'd hoped to preview the Karzoug Statue mini before the packaging went live on paizo.com, but in this case unfortunately we didn't have all our ducks in a row, so the news had to break in this fashion.
Given that, look forward to a MUCH closer look at the Karzoug Statue mini next week, and look for more details on the White Dragon in the weeks to come.
The dragon is not forgotten, and will make its way to you soon, I promise.
|Erik Mona Chief Creative Officer, Publisher
|Steve Geddes
Lots of good guesses here, but I can say there are definitely a couple that no one has mentioned yet.
There are also a couple that people HAVE mentioned that WON'T be in this set, but which WILL be in the next set.
I know it's too early to have anything set in stone, however do you have any ideas as to frequency?
The gap between H&M and RoTRL was eight monthsish, but I heard rumor that 3 sets per year was the goal. Any chance the third set may be released by December/January? Is any view forming as to what frequency would be ideal?
The White Dragon WILL appear in a future release, and I have reason to believe that may be sooner rather than later...
...I promise that the Karzoug Statue (really a Huge Stone Golem) is a _fantastic_ miniature both in terms of sculpt and paint job...
...I'd hoped to preview the Karzoug Statue mini before the packaging went live on paizo.com, but in this case unfortunately we didn't have all our ducks in a row, so the news had to break in this fashion.
Given that, look forward to a MUCH closer...
Dissapointed at no white? Hmm maybe but we get a cool greater stone golem, albeit one that looks like Kourzag, so no I'm not dissapointed, i'm excited. As for the "soon in another format", can we encourage you for more detail? Unlikely I know but...
As for the guesses I'm intregued - I picked through the original quite closely and thought I had the bases covered. Seems you are more tricksy than I thought, but please don't let the hound of tindalso be in the next set, I really want it to be in this one. And a really cool paizo sculpt. lovecraft minis are much needed, not to harp oin a point at all, but the denizens of leng (yay) and the hounds played a role in this path more than others.
If the sets are likely to go 3/year will they be 60 or 40 mini sets, just to get a handle on the price.
ALso is there a chance of getting pics of the rune giant from more angles?
Really impressed with the line-up so far (apart from maybe way too many giants). But those humanoids may have convinced me to torture my wallet and order a case...
One can never have too many humanoids and the ones in this set are probably the finest PPP minis that have come out in years.
Yay Paizo!
|baron arem heshvaun
With a repaint Jakardros could be This guy
Would that mean one of the blonde Iconics with daddy issues would turn traitor?
|Erik Mona Chief Creative Officer, Publisher
Anguish wrote:Thehigher cause wrote:I agree 100%. If would prefer more monster, less humanoids. Also more monsters Pathfinder specific, not done in other lines.
Meh. I'm going to register the opposite vote.
I only tend to use a monster once unless it's a wolf/orc/ogre type iconic monster. I don't need the freak of the week. I've got dozens of those.
On the other hand, I don't like to hear "wasn't that the bartender from the last town?" when re-using a mini for a bandit or major NPC. More, more, more of what gets encountered; people.
I can reuse the same monster over and over. They aren't usually memorable. NPCs on the other hand get retired after being used as a PC or major NPC because they get associated with that character.
I just wish they weren't doing a second bow-user and 2 TWF. I like variety.
I want more types of monsters. I only need ten or so humanoids to represent any given humanoid, but there's only so many times I can let the same mini stand in for "large quadrapedal creature" before they start to blend together. I love the idea of putting a set together based on an adventure supplement. I adore the idea of smaller packs of specific minis, bundled with a small module that uses those creatures.
That's in the works, right? That's why so many goblin sculpts, so that the goblin module can come packaged with all the minis except the PCs? I'm not even sure what the price point should be...
|Kor - Orc Scrollkeeper
Guys, as long as these miniatures are based on Adventure Paths, we're going to pick the appropriate creatures to run the campaign, whether they're humanoids OR monsters.
I get it. I get that a lot of people don't want any more humanoid miniatures.
So noted.
Myself, I like about a 50/50 split. For DM's who collect/buy these, I can understand the desire to have monsters. As a player though, I much prefer ample choices of PC's for me to select my character from -- as do the other people I play with. As a DM, I like the ability to put down a bunch of NPC's that do not all look alike, and somewhat resemble the NPC I want them to represent.
While I am sorry to see that the Huge White won't make it, it is also a mini which would be quite easy to release on its own. Perhaps it could star as the main villain in the next Encounter Pack even?
Otherwise, put me in the camp of those who like to see a healthy dose of humanoids in sets. Monsters are great, to be sure, but more often than not they fall in a relatively small CR niche where they can be used. Humanoids, on the other hand, can be adjusted easily thanks to the presence or lack of class levels. Also, I like a broad selection to choose from, both for NPCs and PCs.
I don't plan on running RotRL at this point, so I am not doing to dish out the $500 to buy a case, but I do plan on buying many singles when it comes out. Humanoids will probably be the largest component of that buy, because they are the most flexible.
Now Erik, you have given a few hints that reefclaws might be in the next set. Which raises the question of what the name of the set will be. I don't suppose it would rhyme with Dull & Cackles now, would it? If so, that would be great. Not simply because that is the AP I plan on running next, but because DDM never made a lot of aquatic monsters, and a set containing a lot of aquatic foes would appeal to long time collecters and plastic newbies like myself.
Whatever may come, thank you for taking the lead from WotC. In more ways than one, in fact. I am a new Paizo customer, and continue to be impressed by everything I see and hear about Paizo as time goes on.
|Deane Beman
I get it. I get that a lot of people don't want any more humanoid miniatures.
But there are also a lot of other people who do...
Sadly my PC miniature needs were not met by Merisel, Ezren, Valeros & Kyra. Until we have a reasonable mix of race, class & gender minis suitable for PCs then I say bring them on!
|Erik Mona Chief Creative Officer, Publisher
DonalGraeme wrote:Now Erik, you have given a few hints that reefclaws might be in the next set.
I have?
Definitely need a reefclaw. When I ran Black Fang's Dungeon, it's the only mini that wasn't in the Heroes & Monsters set.
DonalGraeme wrote:Now Erik, you have given a few hints that reefclaws might be in the next set.
I have?
Perhaps I should have chosen my wording more carefully. Let me try again:
Now Erik, you have indicated (both here and on the Plasticrypt forum) some folks have made accurate guesses about what will be in the next set, and reefclaws happen to be among those people have guessed/wished for the next set.
That work better?
Though I'm rooting for monsters as much as the next mini collector, I LOVE miniatures that represent specific NPCs from the AP. As someone whose ran this once and is currently planning on getting the re-release once its out, I prefer being able to get these kind of minis rather than either having to get, assemble, and paint the Reaper mini (if it exists) or mod an existing D+D mini. I hope you guys keep doing this with future minis sets.
Now, to cross fingers for a Champion of Greed mini. Seriously, out of all the D+D and other minis I know of out there, (and I have extensive knowledge) THERE ISN'T A SINGLE LARGE SIZED FEMALE MINI! Even if its not in this set, please make this happen. I've played with plenty of female players, many of whom play fighter types. What to do if they enlarge themselves....
|Uninvited Ghost
PLEASE do not make big decisions based on feedback unless it's from a large sample size.
I have thousands of DDM, bought from the first set to the last. One thing I really like about the Pathfinder Minis is the more rigid plastic. I have so many goofy bendy DDMs that I spend so much time heating and cooling, never getting back into a good shape. I would much much much rather have the occasional broken sword than 100% chance of curved bendy ones.
|Big Jay
Lots of good guesses here, but I can say there are definitely a couple that no one has mentioned yet.
There are also a couple that people HAVE mentioned that WON'T be in this set, but which WILL be in the next set.
Okay for those not mentioned I would say Sheriff Hemlock (on my realy want list), Sandpoint Devil, Attic Whisperer maybe Boggard and a clear plastic ghost of some kind... lamia cleric, Clockwork Librarian, some giant (ice, hill zombie,runeslave Marsh dont know),Shining Child, Totenmaske, Smoke Haunt,Skul Ripper, Stone Golem, Black Monk mummy, Yethazmari, Witchfire, ice devil end thats all. Think I named every thing that could possibly make it in the final spot.
Eee! I love these three! The Black Arrow characters instantly appealed to me in RotRL, and it's great to see them here in mini form. Something about the world-weary commander, the good-natured big bruiser, and the smooth guy out to save his own neck is just so classic, I couldn't resist them. Totally buying these dudes when possible. I love the idea of having figures for an entire AP. I hope one day to have more dolls to play with figures to collect from my favorite AP, Kingmaker, as well.
Kaven Windstrike: I KNOW I cant be the only person who's initial reaction to the picture was "Its Han Solo!!".
I LOVE this miniature. My friends and I are starting a new campain in a few months and one of us wants to be a Swashbuckler. Im sure I just found his mini. Thank you. :)
I thought, "Han Solo!" too! :)
|Vic Wertz Chief Technical Officer
PLEASE do not make big decisions based on feedback unless it's from a large sample size.
I have thousands of DDM, bought from the first set to the last. One thing I really like about the Pathfinder Minis is the more rigid plastic. I have so many goofy bendy DDMs that I spend so much time heating and cooling, never getting back into a good shape. I would much much much rather have the occasional broken sword than 100% chance of curved bendy ones.
There are complaints about both conditions. But the prime issue is transportation. You can manage if the mini is bent when you show up to play at your FLGS. If it is broken, that is more difficult. No doubt that neither condition is good. But to break a mini with casual handling in the middle of a session is a bummer. IMHO and in the opinion of many others that have posted on this board and other boards the brittle plastic is an issue for Wizkids and Paizo.
There are many types of plastic out there. I am hopeful that a type can be found that is firm without being brittle. It would be good for Wizkids too. I am sure it is a nightmare having to replace broken minis found in boosters.


text:
Number Should not Start with 0 and -,Alphabet is not Allowed
An updated database of over 600 articles and counting gives you rich information about the following topics. These can keep you updated and also take some informed decisions irrespective of whether you are a small provider or an established head doctor of a hospital.
Here at MedicalBillersandCoders.com we try to provide solutions to this evergreen issue for doctors, medical administrative staff, billing and coding professionals to deal with the conventional as well as the latest trends in healthcare industry.
Obamacare is putting a lot of pressure on the profit margins of medical insurance companies. Due to this pressure insurers are denying more claims and increasing their interference between the patients and physicians as far as the medically necessary care is concerned. Claims denials have also increased due to the complexities in coding and billing.
Physicians have to follow a time consuming procedure of dealing with an insurer in case of claims denials; however, they can resolve and receive payments by appealing those denied claims. The success of appeals procedure can help physicians maximize their revenue.
Most Common Reasons for Payment Denials
Mentioned below are some of the common cases of insurance claims denials that need to be avoided:
These are some of the reasons due to which medical insurance companies deny claims. However, an error-free appeal procedure can ensure payment for the denied claims due to reasons other than simple errors during registration.
Evaluate Authenticity of Reasons for Denial
The evaluation of a denied claim is the most important part of filing an appeal. This helps in determining whether following up on the claim is worth spending the time and money. If the billing team feels that the claim was wrongfully denied by the insurance company, an attempt should be made to contest the insurer's decision.
Avoid Delays in Appealing Denied Insurance Claims
There should be no delay in submitting the appeal. It should be done within seven days from receiving the denial notice. If the appeal submission is delayed, the chances of getting it approved will reduce. The failure to resolve denied claims right away results in the claims being left in AR for too long, affecting the cash flow for physicians.
Ask for the Patients' Assistance
Practices shouldn't refrain from taking the patient's help when it comes to appealing a denied claim. Since the patient would want the insurance company to pay for the rendered services, it would be great if they too call the company on behalf of the practice. Many patients don't mind calling up the insurance company in case of a denied claim as long as they don't have to pay the claim on their own.
At times, medical offices receive denials that are against the conditions of the contract set between the insurance company and the practice. Therefore, it is important to understand all the details related to "Covered Services" and "Compensation" before appealing the claims that shouldn't have been denied by the insurer.
Other Solutions to Consider
If your practice can't pursue appeals due to lack of time, money and skilled staff, outsourcing can be an effective solution. Many practices have outsourced their billing requirements to companies like MedicalBillersandCoders.com that have a team of skilled billers and coders. MBC has been helping practices all over the US for over 40 specialties to sail through claims denials issues. Their team handles all aspects of successful claims filing to ensure maximum revenue for practices.
Category :
Accounts Receivables / Claims Denials
We Serve...
Find us through more than forty specialties to help reduce your AR days to an average of 21 days, augmenting your revenue.
About MBC
Medical Billers and Coders is the largest consortium in the United States. We can help you save 35% of your cost in medical billing services and reduce your Account Receivables (AR) days to an average of 21 days.
Know the effects of ICD-10 and how to mitigate revenue loss.
A pictorial way to understand various Billing and Healthcare concept
Our Services
Medical billing is our core focus. For more than a decade, we have been providing medical billing services to various specialties across the 50 States of USA.
The impending transition to ICD-10, one of the most crucial changes in healthcare, the new coding system is here. MBC's professional services in ICD-10 can help you with end-to-end solutions for a successful implementation.
Empower your Knowledge
We are constantly working towards acquiring the latest industry trends to ensure accurate, efficient and complete healthcare services to our clients.


text:
Own Jason Segel's Former Hollywood Hills CompoundPhoto: ShutterStockJason Segel's former vintage Hollywood Hills home can be yours for $10.5 million. The "How I Met Your Mother" star
Jennifer Lukus DRE 01846137's Blog Posts
Tina and Ike Turner's Former Home on the MarketTina Turner left a good house in the city, and after all these years, it's back on the market — looking a lot like the year is still 1977.�
Bamboo sits at the intersection of two styles: eco-friendly design and tropical British Colonial style, which has seen a resurgence of late. For the past several years, bamboo floors and even
Angry Birds and the Jungle Book are playing at the Temeku Movie Theater and it is only $3.75 for morning and afternoon movies and $4.50 for evening movies. It is located at: 26463 Ynez Road,,
Learn to Paint while Sipping Wine with Jill Roberts at South Coast Winery in Temecula. Sunday July 31. 10:00 am -1:00 pm. $70 for Wine Club Members, $75 for non wine-club members. Class includes all
People in Seattle know their houseboats, and technically this is not a houseboat. It's not even a floating home, because it's not connected to the city sewer. This gorgeous, 1,100-square
SUMMER SUNSET CONCERT SERIESJune 2-July 28, 7:00pm, Temecula AmphitheaterEvery Thursday night this Summer a new band will be playing at the Temecula Amphitheater. Gates open at 5:00pm and


text:
You should have arrived here from the lovely Lisa Spangler.
I made this card, using Boutique Borders to make the plaid background, Arctic Penguins for the cute little family, and the sentiment comes from Alpine Carolers. The banner die cut is made from the Bold Banner creative cut.
* There will be a 10% discount code active for the 3 day blog hop (HAPPY2ME)
* Purchases over $100 will receive the HAPPY EVERYTHING stamp set free.
* Purchases over $150 will receive the HAPPY EVERYTHING stamp set and HAPPY SCRIPT die free.
* We are giving away THREE $25 gift certificates each day of the Stampede, 9 winners total. Winners will be chosen randomly based on comments left on the designer's blogs, so make sure to leave lots of love for everyone along the stampede.
* Exclusive prize giveaways on Facebook, Twitter, and Instagram, 3 winners total.
Thanks for stopping by!!! And, congratulations to Kryssi and Angela, the Mamas behind Mama Elephant. You two are amazing! The love that you put into your products gets passed on to us when we use them. And then that love is shared to the people who get the cards we make. Ummm...cheesy, but true!


text:
Sponsor a Page
|Sponsoring a page gets you a banner with what ever you want to
say like the one below. It can be you name, you company name,
website name, blog name or what ever you want. All sponsorships are
subject to approval and may be rejected for any reason. If approved
banner will be placed within 2 business days of receipt of payment.
Exception will be made if problems arise after payment.
Immediate rejection will be result from an inappropriate massage or content at linked site. Should the sites content be deemed inappropriate after purchase, the sponsorship will be terminated without any refunds. This is mainly to prevent a sponsorship from being acquired by fraud. Inappropriate massage or content includes anti-Biblical, anti-Creationist, ant-Christian, and sexually explicit content. It also includes any massage or content deemed contrary to mission of the Genesis Science Mission and its related sites.
* indicates required fields.
Custom Search


text:
Pediatric Laboratories
- Microbiological Lab
Taking blood samples and washing off babies takes place at a specially designed space. Reliable cultures of urine and other biological fluids is also available. All tests are done on children over 2 months old.
- Cardiology Lab
ECG & TRIPLEX for children over 4 years old.
- X-Ray & Ultrasound lab
X-Rays and Ultrasounds are done on children over 30 days old after a consultation with the radiologist.


text:
Monday, April 26, 2010
Combat Baby Come Back Baby
A few weeks ago I went over to Williamson PR to check out their new crop of fall collections and immediately fell in love with a line called Candela. Designed by Natalia Kuks-Jacobs and Gabriela Perezutti-Isacson, the pieces are an interesting mix of romantic, old-fashioned even, but with a bit of a hard edge. For fall 2010, they were inspired by gorgeous American photographer Lee Miller, who started off as a successful model in the 1920s before going on to cover World War II for Vogue to much acclaim. This inspiration produced two completely perfect pieces of outerwear, pictured above.
Here, some images from their lookbook. You can buy their amazing spring shoes now at Shopbop or Revolve:
13 comments:
michael kors outlet online are tory burch sandals honest roshe run with burberry you? nike air max This uhren is oakley not oakley canada easy.
As hermes birkin saturated adidas superstar fats north face canada will ray ban outlet generally asics solidify uggs outlet when abercrombie and fitch chilled cheap nfl jerseys I nike shoes for women would hilfiger outlet expect celine handbags that barbour mens jackets if instyler ionic styler a true religion jeans outlet curry swarovski uk with uggs ghee gucci is michael kors refrigerated michael kors outlet online the air huarache fats mercurial superfly should burberry outlet online be babyliss pro solid ray ban sunglasses on louboutin shoes the baseball bats top jordan release by coach handbags factory the louis vuitton next jordans for sale day. tiffany I nike free am nike air max informed northface by raybans.com a pandora bracelet friend uggs outlet though, abercrombie.com that hollister online the baseball jerseys fat michael kors bags may
cheap air jordans
mont blanc pens
mulberry handbags
louis vuitton bags
cheap oakley sunglasses
nike huarache
replica watches
michael kors outlet online
toms wedges
louis vuitton handbags
ralph lauren uk
uggs boots for women
ray ban sunglasses outlet
nike air max
celine handbags
hollister co
soccer shoes
michael kors outlet clearance
louis vuitton outlet
uggs on sale
pandora jewelry
fake oakley sunglasses
cheap nfl jerseys
coach factory outlet
oakley sunglasses
jordan concords
coach factory outlet
michael kors handbags
beats by dre
burberry outlet
michael kors bag
celine outlet
louis vuitton outlet stores
michael kors outlet
canada goose jackets
oakley sunglasses cheap
polo ralph lauren
louis vuitton handbags
abercrombie and fitch
coach factory outlet
nike outlet storeReplyDelete
michael kors wholesale
tiffany and co jewelry
pandora jewelry
tiffany jewelry
ralph lauren outlet
rolex watches
nike air force 1
coach outlet online
michael kors handbags clearance
coach outlet store
michael kors uk
louis vuitton outlet
babyliss flat iron
cheap jordans
rolex watches
cheap replica watches
ralph lauren polo
ugg outlet
ray ban sunglasses
louis vuitton outlet
true religion jeans
ray ban sunglasses
ghd hair straighteners
ralph lauren polo shirts
coach outlet canada
coach outlet
swarovski crystal
beats by dr dre
prada shoes
ray-ban sunglasses
oakley sunglasses
michael kors outlet online
mcm outlet
prada handbags
cheap snapbacks
true religion outlet
true religion jeans
coach outlet online
true religion outlet
cheap jordansReplyDelete
timberland boots
michael kors uk
michael kors handbags sale
mulberry outlet
nike air max 90
cheap mlb jerseys
coach outlet online
louis vuitton bags cheap
michael kors handbags
fitflop clearance
tiffany outlet
mbt shoes outlet
toms outlet
nike huarache
michael kors handbags wholesale
pandora jewelry
louis vuitton handbags
michael kors outlet
true religion outlet
michael kors outlet online
louboutin pas cher
true religion jeans outlet
lacoste pas cher
coach outlet store
air force 1 shoes
true religion jeans
swarovski jewelry
rolex watches for sale
nike air force 1
cheap nhl jerseys
michael kors wholesale
louis vuitton handbags outlet
longchamp pliage
michael kors outlet
chrome hearts outlet onlineReplyDelete
true religion jeans
mulberry handbags sale
louis vuitton handbags
louis vuitton outlet stores
tory burch outlet
burberry outlet store
reebok shoes
hermes outlet
adidas outlet store
rolex uk
michael kors uk
versace sunglasses on sale
christian louboutin online
rolex watches,rolex watches,swiss watches,watches for men,watches for women,omega watches,replica watches,rolex watches for sale,rolex replica,rolex watch,cartier watches,rolex submariner,fake rolex,rolex replica watches,replica rolex
fitflops clearance
ray-ban sunglasses
true religion jeans
lacoste shirts
ralph lauren outlet
michael kors handbags
mlb jerseys
polo shirts
ferragamo outlet
cheap oakley sunglasses
mulberry outlet
coach outlet online
cheap replica watches
cartier watches for sale
ralph lauren uk
coach outlet
montblanc pens
ray ban sunglasses
cheap oakley sunglasses
cartier sunglasses


text:
About a week ago we attended a cute little "Minnie Mouse" themed birthday party. Besides the adorable birthday girl, they were handing out these headbands. Aren't they the darnest thing? I didn't make them but I definitely wanted to wear them. I thought these would be perfect for a quick Disney trip. My girlfriend has never been and I feel like a surprise trip is definitely due!
How To: Minnie Mouse Ears
1. Glue both ears onto the headband by placing the headband between the two circles and fold in.
2. Place the bow in the middle.
Voala! This also looks pretty cute without the ears. I felt "the ears" were a sweet effect because we all sort of got a little "Yoo Hoo" in our step from time to time.
Look coming soon! <3
with love, Teresita.


text:
|Element Dress// Forever 21 Belt// Gianni Bini Shoes// H&M Clutch
The year 2013 has been without a doubt one of the most life-changing years for me from spending my 1st full year in California to taking on a new career and having the opportunity to travel around the country. As the chapter closes on one capriciously momentous year and sights are set onto the next, I can't even begin to imagine what 2014 has in store. Being someone who never put much stock into the mercurially afflicted idea of New Year's resolutions, if anything, 2014 represents a progression of a time ripe with exciting opportunities. Aside from all of the effervescent glitz and glam that most of us will don tonight, I like to coordinate my NYE look on my outlook for the year ahead. So, this year I opted for a bright ecru lace dress and polished it off with a ultra-femme bow belt and bubblegum pink pumps. From this point of view, the future is looking pretty bright!