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Guide to Choosing the Best Home Warranty
Appliances and systems aren't built to last forever. While your house insurance will cover you in case disaster strikes your property, a home warranty steps in with repairs and replacements when the inevitable more mundane wear and tear takes its toll.
You're right to look for a home warranty, offering peace of mind in the event of an emergency. This policy will keep the cost down to an affordable monthly or annual fee which can be factored into your budget, with no surprises.
Which Factors Should I Consider Before Choosing my Home Warranty?
There are dozens of home warranty companies in the United States, and they are not all created equally. There are a few key considerations to check off your list before you settle on the right one for your home.
Ensure Value for Money
The general price bracket of a home warranty is between $350-$500 per year. Anything higher than this, and users should make sure they know why they are paying a premium. Perhaps the company offers flexible coverage, faster call-outs, or no service fees to explain the high price tag, which is fine if you value those factors more than a lower annual cost. If you're simply looking for appliance cover for example, Choice Home Warranty starts from as little as $33 per month.
The service fee for call outs can range from $60-$100. Getting a certified and trained contractor out quickly in the case of a breakdown can easily be three times this service fee, and a repair or replacement of an item like a refrigerator can be $600 -$1000 alone. Added to this the peace of mind of knowing that you have the help you need when an inevitable failure occurs, and you can see why homeowners and landlords opt for a home warranty for value for money.
As studies have shown that having a home warranty can help sell a house faster and at a higher price, a home warranty is also a sensible investment for anyone looking to sell their home. It's always worth asking when you sign up whether there are special joining offers. Select Home Warranty for example, offer two free months on sign up.
Guarantee Experienced and Specialist Support
In the vast majority of cases, your home warranty company will work with their own vetted and licensed contractors. There are pros and cons to this, and it will suit some customers more than others. If for example you prefer to research and review your own service providers, this might cause you more anxiety than peace of mind. If however, you prefer to have the hassle and research taken off of your hands, it's a perfect partnership. Some companies such as Advanced Home Warranty will allow you to pick your own contractor in certain situations.
It's worth asking around to make sure that you are choosing a home warranty where the service provided meets your needs. 24/7 coverage is a must-have, as well as a reasonable wait time for contractors. Will they send someone out within 24 or 48 hours, even at weekends or over public holidays? How do they vet the companies they use, and do they offer anything unique, such as female contractors on request?
Take Care to Read the Small Print
It's worth taking a look at your contract thoroughly or having a frank chat with customer service before you sign on the dotted line, to make sure that you're getting exactly what you think you're signing up for. Some of the common issues customers have with home warranties come from misunderstandings over coverage or terms. Here are some elements to smooth out from day 1.
You might be able to pick and choose the systems and appliances you want on your policy, but that doesn't mean that they are covered in all cases. Take a waste disposal for example. While this is a common item to see on a home warranty coverage list, it may only be covered for usual wear and tear, rather than if your toddler puts a non-food item inside. There will always be a claims limit to consider, too. Most companies cap this at about $2,000 or $3,000, while AFC Home Club goes above and beyond, offering as much as $7,500.
Pre-existing conditions
If you're buying a home with old appliances, check that your coverage will include repairs and replacements for these. If your washing machine is already out of working order when you sign up for your home warranty, it's unlikely to be included in your coverage.
Waiting period
Some companies will have a waiting time from the day you sign up, to when you can make a claim. This could be 2 or 3 weeks. If you're buying a home which already has coverage and are simply changing the name on the policy this may not be the case, and it might also be waived if you are moving your policy from one company to another.
Make sure you know what the process should be in the event of a breakdown. In most cases you will need to get in touch with your warranty and provider and let them take the reins from there. If you call out your own service contractors, you may not be eligible for reimbursements or further support on that issue.
Emergencies and unexpected fixes
A home warranty is there to cover you in case of emergency. That means that routine service checks and updates are usually not included either. If your air conditioning unit needs a regular maintenance check and cleaning for example, this is unlikely to be covered. Some companies such as Landmark offer upgrades like their 'No Fault' policy which allows you to claim on items which were installed improperly or are broken due to code violations or mismatched systems.
Visit Landmark Site
Where Can I Find This Information on the Top Home Warranty Companies?
One option is to visit every home warranty website, call for quotes, and discuss these considerations with their customer service teams. But this can be an arduous task. Our experts in home warranty can give you the lowdown on what makes each of the top home warranty companies in the United States shine. Whether you're looking for the most experienced contractors, the most all-inclusive coverage, or just the lowest price tag, our home warranty companies comparison chart is a great place to begin.
Advertising Disclosure
This site is a free online resource that strives to offer helpful content and comparison features to its visitors. Please be advised that the operator of this site accepts advertising compensation from companies that appear on the site, and such compensation impacts the location and order in which the companies (and/or their products) are presented, and in some cases may also impact the rating that is assigned to them. To the extent that ratings appear on this site, such rating is determined by our subjective opinion and based on a methodology that aggregates our analysis of brand market share and reputation, each brand's conversion rates, compensation paid to us and general consumer interest. Company listings on this page DO NOT imply endorsement. We do not feature all providers on the market. Except as expressly set forth in our Terms of Use, all representations and warranties regarding the information presented on this page are disclaimed. The information, including pricing, which appears on this site is subject to change at any time.


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When comparing Visual Basic vs Rebol, the Slant community recommends Rebol for most people. In the question"What is the best programming language to learn first?" Rebol is ranked 50th while Visual Basic is ranked 59th. The most important reason people chose Rebol is:
Almost natural language, for example: `write %out.html read http://google.com`.
Ranked in these QuestionsQuestion Ranking
Pro Capable language
Compiling to the same intermediate language as C# and compiling against the same .NET libraries, the differences between C# and VB are largely syntactic. Most VB features even have a 1:1 correspondence with C#.
Pro Huge community
Being Microsoft's introductory language, visual basic has a strong and large community.
Pro English-like syntax
Pro Flexible, won't get in the way of a beginner
A beginner developer can worry about the code logic first, and then learn the language. Visual Basic lets you do basically anything if you really want, while still having the power to be a fully statically-typed and object oriented.
Pro Sibling to C#
If you don't want to start with C# because of its high learning floor, you can start with VB. It is dead easy to switch between the two.
Pro Very simple and efficient language (in terms of number of lines of code)
Pro Easy to deal with Win32API
Using platform invoke, it's very easy to use the win32 api in vb.net. Neat and clean syntax. Not a mess like c# (meaning those "{}"s).
Pro Human friendly
Almost natural language, for example:
write %out.html read http://google.com.
Pro Homoiconic
Code is data, and data can be code. Rebol is based on a simple block data structure, used both for data and for the code itself. Blocks can be manipulated programmatically, and then evaluated as code. This makes metaprogramming easy in Rebol.
Pro Graphical user interface
Beginners are usually stuck making command-line applications in other languages, because GUIs are too hard. Rebol GUIs are easy enough to start with.
Pro Very simple syntax
Rebol's name came from "Relative Expression Based Object Language". Rebol is a functional language and everything is an expression that returns a value. Things that have to be baked into the grammar in other languages are simple function calls with block arguments in Rebol.
Pro Domain specific languages
Rebol's simple homoiconic syntax makes it easy to create "Rebol dialects"--domain-specific languages optimized for a particular purpose. The Rebol distribution includes many of these, and users are free to create more. These DSLs make tasks that would be complicated to express in other languages easy.
Con Bad reputation
Being a visual basic programmer, you're unlikely to be taken seriously by other programmers. According to the 2015 Stack Overflow survey, it was voted the second most dreaded language among developers.
Con Tends to get clunky due to prebuilt assets
Con No separators
A function call expression has no parentheses, and there are no separators between sequenced expressions, not even newlines. This means that you have to know the arity of every function in an expression to know how to parse it. It also means you can run into subtle, hard-to-find bugs if you don't provide enough arguments, since the result of the next expression will be passed instead.
Rebol 2's core is free (gratis) for commercial use, but the license prohibits modification, a violation of the DFSG. Rebol 3 is Free Software (Apache 2.0), but isn't production ready.

subset: commoncrawl


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South Korean retail firm GS Shop and early-stage venture capital firm Bon Angels have participated in a $4.5- million bridge funding for Vietnamese accommodations startup Luxstay, according to a press statement.
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Dispelling Myths for Poison Prevention Week
ST. PAUL, Minn. – This is National Poison Prevention Week - and in Minnesota, the goal is to dispel some common myths about whether and when to call the Poison Help Hotline or head to an emergency room.
More than 40,000 calls are made to the Minnesota Poison Control System every year, but according to Poison-Control Educator Lauren Prnjat, 90 percent of those cases could be handled over the phone, without calling an ambulance.
"People will be able to stay home, just with some treatment recommendations from the Poison Center," she said.
She cited another big misconception – that poison doesn't hurt many people – when in fact, it is the biggest cause of unintentional injury in the country.
Minnesota has a free, online Poison Prevention Training Program that takes about two hours to complete, and doesn't have to be finished all at once.
Prnjat said about 60 percent of accidental poisonings involve medications, which is why she suggests learning more about where they should be stored.
"We really recommend storing all medicines and other potentially poisonous products up high, out of sight and locked up, just so little ones don't accidentally get into it," she added.
It's just as important to dispose of medications and other hazardous products properly.
If you suspect someone has ingested something poisonous, call the Poison Help Hotline at 800-222-1222. It is available 24-7, treats cases like a medical record and there's no limit to the number of times a person can call.
National Poison Prevention Week runs through Sat., March 24.


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Human monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-κB in the nucleus secondary to increased IκBα, IκBβ, and IκBɛ degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IκB, we have sought to identify the regulatory domains of IκBα targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IκBα, we determined that persistent HIV infection of these cells targets the NH2 but not the COOH terminus of IκBα. Further analysis demonstrated that phosphorylation at S32 and S36 is necessary for HIV-dependent IκBα degradation and NF-κB activation. Of the putative N-terminal IκBα kinases, we demonstrated that the Iκκ complex, but not p90rsk, is activated by HIV infection and mediates HIV-dependent NF-κB activation. Analysis of viral replication in cells that constitutively express IκBα negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-κB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.
The Rel family of transcription factors plays an important role in the transactivation of several viral genes, including those of human immunodeficiency virus (HIV) type 1 (HIV-1) (25, 38). HIV-1 replication is regulated, in part, at the transcriptional level through the interplay of viral regulatory proteins with cellular transcription factors interacting with the viral long terminal repeat (LTR) (39). Since the identification and functional characterization of NF-κB cis-acting sequences within the HIV LTR (38), multiple studies have addressed the essential or dispensable role that this transcription factor plays in the reactivation of HIV from a true latent state and in the control of viral persistence (1, 10, 27, 31, 54, 55). Unfortunately, these studies have yielded conflicting results as to the role of NF-κB in these two steps of the viral life cycle in infected host cells. Differences in the type of host cells studied, HIV strain or genetic constructs used, and methodological approaches may explain these conflicting results.
Understanding the potential impact of NF-κB on the regulation of HIV latency has again become a priority, as recent studies suggest that NF-κB controls the reactivation of latent HIV in T cells from HIV-infected patients undergoing highly active antiretroviral therapy (19). An additional reservoir of HIV, separate from that of T cells harboring latent HIV, are cells of the monocyte lineage in which persistent viral replication is observed (36). During all stages of HIV infection, tissue macrophages provide a unique viral reservoir. In these cells, HIV persistently replicates in the absence of cytopathicity, escapes immune surveillance, and spreads via cell-to-cell contact (reviewed in reference 36). The important role of macrophages in AIDS pathogenesis has prompted the investigation of the molecular mechanisms which regulate HIV-1 persistence in these immune cells; one of these mechanisms is thought to be NF-κB dependent. Human macrophages express a constitutive level of NF-κB in the nuclei in the absence of exogenous cellular activation (25). This constitutive pool of nuclear NF-κB may be sufficient to allow for the initiation of HIV transcription immediately following infection. In addition, NF-κB may be required to further sustain persistent HIV replication, as multiple studies have demonstrated that persistent HIV replication in human macrophages or monocytes further upregulates NF-κB activity (2, 34, 40, 43, 48). However, the mechanisms by which HIV infection induces the activation of NF-κB in cells of the monocyte lineage remains unknown. Their identification would greatly enhance the understanding of this process and allow future testing of whether inhibition of the virus-induced activation of NF-κB may decrease viral persistence in cells of the monocyte lineage, hence eliminating an important reservoir of HIV replication in infected patients.
NF-κB is a heterodimeric protein composed of different combinations of members of the Rel family of transcription factors. A well-characterized form of NF-κB is a heterodimer of p50 and Rel-A (p65) (reviewed in references 3 and4). In the majority of cells studied, NF-κB is anchored in the cytosol by an inhibitory protein, IκB. An extensively studied IκB molecule, IκBα, has previously been shown to physically interact with NF-κB and to mask the nuclear localization signal of p50 and Rel-A (6). Following cell activation by one of an array of extracellular stimuli, IκBα undergoes a hyperphosphorylation event that renders the inhibitory molecule susceptible to degradation (7, 13, 47). This process results in the release of NF-κB, which undergoes nuclear translocation and drives gene transcription. Significant advances in the understanding of the molecular mechanisms and the structure-function of the phosphorylation and degradation of IκBα have recently been made. IκBα is constitutively phosphorylated at its COOH terminus by protein kinase-casein kinase II (PK-CK2) (5, 33, 35, 45). While the exact function of this phosphorylation is poorly understood, it appears that phosphorylation at the COOH terminus may play a role in the constitutively rapid protein turnover of IκBα in resting cells, thus potentially favoring a low degree of continuous NF-κB translocation. On the contrary, the N terminus contains two series (S32 and S36) which are required for stimulus-dependent phosphorylation (8, 9, 11, 15, 46, 49, 50, 52) by specific kinases, such as the ones present in the Iκκ complex (Iκκα and Iκκβ) or p90rsk(12, 16, 20, 24, 37, 42, 44, 53, 57). Phosphorylation at these sites primes IκBα to undergo ubiquitination and subsequent degradation by the proteosome.
Our group has previously determined that a mechanism whereby HIV infection results in an increase in the nuclear translocation of NF-κB involves modification and enhancement of IκBα turnover (34). The half-life of IκBα in HIV-infected cells is reduced by at least 50% compared to that in uninfected cells, and this fact directly correlates with increased levels of the nuclear pool of NF-κB in HIV-infected cells. That IκBα is the target of persistent HIV infection in monocytic cells has been further confirmed by other groups (14, 27); one of those groups further demonstrated that inhibition of IκBα degradation with proteosome inhibitors decreases HIV-induced NF-κB activation (27). What remain to be elucidated are the molecular mechanisms whereby HIV infection targets IκBα. Potential mechanisms regulated by HIV infection could target the COOH terminus of IκBα, favoring an enhanced "basal" turnover of this inhibitor molecule by activating PK-CK2 or the proteolytic machinery. Alternatively, HIV infection could result in the activation of other IκBα kinases that target S32 and S36, thus continuously priming IκBα to be degraded via the proteosome. Lastly, HIV could target other regulatory sites of IκBα or even other molecules, such as Rel-A, that could result in the dissociation of NF-κB from IκBα, thus rendering IκBα less stable.
To investigate these possibilities, we have used a cell model of monocytic cells in which persistent HIV replication results in NF-κB activation and a variety of genetically modified tagged IκBα molecules can be constitutively overexpressed. Our results indicate that HIV infection targets the NH2 terminus of IκBα, specifically S32 and S36, causing the enhanced degradation of IκBα and hence increased NF-κB nuclear translocation. The Iκκ complex kinase activity is selectively activated and is shown to mediate increased NF-κB activation in HIV-infected cells. In addition, we demonstrate that HIV-mediated NF-κB activation is not necessary to maintain viral persistence in monocytic cells.
Reagents and antibodies.Tumor necrosis factor (TNF) was purchased from Genzyme (Cambridge, Mass.) and stored in aliquots at −70°C. Cycloheximide was purchased from Sigma (St. Louis, Mo.) and stored at −20°C. Calpain inhibitor I (N -acetyl-Leu-Leu-norleucinal or ALLN) was purchased from Boehringer Mannheim Biochemicals (Indianapolis, Ind.), solubilized in ethyl alcohol, and stored in aliquots at −20°C. Bay 11-7082 (41) was purchased from Biomol (Plymouth Meeting, Pa.), solubilized in ethyl alcohol, and stored at −20°C. G418 was purchased from Calbiochem-Novabiochem Corporation (La Jolla, Calif.), solubilized in RPMI medium, and stored in aliquots at −20°C.
The expression of the Flag-tagged IκB constructs was monitored with an anti-Flag monoclonal antibody (Kodak, New Haven, Conn.). To control for equal loading of proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis, an anti–β-actin polyclonal antibody (Sigma) and an anti-p90rsk antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) were used. Polyclonal anti-IκBα serum was generated with a glutathione S-transferase (GST)–MAD3 fusion protein (34). The viral envelope protein gp120 was detected with an anti-gp120 polyclonal antibody (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Md.). The identity of the complexes binding DNA in the gel shift assays was determined with polyclonal antibodies against the different members of the Rel family (Santa Cruz Biotechnology). Antibodies against p90rsk, Iκκα, Iκκβ, Raf-1, and NF-κB were purchased from Santa Cruz Biotechnology.
DNA constructs.pCMV2-FLAG-IκBα-wt consisted of the full-length "wild-type" sequence of human IκBα (26) cloned into the SmaI-HindIII sites of pCMV2FLAG (Kodak) to generate N-terminally Flag-tagged IκBα-wt (Flag-IκBα-wt). Flag-IκBα-wt was used as a template for subsequent mutations and deletions by PCR-based techniques. Flag-IκBα-ΔN consisted of an N-terminal deletion lacking the first 37 amino acids. This construct was generated with the sense primer wt-FLAG (5′CGGAATTCATGGACTACAAAGACGAT3′) and the antisense primer wt-B (5′GGAATTCCTCATAACGTCAGACGCTG3′).EcoRI sites were created upstream and downstream of the coding sequence. Flag-IκBα-ΔC consisted of a C-terminal deletion lacking the last 40 amino acids and was generated with the sense primer wt-FLAG and the antisense primer ΔC (5′GCGAATTCTCAAAGGTTTTCTAGTGTC3′). This construct contained an EcoRI site downstream of the coding sequence. Flag-IκBα-2N consisted of the full-length sequence of IκBα-wt in which S32 and S36 were mutated to alanine residues. To generate these mutations, a sense primer with the sequence 5′GACGCAGGCCTGGACGCAATG3′ and an antisense primer with the sequence 5′CATTGCGTCCAGGCCTGCGTC3′ were used. Flag-IκBα-4C was created by mutation of S283, S288, S293, and T291 to alanine residues in the PEST sequence (35), cloning into theHindIII-EcoRI site of pCMV2FLAG, and then PCR amplifying with the sense primer wt-FLAG and the antisense primer wt-B. Flag-IκBα-wt, Flag-IκBα-ΔN, Flag-IκBα-ΔC, Flag-IκBα-2N, and Flag-IκBα-4C were then digested and cloned into the EcoRI site of SFFV-Neo under the transcriptional regulation of the Friend spleen focus-forming virus (SFFV) 5′ LTR (22). All of the cloning was verified by DNA sequencing.
Plasmid κB-luc contains three tandem copies of the κB motif of the HIV LTR cloned upstream of the minimal conalbumin-luciferase (con-luc) promoter reporter gene. Plasmid pBLCAT 2 is a mammalian reporter vector designed for the expression of chloramphenicol acetyltransferase (CAT) in mammalian cells transcribed by the minimal thymidine kinase (TK) promoter (Promega, Madison, Wis.). Plasmids Iκκα wt and kinase dead were kind gifts from Alain Israel, Institute Pasteur, Paris, France. Plasmids Iκκβ wt and kinase dead were obtained from M. Roth (Tularik, San Jose, Calif.). Iκκα kinase dead was generated by mutation of aspartic acid 144 to asparagine. Iκκβ kinase dead was created by mutation of lysine 44 to alanine. pcDNA3-Iκκ expression vectors were generated by cloning the cDNA of wild-type Iκκα or Iκκβ or its respective mutant into the cytomegalovirus expression vector pcDNA3 (Invitrogen).
Gene transfection and generation of cell lines.The U937 promonocytic cell line was purchased from the American Type Culture Collection and grown in RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum (Intergen), 1% glutamine, and 1% penicillin-streptomycin. To generate IκBα-expressing cell lines, 107 freshly thawed and exponentially growing U937 cells were resuspended in RPMI 1640 and electroporated with 20 μg of previously linearized DNA by use of a BTX cell electroporator at 250 V for 10 ms. U937 cells electroporated without DNA were used as controls. At 24 h after transfection, cells were resuspended in selection medium containing 5% fetal bovine serum and 700 μg of G418 per ml. After 3 to 4 weeks, upon the incipient growth of neomycin-resistant bulk cultures, cells were cloned by limiting dilution (30). Stable integration and expression of the transfected genes within each monoclonal population were verified by serial passages of the cultures in the absence of the selective antibiotic and by immunoblotting with anti-Flag antibodies.
Separate clones expressing equal levels of Flag-IκBα constructs were selected, and their CD4 surface expression was verified by flow cytometry analysis. Thereafter, three clones expressing each of the Flag-IκBα constructs were pooled, and exponentially growing cells were mock or HIV infected. The level of expression of each of the tagged IκBα constructs was confirmed before and during the period of HIV infection by immunoblotting of cytosolic extracts with anti-Flag antibodies.
Transient transfection of U937 cells was performed as follows. A total of 107 exponentially growing U937 cells were incubated with 4 μg of the con-luc or κB-con-luc reporter construct, 6 μg of the pDNA3-Iκκ construct, 4 μg of the pBLCAT2 reporter, and 300 μg of DEAE-dextran (Pharmacia, Piscataway, N.J.) per ml for 90 min at room temperature. Dimethyl sulfoxide (10%) was then added for 3 min, followed by extensive washing and plating at 0.5 × 107/cells/ml. Two days later, cells were harvested. Luciferase levels were measured with the Promega luciferase assay system, and CAT activity was measured with a CAT enzyme-linked immunosorbent assay kit (Boehringer).
HIV infection and measurement of HIV replication.U937 cells expressing SFFV, Flag-IκBα-wt, Flag-IκBα-ΔN, Flag-IκBα-ΔC, Flag-IκBα-2N, and Flag-IκBα-4C were infected with the HIV LAV-Bru strain as previously described (2, 34, 40). Briefly, 107 exponentially growing U937 cells were sedimented by low-speed centrifugation and resuspended overnight in 10 ml of infective supernatant containing 360 ng of p24 per ml. Mock-infected cells were used as a control. After 24 h, cells were extensively washed and resuspended in culture medium. Cells were passaged twice a week at 0.25 × 106 cells/ml and used from day 30 through day 90 postinfection. During this period, cell supernatants were collected, precleared by centrifugation at 1,500 rpm for 5 min at 4°C, and stored for future analysis of HIV p24 antigen content by an enzyme-linked immunosorbent assay (Coulter-Immunotech Immunology, Westbrook, Maine). At least eight consecutive infections were used for each of these experiments. All the cell lines studied maintained HIV persistence and 100% viability during the study period, except for the U937 clones expressing Flag-IκBα-ΔC, which maintained viability and normal growth while uninfected which underwent immediate and massive cytopathicity upon HIV infection in four consecutive attempts. Therefore, a U937 cell line expressing Flag-IκBα-ΔC could not support a persistent HIV infection. In addition, in some experiments, HIV gp120 expression was determined by immunoblotting with anti-gp120 antibodies.
Nuclear and cytosolic extracts, electrophoretic mobility shift assays, and immunoblotting.Nuclear and cytosolic extracts were prepared by a modification of the method of Dignam et al. (17). A total of 107 cells were washed with ice-cold phosphate-buffered saline and then with buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl). Cells were then lysed for 10 min on ice in the same buffer containing 0.1% Nonidet P-40, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg of aprotinin per ml, 2 μg of leupeptin per ml, and 2 μg of pepstatin per ml. After centrifugation, cells were washed twice with buffer A. Nuclei were pelleted by centrifugation, lysed by resuspension in 25 μl of buffer C (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, DTT, PMSF, aprotinin, leupeptin, pepstatin) and rotated at 4°C for 30 min. After centrifugation, the supernatants were diluted in 50 μl of buffer D (20 mM HEPES, 20% glycerol, 0.05 M KCl, 0.2 M EDTA, DTT, PMSF, aprotinin, leupeptin, pepstatin) and stored at −70°C.
For electrophoretic mobility shift assays, 6 μg of nuclear extract was incubated with a [γ-32P]ATP-labeled double-stranded NF-κB oligonucleotide probe in 15 μl of DNA binding buffer for 15 min at room temperature as previously described (2, 34, 40). Components of the HIV-induced DNA binding protein complexes were identified by incubation of the extract with specific polyclonal antibodies against p50 and Rel-A prior to addition of the labeled probe. The resulting protein-DNA complexes were resolved on a 5% polyacrylamide gel and visualized by autoradiography.
To characterize the level of expression of the Flag-IκBα constructs in uninfected and infected cells, 40 μg of cytosolic protein was analyzed by SDS–10% PAGE. Proteins were transferred to Immobilon-P membranes (Millipore) by standard procedures and blotted with an anti-Flag monoclonal antibody, followed by incubation with rabbit anti-mouse immunoglobulin G (Pierce) and then horseradish peroxidase (Amersham, Buckinghamshire, England). Immunoreactive proteins were detected with an ECL Western blotting detection kit (Amersham). β-Actin and p90rsk were used as internal controls for equal loading in all experiments.
Preparation of recombinant IκBα.The IκBα-MAD3 cDNA (26) plasmid was obtained from Cetus Corporation and was used as a template for subsequent PCR amplification.
The amino-terminal IκBα-MAD3 (positions 1 to 54) sequence was amplified with wild-type primer A (5′CGGGATCCATGTTCCAGGCGGCCGAG3′) as the sense primer, creating a BamHI site upstream of the coding sequence, and wild-type primer B (5′GGAATTCCTCAGCGGATCTCCTGCAGCT3′) as the antisense primer, creating an EcoRI site downstream of the coding sequence. An S32/36A double mutant was amplified from the full-length cDNA by use of primers to create alanines at S32 and S36. Following digestion withBamHI-EcoRI, these sequences were ligated into pGEX-KG (derived from pGEX-2T, from Pharmacia, Piscataway, N.J.). These constructs were transformed into Escherichia coli DH5α cells, which were grown exponentially. After 60 min of stimulation with isopropylthiogalactopyranoside (Sigma), cells were lysed. Proteins were isolated by affinity chromatography on glutathione-bonded 4% cross-linked agarose (Sigma). The purity of GST-IκBα (positions 1 to 54) containing the first 54 amino acids of IκBα and GST-IκBα (positions 1 to 54) containing S32/36A was analyzed by SDS–10% PAGE and subsequent Coomassie blue staining. The purity of both proteins was greater than 90%.
Immunoprecipitation of IκBα kinases and in vitro kinase assays.Whole-cell extracts were prepared for immunoprecipitation and in vitro kinase assays as follows. Aliquots of 107exponentially growing U937 cells were washed twice with cold phosphate-buffered saline, resuspended in lysis buffer containing 40 mM Tris-HCl (pH 8), 0.3 M NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM NaF, 10 mM p-nitrophenyl phosphate (PNPP), 10 mM β-glycerolphosphate, 300 μM sodium orthovanadate, 1 mM DDT, 2 μM PMSF, 10 μg of aprotinin per ml, 1 μg of leupeptin per ml, and 1 μg of pepstatin per ml, and incubated on ice. Cells were then centrifuged at 12,000 × g for 15 min at 4°C. The resultant supernatant contained total cellular proteins, which were quantitated with a Bio-Rad protein assay.
For immunoprecipitation of the Iκκ complex, p90rsk, or Raf-1, 100 μg of cell extract was incubated with anti-Iκκα, anti-Iκκβ, anti-p90rsk, or anti–Raf-1 antibodies for 1 h at 4°C, after which protein A-agarose beads (Life Technologies, Gaithersburg, Md.) were added for 1 h. The beads were then washed three times with 0.5 M NaCl-based lysis buffer, followed by one wash with a buffer containing 50 mM Tris-HCl (pH 7.4) and 40 mM NaCl. The washed beads were then incubated in 15 μl of kinase buffer (20 mM HEPES [pH 7.4], 2 mM MgCl, 2 mM MnCl, 10 μM ATP, 10 mM NaF, 10 mM PNPP, 10 mM β-glycerolphosphate, 300 μM sodium orthovanadate, 2 μM PMSF, 10 μg of aprotinin per ml, 1 μg of leupeptin per ml, 1 μg of pepstatin per ml, 1 mM DTT) with 2 μg of GST-IκBα (positions 1 to 54) or GST-IκBα (positions 1 to 54) containing S32/36A and 0.1 μCi of [γ-32P]ATP. The kinase reaction was performed for 30 min at 30°C, and samples were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and exposed to film.
Increased degradation of Flag-IκBα-wt in HIV-infected cells.To confirm the expression and functionality of the Flag-IκBα constructs, pooled clones expressing equal levels of Flag-IκB constructs were treated or not treated with TNF, followed by the analysis of the cytosolic extracts by SDS-PAGE and immunoblotting with anti-Flag antibodies. As shown in Fig.1A, TNF stimulation led to the rapid hyperphosphorylation and subsequent degradation of Flag-IκBα-wt. In contrast, Flag-IκBα-ΔN and Flag-IκBα-2N were refractory to TNF-induced hyperphosphorylation and subsequent degradation. Flag-IκBα-4C behaved similarly to Flag-IκBα-wt in that it was susceptible to TNF-mediated hyperphosphorylation and degradation. These results confirm that the constitutively overexpressed Flag-IκBα molecules are regulated as previously described for native IκBα and highlight the functional relevance of the N terminus containing S32 and S36 in TNF-induced IκBα hyperphosphorylation and degradation.
As expected (34), persistent HIV infection of U937 cells resulted in decreased cytosolic levels of native IκBα. Moreover, IκBβ and IκBɛ protein levels were also significantly decreased in HIV-infected cells compared to uninfected cells (Fig. 1B).
Having determined that overexpressed Flag-IκBα constructs function similarly to native IκBα upon stimulation with known inducers of NF-κB and that HIV infection of U937 cells results in decreased steady-state levels of endogenous IκB, we next investigated whether Flag-IκBα-wt is also a target of HIV infection. Immunoblotting of cytosolic fractions from mock- and HIV-infected cells expressing Flag-IκBα-wt was performed with anti-Flag antibodies. U937 cells transfected with the parental empty retrovirus vector (SFFV) were also mock or HIV infected and used as controls. As shown in Fig. 1C, the steady-state protein levels of Flag-IκBα-wt were decreased in the cytosolic fractions of HIV-infected cells compared to mock-infected cells, confirming that HIV infection decreases the cytosolic levels of Flag-IκBα and indicating that tagged IκBα constructs can be used to study the regulatory domain(s) targeted by persistent HIV infection in monocytes.
Having previously demonstrated that the decreased level of native IκBα is a result of the enhanced rate of IκBα degradation in persistently HIV-infected monocytes, we investigated whether this process also accounted for the decreased level of Flag-IκBα-wt in infected cells. The half-life of Flag-IκBα-wt was estimated by immunoblotting Flag-IκBα-wt from cytosolic fractions from mock- and HIV-infected cells treated for different time periods with cycloheximide. As shown in Fig. 1D, the turnover of Flag-IκBα-wt was increased in HIV-infected cells compared to mock-infected cells. The half-lives of Flag-IκBα-wt calculated from Fig. 1D were found to be approximately 60 min in HIV-infected cells and 128 min in uninfected cells (Fig. 1E).
The NH2 terminus but not the PEST sequence present in the COOH terminus of IκBα is necessary for IκBα degradation by HIV infection.To characterize which of the regulatory domains of IκBα is targeted by HIV infection, we first focused on the NH2-terminal domain of IκBα. We analyzed the turnover and half-life of Flag-IκBα-ΔN. U937 cells stably transfected with the empty retrovirus vector (SFFV), Flag-IκBα-wt, or Flag-IκBα-ΔN were mock or HIV infected. The half-lives of these constructs were measured by analyzing the levels of the Flag-IκBα constructs in cytosolic extracts from cell cultures treated for different time periods with cycloheximide (as for Fig. 1). As shown in Fig. 2A, Flag-IκBα-ΔN was very stable not only in mock-infected but also in HIV-infected U937 cells, with the resulting half-lives being estimated at greater than 4 h (Fig. 2B). The enhanced stability of Flag-IκBα-ΔN in both mock- and HIV-infected cells contrasts with the more rapid turnover of Flag-IκBα-wt in mock-infected cells and even more rapid turnover in HIV-infected cells (Fig. 1D and Fig. 2A). These results indicate that the increased degradation of IκBα that ensues in HIV-infected cells appears to be dependent on the NH2-terminal domain of the molecule. In addition, these results highlight the potential relevance of this IκBα domain in the regulation of the basal turnover of IκBα in unstimulated transformed cells.
Previous studies have demonstrated that mutation of S283, S288, T291, and S293 to alanines eliminates the constitutive phosphorylation of IκBα mediated by PK-CK2 and may influence the turnover of IκBα (5, 33, 35, 45). Based on this information, we analyzed the turnover and half-life of Flag-IκBα-4C in mock- or HIV-infected U937 cells and compared them to those of Flag-IκBα-wt. In mock-infected U937 cells, the basal turnover of Flag-IκBα-4C was slightly longer than that of Flag-IκBα-wt (Fig. 3), suggesting a potential role of the C-terminal amino acids S283, S288, T291, and S293 in the basal turnover of IκBα in unstimulated monocytic cells. The half-life of Flag-IκBα-4C was shorter in HIV-infected cells than in mock-infected cells but was similar to the half-life of Flag-IκBα-wt in HIV-infected cells (Fig. 3). These results demonstrate that the amino acids present in the PEST sequence of IκBα are not involved in the HIV-mediated degradation and turnover of IκBα.
HIV-induced degradation of Flag-IκBα requires phosphorylation at the NH2-terminal residues S32 and S36.Several studies have identified S32and S36 as targets of inducible IκBα kinases (8, 9, 11, 15, 46, 49, 50, 52). As shown in Fig. 1A, mutation of S32 and S36 to alanines yields an IκBα construct that is refractory to the hyperphosphorylation and subsequent degradation triggered by TNF in U937 cells. Having identified the NH2-terminal domain of IκBα as a target of HIV-induced degradation, we next questioned whether S32 and S36 could be the amino acids that are targeted by HIV infection. For this, we investigated the half-life and turnover of Flag-IκBα-2N in mock- and HIV-infected U937 cells and compared them to the half-life and turnover of Flag-IκBα-wt. Following the same experimental design as that used for Fig. 2 and 3, we observed that in mock-infected cells, mutation of S32 and S36 to alanines significantly prolonged the half-life of IκBα (greater than 5 h) compared to the more rapid turnover of Flag-IκBα-wt (Fig. 4). This very low rate of basal degradation of Flag-IκBα-2N is similar to that observed for Flag-IκBα-ΔN (Fig. 2). Relevant to the focus of this study, we demonstrate that the half-life of Flag-IκBα-wt is significantly reduced in HIV-infected cells compared to mock-infected cells and that Flag-IκBα-2N was refractory to HIV-mediated IκBα degradation (Fig. 4). These results confirm that S32 and S36 are the IκBα amino acids targeted by persistent HIV infection to result in enhanced degradation of IκBα.
S32 and S36 are the targets of IκBα kinases that are activated by a variety of stimuli, such as inflammatory cytokines, and phosphorylation at these amino acids renders IκBα susceptible to degradation by the proteosome (8, 9, 11, 12, 15, 16, 24, 29, 37, 42, 43, 46, 49, 50, 52, 53, 57). To investigate whether HIV infection results in the hyperphosphorylation of IκBα, mock- or HIV-infected U937 cells expressing Flag-IκBα-wt were treated with the proteosome inhibitor ALLN for 3 h, after which cytosolic extracts were separated by SDS-PAGE and immunoblotted with anti-Flag antibodies. To control for the accurate detection of hyperphosphorylated IκBα, mock-infected Flag-IκBα-wt-expressing U937 cells were treated or not treated with TNF and/or a pharmacological inhibitor previously shown to inhibit the TNF-induced hyperphosphorylation of IκBα (Bay 11-7082) (41). As shown in Fig. 5 (upper panel), a more slowly migrating form of Flag-IκBα-wt was observed in mock-infected, TNF-treated cells, specifically in the presence of ALLN (lanes 3 and 4). In HIV-infected Flag-IκBα-wt-expressing U937 cells, a more slowly migrating form of Flag-IκBα-wt was observed only when ALLN was used (compare lanes 6 with lane 5). These effects are dependent on the presence of S32 and S36 in the Flag-IκBα construct, as their mutation to alanines abrogated both TNF-induced and HIV-dependent IκBα hyperphosphorylation (Fig. 5, lower panel). Altogether, these results indicate that the enhanced degradation of IκBα that is observed in HIV-infected monocytes is a result of specific hyperphosphorylation of IκBα at S32 and S36. Whether the differences in the kinetics of IκBα hyperphosphorylation at S32 and S36between transient stimuli, such as TNF, and chronic stimuli, such as persistent HIV infection, are due to the use of different IκBα kinases or simply different upstream control mechanisms is currently unknown.
The Iκκ complex mediates HIV-dependent IκBα degradation and NF-κB activation.Two kinases in the Iκκ complex (Iκκα and Iκκβ) have recently been shown to phosphorylate S32 and S36 of IκBα and to be the targets of inflammatory cytokines, such as TNF and interleukin 1 (12, 16, 29, 37, 42, 53, 57). Having identified S32 and S36 as the regulatory amino acids of IκBα which are targeted by HIV, we questioned whether the Iκκ complex is activated by HIV infection and mediates the increased levels of nuclear NF-κB activation in infected cells. Mock-infected and persistently HIV-infected U937 cells were lysed, followed by immunoprecipitation of the Iκκ complex, p90rsk, or Raf-1. The kinase activities of these immunoprecipitates were analyzed in an in vitro kinase reaction with GST-IκBα (positions 1 to 54) or GST-IκBα (positions 1 to 54) containing S32/36A as a substrate. In HIV-infected samples, increased IκBα kinase activity was present in the Iκκ complex immunoprecipitate but not in the p90rsk (Fig.6A) or the Raf-1 (data not shown) immunoprecipitate. This kinase activity was specific for S32 and S36, as their mutation eliminated the basal and HIV-induced Iκκ complex activity. Also, as shown in Fig.6A, there was no difference in the amounts of Iκκ complex immunoprecipitated with anti-Iκκα antibodies in mock- and HIV-infected U937 cells, thus eliminating the possibility that HIV infection simply increases the pool of Iκκ kinases. These data indicate that HIV infection activates the Iκκ complex, resulting in phosphorylation at S32 and S36.
The potential relevance of the Iκκ complex in mediating the HIV-dependent activation of NF-κB was further analyzed in transient transfection experiments. Transcription from an NF-κB-dependent luciferase reporter gene was analyzed with both mock- and HIV-infected U937 cells in the presence or absence of wild-type or dominant negative forms of Iκκα and Iκκβ. A minimal TK promoter driving the expression of CAT was used to normalize for transfection efficiency differences that might be present between mock- and HIV-infected cells. The results of these experiments demonstrated that the increased NF-κB activity that is observed in HIV-infected cells is reduced by an Iκκβ dominant negative expression vector but not by a dominant negative form of Iκκα or the wild-type form of either kinase (Fig. 6B). Altogether, these studies demonstrate that the Iκκ complex is activated by HIV infection and mediates virus-induced IκBα hyperphosphorylation and NF-κB activation.
HIV-1 replication in U937 cells expressing different transdominant mutants of IκBα.Having determined that S32 and S36 of IκBα are required for HIV-mediated IκBα degradation, we next questioned whether U937 cells expressing Flag-IκBα-ΔN or Flag-IκBα-2N (constructs that are refractory to HIV-dependent degradation) would inhibit HIV-mediated NF-κB activation, and if so, whether this inhibition would result in decreased viral replication. Nuclear extracts and cell-free supernatants were obtained from mock- or HIV-infected cells stably transfected with the SFFV vector, Flag-IκBα-ΔN or Flag-IκBα-2N at the same time as the cytosolic fractions were analyzed to determine the half-lives of the respective IκBα constructs (Fig. 2 and 4). Nuclear extracts were analyzed by a gel shift assay with an oligonucleotide containing NF-κB DNA binding motifs, and viral replication was monitored by measuring p24 levels in culture supernatants. As shown in Fig. 7A and B, left panels, HIV infection of SFFV-expressing U937 cells led to nuclear translocation of a DNA binding protein complex composed of p50 and Rel-A (p65); this finding was not observed in HIV-infected cells expressing either Flag-IκBα-ΔN or Flag-IκBα-2N (Fig. 7A and B, right panels). These observations directly correlate with the inability of these two Flag-IκBα constructs to undergo HIV-mediated degradation, as demonstrated in Fig. 2 and 4.
The levels of HIV replication in the cells expressing SFFV, Flag-IκBα-ΔN, or Flag-IκBα-2N were then analyzed by measuring HIV p24 levels in supernatants from the same cultures as those used to study the IκBα half-life (Fig. 2 and 4) and NF-κB nuclear translocation (Fig. 7A and B). The results of these experiments indicated that there was no significant reduction in the levels of p24 in supernatants of U937 cells expressing Flag-IκBα-ΔN or Flag-IκBα-2N compared to supernatants of control cultures (SFFV expressing) (Fig. 7C).
Using an HIV-susceptible promonocytic cell line which can support persistent viral replication, we have determined that the IκBα residues S32 and S36 and the Iκκ complex (12, 16, 29, 37, 42, 53, 57) are required to mediate HIV-dependent IκBα degradation and, hence, NF-κB activation. The identification of a transdominant negative IκBα molecule which is refractory to HIV-dependent degradation and thus is capable of blocking the HIV-mediated activation of NF-κB extends and confirms previous studies from our group indicating that IκBα is a target molecule and that persistent HIV infection leads to increased NF-κB activation (34). In addition, it provides supporting data that HIV-mediated NF-κB (p50/p65) activation is not necessary to support viral persistence in the U937 monocytic cell line.
The use of pooled clones of monocytic cells that constitutively express genetically modified IκBα constructs has proven to be a valuable tool with which to study the role of NF-κB replication in HIV persistence. Different from punctual stimuli (inflammatory cytokines or transient expression of human T-cell leukemia virus type 1tax), the activation of NF-κB by HIV infection is dependent on the establishment of viral persistence, achieved only after 6 to 10 days of viral infection (2, 34, 40). Due to this unique virus-host cell interaction, the experimental approaches which can be utilized to address the mechanisms by which HIV activates NF-κB have been significantly limited. Previous attempts have used nonmonocytic cell lines which are highly susceptible to gene transfection, such as 293 or COS-7 cells (28, 54, 55). However, such studies, rather than focusing on HIV persistence in stimulating NF-κB activation, have addressed the role of IκBα in controlling the reactivation of HIV from latency or in inhibiting the initiation of viral replication.
The use of our genetically modified monocytic cells has allowed us to address the mechanism(s) by which HIV leads to NF-κB activation and then to study the role of IκBα in controlling viral persistence. To ensure the relevance of this model, significant efforts were made to verify the maintenance of stable IκBα expression and CD4 expression and the functionality of the tagged overexpressed IκBα clones throughout the infections (months). In addition, as was the case for the Flag-IκBα-2N construct, experiments were repeated with each individual clone separately to verify that the results obtained with a pool of three clones were not due to the overgrowth of a single clone. The level of expression of Flag-IκBα-ΔN and Flag-IκBα-2N was significantly higher in HIV-infected cells than in uninfected cells. This result may be due to increased transcription from the SFFV retrovirus promoter in HIV-infected U937 cells. As both the IκBα N-terminal deletion and the IκBα N-terminal mutation are refractory to HIV-induced degradation, over time the steady-state Flag-IκBα-ΔN and Flag-IκBα-2N protein levels may lead to an increase in the levels of the transgene.
The reduced half-life of IκBα observed in HIV-infected cells differs significantly from the very short half-life of IκBα observed following TNF stimulation. This observation initially led to the hypothesis that sites other than S32 and S36 are targeted by HIV infection. However, the finding that S32 and S36 are required for enhanced IκBα turnover in HIV-infected cells, together with the observation that the Iκκ complex (12, 16, 29, 37, 42, 53) is activated and mediates NF-κB activation in HIV-infected cells, demonstrates a shared utilization of this kinase complex and this IκBα domain in mediating NF-κB activation by unrelated stimuli. What accounts for the significant difference in IκBα half-lives with two separate stimuli, i.e., TNF (5 to 10 min) and HIV (50 to 60 min), which share the same IκBα regulatory domain and kinase complex, is unknown. We have previously demonstrated that within an HIV-infected U937 cell culture, ≥90% of cells express intracytoplasmic HIV p24, thus excluding the possibility that a small subpopulation that is actively HIV infected results in a dilution effect (34).
From the available data, we conclude that it is a lower degree of Iκκ complex activation by HIV infection that correlates with the smaller amount of hyperphosphorylated IκBα and the slower IκBα turnover in HIV-infected cells than in TNF-treated cells. Whether the lower degree of Iκκ activation is secondary to the utilization of different secondary messengers that lie upstream of Iκκ is unknown. It is also plausible that HIV infection targets regulatory processes that control the basal level of Iκκ activity rather than its "inducible" activity. Recent data indicate that protein phosphatase 2A (PP2A) dephosphorylates Iκκα, resulting in a decrease in its kinase activity (16) and explaining the NF-κB-activating function of the PP2A inhibitor okadaic acid (16). It is theoretically possible that HIV infection inhibits PP2A, resulting in a higher "basal" Iκκ activity which is separate from the TNF-inducible Iκκ activity. Previous studies from our group have identified p21ras (21) and the atypical protein kinase C isoforms ξ and ι (20) as essential components of NF-κB activation mediated by HIV infection. Whether these secondary messengers target the Iκκ complex is unknown, but recent advances in the characterization of Iκκ complex regulation will now enable the study of the role of these secondary messengers in HIV-induced NF-κB activation and their linkage to the activation of the Iκκ complex.
The apparent lack of dependence of viral persistence on HIV-mediated NF-κB (p50/p56) activation is a significant conclusion from this study. While several groups, including ours, have consistently demonstrated that persistent HIV infection of monocytic cells results in the selective activation of NF-κB (p50/p65) (2, 34, 40, 43), it has not been possible to clearly demonstrate that the HIV-dependent activation of the p50/p65 heterodimer is necessary to maintain viral persistence in such cells. Attempts to test this question have been made with proteosome inhibitors (14, 27). These compounds were shown to inhibit HIV-dependent IκBα degradation and, hence, p50/p65 heterodimer nuclear translocation, which correlated with a reduction in HIV replication. Because proteosome inhibitors may inhibit a variety of additional cell functions and, potentially, specific steps of the HIV cycle, the role of HIV-dependent activation of NF-κB in regulating viral persistence in monocytic cells remains to be fully clarified. The use of genetic approaches such as the one described in this study allows for more specific inhibition of NF-κB. Interestingly, overexpression of transdominant mutants of Flag-IκBα is sufficient to inhibit the nuclear translocation of additional NF-κB (p50/p65) complexes that may result from the enhanced HIV-dependent degradation of IκBβ and/or IκBɛ, indicating that an IκBα negative dominant molecule overrides the functional impact of the two other IκB molecules, at least in HIV-infected monocytic cells.
Our results indicate an apparent dispensable role of HIV-triggered NF-κB (p50/p65) activation in maintaining viral persistence in U937 cells. Whereas it is still possible that HIV-induced NF-κB (p50/p65) activation is indeed involved in controlling viral persistence, inhibition of this mechanism may have allowed for the utilization of complementary mechanisms to maintain viral persistence in the absence of constitutive or HIV-induced nuclear translocation of the p50/p65 heterodimer. Other members of the NF-κB family or alternative transcription factors, such as Sp1, may be constitutively present in the nuclei of host cells or selectively activated by HIV infection (51) and thus may compensate for the lack of nuclear translocation of p50/p65 dimers observed in the clones expressing IκBα negative transdominant molecules. Rel-B nuclear translocation is thought to be refractory to IκBα inhibition (18, 32). Therefore, either the constitutive presence of Rel-B in nuclei or its potential nuclear translocation following HIV infection might serve to compensate for a lack of HIV-induced p50/p65 in the nuclei of infected cells expressing IκBα negative transdominant molecules. While our gel shift assay experiments with NF-κB DNA concatemers did not demonstrate any NF-κB DNA binding activity in HIV-infected cells expressing IκBα S32 and S36 mutants, the detection of DNA binding activity of Rel-B may have been elusive, as previously suggested. Infection of U937 cells which express an IκBα S32/36A mutant with HIV provirus lacking the NF-κB cis-acting motifs could help clarify the potential role of nuclear proteins which could bind and regulate transcription through the NF-κB cis-acting sequences (23).
While the above hypothesis can be adequately tested with U937 cells, it is ultimately necessary to test the role of the HIV-mediated activation of NF-κB in regulating viral persistence within true physiological host cells, such as human macrophages. In these cells, persistent HIV infection results in the continuous activation of NF-κB (34); thus, it is mandatory to test whether its inhibition alters viral persistence in these host cells. Unfortunately, the current lack of specific inhibitors of IκBα phosphorylation at S32 and S36 and the limitation of applying genetic approaches, such as those used here with U937 cells, to primary human macrophages preclude the conclusion that the observations derived from promonocytic cells apply to human macrophages.
With the identification of the Iκκ complex and IκBα S32 and S36 as targets of persistent HIV infection in monocytes, HIV infection can now be added to the growing list of NF-κB activators that utilize this recently identified complex of N-terminal IκBα kinases. In addition, differences in the degree of Iκκ activation and hence in IκBα turnover between a "chronic" stimulus, such as persistent HIV infection, and other, more "punctual" ones suggest that there may be different means of activating the Iκκ complex within the same cell. Lastly, using genetically modified IκBα constructs, we have been able to demonstrate that the HIV-mediated activation of NF-κB is not necessary to maintain viral persistence. Thus, future efforts should be directed at exploring the complementary role of other NF-κB family members or additional transcription factors in regulating viral persistence in human macrophages as an important cell reservoir of HIV infection.
- Received 16 September 1998.
- Accepted 27 January 1999.
- Copyright © 1999 American Society for Microbiology


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They have been hiding in the back of my closet for 3 1/2 years (in which I have moved twice). I just couldn't bring myself to part with them, hoping against hope that someday I could again wear them proudly. I have longingly gazed upon their well-worn seams and subtle wash, even trying them on ocassionally only to be disappointed. At times I feared I would never wear them again.
Today was the day. Yes, you heard me right. I am wearing my pre-pregnancy jeans. Pre-Leah-pregnancy jeans.
And they look dang good.
Chloe + Isabel Changed My Life
2 years ago


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By JetBoy
Friday 21st January, 1966
Mommy woke me up with a kiss. I put my arms around her neck and kissed her back with my tongue. Then her mouth kind of slid down my neck and went between my titties. I loved feeling her lips on my nipples. It made me laugh when she gave them little play bites.
Her hand went down until it was between my legs. I was holding my breath waiting for Mommy to use her fingers on me. But then she didnt do it! She just smiled at me and touched the tops of my legs instead. I was wiggling around under her and wanting so bad to feel her fingers. Then she did touch me down there. I made happy sounds in her mouth while she kissed me, and pretty soon I was coming.
I hugged her after and told her she was the best Mommy a girl could have. She gave me a funny smile and looked right at me as she lay down and pulled up her skirt.
She wasnt wearing panties! Her legs went apart to show me her pussy. I stared at her lovely curly hair and the shiny pink parts of her. It was like a pretty flower in the morning when it's all wet with dew.
She said, You know what to do, Susie. Her voice was so soft.
My hand was all shaky when I put it on her leg. Mommy's skin was warm and smooth. When my fingers touched her dark curly hair I thought my heart would burst, my chest felt so tight.
Her hand showed me where to go, she helped me find her hole. I knew what she wanted. I put two fingers into her, feeling them go inside her body. She was so juicy and hot in there!
She said, Put them in all the way. Mommy had opened her top, and I could see her titties go up and down when she breathed. I wanted to kiss them.
I pushed my fingers as deep into Mommy's pussy as they would go. She said, Now move them in and out. Her voice was all funny like she was running fast. I did what she told me, my arm moving back and forth. Her pussy got really wet, much more than I do.
I guess she was really excited cause she came right then, shivering all over like the room was cold, it wasn't though. She was moaning and breathing hard, and I just kept sliding my fingers in and out like she wanted. Then she put her hand on top of mine, asking me to stop cause she had enough.
I was thrilled and VERY proud. I had made Mommy come all by myself!!
Thank you angel, she said, smiling at me before giving me a mouth open kiss. I stuck my tongue into her mouth to play with hers, and we kissed for a long time before moving apart.
Then Mommy did something that really suprised me. She brought my hand up to her face and licked my wet fingers! I'm sure my mouth was hanging open while I watched her taste her pussy. I never would of thought of doing something like that, but she sure looked like she was enjoying it.
She let my hand go with a little kiss and smiled at me. I wanted to taste too, so I raised my fingers to my mouth and licked one.
Last year at Christmas Mommy had been drinking wine and I asked her for a sip. The taste of her pussy is kind of like wine. Strong and sweet with a smell that made my head feel light. I liked it.
Then Mommy gave me a serious look and told me that I cant tell anyone at all about what we just did, specially Daddy. She said that it had to be our special secret or we couldnt make love any more.
My heart started beating fast when she said that. I didnt think about it until then, but that's what we had been doing. Making love. Its funny how I only understood it when Mommy said the words.
Tomorrow Mommy is taking me back to the ladies shop. She says they have more panties and things.
I keep putting my fingers in my mouth to get more of the taste of Mommy's pussy. Its pretty much gone now, but I can still smell her a little bit.
I wonder if I taste like her? I think after I close this and turn out the light, I'll touch myself and find out if I do or not.
Saturday 22nd January, 1966
The shop hadnt changed since our last visit, though I had so many new things happen since then that it seemed different any how. Like before, Miss Decker put the CLOSED sign up in the window and locked the door shut. Then she took my hand and brought Mommy and me into the little back room.
She asked how I was when we sat down, smiling at me. But it was Mommy who answered her, and she made my face go red as she told Miss Decker about how much I was growing.
Like last time, Mommy told me to stand and take all my clothes off so Miss Decker could measure me again. She did, then said that my breasts had grown a bit since the last time! Mommy drew me to her and kissed my mouth before telling me what a lovely woman I was going to be. Her hand touched my nipples, and it made me tingle.
Miss Decker got out a new set of bras and panties for us, and like last time I marched around in all the different things just like a fashion model. I didnt mind at all being naked in front of them, and I loved it when they reached out to touch me sometimes.
Mommy picked out three new pairs of bras and panties for me. Then she told me she needed to talk to Miss Decker, and for me to go out to the front of the store and wait for her there.
I went out with the bag of all my new things and sat in a chair that was by a table full of grown up panties. After I waited for a few minutes, I saw a clock that said it was almost 4. That's when my favorite radio show is on, called The British Are Coming. They play lots of the songs from England that I like, the Hollies and the Beatles and Jerry and the Pacemakers and stuff like that. I thought I could maybe listen to the radio in the car, so I went to ask Mommy if she would let me have the keys.
When I touched the doorknob to the back room, I heard Mommy gasp, and I stopped right where I was. Instead of walking in I opened the door just a little to peek in and see what was going on.
I couldn't believe what I saw! Mommy was standing with one foot on a chair, just wearing her top and nothing else. Miss Decker was on her knees and her face was in Mommy's pussy, kissing her down there!
I watched for a long time, feeling it get all warm between my legs. Mommy was rubbing her titties through her top that was half open, with one hand. The other hand was holding Miss Decker's head to her pussy.
Then she suddenly threw her head back, crying out Oh, yes! Yes! Yes! before she went all stiff, biting on her lip when she came. Her face was beautiful when she did it.
Then Miss Decker got up, and she and Mommy kissed for a long time, using their tongues. I felt a shiver when I knew they were tasting Mommy's pussy together.
Then Mommy turned around and pushed Miss Decker back against the wall, really hard. I was so suprised that I made a little squeak, but I was lucky and they didnt hear me. Mommy lifted Miss Decker's dress and reached up under it, touching her pussy the way she did mine. Miss Decker moaned, and Mommy made her be quiet with a kiss.
I wanted to touch myself while I watched them make love, but decided not to move. I didnt want to get caught.
Miss Decker must have been very excited cause she came quick, breathing hard and her body shaking all over. Mommy took her hand from under Miss Decker's dress and my heart beat even faster when I saw her lick her fingers. They kissed more, speaking softly in between kisses. I tried to hear, but couldnt.
Then Mommy started to button up the front of her dress, and then I knew that she was about to leave. I had to move fast before she saw me!
I grabbed my bags and ran out of the store and got in the car. I was there waiting when Mommy got there like nothing had happened.
I keep thinking about Miss Decker kissing Mommy's pussy. It seems kind of icky, putting your mouth where someone pees from. But Mommy liked it so much that I guess it has to feel really good.
Monday 24th January, 1966
I was already awake when Mommy came to me this morning. We kissed for a long time, then we touched each others pussies until we both came together. That was amazing!
After that I asked if I could kiss and suck on her nipples, like she did to me the other day.
Mommy said that of course I could. She lay me down in her lap with her breasts in to my face and offered me one. I took a nipple into my mouth and nursed from my mother, like I was a baby again. I really liked that. Then we kissed some more until I had to get ready for school.
Claire got to see me in my pretty new underthings today. I showed off for her, turning so she could see how with my new panties you could see almost half of my bottom. She smiled and said she wanted to have me over to play again.
I want to kiss her REALLY bad. If I dont chicken out, I'm gonna try to the next time I'm over at her house.
Tuesday 25th January, 1966
Mommy woke me, smiling down and brushing my hair back with her fingers. I grinned and rolled over on my back, ready for her.
Hows my little angel? she asked me.
I giggled and pulled the covers away, surprising her when she saw that I was completely naked! I had decided not to wear a nightie or panties to bed any more. I could tell Mommy liked that.
I loved it when she looked me up and down with a look in her eyes like she wanted to do something sexy. I opened my legs for her, putting my hand on my pussy and gently touching it. I wanted her to see.
She said, Let Mommy do that for you.
It felt so nice with Mommy fingering me. I came really fast, and hard too. Then we snuggled and I thanked her for making me feel so good. Then I gave her a hot mouth open kiss with my tongue. I wanted to touch her pussy, too, but there wasnt time.
Wednesday 26th January, 1966
Claire and I played together today at school. But during lunch I couldnt stand it any more. I took her behind the bike shed and pushed her against the wooden wall.
Have you ever kissed another girl? I asked her.
She shook her head and licked her lips with a sort of funny look on her face.
Would you like to try? I asked.
With you? she whispered.
I nodded, and I felt all hot and crazy inside when she nodded too.
It started sort of shy, our lips hardly touching. But then it grew and grew until we were hugging each other as our tongues moved together. It was cold out, but kissing her made me feel hot all over.
Her hips were pushing mine. I wanted to touch her between the legs but I wasnt sure if Claire would want it.
We stopped before anyone else came around and caught us, and then Claire said she would come over soon for dinner. Both of us were blushing and really, really happy!
I'm going to ask Mommy if Claire can come home with me after school and have supper with us Friday night. Wow, just thinking about Claire and me naked and in my bed, is getting my panties wet. I wonder what Mommy would do if she caught us doing stuff together? She wouldnt mind, I bet.
Thursday 27th January, 1966
Mommy and I were cuddling together this morning all warm and naked after making love, and I asked her if I could have Claire spend the night on Friday. She smiled and said yes.
Later a card arrived in the post, a valentine. Its my very first one, and I know I blushed when I opened it.
It was so sweet! She wrote about how she thought I was beautiful, and how she loved the time we spent together at school. I knew she meant when we kissed the other day, and that made me feel warm all over.
Mommy laughed and asked who it was from. I told her, It's a secret, but I let her see it anyway.
She gave me a BIG smile when she read the card, and asked me if Claire was my girlfriend now. I told her that I didn't know but hoped so. Mommy hugged me then and said she was proud of me, and how glad she was that I wanted to be with Claire instead of some boy. Girls are better, she said.
I asked her if that was how she felt, why she was with Daddy instead of a woman?
Mommy told me that she did love Daddy, but she just loves women more. Also without him she couldnt have had me, and she loved me most of all! I had to give her a big kiss when she told me that.
Before leaving for school I made Claire a valentine where I wrote about how pretty she is, and how much I like her. I wanted to write about wanting to see her with all her clothes off, but I decided not to in case her mom or somebody reads it.
Claire squealed and hugged me tight when I gave her the valentine. I invited her to come over tomorrow afternoon and stay for supper. We were both so excited that we could hardly talk, just look at each other and giggle like we were sharing the best secret in the whole world!
We snuck away to the top of the field at lunchtime, hid behind the hedge and kissed. I touched her titties too, which made her blush. I liked feeling her, and I like her letting me do it. I told Claire about wanting to see her with no clothes on, and she just wrapped her arms round me and whispered, I want to see you too.
I cant wait for tomorrow.
Friday 28th January, 1966
Mommy came in early and got in bed with me. I love cuddling her, kissing while her big round titties touch mine. Her hands moved all over my legs and my bottom, then she pushed my legs open so she could touch my pussy. I was wet and ready for her, and she put a finger inside me, rolling it around until she made me come.
I was still breathing hard when Mommy asked me to tell more about Claire. My face got warm then, but I told her how we kissed and I touched her titties and how I wanted to do everything to Claire that I learned from her.
Mommy kissed my cheek and said she thought that was very sweet. Then she reminded me to keep our secret and be careful not to tell Claire any thing about what she and I do together. Mommies and their little girls arent sposed to make love, she told me.
I said, But we do, dont we?
She hugged me to her and whispered, We do, angel. The way she said that made me feel so happy I thought I might cry. But I didnt let myself. I'm too old for that.
Then she layed back and opened her legs wide for me, showing her pussy. I bent over to kiss her titties as I put my hand on it, feeling how wet she was. Its like heaven, sucking Mommy's nipples while I push my fingers in and out of her. I love the way her nipples get so big in my mouth!
Soon she was wiggling on the bed, calling out my name and saying how much she loves me while I made her come. She got pretty loud too.
After she came, she said I was getting better and better every day at making love. I was so proud!
School seemed to last forever yesterday, but the bell finaly rang and we were free. When I saw Claire waiting for me outside she grabbed my hand and we ran nearly all the way to my house.
I invited her inside and introduced Claire to Mommy, who took her hand and said she was happy to meet her. I knew she thought Claire was pretty, and that made me even more excited!
I told Mommy we were going up to my room to play and we ran up stairs. I closed the door behind us. I was dying to touch her all day, and now we were together with no one else there. It felt as if I was about to burst, I was so excited!
Sitting on my bed we kissed really long and deep with our tongues playing. She put both her arms round my neck, and I hugged her too. Then she fell back on the bed and I was on top of her, opening up her shirt.
I put my hand inside and felt her titties, and how when my fingers touch her nipples they get bigger. That hot feeling was starting in my panties again, and all I could think was how I wanted a good look at Claire with her clothes off.
Can I see them? I asked Claire after our kiss stopped. She said I could!
It was so nice to watch Claire take off her school shirt, then her training bra. She was a little bit shy, her cheeks were pink when she dropped her things on the floor. Then she stood there letting me have a good long look at her. She's even prettier than I thought!
I didnt even think about it, I just bent down and licked at her nipple, making Claire squeal cause she was so suprised. I took one in my mouth and sucked on it, the way Mommy likes to do to me.
Claire was breathing hard and holding my face to her titties. I licked and sucked her nipples for a good long time. Then I sat up and took off my top and my bra.
I was dying to feel her mouth on me. I said, Do it to me now.
Then I was lying on my back watching her bend down, then closing my eyes when her mouth covered my nipple. It felt so good! Then her hand touched my other titty, and I started to rub my legs together cause my pussy felt hot. I held her head to me and the good feelings got bigger and bigger.
I couldnt wait any longer, I wanted to do everything with Claire. I pushed her away and I stood up. I was breathing hard and shaking all over. Take your clothes off, I said while I was tearing off my skirt. All I could think about was that I needed to be naked for Claire.
She stared at me for a little bit, then she was undressing too. I kicked my panties away, and I saw her eyes get big as she looked at my pussy.
Then Claire took her own panties off, and I was excited when she showed me her pussy. She was just starting to grow some hair down there, more than me. It looked soft and pretty.
I told her to lay down. Claire did what I said and spread her legs apart, watching me when I looked at her down there. Her pussy was all wet, and open.
She asked me what I was going to do, and acted all shy when she said that. Covering her pussy with both hands.
I didnt answer her. I couldnt. I got on the bed and climbed on top of her and we kissed using our tongues. Then I turned around and got between her legs and started doing what Aunt Evie did to Mommy that time I got to watch them make love. I put my pussy to Claire's and moved on her, rubbing our girl parts together. She looked a little scared at first, but it felt so nice that she started pushing back.
We watched each other while we were doing it, and our eyes were really wide like we couldnt believe that it could be so good. But it was. The feelings were getting big down deep in my belly. We were both slippery wet, and sort of fighting to get closer together. Our hands were pulling hard and we huffed and puffed, trying so hard to get there.
When it did come it felt like fireworks blowing up. I was pushing into Claire, holding her tight. And she was making funny sounds in my ear, moving under me while it was happening to her. Then we fell away from each other and lay still. We both were breathing hard like we just ran a long way.
She took my hand and squeezed it. That was the best ever, she said.
I turned to Claire and we kissed some more. Then we rested some in each other's arms. After a while we got up and put all our clothes back on, to be ready for when Mommy called us down for supper.
When I heard Mommy's voice call I grabbed Claire's hand. We ran down the stairs together, giggling and making lots of noise.
Mommy smiled at us as we ran into the kitchen. She asked us if we were having fun playing. Then her eyes met mine and I saw that she knew what Claire and I had been doing up stairs, and I could tell that it made her feel excited. It sure did me!
I got a weird thought then. In my head I saw Mommy standing outside my door with a hand up under her dress, touching her pussy while she listened to me and Claire playing sexy games. Then I thought of her coming into the room while we were naked and making love, watching us doing it. THEN I thought about Mommy taking her clothes off and getting on the bed with me and Claire!
All the time we ate, I thought about being with Mommy and Claire at the same time. It made me feel all hot down in my panties, and I couldnt sit still.
Daddy was there for supper, and he was very nice and polite to Claire, though I could tell he was thinking about work like he always is. He is going hunting with his friends tomorrow so maybe his mind is on that too. It's funny, all the things going on between me and Mommy that Daddy doesnt know about.
I wanted to go back upstairs with Claire again after supper but there wasnt time. But then I got to walk her home, and we snuck behind a big tree to kiss for a while before I said good night.
I'm in bed now, writing about the great day I had. My pussy is all sticky and wet from that idea I had at dinner, about making love to Claire and Mommy at the same time. What REALLY makes me excited is when I think about them kissing and touching each other while I get to watch.
Oh wow, it's really late! Thats all for tonight.
Continue on to Chapter 4


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So, we like to draw and so do our friends. People send us pics all the time, doodles, sketches, full blown cartoons. We will most likely do one of these posts a month, so SEND US YOUR ARTWORK! Email direct to firstname.lastname@example.com and enjoy the pics!
Email your pics to firstname.lastname@example.com


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Tag Archives: Goldthwait
"The Leakeys of White Mountain Geology": Hanover New Hampshire's James Walter Goldthwait (1880-1947), Richard Parker Goldthwait (1911-1992) and Lawrence Goldthwait (1914-2001)
For countless years the Goldthwait name was synonymous with geology and geography, not only in New Hampshire, but throughout the … Continue reading
Posted in New Hampshire Men Tagged earth, geography, geology, glacier, Goldthwait, Harvard, Ice Age, Leakeys, Mount Washington, New Hampshire, professor, research, science, White Mountain 2 Comments


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Kids & Pizza Napoletana
Ending the year doing a Neapolitan Kids Pizza Class at Kaurilands School, great enthusiasm and so much fun.
Best of all, they never asked for a rolling pin to open the pizza base and NO Hawiian pizza 😏 😉
Making the dough
Showing the fresh yeast
Getting instructions from kids, each step, how to make Neapolitan pizza dough.
Ready for topping
Showing how to recognise when ( il Panetto ) the pizza base its ready to be used
Making Pizza bases
Showing the ( Staglio ) making the pizza base
Time to get messy


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Mar 6, 2019 2:43:03 PM / by
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Early mornings. Late nights. Lots of running around in between... The life of running a startup!
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BNP chairperson Khaleda Zia has filed an appeal with the High Court (HC) challenging her conviction by a trial court in the much-talked-about Zia Charitable Trust corruption case.
Khaleda's lawyer barrister Nowshad Jamir filed the appeal on Sunday on behalf of Khaleda after receiving the copy of the full verdict of the case.
The appeal also sought her bail.
On October 29, a Special Court-5 pronounced the verdict sentencing her to seven years' imprisonment in her absence when she was undergoing treatment at Bangbandhu Sheikh Mujib Medical University (BSMMU) under a special medical board formed by the government.
The court also fined her Tk 10 lakh each, in default, to suffer six months more in jail. It also ordered confiscation of the 42-katha land bought against the trust's name.
On August 8, 2011, the ACC filed the Zia Charitable Trust graft case with Tejgaon Police Station accusing four people, including Khaleda Zia, of abusing power in raising funds for the trust from unknown sources.
The former Prime Minister has been serving five years' rigorous imprisonment at Old Central Jail, as she was convicted in the Zia Orphanage Trust graft case on February 8 last.


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Sunset Reservoir Rehabilitation - 2016079
Bidding Closed
Prebid Date7/23/19 1:30 pm
Bid Date8/5/19 1:00 pm
Company & Contacts
City of Spokane Engineering Dept.
Cindy Kinzer, email@example.com (509) 625-6700
Spokane, WA
Project Estimate: $890,000.00 to $1,000,000.00
Bidders may download electronic files at no cost. Printed documents are available, at Bidders expense, by choosing the "Order" option.
07/23 – A non-mandatory pre-bid conference will be held onsite at the Sunset Reservoir tank. The tank site is located approximately 600 feet west of S. Pepper Tree Lane/S. Carousel Lane and is addressed near 3950 W. Canyon Drive at 1:30 p.m. on July 23, 2019.
Additional project information including the Engineer's estimated cost range for the project, bid results (after bid opening), as well as information about other City projects are available by following this link for The City of Spokane Engineering Services.


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Check Out Sony's Latest Smart Watches
Sony has been known as the leader of tech innovation for a long time. Not only has the brand been identified with its stellar approach Read More
Sony has been known as the leader of tech innovation for a long time. Not only has the brand been identified with its stellar approach Read More
There has been an unprecedented demand for phone watches, with almost everyone who can afford them trying to get smart watch wearables for their needs. Read More
Amidst the plenty of latest brands of newest smartwatch, it has been a tedious task to hunt an appropriate yet cutting-edge smartwatch from an online Read More
When you are planning to buy best watch phone, you need to get some information about them. There are several watch phone is accessible in Read More
In this modern age almost everything has become smart. Our TV's are smart our phones are smart and now our watches are smart too. Thus Read More
Women are equal to men in every respect. If men can have a smart watch exclusively for themselves, women too have an equal right. An Read More
The technology is improving at a tremendous pace today. Every week, you can see some new product make its launch on a worldwide basis. Man Read More
The pebble was one of the major smart watches producers, to hit the business sector, much sooner than the Apple Watch and Android Wear secured Read More
With regards to smart watches, Samsung has had its foot on the gas pedal in the last year. In the wake of propelling the galaxy Read More
We are living in a techno savvy world, where we see a new innovation every day in every sector. We all are well aware of Read More


text: This shop has been compensated by Collective Bias, Inc. and its advertiser. All opinions are mine alone. #MyRadiantLook #CollectiveBias Be RADIANT. Introducing Colgate® Optic White® RADIANT™ Toothpaste I finally got around to writing my beauty routine for you all! I swear it's not 1000 steps and you will walk out the door feeling beautiful, confident … Continue Reading


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How is the Affordable Care Act Different from Medicare?
Release Date
June 28, 2012
If Social Security and Medicare are constitutional, why isn't the individual mandate from the Affordable Care Act also constitutional? Law professor Elizabeth Price Foley explains that the Social Security and Medicare laws are based on a different congressional power than the Affordable Care Act. Social Security and Medicare fall under Congress's power to tax and spend on behalf of U.S. citizens, while the individual mandate is based on the power to regulate commerce.
The question before the Supreme Court is whether the power to regulate commerce includes the power to compel people to participate in commerce. Is it within Congress's constitutional authority to require individuals to buy health care if they do not want to do so?
Professor Elizabeth Price Foley says Congress could have solved the problem of access to health care for the uninsured without using its commerce power. It could have simply invoked the same principle it used with Social Security and Medicare. If that had happened, the Affordable Care Act would not be in a case pending before the Supreme Court.
5 Comments
Chocolate Thunder
I suppose we now know it's the case that SCOTUS found the individual mandate constitutional because of the taxing power, as suggested in this video.
But this is somewhat confusing to me. Would the Framers of the Constitution considered this to be admissible under the power to tax? Also, what limits are there to government if it can use the taxing power in such a way?
Holding the framers in such high esteem is somewhat difficult as they considered slavery acceptable. The only limit I can think of is the ballot box, but unfortunately we know how unproductive that is with the system as it currently exists
Stephen Dincher
The founders would definitely not have considered this admissible. The original constitution outlawed all direct taxes (income, property, and payroll taxes were all unconstitutional until 1908). So it's pretty obvious that there would have been zero approval by the founders for this monstrosity of a bill, and medicare and social security would have been inadmissible as well.
Hunter Markson
Never give the government ideas on how to legally oppress us. You should know better Learn Liberty!
Keith Knight
All theft is immoral, the fact that the state hides behind their threat of monopolized force does not make any action justifiable.
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text: Before Christmas, Lavender class in Year 1 participated in a charity drive for The Toy Project http://thetoyproject.co.uk/. Our parents organised gifts to be donated to this charity on behalf of our class. This was such a wonderful opportunity for the children to participate and understand how we continue to help those in need, especially around the holiday time. A special thanks to all Lavender class parents who participated and donated new and used toys. We will continue this charity drive next year and hopefully continue the gift of giving!


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Greenwood Village
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EFT is effective in reducing fear, anxiety, and pain. It can retrain your brain to release food cravings and habits, and it can remove internal blocks and improve self-esteem and personal performance.
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