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Constitutional Law Group July 11, 2016 Europe, European Union, UK government, UK Parliament
Kenneth Armstrong: Should Turkeys Vote (be)for(e) Christmas? An Early General Election and Article 50
In a piece published last week I set out the argument for why a general election is needed before the UK notifies a decision to withdraw from the European Union in terms of Article 50 TEU.
Writing here, Mike Gordon similarly also redirects constitutional lawyers concerns about the role of parliament in the withdrawal process, towards the same end: an early general election.
The election of a new leader of the Conservative Party – and in consequence the UK's future prime minister – frames and dramatizes the constitutional problem highlighted by others that it will be the membership of the Conservative Party and not MPs that will elect someone to implement the 23rd June referendum result.
The demand made by those calling for an early election is to restore representative democracy to its central place in our constitutional framework by requiring all political parties to set out and contest their vision of a future UK-EU relationship.
But to avoid merely repeating arguments made elsewhere, this contribution advances the argument by suggesting that in certain ways an early general election may actually be in the interest of the political parties and their leaders.
The former leader of the Liberal Democratic Party, Nick Clegg, writing in The Guardian, has recently made the argument that an early general election should be called.
It would be easy to dismiss Clegg's intervention as the self-interested demands of a former leader whose party lost 49 MPs in the last general election in 2015. An early general election might be self-evidently in his party's interest.
However, a plausible argument can be made that the discipline of a general election may not only help give direction to our political parties after a period of turmoil, it may actually help them pursue their own interests.
And with elections to the French Presidency next year already beginning to reveal their significance for Brexit, it would seem odd that elections held in other EU states could determine the outcome of negotiations more than an election in the UK.
Before setting out the political argument, it's useful to set out briefly the fundamental thesis that underlies the claim that a general election ought to be held before Article 50 is triggered.
The referendum held on 23rd June was the start and not the end of a process through which the UK will determine its future relationship with the EU. Whereas David Cameron as Prime Minister had a mandate for a referendum that was promised in his party's manifesto, none of the political parties have a mandate from which to define future UK-EU relations.
The internal leadership contest within the Conservative Party is no substitute for a true electoral mandate. A process is needed that allows all parties – whether of the left or the right; from all the constituent territories of the UK; and whether they were in favour or against EU membership – to set out their positions on where Brexit will take the country.
And only by a political contest can both a new parliament and government play their respective constitutional roles in the Article 50 process.
This proposal respects the outcome of the referendum but returns representative democracy to its central role in the UK's essentially political and facilitative constitutional structure.
But is this proposal actually in the interests of the political parties?
As the governing party, it will be for the next Conservative Prime Minister to trigger Article 50. It seems likely that the leadership contest that will elect not just a new leader but also the Prime Minister will be fought between individuals on either side of the Remain/Leave camps.
If a 'Remain' candidate like Theresa May were to win the leadership, agreeing a position in a manifesto that Brexit means Brexit, could help ensure that the referendum was respected in substance and not just in form. It could also increase her credibility not just within her own party, but in the Article 50 negotiations themselves; important given that the Treaty rules weight the bargaining power against the withdrawing state.
If a 'Leave' candidate like Andrea Leadsom were to win the leadership, whatever authority they might have to initiate the UK's divorce from the EU, they would lack any electoral mandate as to what 'Leave' might actually entail.
What 'Leave' means is really important.
One argument against the Electoral Commission's recommendation to change the referendum question from one that would give a Yes/NO answer to Remain/Leave, was that it was not clear what voters would be voting for if they voted 'Leave'.
For some this might simply mean having nothing whatsoever to do with the EU. For others it might mean Swiss or Canadian style trade deals. Some might even have contemplated the European Economic Area or 'Nordic' model. But having voted for Leave, we simply do not know which of these alternative models might be sought, as Oliver Letwin made clear in his appearance between the Common Foreign Affairs Committee on 5 July.
To put it at its simplest, a majority of the electorate has voted to leave the EU but a majority has not expressed its wishes as to where that takes the UK in its future relationship with the EU.
An early general election fought on the basis of a specific model of future UK-EU relations might also help a new Prime Minister avoid the risks associated with an outcome of future negotiations which a majority of the electorate would not support and which might give rise to demands for a referendum on the new relationship. A future referendum that rejected a UK-EU deal would likely lead to the fall of the government and leave far greater long-term uncertainty than an early general election.
For supporters of the UK Independence Party, a general election would be a means of seeking to reflect their preferences in the political process going forward. While Nigel Farage was a key figure promoting Brexit, he was not directly part of the official Vote Leave campaign. In the aftermath of the referendum result, it was suggested that Farage should have a role in the future negotiations once Article 50 was triggered. With the announcement of his departure as leader of his party, a new leader will emerge and a general election would be an opportunity for UKIP supporters to seek more direct influence over the Article 50 process through the election of representatives to a new parliament.
The situation in the Labour Party is more complex. The Labour leader Jeremy Corbyn has lost the confidence of his parliamentary colleagues. A leadership election may well ensue. The contests within the party are not just about leadership but also between different political wings and factions.
Left-wing Brexit supporters or sympathisers criticised the power taken from national control and argued that outside the EU, a Labour government could tackle the politics of austerity.
Those Labour supporters who backed the Remain campaign tended to see European cooperation as a mechanism of managing the forces of globalisation and a means of providing regulatory standards for workers, consumers and to protect the environment.
A general election would be an opportunity for the Labour Party to define a clearer vision of what Brexit means from a Left perspective. This would also give the party an opportunity to speak directly to the aspirations and fears of younger voters who not only are an important constituency of its membership but were also more likely to have voted for the UK to remain in the EU.
In short, if the referendum campaign suffered from a failure to articulate both Left and Right versions of Remain/Leave, then a general election could compensate by contesting these different versions of UK-EU relations.
The Scottish National Party was the big winner in the 2015 general election, winning 56 of the 59 constituency seats in Scotland. In itself, this result tells us how much referendums change politics and why general elections are important in reflecting those changed realities.
With such dominance of the Scottish seats that it contests, the SNP may have little incentive to go into another general election compared with the position of the Liberal Democrats. But for the same reasons as apply to UKIP – the desire to have voice and influence in future UK-EU negotiations – an election in which the SNP set out a clear vision of Scotland's place in Europe might go some way to allow the SNP to navigate between respecting the outcome of the Scottish independence referendum and protecting Scotland's relationship with the EU.
More radically, it could be the pretext for seeking an electoral mandate for a second independence referendum given that 62% of voters in Scotland voted for the UK to remain in the EU. Although a general election would not be without its risks – the SNP lost seats at the more recent elections to the Holyrood parliament – the lure of a second independence referendum might yet make an election an attractive prospect.
Like Scotland, a (smaller) majority of voters in Northern Ireland backed continued UK membership of the EU.
Brexit has significant implications for Northern Ireland and key questions need to be answered about how to respect the Good Friday Agreement and whether border controls will be needed between a Northern Ireland outside of the EU and an Ireland that remains a Member State. Political leaders have already set out rather different visions of what future negotiations should look like, with the SDLP suggesting that talks include representatives of the Irish government. A general election is the obvious mechanism through which to allow these different visions to be contested.
In Wales, the key issue for the future is the significant amount of EU funding which it receives under the EU Cohesion Funds. Understanding how the withdrawal of such funds will affect Wales and the positions of the different parties in terms how such a loss might be compensated would be at the centre of a Welsh general election campaign.
European leaders may well complain that a general election would further delay formal withdrawal under Article 50.
This proposal envisages that a general election could be held in late Autumn with a view to a UK Prime Minister being in a position to notify the December meeting of the European Council of the UK's intentions under Article 50. It is not, therefore, necessarily the case that a general election would unduly delay the start of the withdrawal process.
There is one final reason why I believe a general election might be in the interest of the political parties.
No one can be in any doubt about the enormous ramifications of the referendum result. Politics is in a state of flux and, to a certain extent, in disarray. With leaders resigning or under threat of replacement there is a growing sense of a political vacuum emerging between the referendum result and its implementation with no one having or taking responsibility for taking things forward.
The discipline of a general election is perhaps the most natural response to focus the minds of our political parties and to fill the vacuum.
Kenneth Armstrong is Professor of European Law, University of Cambridge.
(Suggested citation: K. Armstrong, 'Should Turkeys Vote (be)for(e) Christmas? An Early General Election and Article 50', U.K. Const. L. Blog (11th Jul 2016) (available at https://ukconstitutionallaw.org/))
Posted in Europe, European Union, UK government, UK Parliament and tagged Article 50 TEU, Brexit, general election, UK EU Referendum. Bookmark the permalink.
John Andrews	 on July 11, 2016 at 11:11 am
I'm increasingly feeling some sympathy with the old "what do you think about the discovery of 3 lawyers dead in the sea?" "A slow start". These posts are too much about hypothetical issues and personal judgements.
R W Barltrop (@r_barltrop)	 on July 11, 2016 at 4:07 pm
Dear Professor Armstrong,
You write: "To put it at its simplest, a majority of the electorate has voted to leave the EU but a majority has not expressed its wishes as to where that takes the UK in its future relationship with the EU."
Is the first part of the sentence correct? Isn't the core definition of "electorate" those who have the right to vote and are registered to vote?
In the 23 June referendum a majority of those who cast ballots voted to leave. But they were not a majority of the electorate: they were 17.4m (37.4%) of the electorate of 46.5m. Meanwhile, 16.1m (34.7%) of the electorate voted Remain, and 12.9m (27.9%) of the electorate did not vote. Put another way, 62.6% of the electorate did not vote Leave.
This consideration surely only strengthens your point that "a majority of the electorate has not expressed its wishes as to where [the Leave vote] takes the UK in its future relationship with the EU"?
Thomas Adams: Is There a Mandate Here?
Yossi Nehushtan: Why Is It Illegal for the Prime Minister to Perceive the EU Referendum's Result as Morally-Politically Authoritative?


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Joshua David Willingham (born February 17, 1979) is a former American professional baseball left fielder. He played  in Major League Baseball (MLB) for the Florida Marlins, Washington Nationals, Oakland Athletics, Minnesota Twins and Kansas City Royals.

Early years
He was born in Florence, Alabama to David and Denise Willingham where he played high school baseball and basketball at Mars Hill Bible School. Willingham attended college at the University of North Alabama. He played shortstop and was a two-time All-Gulf South Conference selection (1999, 2000) and a 2-time American Baseball Coaches Association All American. He is a member of Sigma Chi fraternity, Eta Rho Chapter.

Professional career

Florida Marlins

The Florida Marlins selected Willingham in the 17th round, with the 491st overall selection, of the 2000 Major League Baseball draft.  He made his MLB debut on July 6, 2004, against the Pittsburgh Pirates but did not garner regular playing time until 2006.

After struggling to find a fielding position for Willingham, as his advanced hitting abilities did not allow him the opportunity to develop defensively, the Marlins made Willingham their starting left-fielder during the 2006 season in wake of the Marlins's "market correction" in the 2005 offseason.

On April 4, 2006, Willingham recorded his first career multi-homer game, launching two against the Houston Astros. He ended the 2006 season with a solid line of .277/.356/.496 with 26 home runs and 74 runs batted in.

2007 proved to be another successful season for Willingham in his sophomore season.  He maintained a solid batting average while hitting 21 home runs and 89 runs batted in.  He became the team's regular left fielder in 2007, making just three errors in 223 total chances.

Willingham saw a reduced amount of playing time in 2008 due to a 50-game stint on the disabled list, appearing in just over 100 games as opposed to over 140 in the previous two years.  His production suffered as his batting average regressed (.258) and his home run totals dipped to 15 on the season. He homered in 4 straight games for first time in career, September 21–26.

Washington Nationals

On November 11, 2008, Willingham was traded to the Washington Nationals, along with pitcher Scott Olsen for Emilio Bonifacio, Jake Smolinski, and P.J. Dean. Willingham began 2009 as the Nationals' fifth outfielder behind Adam Dunn, Elijah Dukes, Austin Kearns, and Lastings Milledge.  In May, however, Kearns was sidelined with a fractured foot, giving Willingham a starting role.  Dunn was eventually moved to first base and Milledge was traded for then-Pittsburgh Pirates outfielder Nyjer Morgan.  Willingham was very productive, proving himself worthy of the everyday left fielder position. On July 27, 2009, Willingham became the 13th player in MLB history to hit two grand slams in the same game and the 7th in MLB history to accomplish this feat in back to back innings. He ended the season with a line of .260/.367/.496 with 24 home runs and 61 RBIs.  Having secured the starting left field job, Willingham was productive in the Nationals lineup in 2010. He batted 5th behind sluggers Ryan Zimmerman and Adam Dunn.

Oakland Athletics

On December 16, 2010, Willingham was traded to the Oakland Athletics for pitcher Henry Rodríguez and outfielder Corey Brown. His 2011 batting average of .246 was his lowest since his rookie season, but he reached career pinnacles in home runs (29) and RBIs (98). Willingham won the 2011 Catfish Hunter Award.

Minnesota Twins
On December 15, 2011, Willingham signed a 3-year deal with the Minnesota Twins worth $21 million.

Willingham was named AL Player of the Week for the week of April 9–15. Willingham hit 10-22 with 3 HR in 3 consecutive games and 4 RBI, getting a hit in every single game.  Willingham singled in the 9th inning of a game on April 21, 2012 to tie Kirby Puckett's franchise record of getting a hit in the first 15 games of the season. On May 29, Willingham hit a two-out, three-run, walk-off HR against the Athletics. By mid-June, Willingham had hit 14 home runs before finishing the season with a career high 35 Willingham's home run total tied him with Bob Allison's 35 home runs in 1963 for the most by a Minnesota Twin (following their move in 1961) not named Harmon Killebrew.

Kansas City Royals
On August 11, 2014, Willingham was traded to the Kansas City Royals for RHP Jason Adam. In September, he announced that the 2014 season would be his last. He hit a bloop single in the 2014 American League Wild Card Game and was taken out for pinch runner Jarrod Dyson. That would turn out to be the game-tying run that would send the game to extra innings.

Willingham officially announced his retirement on November 24, 2014.

Personal
Willingham is married to his high school sweetheart, Ginger Jaynes Willingham.  They have three sons: Rhett, Ryder, and Rogan.  Josh and Ginger started a charitable foundation and fundraising golf tournament named in honor of his younger brother, Jon Willingham, who died in a car accident in 2009.

On June 9, 2021, Willingham was announced as the head coach at his high school alma mater, Mars Hill Bible School in Florence, AL.

References

External links

1979 births
Living people
North Alabama Lions baseball players
North Alabama Lions football players
Florida Marlins players
Washington Nationals players
Oakland Athletics players
Minnesota Twins players
Kansas City Royals players
Utica Blue Sox players
Kane County Cougars players
Jupiter Hammerheads players
Gulf Coast Marlins players
Carolina Mudcats players
Albuquerque Isotopes players
Sacramento River Cats players
Rochester Red Wings players
Baseball players from Alabama
Major League Baseball left fielders
Sportspeople from Florence, Alabama
American members of the Churches of Christ
Silver Slugger Award winners

redpajama_set_name: RedPajamaWikipedia


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CHEMOKINES, CYTOKINES, AND INTERLEUKINS| February 1, 2008
TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts
Claudia Bossen,
Claudia Bossen
1Department of Biochemistry, University of Lausanne, Epalinges, Switzerland;
Teresa G. Cachero,
Teresa G. Cachero
2BiogenIdec, Cambridge, MA;
Aubry Tardivel,
Aubry Tardivel
Karine Ingold,
Karine Ingold
Laure Willen,
Laure Willen
Max Dobles,
Max Dobles
Martin L. Scott,
Martin L. Scott
Aris Maquelin,
Aris Maquelin
Elodie Belnoue,
Elodie Belnoue
3Department of Pathology-Immunology and Pediatrics, University of Geneva, Geneva, Switzerland; and
Claire-Anne Siegrist,
Claire-Anne Siegrist
Stéphane Chevrier,
Stéphane Chevrier
Hans Acha-Orbea,
Hans Acha-Orbea
Helen Leung,
Helen Leung
4Garvan Institute of Medical Research, Darlinghurst, Australia
Fabienne Mackay,
Fabienne Mackay
Jürg Tschopp,
Jürg Tschopp
Pascal Schneider
https://doi.org/10.1182/blood-2007-09-110874
A related article has been published: Different partners in different places?
Claudia Bossen, Teresa G. Cachero, Aubry Tardivel, Karine Ingold, Laure Willen, Max Dobles, Martin L. Scott, Aris Maquelin, Elodie Belnoue, Claire-Anne Siegrist, Stéphane Chevrier, Hans Acha-Orbea, Helen Leung, Fabienne Mackay, Jürg Tschopp, Pascal Schneider; TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts. Blood 2008; 111 (3): 1004–1012. doi: https://doi.org/10.1182/blood-2007-09-110874
The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R–dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.
b-lymphocytes, ligands, mice, plasmablast, oligomers, antibodies, agonists, signal transduction, binding (molecular function)
B-cell activating factor of the tumor necrosis factor (TNF) family (BAFF, also known as BLyS) and a proliferation-inducing ligand (APRIL) are 2 members of the TNF family mainly involved in B-cell physiology.1-3 BAFF is expressed by myeloid cells and by unidentified radiation-resistant cells, possibly stromal cells of secondary lymphoid organs.4-6 BAFF is either expressed at the cell surface or released into a soluble form through cleavage by an uncharacterized furin.7-9 Members of the TNF family usually assemble as trimers,10 but soluble BAFF was crystallized both as a trimer and as a virus-like structure resulting from the ordered assembly of 20 trimers through an unusually long loop of BAFF between β-sheets D and E (DE loop).11,12 Although the physiologic relevance of BAFF 60-mer has not been studied, recent work showed that BAFF 60-mer was also produced by cells expressing BAFF endogenously and that the 60-mer was moderately more potent than the 3-mer at costimulating BCR-induced thymidine uptake in primary B cells.13 APRIL, a close homolog of BAFF, can also costimulate B cells but requires oligomerization to do so.14
BAFF and APRIL share 2 receptors, namely, TACI (transmembrane activator and CAML interactor) and BCMA (B-cell maturation antigen). In addition, BAFF binds to a third receptor, BAFF-R, also known as BR3. The role of BAFF in peripheral B-cell development is well recognized. B cells are generated in the bone marrow, which they exit at an immature stage. Subsequent maturation takes place in the spleen, where B cells evolve through the transitional T1 and T2 stages to mature follicular B cells or marginal zone B cells. Encounter with antigen leads to B-cell activation and differentiation into memory B cells or antibody secreting plasmocytes, whose survival in the bone marrow requires BAFF and/or APRIL.14,15
In BAFF-deficient mice, the reduced expression of CD21 and CD23 by B cells was initially interpreted as a blockade at the T1 stage, but it was shown later that subsequent maturation steps also occurred in BAFF-deficient mice, yet at reduced frequency.16 This finding is in line with the broad survival function of BAFF described for both transitional and mature B cells,17,18 and with the fact that BAFF can be func-tionally replaced by enforced expression of the antiapoptotic factor Bcl-2 in B cells, further pointing to its pro-survival function.19 BAFF-R deficiency recapitulates most of the phenotype of BAFF-deficient mice but affects T-dependent and -independent humoral responses less severely,20 suggesting that, although BAFF-R transmits important BAFF signals, there are other functional receptors for BAFF in vivo.
The role of APRIL in B cell physiology is less well established. One strain of APRIL-deficient mice had lowered IgA serum levels and impaired mucosal IgA responses21 that were not observed in a second APRIL-deficient strain.22 Reduced IgA serum levels are also characteristic of TACI-deficient mice, indicating that APRIL might mediate IgA class switch, or survival of switched IgA-secreting cells, through TACI.23 TACI is generally perceived as a negative regulator of B cells because TACI−/− mice have an enlarged B-cell pool that may be linked to autoimmune manifestations.23,24 TACI-deficient mice are also characterized by reduced antibody responses to T-independent antigens, a feature that is also observed in BAFF-deficient mice.20,23 Interestingly, approximately 10% of patients with common variable immunodeficiency (CVID) have mutations in TACI, suggesting a role for TACI in the production of antibodies of various isotypes.25,26
The third receptor, BCMA, is apparently dispensable for early B-cell differentiation stages but likely plays a role for long-term maintenance of bone marrow plasma cells.15
To date, the signaling impact of ligand oligomerization has not been investigated in these systems. Here, we studied the ability of BAFF 3-mer and higher-order oligomers to signal through BAFF-R and TACI in primary mouse B cells. In contrast to BAFF-R, TACI was unresponsive to BAFF 3-mer yet provided survival and differentiation signals when triggered by oligomeric forms of BAFF or APRIL. BAFF 60-mer is one form of oligomeric BAFF that can activate TACI and that exists naturally in vivo.
Mice, cell lines, and reagents
C57/Bl6 mice, 6 to 8 weeks old, were obtained from Harlan Netherlands (Horst, The Netherlands). TACI−/−, BCMA−/−, TACI−/− × BCMA−/−,20 and BAFF transgenic mice27 have been described previously. Mice were handled according to institutional and Swiss Federal Office guidelines, as well as under the authorization of the Veterinarian Service of Canton de Vaud.
Jurkat-BCMA:Fas cells (Jurkat-2309 clone 13) were generated as described in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). Myc-BAFF, BAFF H218A, BAFF A134 and BAFF N242Q, Flag-BAFF, BCMA-Fc, TACI-Fc and BAFF-R-Fc have been described previously.13,28 Flag-tagged APRIL containing amino acid 92-233 of human APRIL was purified from supernatants of transiently transfected 293T cells (Document S1). The hamster mAb B9C11 anti–mouse BAFF-R was kindly provided by Dr Melissa Starovasnik (Genentech, San Francisco, CA). Commercial reagents were as follows: rat IgM Buffy-2 anti–human BAFF, rat IgG1 5A8 anti–mouse BAFF, and rat IgG1 1C9 anti–mouse BAFF (Alexis, Lausen, Switzerland), anti-Flag M2 mAb and biotinylated M2 mAb (Sigma-Aldrich, St Louis, MO), carboxy fluorescein diacetate succinimide ester (CFSE; Invitrogen, Carlsbad, CA), and mouse interleukin-4 (IL-4) and mouse IL-6 (PeproTech, Rocky Hill, NJ).
Cytotoxicity assay
The cytotoxicity assay using BCMA:Fas Jurkat cells was performed as described for FasL on Jurkat cells.29 When stated, the assay was performed in the presence of 1.5 μg/mL of cycloheximide to increase the susceptibility of the cells to apoptosis. In some instances, BCMA-Fc was added at a final concentration of 2.5 μg/mL to assess the specificity of the assay.
For membrane–bound BAFF, 293T cells were transfected with an uncleavable form of full-length mouse BAFF (R125A and R126A) and incubated for 16 hours with a fixed number of Jurkat BCMA:Fas target cells and various ratio of effector cells. Cell death was assessed by fluorescence-activated cell sorting (FACS) based on the forward and side scatter profile, which allows discriminating 293T cells from live Jurkat cells from dead Jurkat cells. To monitor BAFF in transfected 293T cells, cells were lysed in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton-X100, complete protease inhibitor cocktail (Roche), and BAFF was quantified in the lysate by enzyme linked immunosorbent assay (ELISA; Document S1).
Gel permeation chromatography
293T cells were transiently transfected with a full-length mouse BAFF cDNA and grown for 7 days in carbonate-buffered medium. Cell supernatants or mouse plasma (200 μL) were fractionated by gel filtration on Superdex-200 as previously described13 and analyzed by Western blotting using Buffy-2 mAb, or by ELISA (Document S1). Fractions were also tested for their ability to kill Jurkat BCMA:Fas cells.
Characterization of ligand-receptor interactions
Immunoprecipitation of TACI-Fc with protein A-Sepharose followed by Western blot analysis, and detection of ligand-receptor interactions by ELISA were performed according to standard techniques (Document S1).
Splenocyte survival, costimulation, and MHC class II up-regulation assays
A detailed protocol is provided in Document S1. Briefly, B220-purified B cells were grown for 66 hours in the presence of ligands, anti-Flag, and anti-BAFF-R antibodies as required. Viable cells were identified based on the forward and side scatter profile. B-cell proliferation in response to an antimouse μ chain F(ab′)2 antibody was monitored by thymidine incorporation. In some experiments, B cells were stained with CFSE before cultivation. MHC class II up-regulation was monitored by FACS analysis.
Ig production assay
Purified splenic B cells (106/well in 1 mL of complete RPMI, 24-well plates) were grown for 6 days in the presence of the different ligands at 100 ng/mL. When specified, IgA+ B cells were depleted by incubation with biotinylated goat antimouse IgA (1:500; Invitrogen) for 10 minutes at 4°C followed by streptavidin-conjugated magnetic beads (Miltenyi Biotech, Auburn, CA). IgA and IgM titers in culture supernatants were quantified by ELISA (Document S1).
Plasmablast survival assays
Ten- to 12-week-old mice were injected subcutaneously in hind footpads with 107-108 mouse mammary tumor virus (MMTV) particles in infected milk.30 MMTV (SW) encodes a Vβ6-specific superantigen. Synchronized plasmablasts were isolated from the draining popliteal lymph nodes 6 days after infection, and purified with phycoerythrin-conjugated antimouse CD138 (BD Biosciences, San Jose, CA) and anti-phycoerythrin magnetic beads (Miltenyi Biotech). Purified plasmablasts (105/well in 200 μL of complete RPMI) were incubated for 44 hours with different ligands at 500 ng/mL. Number of viable plasmablasts were analyzed by IgG2a/c enzyme-linked immunospot (ELISPOT).
Survival of CD138+, tetanus toxoid-specific splenic plasmablasts, obtained at the peak of a booster response, was measured in the presence or absence of various BAFF and APRIL proteins, essentially as described31,32 (Document S1).
Natural and engineered BAFF oligomers are more potent than BAFF 3-mer at delivering multimerization-dependent signals
Some TNF family ligands are not biologically active as soluble trimers, but gain activity when oligomerized by fusion with the Fc portion of an immunoglobulin, or by addition of cross-linking antibodies, thereby mimicking the membrane-bound form of the ligand.29,33 It is not known whether the activity of BAFF is conditioned by its level of oligomerization, such as those found in BAFF 3-mer and BAFF 60-mer.13,34 To monitor the activity of BAFF 3-mer and oligomers, a surrogate cell death assay was developed, in which the oligomerization-dependent apoptotic Fas pathway can be initiated by BAFF. For this purpose, a fusion protein consisting of the extracellular domain of BCMA fused to the transmembrane and intracellular domains of Fas was expressed in Jurkat T cells. Clones that underwent apoptosis in response to BAFF were selected. Ligands tested included BAFF 3-mers (myc-BAFF, Flag-BAFF, and BAFF with mutation H218A preventing 60-mer formation) and various BAFF oligomers (ie, containing 2 or more BAFF 3-mers within the same complex) such as Fc-BAFF, Flag-BAFF plus anti-Flag, or BAFF 60-mers (BAFF starting at the natural processing site A134, with or without mutation N242Q that prevents N-glycosylation; Figure S1). Stimulation of Jurkat BCMA:Fas cells revealed that all BAFF oligomers were 2 to 3 orders of magnitude more potent than BAFF 3-mers at inducing cell-death through the surrogate, multimerization-dependent Fas signaling pathway (Figure 1A).
BAFF 3-mer and 60-mer bind BCMA and TACI, but only BAFF 60-mer, BAFF oligomers, and membrane-bound BAFF activate a reporter signaling pathway dependent on ligand oligomerization. (A) Jurkat T cells expressing a BCMA:Fas chimerical receptor were exposed for 16 hours to various concentrations of BAFF 3-mer or 60-mer. Cells were also exposed to Flag-BAFF (3-mer) alone or in the presence of a cross-linking anti-Flag antibody, or to Fc-BAFF (6-mer). Cell viability was measured with the PMS/MTS assay. (B) Jurkat BCMA:Fas cells were incubated with various ratio of 293T cells transfected with full-length BAFF R125A R126A (293T-BAFF uncleaved) or with empty plasmid (293T mock). Myc-BAFF (3-mer) and BAFF A134 (60-mer) were used instead of cells to calibrate the assay. BAFF present in transfected 293T cells was quantified by ELISA. The highest concentration used (40 ng/mL) corresponds to an effector to target ratio of 1:1. Maximal cell death obtained with this assay was approximately 50%. (C) BAFF 3-mer and BAFF 60-mer were immunoprecipitated with TACI-Fc. Inputs and immunoprecipitates (IP) were analyzed by Western blotting using an anti-BAFF antibody (Buffy-2). The same blot was reprobed with an anti-Fc antibody (bottom). The higher band of BAFF carries an N-linked oligosaccharide on asparagine 242, a site that is not used in mammalian cells.9 Note that recombinant TACI-Fc is partially processed at Lys108 and Arg110.28 (D) Recombinant BCMA-Fc, TACI-Fc, and BAFF-R-Fc were immobilized on ELISA plates and exposed to a fixed concentration of Flag-BAFF, whose binding was visualized with anti-Flag secondary reagents. BAFF 3-mer and BAFF 60-mer were added at the indicated concentrations to compete with Flag-BAFF binding and therefore reduce the ELISA signal.
To determine whether membrane–bound BAFF can trigger a multimerization-dependent pathway, a noncleavable form of full-length BAFF with mutations R125A and R126A in the furin-processing site was generated (Figure S2). Cells expressing noncleavable membrane-bound BAFF were at least 50-fold more active than soluble BAFF 3-mer on BCMA:Fas Jurkat cells, indicating that membrane-bound BAFF can indeed be assimilated to an oligomeric ligand (Figure 1B). Membrane-bound BAFF was apparently less active that soluble BAFF 60-mer in this assay (Figure 1B), possibly because only a fraction of membrane-bound BAFF is expressed at the cell surface.
To determine whether initiation of productive signaling through unmodified BCMA was also sensitive to BAFF oligomerization, BCMA-transfected 293T cells were exposed to BAFF 3-mer or BAFF 60-mer, and monitored for the activation of a NF-κB reporter system. In this assay, BAFF 60-mer readily induced a robust, BCMA-dependent NF-κB signal, whereas BAFF 3-mer was roughly 1000-fold less active (Figure S3).
The difference between BAFF 3-mer and BAFF 60-mer observed in both assays could not be accounted for by differential binding to the receptor, as both proteins bound TACI-Fc in immunoprecipitation experiments (Figure 1C) and similarly bound BCMA, TACI, and BAFF-R in an ELISA assay (Figure 1D). Only BAFF N242Q displayed a comparatively reduced affinity (Figure 1D), which probably explains why it was somewhat less active than BAFF A134 (Figure 1A).
These results indicate that BAFF oligomers, including membrane-bound BAFF and BAFF 60-mer, are activators of multimerization-dependent signaling pathways.
The antimouse BAFF-R antibody B9C11 antagonizes BAFF–BAFF-R signals
Receptor-deficient mice were used to study the impact of ligand oligomerization on BAFF-R and TACI signaling in primary B cells. However, instead of using BAFF-R−/− B cells that are not only scarce but also immature, BAFF-R-expressing B cells were treated with a blocking anti-BAFF-R antibody (B9C11). Despite a weak agonist activity, B9C11 completely abrogated the pro-survival effects of Myc-BAFF (3-mer), BAFF H218A (3-mer), and BAFF N242Q (60-mer), and also antagonized the pro-survival effects of BAFF A134 (60-mer) at concentrations less than 20 ng/mL (Figure 2M-P). Thus, B9C11 prevents BAFF signaling through BAFF-R and can therefore be used to discriminate BAFF signals delivered through BAFF-R from those given through TACI and/or BCMA.
Costimulation of thymidine incorporation in BCR-stimulated B cells by BAFF and APRIL is mediated by both BAFF-R and TACI. TACI responds to oligomerized ligands only. Purified B splenocytes of various genotypes were cultured under BCR-stimulating conditions, with or without anti–BAFF-R B9C11, and with the indicated concentrations of BAFF and APRIL. After 48 hours, cells were pulsed for 16 hours with thymidine, harvested, and counted. Points represent mean plus or minus SD of triplicate cultures. Note that B9C11 abolishes BAFF-mediated effects through BAFF-R. At the concentration used, B9C11 totally blocked BAFF A134 at concentrations up to 10 ng/mL, but only partially at higher concentrations.
BAFF 3-mer, BAFF oligomers, and APRIL costimulate B cells through distinct sets of receptors that respond differentially to ligand oligomerization
The ability of BAFF 3-mers and oligomers to costimulate thymidine incorporation in BCR-stimulated primary B cells was tested in vitro. For this purpose, ligands were titrated on purified B splenocytes of wild-type, BCMA−/−, TACI−/−, and BCMA−/− × TACI−/− mice, in the presence of stimulating anti-IgM antibodies and with or without addition of the B9C11 anti–BAFF-R antibody at a blocking concentration. Previous work demonstrated that anti-IgM stimulation up-regulates TACI in these BAFF-R–positive cells.35 Because APRIL binds to TACI and BCMA, but not to BAFF-R, recombinant Flag-tagged APRIL was also tested, in the presence or absence of cross-linking anti-Flag antibody.14 We selected human APRIL because it does not bind at all to murine BAFF-R, in contrast to mouse APRIL.36
Thymidine incorporation in BCR-stimulated wild-type B cells was costimulated in a dose-dependent manner by BAFF 3-mer, BAFF 60-mer, and cross-linked APRIL (Figure 2A,B,Q). Although CFSE-labeled cells underwent division in response to anti-BCR stimulation, costimulation with BAFF, or cross-linked APRIL increased the number of viable cells without affecting the division pattern (Figure S4). BAFF also enhanced B-cell survival in the absence of B-cell receptor stimulation (Figure S4). These results suggest that the increased thymidine incorporation mediated by BAFF and APRIL costimulation is the result of mainly, if not entirely, increased B-cell survival.
APRIL-mediated effects were entirely TACI-dependent and insensitive to the presence of the B9C11 anti-BAFF-R antibody (Figure 2S,U,V). In contrast, the survival effect of BAFF 3-mer was abrogated in the presence of B9C11, irrespective of the presence or absence of TACI and BCMA (Figure 2M,O). Thus, the same biologic outcome, B-cell survival, was mediated through BAFF-R in response to BAFF 3-mer, and through TACI in response to cross-linked APRIL.
The survival effects of BAFF 60-mers were only reduced by the blocking anti-BAFF-R antibody or by TACI deficiency in isolation but was abrogated by the combination of both, demonstrating that BAFF 60-mer activated both BAFF-R and TACI (Figure 2A,G,K,M). Interestingly, TACI signals were only induced by oligomeric ligands, ie, BAFF 60-mer or cross-linked APRIL, but not by Flag-tagged APRIL or 2 different BAFF 3-mers. In contrast, BAFF-R signals were triggered by both BAFF 3-mer and BAFF 60-mer.
In addition to BAFF 60-mer and cross-linked APRIL, TACI signaling was also efficiently triggered by Fc-BAFF and cross-linked Flag-BAFF, but not by Flag-BAFF alone, indicating that TACI responds to oligomeric, but not trimeric, ligands (Figure S5). Moreover, signaling of BAFF N242Q (60-mer) through TACI could be inhibited in a dose-dependent manner by an excess of BAFF H218A (3-mer; Figure S6).
Taken together, these results indicate that costimulation of BCR-stimulated B cells results from increased cell survival rather than increased cell proliferation and that different ligands functionally engage different sets of receptors: BAFF 3-mer signals through BAFF-R only, cross-linked APRIL through TACI only, and BAFF 60-mer through both BAFF-R and TACI, the latter receptor responding to oligomeric ligands only. BCMA was not involved in B-cell costimulation.
TACI-mediated, but not BAFF-R–mediated MHC class II up-regulation requires oligomeric ligands
Murine B cells treated with IL-4 and IL-6 up-regulate MHC class II molecules and can present antigens in response to APRIL.37 MHC class II was up-regulated in B cells not only in response to cross-linked APRIL, but also in response to BAFF 3-mer and BAFF 60-mer, but not Flag-APRIL alone (Figure 3). The effect of APRIL was TACI-dependent, whereas that of BAFF 3-mer and BAFF 60-mer was also seen in B cells expressing BAFF-R only (Figure 3). Blocking of BAFF-R with B9C11 completely abrogated the activity of Myc-BAFF (3-mer) and Flag-BAFF (3-mer), but only reduced that of cross-linked BAFF, Fc-BAFF, and BAFF 60-mer, indicating that BAFF oligomers can also signal MHC class II up-regulation through TACI (Figure S7). Thus, as shown for B-cell costimulation, ligand-mediated up-regulation of MHC class II in B cells can be mediated by BAFF-R and TACI, the latter receptor responding to oligomeric ligands. BCMA was not required for MHC class II up-regulation.
BAFF-R– and TACI-dependent up-regulation of MHC class II in B cells. (A) Purified wild-type and TACI−/− B splenocytes were stimulated plus or minus cross-linked APRIL in the presence of IL-4 and IL-6, and surface expression of MHC class II was monitored by FACS. (B) Experiment was performed as in panel A with B cells of the indicated genotype, and with BAFF and APRIL at the indicated concentrations. MFI indicates mean fluorescence intensity.
Increased immunoglobulin production by B splenocytes and plasmablast survival require TACI and oligomerized ligands
BAFF and APRIL enhance immunoglobulin production in in vitro cultures of purified B splenocytes.21 IgM production in supernatants of B splenocytes cultured for 6 days was increased 3- and 6-fold in response to cross-linked APRIL and BAFF 60-mer, respectively, an effect that was mainly mediated by TACI (Figure 4A). In contrast, BAFF 3-mer only induced a modest, BAFF-R–dependent response. Although IgA secretion paralleled that of IgM in this assay, this was the result of increased survival of preexisting IgA-positive cells rather than isotype switch (data not shown). We conclude that increased IgM and IgA production in this assay was predominantly mediated by TACI in response to oligomerized ligands, with a possible marginal contribution of BAFF-R.
Enhanced IgM secretion by splenocytes and plasmablast survival require TACI and oligomerized TACI ligands. (A) Purified B splenocytes were cultured for 6 days in the presence of 100 ng/mL of the indicated ligands, after which time IgM titers were measured in culture supernatants. The figure shows the fold increase relative to medium only for the same genotype (titers were comparable for the different genotypes). Results are mean plus or minus SD of triplicate cultures. (B) MMTV-induced plasmablasts of the indicated genotypes were purified and cultured for 2 days in the presence of 500 ng/mL of the indicated ligands or 10 ng/mL of IL-6, after which time IgG2a/c-secreting cells were enumerated by ELISPOT. ASC indicates antibody-secreting cell. Data are expressed as fold increase compared with wild-type B cells in medium only, and represent mean plus or minus SD of triplicate (APRIL) or quadruplicate (medium and BAFF) cultures, representative of 2 experiments with similar results. (C) Tetanus toxoid-specific plasmablasts were purified from spleen at the peak of the booster response and cultured for 2 days in the presence of 500 ng/mL of the indicated ligands or 10 ng/mL of IL-6. Tetanus toxoid-specific plasmablasts were enumerated by ELISPOT, and the percentage of survival determined relative to values obtained at day 0. Values are mean plus or minus SD of 2 independent cell preparations (or 3 preparations for wild-type) cultured in triplicates (*P < .05; **P < .01).
Increased immunoglobulin production can result from either enhanced Ig secretion and/or from a higher number of Ig-secreting cells. To specifically address the survival effect of BAFF and APRIL on antibody secreting cells, mice were infected with MMTV and synchronized plasmablasts were isolated from the draining lymph node 6 days after infection.30 Alternatively, mice were immunized with tetanus toxoid, and plasmablasts were isolated from spleens at the peak of the booster response. Purified plasmablasts were cultured for 2 days in vitro, in the presence or absence of BAFF or APRIL, and the number of viable, antibody-secreting cells was quantified by ELISPOT. Under these conditions, numbers of antibody secreting cells were increased 6- to 10-fold in response to BAFF 60-mer or cross-linked APRIL (Figure 4B,C). This effect was impaired in TACI-deficient cells and, to a lesser extent, in BCMA-deficient cells, and was completely abrogated in cells deficient for both TACI and BCMA, indicating that TACI and BCMA, but not BAFF-R, contribute to plasmablast survival. BAFF 3-mer and Flag-APRIL displayed weaker activity than their oligomerized counterparts on responsive B cells, in line with the idea that TACI, and possibly BCMA, require oligomerized ligands. In summary, plasmablast survival is enhanced by oligomeric BAFF and APRIL in a TACI- and possibly BCMA-dependent manner.
Formation of 60-mers is a property of both human and mouse BAFF
If oligomerization of BAFF in the form of a 60-mer were a physiologically relevant feature, as suggested by its specific ability to trigger TACI-dependent responses, it would be expected to be conserved across species. The extended DE loop of BAFF, also known as the flap region,12 is not found in other TNF family members, but its length is precisely conserved in BAFF of fishes, batrachians, birds, and mammals (Figure 5A). Moreover, all residues involved in trimer-trimer interaction, including the histidine residue responsible for pH-dependent assembly of the 60-mer,12,13 are conserved in human, mouse, opossum (marsupial), platypus (monotreme), chicken, and 10 additional bird and mammalian BAFFs (Figure 5A and data not shown).
BAFF 60-mer is conserved across species. (A) Alignment of BAFF from various species, and of human TNF family members, in the region of the DE loop. Opossum:Monodelphis domestica (GenBank:XP_001375808). Platypus:Ornithorynchus anatinus (GenBank:XP_001514753). Chicken:Gallus gallus (GenBank:NM_204327). Frog:Xenopus tropicalis (EMBL:42:1929088:1912327). Fish:Danio rerio(GenBank: XM_684671). (B) Gel permeation chromatography analyses of naturally processed mouse BAFF in supernatants of 293T cells transfected with full-length mouse BAFF. Elutions were performed at pH 7.4 or pH 5, and the elution positions of molecular weight standards (in kDa) are indicated at the top of the figure. Fractions were analyzed by Western blotting using a cross-reactive antihuman BAFF antibody (Buffy-2).
It has been previously demonstrated that addition of a short N-terminal Myc tag to soluble BAFF prevents 60-mer formation.13 Because murine BAFF contains an additional exon encoding 31 amino acid residues close to the mature N-terminus of soluble BAFF9 (Figure S1), it is questionable whether mouse BAFF can form 60-mer at all. However, supernatants of 293T cells transfected with full-length mouse BAFF contained, in addition to BAFF 3-mer, a fair proportion of a high molecular weight ( > 600 kDa) form of processed mouse BAFF, suggesting that it was able to form 60-mers. This conclusion was reinforced by the observation that murine BAFF 60-mer was acid-sensitive and, therefore, no longer detected when analyzed at pH 5 (Figure 5B). We conclude that the formation of a 60-mer is an evolutionarily conserved property of BAFF.
Circulating BAFF 60-mer in BAFF transgenic mice and TACI−/− mice is a major provider of oligomerization-dependent signals
Although BAFF 60-mer has been previously found in supernatants of human U937 cells,13 it has never been described in ex vivo samples. For the purpose of detecting BAFF 60-mer in vivo, plasma of BAFF transgenic mice that express full-length mouse BAFF under the α1-antitrypsin promoter27 was size-fractionated by gel permeation chromatography and analyzed for its BAFF content by ELISA and for its oligomerization-dependent activity with Jurkat BCMA:Fas cells. Although the majority of BAFF eluted with a size of approximately 60 kDa (BAFF 3-mer), approximately 4% of the BAFF protein eluted at a size of approximately 1000 kDa, compatible with that of BAFF 60-mer, and accounted for two-thirds of the total oligomerization-dependent activity (Figure 6A). The activity of this peak was totally abrogated by a soluble decoy receptor and displayed acid sensitivity, further suggesting that it was the result of BAFF 60-mer (Figure 6A and data not shown). In line with these results, BAFF 3-mer isolated from plasma of BAFF transgenic mice induced a robust, B9C11-inhibitable survival response in wild-type B cells, whereas the more diluted BAFF 60-mer also induced a specific response, but of lower intensity (data not shown).
Differentially active forms of trimeric and oligomeric BAFF in mouse plasma. Plasma from BAFF transgenic (A), wild-type (B), and TACI−/− (C) mice were fractionated by gel permeation chromatography. Mouse BAFF was quantified in the fractions by ELISA (■) and expressed with different scales for each genotype. The oligomerization-dependent activity of BAFF in 25 μL, 5 μL, and 2.5 μL of wild-type, BAFF transgenic, and TACI−/− fractions, respectively, was detected with reporter Jurkat BCMA:Fas cells that die in response to oligomerized BAFF (○). For wild-type and TACI−/− fractions, the assay was performed in the presence of cycloheximide (CHX) that enhances the sensitivity of the assay. Where indicated, BCMA-Fc was added to demonstrate the killing specificity (●).
BAFF 60-mer was then sought in mice that are not transgenic for BAFF. As the Fas pathway used in the bioassay is inhibited by short-lived proteins such as FLIP, its sensitivity can be increased by inhibitors of protein synthesis.38 Indeed, cycloheximide increased the detection limit of the assay approximately 10-fold (Figure S8), but this remained insufficient to detect the activity of a putative BAFF 60-mer in the plasma of wild-type mice (Figure 6B). However, under these conditions, BAFF 60-mer was readily detected in the plasma of TACI−/− mice that display approximately 10-fold elevated circulating BAFF levels compared with wild-type mice (Figure 6C).
An additional activity peak of intermediate size and of higher specific activity than BAFF 3-mer was detected in both BAFF transgenic and TACI−/− mice but was not further characterized (Figure 6A,C).
We conclude from these experiments that several forms of soluble BAFF are found in the plasma of BAFF transgenic and nontransgenic mice, including a low-abundance but highly active BAFF 60-mer.
One of most striking finding of this study is the differential ability of BAFF-R and TACI to signal in response to their common ligand BAFF. Although both receptors bind BAFF with high affinity (reviewed by Bossen and Schneider1 ), BAFF-R responded to both BAFF trimers and higher-order BAFF oligomers, whereas TACI showed a strict specificity for the latter. This differential signaling behavior was consistently observed in several assays involving B cells at various stages of differentiation, even if the final outcome of the signaling pathways, such as cell survival or up-regulation of surface markers, was the same for both receptors.
What are the physiologic forms of BAFF that can signal through TACI? An obvious candidate is the membrane-bound form of BAFF that has been identified on myeloid cells.7,8 Thus, productive signaling through TACI may require cell-cell contacts, ensuring that the signal is delivered at specific sites, and not in a systemic manner. BAFF-R, in contrast, may also respond to soluble BAFF 3-mer, which represent the majority of systemic BAFF. This situation is reminiscent of TNF-R2, which responds to membrane-bound TNF, and TNF-R1, which responds to both membrane-bound and trimeric soluble TNF.39 Another candidate activator of TACI signals is BAFF 60-mer, an oligomeric form of BAFF known from crystallographic studies,12,34,40 but for which little or no information exist regarding the function and occurrence in vivo. Our study provides important new elements pointing to possible physiologic implications of BAFF 60-mer. First, BAFF 60-mer formation is evolutionarily conserved. Second, an active form of BAFF 60-mer is found in plasma of BAFF transgenic mice but also in plasma of mice such as TACI−/− with an unmanipulated BAFF gene. Third, BAFF 60-mer and BAFF 3-mer activate different sets of receptors and can therefore be considered as distinct ligands.
The originality of BAFF compared with membrane-bound and soluble TNF is its unique mode of oligomerization as a virus-like particle, its pH-sensitive nature, and the ability to mimic a membrane-bound form of the ligand in a soluble form.
How is it possible to explain that TACI and BAFF-R have different requirements for ligand oligomerization? This may reflect receptor-specific mechanisms of intracellular signaling. TACI binds TNF receptor–associated factors (TRAFs) on its intracellular domain (reviewed by Bossen and Schneider1 ). TRAFs are trimeric intermediates in the signaling pathway of numerous TNF receptor family members.41 By bringing together 3 receptors, a trimeric ligand may allow recruitment to the complex of just one intracellular TRAF, which may not be enough to induce signaling, as oligomerization of trimeric TRAF-2 and TRAF-6 is required to activate downstream signaling events.42 BAFF-R only interacts with TRAF-3, whose mode of action and multimerization requirements may be distinct, making BAFF-R–mediated signals less dependent on ligand oligomerization.
Beside promoting survival, BAFF and APRIL also induced up-regulation of activation markers such as MHC class II, in line with the enhanced MHC class II expression found in B cells of BAFF transgenic mice at various maturation stages.27 Although BCMA was reported to induce MHC class II up-regulation in IL-4– and IL-6–stimulated A20 cells,37 our results demonstrate that MHC class II expression in primary B splenocytes can also be stimulated by BAFF-R and TACI signals, and that stimulation with IL-4 and IL-6 was not essential for this outcome.
It is well documented that BAFF-R induces B-cell survival, and BCMA is strongly suspected to promote survival of long-lived plasma cells.15,43 However, in contrast to BAFF-R and BCMA, TACI is generally described as a negative regulator of B-cell survival because TACI-deficient mice have an enlarged B-cell pool23,24 and because an engineered cell line expressing an ectodysplasia A receptor/TACI chimera could be killed with ectodysplasia A.24 In addition, agonist anti-TACI antibodies inhibited proliferation of a B-cell line,24 and anti-TACI antibodies prevented Ig secretion in BAFF-R– or CD40-stimulated B cells.24,44 How can these results be reconciled with our observation that TACI only mediated positive events such as survival of primary B cells in vitro when engaged by BAFF and/or APRIL? TACI signals triggered with anti-TACI antibodies or with natural ligands may be different, for example, because of a differential impact on TACI-proteoglycan interactions, which appear to modulate TACI signals.45,46 In addition, it has not been demonstrated in vivo that TACI directly transmits negative signals within B cells: B cell hyperplasia in TACI−/− mice may also result from the loss of a BAFF/APRIL inhibitor such as a soluble, decoy form of TACI. TACI can indeed be processed at Arg122, Arg110, and Lys108, and each of these cleavage events is predicted to release functional forms of soluble human and mouse TACI.28 Thus, loss of TACI could remove both a survival signal for TACI-expressing cells and an inhibitor of soluble BAFF, leading to the observed raise of BAFF concentration in TACI−/− mice. As a result, naive B cells (expressing BAFF-R) would survive better or in greater number because of increased BAFF availability, leading to the observed B-cell hyperplasia, whereas plasmablasts and short-lived Ig-secreting cells that preferentially up-regulate TACI and play a key role in T-independent humoral responses3,47 would be specifically penalized by loss of TACI.
Interestingly, T-independent responses are inefficient in TACI−/− mice23 and BAFF−/− mice,20 but not BAFF-R−/− mice,20 suggesting that they rely on a BAFF/TACI axis that, according to our findings, would require an oligomerized form of BAFF (60-mer or membrane-bound). Loss of TACI also leads to decreased IgA levels.23 In this respect, we can speculate that the much elevated IgA levels observed in BAFF transgenic mice27 may be due, at least in part, to BAFF 60-mer acting on TACI. A pro-survival role of TACI is also in line with its up-regulation in several multiple myeloma cell lines.48
Decreased plasmablast survival could contribute to decreased immunoglobulin levels in CVID patients with TACI mutations. Until now, impaired Ig switch has received great attention to explain the link between TACI deficiency and CVID.21,25,26 Nevertheless, IgM levels are often reduced in these patients, indicating that defective Ig switch may not be the sole cause of CVID, in line with an implication of TACI in plasmablast survival.
Inadequate immune responses can lead to autoimmunity, and BAFF overproduction is consistently found in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, or Sjögren syndrome.2 As both short- and long-lived Ig-secreting cells are involved in the pathology of SLE, signaling through both TACI and BCMA might contribute to the disease.49 We have shown that TACI respond to oligomerized ligands, and it will be of interest to determine whether BAFF 60-mer may be produced in excess in these pathologies and whether the specific targeting of BAFF 60-mer may represent an alternative treatment for these autoimmune disorders.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
The authors thank Chantal Tougne (University of Geneva) for expert technical assistance, Susan Kalled (BiogenIdec) for her help in providing reagents, Fang Qian (BiogenIdec) for sharing preliminary results, and Melissa Starovasnik (Genentech, San Francisco, CA) for providing the B9C11 antibody.
This work was supported by grants from the Swiss National Science Foundation, the National Center of Competence in Research in Molecular Oncology, and the Commission for Technology and Innovation program (P.S., J.T.).
Contribution: C.B. performed research, analyzed data, and wrote the paper; T.G.C. designed research and contributed vital reagents; A.T., K.I., L.W., A.M., and S.C. performed research; M.D., M.L.S., E.B., C.-A.S., H.A.-O., H.L., and F.M. contributed vital reagents or techniques; J.T. designed research; and P.S. designed research, analyzed data, and wrote the paper.
Conflict-of-interest disclosure: T.G.C. is a stockholder of BiogenIdec. All other authors declare no competing financial interests.
Correspondence: Pascal Schneider, Biochemistry Department, University of Lausanne, Boveresses 155, CH-1066 Epalinges, Switzerland; e-mail: pascal.schneider@unil.ch.
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Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex.
Ligand-receptor binding revealed by the TNF family member TALL-1.
B cell-activating factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells.
Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human.
J Biol Chem
Legarda-Addison
Varughese
B cell maturation antigen, the receptor for a proliferation–inducing ligand and B cell-activating factor of the TNF family, induces antigen presentation in B cells.
Wajant
NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling.
Douni
The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor.
Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation.
Nat Struct Biol
Thermodynamic characterization of the interaction between TRAF2 and TNFR peptides by isothermal titration calorimetry.
Signaling by proinflammatory cytokines: oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal effector domain.
Genes Dev
Ellyard
BAFF selectively enhances the survival and effector function of CD38+ plasmablasts generated from activated human memory B cells.
Okumura
Kobata
TACI attenuates antibody production costimulated by BAFF-R and CD40.
TACI regulates IgA production by APRIL in collaboration with HSPG.
Elsawa
Mantchev
Selective activation of TACI by syndecan-2.
Yoon
BAFF supports human B cell differentiation in the lymphoid follicles through distinct receptors.
Int Immunol
Darce
Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival.
Short-lived plasmablasts and long-lived plasma cells contribute to chronic humoral autoimmunity in NZB/W mice.
© 2008 by The American Society of Hematology
Document 1. Supplemental methods (PDF, 72.1 KB)- pdf file
zh1004-sup-figures1- jpeg file
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redpajama_set_name: RedPajamaCommonCrawl


Beautiful House with private swiming pool, near Golf club S'era de Pula.
Es Moli nou is a wonderful house, near Costa de los Pinos. The house and mill was reformed in 1997. This located near the beach, golf place, supermarket. The garden is very big and nice for relax.
The Villa has two separate houses in the same building. This advertisement is to rent the main house, located at the top of the building. The owner lives in the house below. Garden and pool are to be used exclusively for the main house rented.
The beach is only 2km, the golf course only 500m.
Roman ruins have now found just 200m.


text:
Front page - The U-boats - Listing of all U-boats
Carl Emmermann
(U-172, U-3037)
Laid down 1 Nov 1940 Deutsche Werft AG, Hamburg (werk 305)
Commissioned 4 Nov 1941 Korvkpt. Karl-Heinz Wolff
4 Nov 1941 - Sep, 1942 KrvKpt. Karl-Heinz Wolff
Sep, 1942 - 15 Jul 1943 KrvKpt. Werner Witte (German Cross in Gold)
4 patrols
4 Nov 1941 - 30 Jun 1942 4. Flottille (training)
1 Jul 1942 - 15 Jul 1943 10. Flottille (active service)
Successes 5 ships sunk, total tonnage 29,091 GRT
3 ships damaged, total tonnage 20,014 GRT
1 ship a total loss, total tonnage 7,129 GRT
Sunk on 15 July 1943 in the North Atlantic south of the Azores, in position 34.02N, 26.01W, by a Fido homing torpedo from an Avenger aircraft (VC-29 USN/T-12), assisted by a Wildcat aircraft (VC-29 USN/F-7), of the US escort carrier USS Santee. 54 dead (all hands lost).
Loss position
See the 9 ships hit by U-509 - View the 4 war patrols
Wolfpack operations
U-509 operated with the following Wolfpacks during its career:
Streitaxt (20 Oct 1942 - 2 Nov 1942)
Schlagetot (9 Nov 1942 - 15 Nov 1942)
Attacks on this boat and other events
While operating against the Allied Torch landings off Morocco the boat operated for three days in waters less than 200 ft (61m) deep, and Witte reported being constantly bombed and depth charged by Allied surface vessels and aircraft. Damage forced her to move to deeper water and then to France. (Sources: Blair, vol 2, page 108)
After sinking the British merchantman Queen Anne, U-509 was fired on by ASW trawler HMS St. Zeno, which also dropped seven depth charges before the boat managed to escape. No damage. (Sources: Blair, vol 2, page 229)
2 recorded attacks on this boat.
Men lost from U-boats
Unlike many other U-boats, which during their service lost men due to accidents and various other causes, U-509 did not suffer any casualties (we know of) until the time of her loss.
We have 1 emblem entry for this boat. See the emblem page for this boat or view emblems individually below.
Hitler's U-boat War, Vol II
Blair, Clay
U-Boat Operations of the Second World War - Vol 1
Wynn, Kenneth
German U-Boat Losses During World War II
Niestle, Axel
Hitler's U-boat War


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text:
Ar-Bo
Antoine-Henri Becquerel - Chemistry Encyclopedia
Antoine-Henri Becquerel
FRENCH PHYSICIST
Antoine-Henri Becquerel was born the son of the physicist Alexandre-Edmond Becquerel, and the grandson of the physicist Antoine-César Becquerel, and it is not surprising that he followed in their footsteps. It is also not surprising that his research interests centered around solar radiation and phosphorescence, as these are phenomena that his father had investigated. He entered the École Polytechnique, in Paris, in 1872, which he left in 1874 and to which he subsequently returned. Becquerel received a doctorate degree from the Faculty of Sciences of Paris in 1888. In 1892, he was appointed professor of applied physics in the Department of Natural History at the Paris Museum, and in 1895, professor of physics at the École Polytechnique.
Becquerel's early work focused on plane-polarized light, the phenomenon of phosphorescence (in which certain compounds glow after being exposed to direct light), and the absorption of light by crystals. But all of his early research became overshadowed by his discovery of natural radioactivity. Although Becquerel did not initially comprehend what he was observing, his landmark discovery of radioactivity paved the way for a new understanding of the atom and atomic structure.
On February 24, 1896, Becquerel attended a meeting of the French Academy of Science and presented a short paper (one of the quickest methods in France at that time for disseminating results). One can well imagine Becquerel's excitement as he reported his results to the members of the academy.
One wraps a Lumiere photographic plate with a bromide emulsion in two sheets of very thick black paper, such that the plate does not become clouded upon being exposed to the Sun for a day. One places on the sheet of paper, on the outside, a slab of the phosphorescent substance, and one exposes the whole to the Sun for several hours. When one then develops the photographic plate, one recognizes that the silhouette of the phosphorescent substance appears in black on the negative. (Becquerel Comptes Rendus )
From this simple experiment, Becquerel concluded that the phosphorescent substance had to be emitting a type of ray that was passing through the paper and reducing the silver in the emulsion. This would seem to make sense, as the production of X rays, discovered a few years earlier by Wilhelm Röntgen, is accompanied by a soft glowing spot at the surface of the cathode ray tube. Becquerel decided to probe his unusual rays a little further. One week later, on March 2, 1896, Becquerel was back before the French Academy with the results of his further experiments. He had continued his experiments using a double sulfate salt of uranium and potassium
French physicist Antoine-Henri Becquerel, co-recipient of the 1903 Nobel Prize in physics, "in recognition of the extraordinary services he has rendered by his discovery of spontaneous radioactivity."
(potassium uranium sulfate monohydrate), which has a strong but short-lived phosphorescence.
He carefully wrapped his photographic plates in black paper, coating the paper with a crust of the uranium double salt, and upon exposure to sunlight he once again observed the "signature" of the phosphorescenceinduced rays. However, repeating the experiment on Wednesday, February 26, and Thursday, February 27, he was frustrated by two days of only intermittent sunlight. And because the Sun made no appearance on the two days following, on March 1 he developed his plates. Expecting to see only a faint silhouette resulting from the wrapped plates' intermittent exposure to sunlight, he was surprised to see that the silhouettes appeared with great intensity.
Becquerel suspected that the rays that produced the silhouettes emanated from the uranium salt itself, and that the small amount of sunlight was of no consequence. He arranged three more experiments, in which photographic plates were kept completely in the dark but put in direct contact with: (1) the salt; (2) a thin sheet of glass; and (3) a thin sheet of aluminum. He surmised that the glass would eliminate any possibility that a silhouette was the consequence of a chemical reaction, and that the aluminum would block the mysterious rays.
Developing the photographic plates, Becquerel observed an intensely defined silhouette on the first two plates, and a clear but considerably weaker silhouette on the third. Because he had double-boxed his plates inside his dark room and had placed the ensembles inside a drawer that he then closed, he was able to conclude that his mysterious rays were not related to phosphorescence and were not induced by sunlight.
It was another four years before Becquerel's radiation became understood as the production of β -rays (high energy electrons), but by then there was no question that Becquerel had discovered the instability of some atomic nuclei, and that he was richly deserving of the 1903 Nobel Prize that he shared with Pierre and Marie Curie.
SEE ALSO Curie, Marie Sklodowska ; RÖntgen, Wilhelm .
Todd W. Whitcombe
Asimov, Isaac (1989). Asimov's Chronology of Science and Discovery. New York: Harper & Row.
Farber, Eduard (1952). The Evolution of Chemistry: A History of Its Ideas, Methods, and Materials. New York: Ronald Press.
Leicester, Henry M. (1956). The Historical Background of Chemistry. New York: Wiley.
Becquerel, Antoine H. Comptes Rendus , translated. "On the Rays Emitted by Phosphorescence." Available from http://webserver.lemoyne.edu/faculty/giunta .
Becquerel, Antoine H. Comptes Rendus translated. "On the Invisible Rays Emitted by Phosphorescent Bodies." Available from http://webserver.lemoyne.edu/faculty/giunta .
Nobel e-Museum. "Antoine Henri Becquerel—Biography." Available from http://www.nobel.se .
Antoine-Henri Becquerel - Chemistry Encyclopedia forum
Bardeen, John Berg, Paul


text:
Crieff Morrisonians Football Club was a football club from the village of Crieff, Perthshire, Scotland.

History

The first recorded activity for the club was at the end of the 1896–97 season, under the name Morrisonian F.C., the name taken from Morrison's School in Crieff.  Indeed the club was originally solely for pupils and ex-pupils, although the restriction was subsequently abandoned.

The club sought to join the Perthshire Association for the 1897–98 season, by now called Crieff Morrisonians, but the application was rejected.

The club entered the Scottish Qualifying Cup from 1898–99 to 1920–21.  The only time it won through to the Scottish Cup was in 1904–05.  Wins over Vale of Teith F.C., Stanley, and Lochee United F.C. were enough to qualify for the first round proper.  In the fourth round of the Qualifying Cup, the club lost 6–2 at home to St Johnstone, and in the Cup itself, the club went down 3–1 at Kirkcaldy United.

The club's greatest achievements were in winning the Perthshire League in 1908–09 and 1910–11, although the competition in the era scarcely had more than half-a-dozen entrants.

The club reached the final of the Perthshire Cup four times, losing on every occasion.  Its closest finals were in 1909–10, losing 2–1 against Dunblane after conceding a last-minute winner via a freak deflection, and 1919–20 losing 3–2 to Blairgowrie Amateurs F.C.; the loss of several players to the junior ranks post-war had cost the team many of its best.

Perhaps the club's finest result was a 4–2 win over St Johnstone en route to the 1909–10 County Cup final, although the Saints turned up two men short.  The club did pick up the Consolation Cup in 1911–12, with a 3–0 win at Vale of Atholl F.C. in the final.  The second XI also won the Perthshire 2nd XI Cup in 1903–04 and 1904–05, the second year by beating St Johnstone 9–3 in the final, Harry Paul - then still a schoolboy at the Academy - being particularly praised for his centre-forward play.  During the match, MacPhail tore his shorts, and, because he lived close to the ground, ran home to change into long trousers.

The club became a limited liability company in May 1914, in order to put the club on a more professional footing, and to clear accumulated debts; the plan was to join the Central Football League, but the timing was unfortunate, as World War 1 put most football competitions in abeyance - indeed the club shut down operations entirely for the duration.

1920–21 proved to be the club's final season.  The Sonians' final appearance in the Scottish Cup ended in ignominy; after a walkover in the first round of the Qualifying Cup, the club scratched to Stenhousemuir F.C. in the second, in return for a £20 "bait".  The club also scratched from the Perthshire Cup in the semi-final in 1920–21 when drawn to play St Johnstone, and, although it entered the same competition for 1921–22, the club was wound up in March 1921.

Colours

The club's colours were green and white.

Ground

The club originally played at the Hollybush farm ground, owned by a Mr Temple.  From 1904 the club moved to Turretbridge Park.

Notable players

Harry Paul, who later joined Queen's Park and became a Scottish international.
James MacGregor, left-back who was the only Crieff player to gain County honours, who later played for Partick Thistle F.C. and Dundee F.C.

External links

Perthshire Cup results
Perthsire League

References

Football clubs in Scotland
Defunct football clubs in Scotland
Association football clubs established in 1897
Association football clubs disestablished in 1921
1897 establishments in Scotland
1921 disestablishments in Scotland


text:
Create Jobseeker account
AFA Home
Professor of Finance and Business Economics - tenure track (rank open), Marshall School of Business
Competitive salary and benefits offered.
Full Professor or Chaired Professor (Research), Ph.D. Candidates (Tenure Track, Research), Seasoned Faculty (Tenure Track, Research)
Applications are invited for full-time tenure track faculty positions (rank open) in the Department of Finance and Business Economics at the USC Gordon S. Marshall School of Business. Appointments will begin Fall semester of 2019.
Applicants are sought in all areas of Finance and Economics.
Preferred qualifications for full-time positions include a doctoral degree or foreign equivalent in an appropriate area of study. Candidates nearing completion of their degree may also be considered. Preference may be given to individuals with relevant experience in teaching at the college/university level and/or evidence of research competence.
Application instructions: Only on-line applications via USC's faculty application portal will be accepted. Applications must include a cover letter, curriculum vitae, and at least one research paper. A minimum of three names and email addresses for recommendation letters are also required. Additional materials are optional.
There are upload fields for "Cover Letter", "CV", Curriculum" and "Other". Each field permits ONE attachment only. PDF files preferred - maximum 5MB each. "Cover Letter" "and "CV" are self-explanatory. Upload your Job Market Paper to "Curriculum". "Other" may be used to upload optional documents (additional research papers, teaching or research statements, teaching evaluations, etc.), however all optional materials must be combined into a single PDF file before uploading.
At the end of the on-line application process, fields will be provided for the names and email addresses of a minimum of 3 and a maximum of 5 references. Email addresses do not need to be unique - if an Admin will be uploading your letters, use individual faculty names but the Admin's email address for each reference.
If you want to be notified when a letter is uploaded, please check the notification box ("I would like to be notified as my references submit their letters.") at the bottom of page 3 before you submit your application!
You will not be able to change your application on-line after submitting it. If you need to update, replace, or add to your application, email additional information or documents to fbe@marshall.usc.edu, with a subject line of "Application Update for Firstname Lastname".
Application links:
Finance: https://facultypositions.usc.edu/FAS/application/position?postingId=REQ20064935
Macroeconomics: https://facultypositions.usc.edu/FAS/application/position?postingId=REQ20064943
Microeconomics: https://facultypositions.usc.edu/FAS/application/position?postingId=REQ20064944
Application Deadline: Nov. 26, 2018. Late applications accepted!
Screening of applications will begin immediately upon receipt and will continue until the positions are filled. Initial interviews will occur at the ASSA/AFA/AEA meetings in Atlanta, Jan. 4-6, 2019.
The Department of Finance and Business Economics faculty are a collaborative and inclusive group of scholars who value diverse pedagogical, curricular, and methodological approaches.
USC is an equal opportunity, affirmative action employer, proudly pluralistic and firmly committed to providing equal opportunity for outstanding persons of every race, gender, creed and background. USC is building a diverse faculty that embraces a range of disciplinary traditions. The University will consider all candidates for employment without regard to race, color, religion, sex, sexual orientation, gender identity, national origin, protected veteran status, disability or any other characteristic protected by law or USC policy. USC will make reasonable accommodations for qualified individuals with known disabilities unless doing so would result in an undue hardship. USC will consider for employment all qualified applicants with criminal histories in a manner consistent with the requirements of the Los Angeles Fair Chance Initiative for Hiring ordinance.
For more information: email fbe@marshall.usc.edu or visit http://www.marshall.usc.edu/fbe
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text:
Robert Bolder
Only Yesterday (1933 film)
Robert Bolder (1859 – 10 December 1937) was an English film actor of the silent era. He appeared in 99 films between 1912 and 1936. He was born in London and died in Los Angeles, California.
His Athletic Wife (1913)
The Nick of Time Baby (1916)
The Beggar Prince (1920)
The Girl in Number 29 (1920)
Black Beauty (1921)
Beyond the Rocks (1922)
Grumpy (1923)
The Dramatic Life of Abraham Lincoln (1924)
Vanity's Price (1924)
Blue Blood (1925)
Tarzan and the Golden Lion (1927)
The Great Impersonation (1935)
Robert Bolder at the Internet Movie Database
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/Robert_Bolder
Only Yesterday may refer to:
Only Yesterday (1933 film), an American drama starring Margaret Sullavan
Only Yesterday (1991 film), a Japanese anime directed by Isao Takahata
"Only Yesterday" (song), a song by The Carpenters
Only Yesterday (album), a greatest hits album by The Carpenters
Only Yesterday: The Carpenters Story, a 2007 British TV documentary
Only Yesterday: An Informal History of the 1920s, a 1931 book by Frederick Lewis Allen, purported inspiration for the 1933 film
Only Yesterday, a 1945 novel by S. Y. Agnon
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/Only_Yesterday
Only Yesterday is a 1933 American Pre-Code drama film about a young woman who becomes pregnant by her boyfriend before he rushes off to fight in World War I. It stars Margaret Sullavan (in her film debut) and John Boles.
The film was based on the novel Briefe einer unbekannten (Letter from an Unknown Woman) by Stefan Zweig, though he was not credited. The on-screen credit claims that it is based on the 1931 nonfiction bestseller Only Yesterday by Frederick Lewis Allen.
The film is set in a time frame close to that of Allen's book but otherwise bears no resemblance to it, and the appropriation of the title may simply have been an attempt to capitalize on the book's fame at the time of the film's release.
Margaret Sullavan as Mary Lane
John Boles as James Stanton Emerson
Edna May Oliver as Leona
Billie Burke as Julia Warren
Benita Hume as Phyllis Emerson
Reginald Denny as Bob
George Meeker as Dave Reynolds
Jimmy Butler as Jim Jr., Mary's son
Noel Francis as Letitia
Bramwell Fletcher as Scott Hughes
This page contains text from Wikipedia, the Free Encyclopedia - https://wn.com/Only_Yesterday_(1933_film)
Only Yesterday (Japanese: おもひでぽろぽろ, Hepburn: Omoide Poro Poro, lit. "memories trickle down") is a 1991 Japanese animated drama film written and directed by Isao Takahata, based on the manga of the same title by Hotaru Okamoto and Yuko Tone.Toshio Suzuki produced the film and Studio Ghibli provided the animation. It was released on July 20, 1991. The ending theme song (愛は花、君はその種子 'Ai wa Hana, Kimi wa sono Tane', lit. "Love is a flower, you are its seed") is a Japanese translation of Amanda McBroom's composition "The Rose."
Only Yesterday is significant among progressive anime films in that it explores a genre traditionally thought to be outside the realm of animated subjects, in this case a realistic drama written for adults, particularly women. The film was, however, a surprise box office success, attracting a large adult audience of both genders.
In 1982, Taeko is 27 years old, unmarried, has lived her whole life in Tokyo and now works at a company there. She decides to take another trip to visit the family of the elder brother of her brother-in-law in the rural countryside to help with the safflower harvest and get away from city life. While traveling at night on a sleeper train to Yamagata, she begins to recall memories of herself as a schoolgirl in 1966, and her intense desire to go on holiday like her classmates, all of whom have family outside of the big city.
rickroberts.net
robertpugh.org
onlyflamenco.com
onlybadnews.com
robertsstagecurtains.com
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robertmonroe.org
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Only Yesterday, Gene Watson
Only Yesterday, Cardigans
Only Yesterday, Lovin' Spoonful
Only Yesterday, Circle Ii Circle
Only Yesterday, Taken By Trees
Only Yesterday, Carpenters
Only Yesterday, Barbra Zinger
Fancy meeting you here
I guess I always knew there'd
Come a time we'd meet this way
It's good to see your face again
How long has it been
It seems like only yesterday.
You know I still remember
Time we spent together
Little things we'd do and say
The night you said goodbye
Stays here on my mind
As if it were only yesterday.
I feel the unrelenting march of time
And every day I fall another day behind
I want no other love, I wish on stars above
Not for tomorrow only yesterday.
--- Instrumental ---
And every day I fall another day behind.
Our love builds on for me
It is my everything
But for you it's only yesterday


text:
Home Page One The Lead Wildfire hazard inspections add to home seller disclosures
Wildfire hazard inspections add to home seller disclosures
Natures Image landscapers Richard Aguirre (right) and Alberto Vargas trim invasive plants in August 2019 in Oro Canyon. Photo by Daniel Langhorne
By Breeana Greenberg, Special to the Independent
New state fire safety requirements for housing sales went into effect on July 1.
A California assembly bill passed in 2019 requires property sold in high fire hazard zones to document that the property meets wildfire protection measures.
According to the assembly bill, more than two million houses in California are located within "high" or "very high" fire hazard severity zones. About 85 percent of Laguna Beach is in the "very high" zone according to a recent fire department press release.
"This is a very challenging situation for a city like Laguna Beach, where nearly 90% of the area of the city is classified as very high hazard severity zone, the highest wildfire risk classification, which is similar to the risk classification of Paradise, [Calif.," said Matt Lawson, chair of the emergency and disaster preparedness committee.
Assembly Bill 38 noted the increase in frequency and severity of wildfires. "As compared with 1986, wildfires in the western United States have occurred nearly four times more often, burning more than six times the land area and lasting almost five times as long."
California's Natural Resources Secretary Wade Crowfoot echoed the growing risk of wildfires at an April press conference in Shaver Lake.
"Consider last wildfire season, five of the six largest wildfires in the state's history were burning at the same time last summer, including the Creek Fire where we are today," Crowfoot said. "So while we ramp up and continue to build our response capacity through CAL FIRE and their partners, clearly, much more needs to be done on a proactive, upfront basis to reduce the risk of catastrophic wildfire."
"Yes, we must deal with the underlying causes of the issues related to climate change, and we are committed," said Gov. Gavin Newsom at the April press conference. "And, yes, we need to do more on forest management, vegetation management, more prescribed burns, more work to get our forest and fuel breaks and homes hardened, and do defensible spaces and prepare and do our best to prevent it."
"We see the impacts of wildfire," Laguna Beach Fire Chief Mike Garcia said. "We're seeing it as we speak, there's a vegetation fire in San Clemente not far from here. Going on right now, we have crews there, we have a crew right now at the Tumbleweed fire and Gorman. We participate in these all the time, and our goal here is to try to make our community as safe as possible. And that is the intent of our legislature with a lot of these bills and these laws that get put into place. But what's difficult is when you have many programs going on and you're trying to take care of your community, it's really trying to staff appropriately, and prepare for taking on these new tasks, and that's what I find a little difficult with the implementation of some of these bills."
As of July 2, the Laguna Beach Fire Department began taking requests from property owners to inspect the area between a structure and flammable grass, trees, or shrubs that surround it, known as the defensible space. The fire department explained in a June 28 memorandum that by trimming brush away from homes and spacing shrubs and trees at a distance from each other, homeowners can give firefighters enough space to safely work during a wildfire.
The city has hired a new defensible space inspector, Justin Day, to conduct property inspections. There will be a three-month grace period in which the city will be waiving the fee for inspections. Starting in October, the fees will need to be paid prior to scheduling an inspection.
"It's our intent to have that fee pay for an inspector or a part-time inspector to conduct that, so we can keep up with the volume and not hinder the progress of selling the person's home and purchasing it," explained Garcia.
Laguna Beach Realtor Chris Tebbutt is encouraging his clients to get their homes inspected as soon as possible. "It makes it easier for buyers to be willing to buy a property, knowing that that has been taken care of," Tebbutt said. "Education is the key to this thing so that all homeowners realize this is a requirement, and that's in addition to the real property report that's already required."
"Because Laguna is in such a high fire zone and has had a history of disastrous fires, it is clear that the city and our fire department are absolutely committed to continuous learning from other similar areas such as Paradise," Tebbutt said.
"In Laguna, we have a very beautiful and unique community," said Garcia. "Trying to implement fire protection will keep the aesthetic beauty of our communities, and it's obviously a concern and a challenge. I believe that with our proposed defensible space plan, we're doing just that."
The Fire Department is currently following CAL FIRE guidelines while working on creating its own set of guidelines. There will be a public workshop at 6 p.m. on July 14 in the City Council Chambers where the fire department will present on the fire prevention plan.
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See public equity investment list I've compiled below from 1977-2012 Berkshire Hathaway shareholder letters. I have a list of buyouts and private investments, as well. Another set of data.
Source: Berkshire Hathaway annual shareholder letters, 1977-2012.
Berkshire's reporting of its positions in this format starts in 1977 because it is the most apples-to-apples format on a GAAP basis compared to now. I will be updating the data to go further back to include pre-1977 investments as well as private acquisitions and post-2012 updates.
Note: I believe the $ units in the list above are in $000s each year until they change at one point, and I need to scrub my numbers to confirm; I think the $ units may have changed during the mid-80's. Regardless, this at least accurately reports the companies underlying the public stock investments.