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|Chiefs QB Alex Smith: QBs 'particular' about their footballs
|05.13.15 at 9:26 am ET
Chiefs quarterback Alex Smith said Tuesday that while he has yet to check out the specifics of the Wells Report and Deflategate, he knows that signal-callers can be "particular" when it comes to the state of the football and how it's broken in.
"As far as breaking a ball in, I've never heard a quarterback ever altering the inflation of a football," he said. "But certainly some quarterbacks prefer brand new balls and some guys prefer them to be worn in a little more. It's kind of team to team, quarterback to quarterback."
"I like them right out of the box," Kansas City backup QB Chase Daniel said. "That's just how I throw them. That's how I have my entire life. I just haven't paid much attention to it."
Smith and Daniels are just the latest current quarterbacks to weigh in on the situations. Peyton and Eli Manning have acknowledged fellow QB Tom Brady as a friend, but mostly steered clear of any sort of definitive statement. Meanwhile, San Francisco quarterback Colin Kaepernick gave a clear defense of Brady via Twitter late last week, saying, "Let it go and let Tom be great."
|Colin Kaepernick throws support behind Tom Brady: 'Let Tom be great'
|05.08.15 at 11:51 pm ET
Colin Kaepernick was the first starting quarterback to weigh in publicly on what Tom Brady is going through, and it appears that the Niners' quarterbacks is firmly in Brady's corner. Kaepernick tweeted out the following Friday evening:
No football in the world is going to help you win by 38! Let it go and let Tom be great! On to the 2015 season #7tormsComing
' Colin Kaepernick (@Kaepernick7) May 8, 2015
Later in the evening, Denver quarterback Peyton Manning, speaking at a fundraiser in Indy, was asked about Brady's situation.
"Like I said, I'll speak it as clearly and slowly as I can. He's my friend, he'll always be my friend," Manning told reporters before a fundraiser for the Indianapolis children's hospital named in his honor. "I don't know what happened, I don't have much more than that for you."
For more Patriots news, check out weei.com/patriots.
|Fantasy Football: Week 16 starts, sits
|12.19.14 at 9:15 am ET
It's bowl week for most fantasy footballers, and we're back with a fresh batch of starts and sits for your perusal. I've posted three starts for the four major positions and a player to avoid for each as well. As always, for information on players not listed in this article, I encourage you to check out my full lineup rankings at Rotobahn.
Don't forget to tune in Sunday morning for another episode of the Fantasy Football Hour with my co-host Jim Hackett. If you have last-minute lineup concerns, you should check out my Sunday chat. It takes place right here at WEEI.com and it runs from 11 a.m. to noon.
If you want to keep track of all our fantasy football content, both here and at Rotobahn, follow me on Twitter. I send out links to all fresh articles and chats as well.
Mark Sanchez, Eagles at Washington
The top nine quarterbacks are pretty well locked in by my math. After that, I see Sanchez as a good option because he has a nice matchup and because of the weapons around him.
Russell Wilson, Seahawks at Cardinals
He's a big-game player, and he'll make enough plays to give you QB1 output. The Cardinals are not fully loaded on either side of the ball right now, so stick with Wilson unless you have a higher-ranked option.
Eli Manning, Giants at Rams
The emergence of Odell Beckham has changed Manning's fortunes for the better. He's a viable quarterback this week in all leagues if you are in need. The Rams are tough, but I expect low-end QB1 output, so start him if you have the need.
Colin Kaepernick, 49ers vs. Chargers
There's just too much going wrong in San Francisco to try him this week. The Harbaugh situation has gone from bad to worse, and it's showing up on the field. You can do better on the average waiver wire. Check the Week 16 rankings for options.
|Fantasy Football: Week 10 starts, sits
|11.07.14 at 9:15 am ET
Welcome to the Week 10 starts and sits. It's money time in most leagues, as weaker teams begin to fall by the wayside and playoff scenarios get more defined. And isn't it annoying that Week 10 features as many byes as any week this season? Six teams are off. Just when you need Rob Gronkowski, T.Y. Hilton and Arian Foster the most, they are kicking back and chilling out. Hopefully you've been reading the waiver wire and accumulating depth because this is the week most of us need it. If you are looking for information on players not listed in this space, head on over to Rotobahn and check out my full lineup rankings.
Don't forget to tune in Sunday morning for another episode of the Fantasy Football Hour with my buddy Jim Hackett. If you have last-minute lineup concerns, you should check out my Sunday chat. It takes place right here at WEEI.com and it runs from 11 a.m. to noon every Sunday.
If you want to keep track of all my fantasy football content, both here and at Rotobahn, follow me on Twitter. I send out links to all fresh content.
Colin Kaepernick, 49ers at Saints
He can run on this team, and it's a game that should force the 49ers to open up the offense somewhat. And that could lead to good output because the matchup is pretty good for San Francisco's offense.
Mark Sanchez, Eagles vs. Panthers
Sanchez is a capable player, as Patriots fans have seen at times, but he has some obvious flaws. Those flaws will hurt him a lot less in Chip Kelly's offense than they did in Brian Schottenheimer's offense. He'll get better protection and he'll have a better ground game to draft behind. He also has a deep group of receivers to target. You can play Sanchez this week and get solid numbers.
Joe Flacco, Ravens vs. Titans
It's a nice home matchup and I expect Flacco to post good numbers against a mediocre Titans defense. He's got enough healthy weapons and a productive ground game to set up some big plays downfield. He's a good Week 10 play in all leagues.
|Adam Schefter on MFB: 'It probably ends when Tom Brady says it ends' in New England
|10.10.14 at 1:37 pm ET
ESPN NFL analyst Adam Schefter joined Middays with MFB on Friday to talk about the Patriots and a potential trade of Tom Brady. To hear the interview, go to the MFB audio on demand page.
Schefter recently was involved in a discussion on an ESPN program about what Brady would be worth on the trade market, after he heard speculation about the quarterback's future. Schefter noted that it was a hypothetical discussion, and that he has no knowledge of Brady being on the market.
"I happened to speak to a lot of different people about that topic last week because I was just curious about it," Schefter said. "That's not to say that New England ever would trade him, that he would agree to go. We were playing the hypothetical game, and if that were to happen after the season. I think that there was one — one executive who told me that he thought that New England would get back two [first-round picks].
Now, again, we debated that, and I spoke to some other people and they were like, "Two ones, no, no, no, no, no.' He was in the minority, clearly. I think I spoke to about a half-dozen front-office/GM types in charge of those kind of things. After talking to them, the conclusion I reached was that if New England were to try to trade Tom Brady, which I don't see happening, if he were to agree to go somewhere, which I don't see happening, if all these things were to come together and somehow all this did happen — and he followed the same path that Joe Montana took one day for the Niners, going to Kansas City, or some of the other great players like that — I think New England would get back at least a one, and maybe something else like a four or a five, a conditional kind of pick."
Schefter said he doesn't know how Brady's tenure will end in New England, but he hopes it's ultimately up to Brady.
"You're talking about such an extraordinary career, arguably the greatest football player of all time, somebody who is immersed in that city and its culture and everything," Schefter said. "How does it end? It probably ends when Tom Brady says it ends.
"It's a different kind of case. Could he be traded? Yeah, that can always happen. I don't think it would surprise me. Could he be cut? It would surprise me, you cut Tom Brady, I don't see that. At some point he'll decide, 'I've had enough.' That would be my favorite for how this plays out. But there are a lot of different ways it could go."
|Fantasy Football: Week 3 starts, sits
|09.19.14 at 11:22 am ET
Welcome to the Week 3 starts and sits. Between injuries, suspensions, deactivations and the like, the average fantasy football GM's head is spinning. I know mine is. As always, I will avoid the easy calls. I'm not here to tell you what to do with players you'd never consider benching.
I'll be back Sunday morning with my partner in crime, Jim Hackett, for another Fantasy Football Hour. Check it out! We're on air from 6:30 to 7:30 a.m. on 93.7 FM. If you sleep in, you can listen right here on WEEI.com. In addition, I'll be here, as always, with a chat on Sunday starting at 11 a.m. Head on over if you have any lineup questions.
If you are looking for info on players not listed in this article, go to Rotobahn and check out my full Lineup Rankings. I'll be updating them over the weekend, as always. And, to keep track of all things fantasy football at both Rotobahn and WEEI, feel free to follow me on Twitter. I always tweet links to new articles.
Russell Wilson, Seahawks vs. Broncos
He'll shine in a rematch of last year's Super Bowl. Wilson's an underrated fantasy QB and he'll give you some touchdowns this week. He's playable in all formats.
Colin Kaepernick, 49ers at Cardinals
Kaepernick's always a solid play and should get it done this weekend in a tough divisional matchup. Yes, he has some injuries to his tight ends, but San Francisco has more offensive depth than in the past. Start Kaepernick if you normally would.
Kirk Cousins, Washington at Eagles
He's not an ideal option because he's got injuries to his tight ends and receivers, but he'll also be playing in a high-scoring game and his team will not be in close-it-out mode at the end. Washington is far more likely to be throwing the football in the fourth quarter. Cousins can help you in deep formats.
Joe Flacco, Ravens at Browns
He's playing decent football, but the Browns are looking tough at home and can take his receivers away for much of the game. Flacco is playable, but this is a week to look for other options if you can.
Derek Carr, Raiders at Patriots
It may be tempting to start him in a deep league because he'll be throwing a lot and playing from behind, but I hate playing rookie quarterbacks against Bill Belichick defenses. I'd avoid doing so if possible.
|Fantasy Football 2014: 20 high-value targets
|07.17.14 at 11:50 am ET
Let's take a look at draft value based on a player's average draft position. It's a good way to get a quick feel for where the big values might be in drafts when August rolls around. As we like to do at Rotobahn, we'll be counting the values down from late selections to the early ones. Jim Hackett and I will get into some of these players in Friday's podcast, so be sure to check that out. For a look at our take on the top 200 fantasy options for 2014, head on over to Rotobahn.com, where you will also find scouting reports on over 60 rookies and early projections for the upcoming season.
ADP data was obtained at Fantasy Football Calculator.
Andrew Hawkins, WR, Browns — ADP 224
He's not being drafted at all in smaller leagues, and you can get him very late in large formats, as his ADP indicates. That's not bad for a player who could be the Browns' top receiver in 2014. Frankly, if he stays healthy, I can't see anybody else on the roster outplaying him. In fairness, that speaks to how underrated Hawkins currently is, but it also speaks to how woefully thin Cleveland is at receiver.
Odell Beckham, WR, Giants — ADP 163
Do not wait until 163 to draft this guy. Beckham, for whatever reason, is being ignored in fantasy circles and we think he's a potential WR3 in 12-team leagues. As we said in his Rotobahn scouting report, Beckham has perhaps the best hands we've seen since we scouted Michael Crabtree. He's also got all the skills you want a receiver to have, including outstanding agility and speed. He should fit right into the Giants offense and could be a fantasy force from Day 1. He's a steal at his current ADP.
Justin Hunter, WR, Titans — ADP 159
Hunter's ADP has risen about 10 spots over the last month … and he'll jump a bit more before most of us draft, but he still projects as a value this season. He should be ready to take a step forward in 2014, and with Kenny Britt now gone, Hunter's snap totals should more than double. We expect him to be a starter or an in-effect starter. He has a chance to post double-digit scores if things go well in Tennessee. Hunter is by far the most explosive athlete the Titans have on offense. He has WR1 upside in long-term leagues and has the upside to be a WR2 this year. He should be off the board somewhere around the 100 mark in our opinion. He's a potential steal right now.
Aaron Dobson, WR, Patriots — ADP 138
This could change, and you may pay a bit more for him in this part of the country, but Dobson is a pretty solid WR3 option in 12-team leagues if he's healthy — and the news on his foot has been positive lately. If he's starting at split end, he's worth a pick closer to 100 overall, and he'd still have some upside there due to his touchdown potential.
Marvin Jones, WR, Bengals — ADP 129
As with Hunter, Jones' ADP has risen a bit this summer and he'll move up another round, and perhaps more in the coming weeks, but we still like him at that price tag. He's a much more complete player than he gets credit for, and he should have no problem holding on to the starter's spot opposite A.J. Green. He looks like a borderline WR3 to us in 12-team leagues with upside to be more.
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Surname Saturday – Barker
This week's Surname Saturday themed post covers my paternal Barker family, who I've traced back to a village in the county of Hertfordshire, England. Continue reading
Posted in Barker, Hertfordshire, Surname Saturday Tagged Barkway, Downton Abbey, Esther Elizabeth Nottage, George Burnell, Hester Hawks, John H Phillips, Mary Ann Barker, Mary Ann Burnell, Mary Head, Peter Barker, Royston, Royston & District Museum, Surname Saturday, Thomas Barker, William Barker 1 Comment
2 million Hertfordshire parish records arrive at FindMyPast
FindMyPast have added 2 million Hertfordshire parish records. Continue reading
Posted in Barker, FindMyPast.co.uk, Head, Hertfordshire Tagged Barker, Barkway 2 Comments

subset: commoncrawl


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Miami Heat v Charlotte Bobcats
A member of the Charlotte LadyCats dance team performs during the... Nieuwsfoto'sBasketbal,Basketbal - NBA Pro,Charlotte - North Carolina,Dansen,Georganiseerde groepen,Horizontaal,Miami Heat - Basketbalteam,North Carolina - Staat,Sport,Sportteam,Sportwedstrijd,Time Warner Cable Arena,Uitvoering,Verenigde StatenPhotographer Kent SmithCollection: National Basketball Association 2013 NBAECHARLOTTE, NC - APRIL 5: A member of the Charlotte LadyCats dance team performs during the game against the Miami Heat at the Time Warner Cable Arena on April 5, 2013 in Charlotte, North Carolina. NOTE TO USER: User expressly acknowledges and agrees that, by downloading and or using this photograph, User is consenting to the terms and conditions of the Getty Images License Agreement. Mandatory Copyright Notice: Copyright 2013 NBAE (Photo by Kent Smith/NBAE via Getty Images)


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State of Origin III - Preview
WE take up the tale in the Year of out Lord 2006, at a time when the King?s colony in the Great Southland has descended upon the fair State of Victoria. Despite supplications to the contrary, General Gallop has determined Ye Olde Docklands to be of a form pleasing to his eye. And so, it was agreed upon, that it should host the third and final State of Origin match for said year.The 78th clash between New South Wales and Queensland is set to take place undercover at Telstra Dome in Melbourne where the Maroons will look to arrest a three-series losing streak that is threatening to debase the very notion of Origin Football?well depending on who you talk to.I suppose if you were going to endanger the existence of this interstate rivalry, then Melbourne would be a fitting place for such an act. But when considering the possible outcomes of this match, I?m not sure there is a more neutral arena for it to be played.Of the four previous Origin matches played in Melbourne, three have been contested at the MCG, with NSW holding the upper hand 2-1. The first ever game south of the Murray was played at Olympic Park back in 1990 and was also won by NSW. But three of the four contests were following games in Sydney, and twice the series was on the line only for NSW to claim victory. This is the first time the decider has been taken out of either of the two native States however.So I think all that points to a wide open game and that is able to be won by either team on their day.The BluesNew South Wales come into this game offering a few changes to the sides that have played in the first two matches.Not since Antony and Cleopatra had we seen such an uneasy, unholy union that was Brett Finch and Braith Anasta. They?ve had their time sheets marked ?Never to be Selected Again? and make way for the future which is Craig Gower and Mark Gasnier, with Matt Cooper slotting into the centres.This will ultimately prove to be the talking point of the match: if NSW win the media will hail the pairing as a success; if they lose it will be a disaster. I think it has the potential to be a great combination if Gower can direct the play and kick well. With a guy of Gasnier?s strikepower running off your shoulder, any half gap can result in mayhem for the opposition. Moreover, if Queensland is expecting this it will bring Brett Hodgson and Ben Hornby into calculations as worthy alternatives.I?m not sure how NSW will introduce Hornby, but I?ve been thinking that perhaps he?ll get a run at first receiver when Danny Buderus comes off with Gower playing acting half. Whatever takes place, NSW will be hoping he can work as well with Gasnier tomorrow night as he does at club level. With Cooper only serving to strengthen the defence of the Blues, the rest of the backline is left unchanged. Which brings us to the forwards ? NSW needs to dominate here if the experiment of Gasnier is to work.Brent Kite, Andrew Ryan and Craig Wing were all given the flick, opening the door for Luke Bailey to start, Luke O?Donnell to be reinstated and Paul Gallen will make his debut from the bench wearing the no.14 jumper, although there are some suggestions that he will start the match or perhaps Mark O?Meley.The MaroonsIn the Queensland camp things haven?t been quite so smooth. Gun centre Justin Hodges was ruled out early in proceedings pushing in-form winger Brent Tate one man in and selectors opting for a bit of a surprise in Rhys Wesser to cover the flank.Wesser played his last match in Game III 2004 and, whilst he has made his mark at fullback, he?ll be up to the task for this one. Queensland opted for Canberra custodian Clinton Schifcofske to fill the boots left behind by the injured Karmichael Hunt (Game II) and the out of sorts Matt Bowen (Game I). This is the first time Queensland has ever used three fullbacks in one series, but injuries have galvanised them in the past and it would be foolish to think this would do anything but in an Origin decider.The big surprise was Josh Hannay?s recall. He hasn?t played since Game III in 2003, and by all reports he?ll be lucky to play in this one. A stomach virus has struck the Cowboy down and Willie Tonga has been brought in as cover. Strangely, his last outing as a cane toad was the same one as Rhys Wesser, and they got belted 36-14 in Sydney.The forwards are much the same with the exception of Lillyman, who makes way for the experience of Tonie Carroll off the bench. I?m not a huge fan of this as I would expect Lillyman to make more impact than Carroll from the bench, but I?d still use Carroll as a starting player.Price, Civoniceva, Webb and the smarts of Smith at hooker will give NSW plenty to think about though.The VerdictFor the first time in a long time I?m really unsure about this game.NSW look the better side on paper but how many times have punters been duped into thinking the Blues are much stronger only to have Queensland relish in the underdog tag and upset them? Too many, that?s how many?Like just about every contest, a good big man will beat a good little man. All the Maroon forwards are strong and mobile with plenty of experience. Meanwhile, NSW have traded in Ryan for Gallen and I?m not sure that?s a good idea.It?s likely to be a bit greasy down there and I think kicking will be very important. When the roof is closed at Telstra Dome the ball travels a little further and this could even things up a little.But it?s a hard game to call. I?m going to choose Queensland by 6 points because their halves have remained the same and whilst they?ve had a few changes elsewhere in the backline, the rest of the team will rally around their men and get the job done.
25 Years Ago - Redcliffe Dolphins
JULY 1981 was kind to the Redcliffe football club, with three successive wins all but assuring the Dolphins of a top two finish to the season. July also meant the completion of the representative season, and saw explosive young centre Chris Close and club leader Arthur Beetson return to the club fray permanently for the final run-up to the semi-finals. July also saw a major return to form of former international halfback Greg Oliphant, who had only been a fringe first-grader for most of the season.
Dragons PL & Flegg sides
PREMIER League Dragonsv South Sydney Rabbitohs1:00pm Sunday 9 July - Oki Jubilee Stadium1. Jason NIGHTINGALE, 2. Josh MORRIS, 3. Beau RYAN, 4. Daryl MILLARD, 5. Tom HEWITT, 6. Richard WILLIAMS, 7. Chris SHEPPARD (c), 8. Charlie LEAENO, 9. George NDAIRA, 10. Chris HOUSTON, 11. Corey PAYNE, 12. Lee TE MAARI, 13. James JIMENEZ, 14. Jamie DUNNING, 15. Jess CAINE, 16. Bryson GOODWIN, 17. Reece BLAYNEY, 18. Tom HABERECHT, 19. Lagi SETU, 20. Jamie KEITHCoach: Steven Price Jersey Flegg Dragonsv South Sydney Rabbitohs11:20am Sunday 9 July - Oki Jubilee Stadium1. Travis ROCHE, 2. Rota SETU, 3. Daniel NICASTRI, 4. Junior SEUMANUTAFA, 5. Luke IRWIN, 6. Matt SEAMARK, 7. Blake DUREAU, 8. Dan HUNT, 9. Brad DAVIDSON (c), 10. Luke YOUNG, 11. Matt PRIOR, 12. James GREHAN, 13. James KEELAN, 14. Jason WHITE, 15. Francis LANGIFISI, 16. Jhi TANNER, 17. Ryan MILLARD, 18. Matt MUNDINE, 19. Peter WAKLEYCoach: Corey Pearson
Dragons team v Rabbitohs - Round 18
DRAGONS Chasing Seven in Row against Rabbitohs
Eastwood wants Kiwi no.6
BRISBANE'S rising New Zealand star Greg Eastwood says he wants to fill the big shoes of good friend Benji Marshall at this year's Tri Nations rugby league series.
Cronulla name team to play Wests
1. Toshio Laiseni 2. Darren Albert 3. Nigel Vagana 4. Beau Scott 5. Luke Covell 6. Adam Dykes 7. Brett Kimmorley 8. James Stosic 9. Kevin Kingston 10. Luke Douglas 11. Greg Bird 12. Lance Thompson 13. Paul Gallen
Roosters team to play Manly
ROOSTERS Shaun Foley, Ryan Cross, Iosia Soliola, Craig wing, Amos Roberts, Braith Anasta, Josh Lewis, Adrian Morley, Brett Finch, David Shillington, Shane Shackleton, Chris Flannery Interchange: Ashley Harrison, Heath L?Estrange, Mickey Paea, Charles Tonga
Tigers side to play Sharkies
WESTS Tigers Coach Tim Sheens has moved Robbie Farah back from half-back to hooker for the vital clash against the Cronulla Sharks at Toyota Park on Sunday, July 9, at 3pm, in round 18 of the Telstra Premiership. Scott Prince returns to his position of half-back with Daniel Fitzhenry back at five-eighth after overcoming a collarbone injury. Stuart Flanagan makes way at hooker for Farah. Shannon McDonnell moves to the wing for the return of NSW Origin full-back Brett Hodgson with Taniela Tuiaki going back to Premier League. Rangi Chase, who made his first grade debut for the Wests Tigers against the Bulldogs, also returns to Premier League. Sam Harris has been promoted from the Balmain-Ryde Eastwood Tigers Premier League team to first grade on the interchange bench. 1 Brett Hodgson (c) 2 Shane Elford 3 Dean Collis 4 Jamaal Lolesi 5 Shannon McDonnell 6 Daniel Fitzhenry 7 Scott Prince 8 Keith Galloway 9 Robbie Farah 10 John Skandalis 11 Ben Galea 12 Todd Payten 13 Dene Halatau 14 Bronson Harrison 15 Chris Heighington 16 Sam Harris 17 Ryan O'Hara
Storm name side to play the Broncos
MELBOURNE Storm coach Craig Bellamy has named his team to play the Broncos at Olympic Park from 7:30pm.
Bronco team to face the Storm
INJURED Test winger Tame Tupou and the suspended Casey McGuire will miss the Ergon Energy Broncos top-of-the-table clash with ladder leaders Melbourne at Olympic Park on Friday night.
Panthers team to play Raiders named
HERE is the Panthers side to play the Raiders at Credit Union Australia Stadium Penrith on Saturday night:
Eels name team to play Knights
PARRAMATTA coach Brian Smith has named his team to play the Newcastle Knights at Parramattas Stadium on Saturday night. Coach Smiths team is;
Rabbitohs team to take on Dragons
THE Real Insurance Rabbitohs are pleased to announce their side to take on the St George Illawarra Dragons in round 18 of the NRL Telstra Premiership this Sunday, 9 July at 3pm at OKI Jubilee Stadium.
Sea Eagles name side to play Roosters
COACH Des Hasler has named a 5 man bench to take on the Roosters this Saturday. The Sea Eagles are still without Stephen Bell but see the return of Travis Burns and Kylie Leuluai to the side. Steven Menzies will play if he comes through Wednesday's State Of Origin match ok.
Warriors name team to play Bulldogs
VODAFONE Warriors Jerome Ropati and Louis Anderson will make their 50th first-grade appearances in Sunday?s NRL clash against the fourth-placed Bulldogs at Telstra Stadium. The two 21-year-olds again line up in a Vodafone Warriors side that?s been all but unchanged since starting a four-match winning streak against the Roosters on June 11. The only change during that spell has come about through captain Steve Price?s State of Origin commitments but they finish for the season tomorrow night when he and his Queensland team-mates face New South Wales in the series-deciding third match at Melbourne?s Telstra Dome. In his absence, the Vodafone Warriors picked up maximum points against Wests Tigers, the Roosters and Penrith while Ruben Wiki was stand-in captain. ?There have been a lot of pleasing aspects lately and one of them is the fact we have been able to win each time Steve was out of the side,? said head coach Ivan Cleary. ?That?s a reflection on the contribution every player has made but we?re also really fortunate to have a leader of Ruben?s ability when Steve?s not there. ?Now Steve?s set to return on a permanent basis, which can only help us at a time when we?ve able keep such a settled side together.? Sunday?s match provides personal milestones for Ropati and Anderson, the former making his debut in 2003 and the latter early in the 2004 season. ?For young players they have done extremely well and they?re certainly showing signs of developing to the next level,? said Cleary. Anderson has crammed in an extraordinary amount of top-level football for a player who turned 21 only a week ago. Besides eyeing his 50th outing for the Vodafone Warriors he has also played 13 tests for the Kiwis already. If the clash at Telstra Stadium will have meaning for Ropati and Anderson, it will also be a special occasion for Price who will play in front of a Bulldogs crowd for the first time since leaving his old club after the 2004 season. In fact, this will be the first time the Vodafone Warriors have played the Bulldogs in Sydney since winning 48-22 in the 2003 finals. That match was the fourth leg of a five-match winning run for the Vodafone Warriors. After beating the Roosters, Newcastle, South Sydney and Penrith in the last four rounds, the Vodafone Warriors can make it five on end again this weekend but Cleary?s more interested in the quality of the performance than the statistics. ?We won but won ugly against Penrith last weekend,? he said. ?It?s not a case of thinking about five in a row when we take on the Bulldogs. For us it?s all about putting together a much-improved overall performance.


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Search Results for: pldt pocket wifi
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PLDT WiFi Zone: Can paid public WiFi survive?
Relevance: 100% Posted on: March 9, 2012
Yesterday, PLDT and Intel held a joint event to present a partnership for the PLDT WiFi Zone and the Intel-powered…
Smart discounts LTE Pocket WiFi, now costs Php4,888
Relevance: 87% Posted on: October 7, 2013
In an effort to bring their own prepaid LTE offering at par with Globe's, Smart has slashed more than Php2,000…
Hands-on: ZTE MF90 LTE Pocket WiFi
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The successor to the ZTE MF80 pocket WiFi from 2 years ago is the new ZTE MF90 which is now…
Smart Bro Pocket WiFi Portable 3G Router
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Smart now has their own version of a portable 3G+WiFi router they called SmartBro Pocket WiFi. This new product competes…
Giveway: Huawei E5151 Pocket WiFi
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Last month, we featured Huawei's new pocket WiFi -- the E5151. The router supports up to 10 devices and has…
Huawei's new 4G/LTE Pocket WiFi line-up
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Huawei makes some of the most interesting little gadgets around and when I visited their booth at CommunicAsia yesterday, they've…
AT&T Unite LTE Pocket WiFi while roaming in the US
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Since I have a back to back event coverage here in the US (CES in Las Vegas and NAIAS in…
Huawei E5151 adds LAN port to Pocket WiFi
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Huawei's newest iteration of their pocket WiFi is slightly different from the previous ones. The E5151 comes with a built-in…
PLDT DSL Subscribers get Free WiFi
Relevance: 80% Posted on: March 25, 2007
Just got my latest PLDT DSL Billing today and found several brochures for a couple of promos on myDSL. One…
ZTE's HSPA+ Pocket WiFi selling for Php7,500
Relevance: 80% Posted on: August 26, 2011
Looks like an HSPA+ capable pocket WiFi is now available in the grey market -- we spotted one a week…


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Hi there at our free ringtones website, where you can get ringtones for free for your cell. Today we have a ringtone of "Continental classic 4" by motorola as well as 326 other ringtones by motorola. "Continental classic 4" is a gorgeous track from the "Phone" album that was created by motorola at their recording studio together with other 1 tracks. You can listen to all 5 motorola – "Continental classic 4" ringtone variations available here and download it for free just in a click. You can also listen to other 16 albums produced by motorola for all time. "Continental classic 4" is referred to , and we bring you a huge amount of albums absolutely for free. And finally, we have collected lyrics for this track and you can view it here as well. So feel free to browse, listen, download ringtones at Audiko.net. Make your phone sound cool!
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All rooms includes: air conditioning, one king-size bed or two double beds, satellite T.V., private bath and shower, hair dryer and safety deposit box. 3 Restaurant and 3 bars Modern building Medium size lobby You will be totally charm by this oceanfront hotel. Its airy and casual beachfront surrounds with expansive views of the Caribbean Sea, Mountains and beautiful tropical gardens.
Located at one of the best zones of Isla de Margarita, El Tirano. Only 20 minutes from commercial zone of Porlamar and 5minutes from Playa El Agua y Playa Parguito. 20 kms to city centre 40 kms to the nearest airport (del caribe international general santiago marino airport) Close to the beach


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Note: If you like your hoodies baggy, go two sizes up
And it's a "Premier", too.
beach surf holden eh
Cool vintage type T.
Thanks ER…
yeah you can park here, no probs.. haha
Back in the 50s (now I'm starting to sound like your old man…), you could park on the beach at Torquay… this, of course, would have been taken in the early 60s.
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Bus passenger in Arizona dies after altercation
PRESCOTT, Ariz. — A Greyhound bus passenger who appeared to be impaired
died Friday following an altercation with an off-duty driver after the vehicle
broke down in north-central Arizona, authorities said.
Yavapai County Sheriff's officials said the name of the 53-year-old man was
being withheld until his family could be notified.
An autopsy was scheduled to determine the cause of death and sheriff's
officials also were awaiting results of a toxicology test on the man.
Deputies were called around 2:45 a.m. Friday about a disorderly passenger who
appeared to be impaired by alcohol, drugs or medication on the bus bound for
They reported finding an unconscious man lying outside the bus along Interstate
17 and he was pronounced dead at the scene.
Deputies interviewed the bus driver, the off-duty driver and several of the
nearly 50 passengers.
They told authorities that after the bus was stopped by mechanical issues, the
passenger began arguing with the driver and allegedly tried to assault him.
The off-duty driver reportedly intervened and the passenger collapsed inside
the bus.
Sheriff's officials didn't release the names of the driver or the off-duty
They said it was unclear if the confrontation resulted in the man's death or
other factors were involved, but no arrests have been made in the case.
A call to Greyhound for comment on the incident wasn't immediately returned
Friday afternoon.


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Mountain "Don't"
In the Internet advertisement, a talking goat (voiced by Tyler, the Creator) warns an apparent mugging victim, a white woman, not to "snitch on a playa" as it is called in for a police line-up with five Black men (who dressed the part in du-rags and bandanas). Following outcry, the video was removed from Mountain Dew's video channel as well as the musician's on Wednesday. (Photo: EntertainmentHeart via YouTube)
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RATINGS: 1578 WEIGHTED AVG: 3.76 SEASONAL: Summer EST. CALORIES: 288 ABV: 9.6%
a stout of great contention We are not the harbingers of truth as some may suggest but it may indeed be argued that our brewing philosophy is tantamount to a dessert with a bellicose past. How, you may ask, would a brewery determine a likeness to hard-coated custard? Our response is simple; its all in the power of history, and of course, the extra finesse needed to top off a contentious treat with definition.
By comprehending the labyrinthine movement of time, one would not think it strange to trace the errant path of an ordinary object such as a cream dessert only to discover that it has been the cause of cultural disputes since the middle ages. The British founders of burnt cream and from Spain, crema catalana, both stand by their creative originality and we respect that, but it was the French Crème Brûlée, amid the strife of contention, that survived to represent our deliciously creamy brew. 9.6% abv 25º plato 195º L 22 oz / 1/6 keg 2-row pale malt / dark caramel malt / vanilla bean / lactose sugar / kettle hops: columbus / aroma hops: horizon
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Le Chateau de Lacan
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Travel Blogs from Brive-la-Gaillarde
Monaco and Monte Carlo
... festivities underway in the city - coloured flags and bunting against the azure colour of the Mediterranean sea, it is no wonder this is the playground for many to enjoy. After a tour of the city, and with a stop on the way back to Eze, a gorgeous French village (separate blog on the way) we returned to Monaco, specifically Monte Carlo in the evening to celebrate Pat's birthday in style. We ate dinner in the old casino, a lavishly built and decorated interior which reminded me ...
A good time with familie
... m walking, I keep tramping though. And what do you know, I get picked up. I'm telling them I'm heading in the direction of Tulle. They don't go there but they can take me to a better place to tramp further. In the car I start my navigation app on my phone. I put in Bassignac-le-Haut, the town where I need to be. To my surprise, where heading exactly that direction. I hand my phone to the guy in the passenger seat. He takes a ...
Canoeing the Dordonge and the Market in Sarlat
... we head to town for the day. We take our bag to buy strawberries, olives, zucchini, tomatoes, and a baguette. We stroll the shops and wish we had room to buy souvenirs and gifts for family at home. There's so many wonderful, unique items in the market but it is just too expensive to ship things back to the states. We stop for a salad for lunch and the people next to us turn out to be from Colorado. What a small world. After ...
Biking near Sarlat and Farewell Dinner
Sally was feeling somewhat better this morning but Anne had a sore throat so four of us, Sally, Karen, Greg and I decided to go biking. Darlene and Ted joined us for the drive to Sarlat where we dropped them off to meander around the town and go to a cafe for a couple of hours. The rest of us drove another five minutes to a car park where the bike rental company had agreed to meet us with our bikes at ...
Boiled lollies to chocolates and feeling weird
... with no appliances, the bath sort of clean, the beds small and authentic farmhouse style, the stairs a bit rickety. We don't have to stay here if you don't want to sweetie. Well where else are we going to go? The Madame have me a pamphlet for a restaurant down the road a few kilometres. It looked like they also had rooms. We left to get some dinner without unpacking the car never to return. The next place was just as bad. We can ...


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In adult mammals such as rats, the blood-testis barrier (BTB) conferred by adjacent Sertoli cells in the seminiferous epithelium segregates post-meiotic germ cell development from the systemic circulation and is one of the tightest blood-tissue barriers. Yet it must "open" transiently at stage VIII of the epithelial cycle to accommodate the migration of preleptotene/leptotene spermatocytes. While this is a vital event of spermatogenesis, the mechanism(s) that regulates BTB dynamics is virtually unknown. Recent studies have suggested that transforming growth factor-β3 (TGF-β3) and tumor necrosis factor α (TNFα) secreted by Sertoli and germ cells into the microenvironment of the BTB are capable of inducing reversible BTB disruption in vivo, apparently by reducing the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB via the p38 mitogen activated protein (MAP) kinase signaling pathway. In this study, local administration of TGF-β3 (200 ng/testis) to the testis reversibly perturbed the BTB integrity in vivo. We next sought to delineate the mechanism by which these cytokines maintain the steady-state level of integral membrane proteins: occludin, junctional adhesion molecule-A (JAM-A) and N-cadherin at the BTB. Primary Sertoli cells cultured in vitro were shown to establish intact tight junctions and functional BTB within two days when assessed by transepithelial electrical resistance (TER) measurement across the cell epithelium. Sertoli cell integral membrane protein internalization at the BTB was assessed by biotinylation of cell surface proteins, to be followed by tracking the endocytosed/biotinylated proteins by using specific antibodies. Both TGF-β3 (3 ng/ml) and TNFα (10 ng/ml) were shown to significantly accelerate the kinetics of internalization of JAM-A, N-cadherin, and occludin versus controls. Treatment of cells with phenylarsine oxide (PAO) at 10 μM that blocks clathrin-mediated endocytosis was shown to inhibit the TGF-β3-induced protein internalization. This inhibition of TGF-β3-mediated protein endocytosis was further validated by silencing of clathrin. The specific effect of TGF-β3 on protein internalization was confirmed by RNAi using specific TGF-β receptor I (TβR1) siRNA duplexes. When TβR1 was knocked down, the TGF-β3-induced increase in the kinetics of JAM-A and occludin endocytosis was abolished, making them indistinguishable from controls, illustrating the specificity of the TGF-β3 effects on protein endocytosis. In summary, this report demonstrates for the first time that BTB dynamics are regulated by TGF-β3 and TNFα via an enhancement of protein endocytosis at the BTB.
Recent studies have shown that blood-testis barrier (BTB) dynamics during spermatogenesis are regulated, at least in part, by cytokines that determine the steady-state levels of integral membrane proteins at the BTB [for reviews, see (Lui and Cheng, 2007; Xia et al., 2005; Yan et al., 2007)]. For instance, it was shown that local administration of either TGF-β3 (Xia et al., 2006) or TNFα (Li et al., 2006) to testes of adult rats reversibly disrupted the BTB integrity when examined by dual-labeled immunofluorescence analysis using markers at the BTB, such as occludin, N-cadherin, JAM-A, and ZO-1. More important, local administration of TNFα to testes at concentration comparable to its endogenous level in the testis was shown to reversibly disrupt the BTB integrity. This was monitored by an in vivo functional assay by tracking the diffusion of a small fluorescent probe, such as fluorescein-5-isothiocyanate (FITC, Mr 389), from the systemic circulation to the adluminal compartment of the seminiferous epithelium following its administration at the jugular vein (Li et al., 2006). These results are consistent with recent observations in other epithelia, such as in small intestine and kidney, wherein cytokines are shown to be important regulators of tight junction (TJ) permeability barrier, such as in small intestine and kidney [for reviews, see (Walsh et al., 2000; Xia et al., 2005)]. These findings are important, since they illustrate that at the microenvironment of the BTB in the seminiferous epithelium, it is likely that cytokines, namely TNFα and TGF-β3 produced by Sertoli and/or germ cells (De et al., 1993; Skinner, 1993; Xia et al., 2006), contribute to the 'restructuring' and/or 'opening' of the BTB to facilitate the transit of preleptotene/leptotene spermatocytes across the BTB. This postulate is also supported by the observations that the expression of these cytokines was shown to be relatively high at or near the BTB during stage VIII of the epithelial cycle by immunohistochemistry (Lui et al., 2003a; Siu et al., 2003a; Xia et al., 2006). However, the mechanism by which cytokines induce a loss in the steady-state levels of integral membrane proteins at the BTB (e.g., occludin, JAM-A, N-cadherin) remains unknown.
Protein endocytosis plays a crucial role in regulating the steady-state levels of proteins at the cell junctions, which can be clathrin- or caveolin-dependent or via macropinocytosis [for reviews, see (Ivanov et al., 2005; Maxfield and McGraw, 2004)]. More importantly, it was postulated that the replacement of the apical ectoplasmic specialization [apical ES, a testis-specific actin-based atypical adherens junction (AJ) type] (Wong et al., 2008b) by apical tubulobulbar complex (apical TBC) to facilitate spermiation at stage VIII of the seminiferous epithelial cycle was mediated via protein internalization (Pelletier and Byers, 1992). Indeed, recent studies have supported this speculation wherein the adhesion domains of nectins 2 and 3 were found to be internalized as membrane vesicles near the TBC at spermiation (Guttman et al., 2004). In addition, recent studies have shown dynamin 2, a large GTPase known to be involved in protein endocytosis (McNiven et al., 2000; Sever et al., 2000) is structurally associated with integral membrane proteins at the BTB in rat testes (Lie et al., 2006). Furthermore, another isoform of dynamin, dynamin 3, was shown to be testis-specific (Kamitani et al., 2002). In light of these findings, we sought to examine if endocytosis is indeed taking place at the BTB of adult rat testes to regulate the steady-state levels of BTB- integral membrane proteins to facilitate the transit of preleptotene/leptotene spermatocytes during the epithelial cycle of spermatogenesis. This is the subject of this report.
Sprague-Dawley rats were purchased from Charles River Laboratories (Kingston, NY) and housed at the Rockefeller University Laboratory Animal Research Center with a 12 hr:12 hr light:dark cycle at 22 °C with access to rat chow and water ad libitum. The use of animals in this study was approved by the Rockefeller University Animal Care and Use Committee (Protocol Numbers: 03017 and 06018).
Primary Sertoli cell culture was prepared as previously described (Cheng et al., 1986; Xia et al., 2006). Freshly isolated Sertoli cells were cultured at 0.5 × 106 cells/cm2 on Matrigel (BD Biosciences)-coated dishes as described (Xia et al., 2006) in serum-free Ham's F12 Nutrient Mixture and Dulbecco modified Eagle medium (F12/DMEM, 1:1, v/v, Sigma) containing HEPES (15 mM) and sodium bicarbonate (1.2 gm/L), supplemented with bovine insulin (10 μg/ml), human transferrin (5 μg/ml), epidermal growth factor (2.5 ng/ml), bacitracin (10 μg/ml), and gentamicin (20 μg/ml) as described (Mruk and Cheng, 1999). On the day of cell isolation, these cultures were designated as day 0. Matrigel was diluted 1:7 with F12/DMEM and dishes were coated 24 h prior to their use. These Sertoli cell cultures were incubated in a CO2 incubator at 35 °C in a humidified atmosphere with 5% air/95% CO2. About 36 hr thereafter, Sertoli cell cultures were subjected to a hypotonic treatment using 20 mM Tris, pH 7.4 at 22 °C for 2.5 min to lyse residual germ cells as described (Galdieri et al., 1981). Cultures were then washed twice in F12/DMEM. These Sertoli cells were contaminated with negligible germ cells when assessed by RT-PCR and/or immunoblotting using specific germ cell markers, such as c-kit receptor as described (Lee et al., 2004). Furthermore, by day 2 and thereafter these cultures were shown to form functional BTB when assessed by transepithelial electrical resistance (TER) measurement (Lui et al., 2001) across the Sertoli cell epithelium when cells were cultured on Matrigel-coated bicameral units. Additionally, functional BTB was detected as manifested by the presence of intact TJ and basal ES in these cultures on day 3 when examined by electron microscopy (Lee and Cheng, 2003; Siu et al., 2005). Indeed, these primary Sertoli cell cultures have been used by different laboratories including ours to study BTB function which mimic many of the functional and ultrastructural features of BTB in vivo (Byers et al., 1986; Chung et al., 2001; Janecki et al., 1991; Janecki et al., 1992). Media were replaced daily until these cultures were used on day 4 for the endocytosis assay to assess the effects of cytokines on the kinetics of endocytosis of integral membrane proteins at the BTB. Media collected on day 4 from selected cultures were used and served as Sertoli cell-conditioned media (SCCM).
GCCM was prepared from freshly isolated total germ cells using a nonenzymatic mechanical method as detailed elsewhere (Aravindan et al., 1996). In short, total germ cells without elongating/elongated spermatids isolated from adult rat testes were cultured in F12/DMEM supplemented with sodium pyruvate (2 mM) and sodium lactate (6 mM) as described (Aravindan et al., 1996) at 35 °C in a humidified atmosphere with 5% CO2/95% air (v/v) for 14–16 hr. Thereafter, media were collected, centrifuged at 800 g for 20 min to remove cellular debris and the supernatant was collected and concentrated using a Millipore YM-10 membrane in an Amicon Model 8050 ultrafiltration unit.
The in vivo BTB integrity assay was performed as earlier described (Li et al., 2006). In short, 200 ng recombinant TGF-β3 (R&D Systems, Minneapolis, MN) was administered locally to adult rat testis (~300 gm b.w.) on day 0 as described (Li et al., 2006). On day 2 and 14, rats (n=3 per treatment group for each time point) was anesthetized by ketamine HCl (60 mg/kg b.w., i.m.) with xylazine (10 mg/kg b.w., i.m.) as an analgesia. Rats without treatment/treated with vehicle (saline), or with CdCl2 for 3 days at 3 mg/kg b.w. via i.p. were served as negative and positive controls, respectively. A small incision of about 0.5–1 cm above the jugular vein was opened and 200 μl of FITC (1 mg/ml) in PBS was administered into the jugular vein using a 28-gauge needle. The incision was then stitched, and rats were allowed to recover. About 60–90 min thereafter, rats were euthanized by CO2 asphyxiation, and testes were immediately removed under aseptic conditions, and frozen in liquid nitrogen. Testes sections (about 8 μm) were obtained in a cryostat. All sections within an experimental set including controls and treatment groups were mounted with or without anti-fade reagent containing 4′,6′-diamidino-2-phenylindole (DAPI; Vector Laboratory, Burlingame, CA), and the green fluorescence for FITC was visualized in an Olympus BX40 fluorescent microscope. At least 100 tubules were randomly selected and acquired using an Olympus DP70 12.5 megapixel digital camera with the QCapture Software Suite (Version 2.56) (Quantitative Imaging Corp, Surrey, BC, Canada). Images were exported to TIFF format images. Distance of the FITC fluorescence that diffused away from the base of each seminiferous tubule (i.e., the relative location of the BTB near the basement membrane) (Df) in ~100 randomly selected tubules from two testes versus radius of the tubule (Rd) in treatment and control groups were computed. For oblique sections of seminiferous tubules, Rd was obtained by averaging the shortest and the longest distance from the basement membrane.
Endocytosis assay was performed essentially as earlier described with minor modifications (Le et al., 1999; Morimoto et al., 2005). Briefly, Sertoli cells cultured at 0.5 × 106 cells/cm2 on Matrigel-coated 6-well dishes for 4 days were washed twice with ice-cold PBS, incubated with 0.5 mg/ml Sulfo-NHS-SS-Biotin (Pierce) in PBS (10 mM sodium phosphate, 0.15M NaCl, pH 7.4 at 22 °C) containing 1 mM CaCl2 and 0.7 mM MgCl2 (PBS/CM) at 4 °C for 30 min to allow biotinylation of cell surface proteins. Excess Sulfo-NHS-SS-Biotin was quenched by 50 mM NH4Cl in PBS/CM at 4°C for 15 min. Then cells were washed twice with ice-cold PBS and incubated with F12/DMEM with (test) or without (control) cytokines (TGF-β3 at 3 ng/ml and TNFα at 10 ng/ml), or with concentrated GCCM at 50 μg/ml, at 35 °C for various time points in a humidified atmosphere with 95% air and 5% CO2 (v/v) to allow internalization of cell surface biotinylated proteins since endocytosis does not occur at 4 °C. At specified time points, cells were washed in cold PBS, incubated with a biotin stripping buffer [50 mM MESNA in 100 mM Tris/HCl (pH 8.6) containing 100 mM NaCl and 2.5 mM CaCl2] at 4 °C for 30 min to remove any remaining biotin or non-endocytosed biotinylated proteins on the cell surface, and quenched with a quenching buffer [5 mg/ml iodoacetamide in PBS/CM] at 4 °C for 15 min. Thereafter, cells were washed twice with ice-cold PBS and lysed in an IP lysis buffer [10 mM Tris, pH 7.4 at 22 °C, containing 0.15 M NaCl, 2 mM PMSF, 1 mM EGTA, 1% NP-40 (v/v), 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml aprotinin and 10% glycerol (v/v)]. Equal amount of cell lysates (about 400 μg protein) between samples in an experiment were incubated with NeutrAvidin beads (Pierce) to pull down biotinylated proteins, washed three times in PBS, and biotinylated proteins were extracted in SDS sample buffer (Xia and Cheng, 2005). Proteins were resolved by SDS-PAGE, to be followed by immunoblot analysis using corresponding specific antibodies (see Table 1) to assess the kinetics of internalization of different integral membrane proteins.
Pre-designed siRNA duplexes targeting TβRI (Genbank Accession Number: NM_012775; Cat. # 16708-48442), dynamin 2 (Genbank Accession Number: NM_013199; Cat. # 16708-197996) and dynamin 3 (Genbank Accession Number: NM_138538; Cat. # 16708-193600) were purchased from Ambion (Austin, TX) and their sequences are listed in Table 2. In short, Sertoli cells at 0.5 × 106 cells/cm2 were cultured for 2 days with negligible contamination of germ cells to allow the formation of an intact cell epithelium with functional TJ-barrier and the ultrastructures of BTB when examined by transepithelial electrical resistance (TER) and electron microscopy as described (Lui et al., 2003a; Siu et al., 2005). Thereafter, cells were transfected with specific siRNA duplex at 200 nM, using Oligofectamine (Invitrogen) for ~24 hr using protocols supplied by the vendor. Endocytosis assay was performed 2 days after transfection as described above and cultures were terminated at specified time point to assay changes in the kinetics of protein endocytosis. In selected experiments, cells that were transfected with specific siRNA duplex versus scrambled control siRNA duplex were harvested three days later for lysate preparation to confirm specific target protein knock down by immunoblot analysis.
Phenylarsine oxide (PAO) is an inhibitor of clathrin-dependent endocytic pathway (Ivanov et al., 2004). A recent report using specific inhibitors to clathrin (e.g., phenylarsine oxide, PAO) or caveolin (e.g., cholesterol oxidase, CO) have shown that protein endocytosis at the BTB using Sertoli cells cultured in vitro is likely mediated via the clathrin-dependent pathway (Yan et al., 2008). However, results derived from studies using specific inhibitors to study protein endocytosis should be cautiously interpreted (Ivanov, 2008); since PAO, besides blocking clathrin-mediated endocytosis, was shown to block macropinocytosis, phagocytosis (Frost et al., 1989; Massol et al., 1998), protein tyrosine phosphatase and Rho GTPase activity (Gerhard et al., 2003; Retta et al., 1996). PAO (Sigma-Aldrich) was used at 10 μM to examine its effects on TGF-β3-mediated enhanced endocytosis as described (Yan et al., 2008). Furthermore, to validate the earlier findings using inhibitors (Yan et al., 2008), we have re-examined the regulation of protein endocytosis at the BTB by cytokines (e.g., TGF-β3) using a more selective tool in which clathrin was knocked down by RNAi using specific clathrin siRNA vs. control scrambled duplexes. In short, Sertoli cells were isolated from 20-day-old rat testes (Day 0) as described above and plated at a density of 0.3 × 106 cells/cm2 on Matrigel-coated dishes where ultrastructures of BTB, such as TJ and basal ES, were detected by electron microscopy as described (Siu et al., 2005). Two days after isolation (Day 2), cells were transfected with 100 nM siRNA (ON-TARGET plus set of 4 duplexes LQ-090659-01; Dharmacon) against rat clathrin heavy chain (Genbank Accession Number: NM_019299). On-target plus siControl non-targeting pool (D-001810-10; Dharmacon) was used as the corresponding control. 10 μl of TransIT-TKO transfection reagent (Mirus Bio) was used to transfect a total volume of 2 ml reaction mixture per well in 6-well culture dishes. Two days after transfection (Day 4), endocytosis assay was performed as described above. Biotinylated proteins were pulled down by NeutrAvidin beads (Pierce), washed four times with IP lysis buffer and extracted in SDS-sample buffer. Cell lysate was also harvested to assess the steady-state protein level of clathrin heavy chain after silencing. Under our experimental conditions, a ~50–60% reduction in clathrin protein level was observed. In preliminary experiments, a cell density of 0.5 × 106 cells/cm2 was also used. However, at this higher cell density, Sertoli cells were required to be transfected twice in order to achieve acceptable knockdown when assessed by immunoblotting as previously described (Wong et al., 2008a), and endocytosis assay was performed 24 h after the second transfection (Day 8). Yet, endocytosis assays using Sertoli cells at higher density did not yield satisfactory result due to the diminished level of junction proteins on Day 8 of cells in culture (unpublished observations). Thus, the silencing condition was subsequently modified by using a lower cell density at 0.3 × 106 cells/cm2 vs. 0.5 × 106 cells/cm2, but BTB ultrastructures were visible by electron microscopy, and functional TJ-barrier was still formed, and Sertoli cells were transfected only once with the corresponding siRNA or control duplexes on Day 2, with the endocytosis assay performing on Day 4 to examine the effects on the kinetics of protein endocytosis in the presence or absence of TGF-β3.
Sertoli cells were cultured on coverslips at 0.5–1 × 105 cells/cm2 for dual-labeled immunofluorescence analysis. At specified time points following treatment versus controls, Sertoli cells were fixed in 4% paraformaldehyde (v/v) for 10 min, rinsed and treated with 10 mM glycine for 5 min to quench the aldehyde groups. Cells were permeabilized with 0.2% Triton X-100, rinsed and incubated with 10% normal goat serum in PBS for 15 min. For double-label immunofluorescence analysis, cells were incubated for 1 hr with the primary antibodies at appropriate dilutions (see Table 1), rinsed, and incubated for 1 hr with the relevant fluorescent secondary antibodies. Negative controls to assess staining specificity included incubation with normal IgG of the corresponding animal species of the primary antibodies or the omission of the primary antibodies. After extensive washing, coverslips were mounted on slides using Vectorshield mounting medium with DAPI (Vector Laboratory, Burlingame, CA). Images were examined and obtained by confocal microscopy and was performed at The Rockefeller University Bio-Imaging Resource Center with an inverted Zeiss LSM 510 Laser Scanning confocal microscope. Images were acquired using the Zeiss LSM 510 (v. 3.2) software and exported in TIFF format. Images were merged and analyzed using Adobe PhotoShop (Version 7.0).
Recombinant human TGF-β3 purchased from Calbiochem (Cat no. PF073) was resuspended and dissolved in 4 mM HCl containing 0.1% BSA (w/v). Just prior to its use, it was diluted in PBS to a concentration of 1 ng/μl. Adult rats (~300 gm b.w.) received 200 ng of recombinant TGF-β3 in a total volume of 200 μl per testis via intratesticular injection using a 28-gauge needle as described (Li et al., 2006) versus vehicle control with n = 2 rat per treatment or control group. Two days after TGF-β3 administration, rats were euthanized by CO2 asphyxiation, testes were removed and processed for electron microscopy as described (Li et al., 2006; Yan and Cheng, 2006). Electron micrographs were obtained using a JEOL 100CXII Electron Microscope (Peabody, CA) at 80 kV at the Rockefeller University Bio-Imaging Resource Center.
Immunoblotting and immunoprecipitation using the corresponding antibodies against different target proteins (see Table 1) were carried out as previously described (Xia and Cheng, 2005; Xia et al., 2006). Protein concentration in lysates was estimated by Coomassie blue-dye binding assay using BSA as a standard as described (Bradford, 1976). Proteins in GCCM were concentrated by ultrafiltration using an Amicon 8050 unit with YM-10 filters. Prior to their inclusion in the Sertoli cell cultures, GCCM was filtered via a 0.2-μm sterile filter unit and protein concentration was estimated and the desired concentration was appropriately adjusted by F12/DMEM under sterile conditions.
Each experiment was repeated at least three times using different batches of primary Sertoli cell cultures. Each time point in an experimental set contained triplicate cultures. Results of a treated sample group were compared to the corresponding control by ANOVA to be followed by Tukey's Honest Significant Test or Dunnett's test using the GB-STAT Statistical Analysis Software package (Version 7.0) (Dynamic Microsystems Inc., Silver Spring, MD).
As shown in Fig. 1, by local administration of recombinant human TGF-β3 (200 ng/testis) to adult rats (~300 gm b.w. at 100 days of age), the BTB integrity was shown to be disrupted by day 2 (Fig. 1A–B vs. D). This conclusion was reached since the BTB failed to restrict the FITC green fluorescence in the basal compartment of the seminiferous epithelium (see controls in Fig. 1A–B), resulting in the diffusion of FITC across the barrier. For instance, green fluorescence was detected in the seminiferous epithelium beyond the BTB in TGF-β3-treated rats, similar to rats treated with CdCl2 except that it was not that extensive (Fig. 1D vs. C, and A–B). Cadmium is an environmental toxicant known to disrupt BTB integrity irreversibly in rats (Hew et al., 1993; Setchell and Waites, 1970; Wong et al., 2004). As shown in Fig. 1E, the TGF-β3-induced disruptive effect on the BTB integrity is transient, since by 14 days after the treatment, the BTB was shown to restrict the diffusion of the FITC across the barrier, similar to control rats (Fig. 1E vs. A–B). To further validate the effects of TGF-β3 on the BTB following its local administration into the testis, electron microscopy was performed as shown in Fig. 2. The BTB in adult rat testes is created by TJ (see the 'kisses' denoted by the black arrows) at the Sertoli-Sertoli cell interface which is also present side-by-side with the basal ES (Fig. 2A). Basal ES is typified by the presence of actin filament bundles (see black arrowheads) sandwiched between the endoplasmic reticulum (ER) and the plasma membrane (note: the two apposing Sertoli cell plasma membranes are denoted by the two opposing arrowheads) (see Fig. 2A), which co-exist with TJ. However, in rats treated with TGF-β3 for 2 days, distinguishable ultrastructural damages were visible at the Sertoli-Sertoli cell interface (see white arrowheads in Fig. 2B), and both TJ and basal ES were no longer detactable at the BTB in this rat testis (Fig. 2B vs. 2A2A).
It is likely that at stage VIII of the seminiferous epithelial cycle of spermatogenesis, TGF-β3 is secreted by Sertoli and/or germ cells into the BTB microenvironment near the basement membrane of the seminiferous epithelium, contributing to its 'restructuring' (and/or 'opening') to allow the transit of preleptotene/leptotene spermatocytes across the barrier. Thus, it is important to determine if similar level of TGF-β3 can be reached in the testis endogenously. Fig. 3A–F summarizes the results of a study using a solid-phase based immunoblot assay to assess the relative levels of TGF-β3 and TNFα in SCCM, GCCM versus testes (Fig. 2A, C) by plotting the relative amount of corresponding cytokine in these media/lysates against the recombinant proteins. As shown in Fig. 3B, D and summarized in Fig. 3E, F, it was estimated that SCCM, GCCM and testis lysates (in about 300 μg total protein) contained 7.5±0.7, 5.8±0.4, and 2.5±0.5 ng of TGF-β3 (n = 3) versus 1.5±0.2, 4.5±0.6, and 1.9±0.3 ng of TNFα (n = 3), respectively. This was equivalent to about 1.2±0.3 and 0.91±0.13 μg (n = 3) TGF-β3 and TNFα/testis, respectively, assuming the testis weight of an adult rat is ~1.6 gm, which was used to prepare testis lysates using a testis:IP buffer ratio of 1:3 (w:v), and protein concentration was obtained by Bradford reagent (Bradford, 1976). Thus, these results illustrate that the in vivo effects of TGF-β3 on the BTB integrity as shown in Fig. 1D and Fig. 2 are physiologically relevant since this concentration of TGF-β3 or a combination of cytokines (e.g., TGF-β3 and TNFα) can possibly be achieved at the BTB microenvironment at the site between Sertoli cells and preleptotene/leptotene spermatocytes, which transiently disrupts the BTB to facilitate the transit of preleptotene spermatocytes.
Figure 4A summarizes the results of a representative set of experiments by monitoring the kinetics of endocytosis of several integral membrane proteins at the BTB: occludin, JAM-A, and N-cadherin. It was noted that the presence of TGF-β3 and TNFα enhanced the kinetics of endocytosis of biotinylated occludin, JAM-A and N-cadherin (Fig. 4A, B:a, b, c) from the Sertoli cell surface since significantly higher levels of these biotinylated integral membrane proteins were detected in the cytosol by 10 to 60 min (Fig. 4A, B). GCCM at 50 μg/ml was also effective in accelerating the internalization of biotinylated cell surface integral membrane proteins (Fig. 4A, B) possibly due to the presence of cytokines, in particular TNFα, in GCCM (see Fig. 3). However, the steady-state levels of the three BTB integral membrane proteins: occludin, JAM-A, and N-cadherin, in lysates from these cultures subjected to biotinylation and endocytosis assay with or without treatment with TGF-β3, TNF-α or GCCM vs. controls did not alter considerably (Fig. 4A, bottom four panels). These results were further verified by fluorescent microscopy as shown in Fig. 5A, B. For instance, in control Sertoli cell cultures without treatment with any cytokine, biotinylated proteins mostly localized at the cell surface (green fluorescence, FITC-streptavidin that bound to biotin), co-localizing with either occludin (red fluorescence) (Fig. 5A:a, b, c) or JAM-A (red fluorescence) (Fig. 5B:a, b, c) (see white arrowheads). By 30 min, cell surface biotinylated occludin (Fig. 5A:d, e, f) and JAM-A (Fig. 5B: d, e, f) were internalized (see white arrowheads); however, more occludin (Fig. 5A: h, i) and JAM-A (Fig. 5B: h, i) in the TGF-β3 treated Sertoli cell cultures were found to be internalized, moving away from the cell-cell interface (Fig. 5A: g, h, i and Fig. 5B: g, h, i). Furthermore, 3-dimensional projections of selected images (f & i) were reconstructed from a series of confocal images (z-stack) using the Imaris software (see f' and i' corresponding to f and i in Panels A and B), from which the protein internalization was better visualized. It is obvious that based on this analysis, TGF-β3 was capable of disrupting JAM-A- & occludin-based TJ fibrils via enhanced endocytosis, consistent with results shown in Fig. 4A, B.
To further validate the effects of TGF-β3 and to assess the role of dynamins in mediating protein endocytosis at the BTB, RNAi was used to knock down TβR1, dynamin 2 or dynamins 2 and 3. Treatment of Sertoli cells with either TβR1- or dynamin 2-specific siRNA duplex versus scrambled siRNA duplex (controls) was shown to specifically knockdown the steady-state level of the corresponding protein by ~50–60% without affecting the other protein, illustrating the specificity of this technique (Fig. 6A, B). Interestingly, partial knockdown of dynamin 2 in Sertoli cells by RNAi had no apparent effects on the endocytosis of JAM-A at the BTB (Fig. 6C, D). However, treatment of Sertoli cells with TβR1 siRNA duplex was shown to abolish the TGF-β3-induced acceleration of endocytosis of biotinylated JAM-A, but not TNFα-induced acceleration of protein endocytosis (Fig. 6E, F). Interestingly, while RNAi of dynamin 2 alone had no apparent effects on protein endocytosis (Fig. 6C, D), when both dynamins 2 and 3 were knocked down, protein internalization was disrupted significantly at the 60-min time point (Fig. 6G, H vs. C, D). Furthermore, silencing of dynamins 2 and 3 also disrupted the TGF-β3- and TNFα-induced acceleration of protein endocytosis at both 10- and 60-min (Fig. 6G, H).
Phenylarsine oxide (PAO) is a known inhibitor of clathrin-dependent pathway of protein endocytosis (Ivanov et al., 2004). The presence of PAO in the Sertoli cell cultures at 10 μM alone was shown to block the endogenous internalization of occludin, JAM-A and N-cadherin (Fig. 7A, B), illustrating the endocytosis of these proteins at the BTB is likely mediated via the clathrin-dependent pathway. The presence of PAO also abolished the TGF-β3-induced acceleration of protein endocytosis (Fig. 7A, B). However, PAO is known to block macropinocytosis, phagocytosis (Frost et al., 1989; Massol et al., 1998), protein tyrosine phosphatase and Rho GTPase activity (Gerhard et al., 2003; Retta et al., 1996). To further validate these data, a more specific approach by targeting clathrin was used. Specific siRNA duplex was used to knock down clathrin protein level by ~50–60% in primary Sertoli cells with established BTB as shown in Fig. 8A, B. Consistent with results obtained by using inhibitor such as PAO (see Fig. 7), the knockdown of clathrin by RNAi significantly blocked the accelerated endocytosis of JAM-A induced by TGF-β3 (Fig. 8C, D). While the use of RNAi to silence clathrin failed to block protein endocytosis as effective as an inhibitor (Fig. 8 vs. Fig. 7) since we only managed to knock-down ~60% of the clathrin steady-state protein level in these primary Sertoli cell cultures and some JAM-A continued to be endocytosed in the transfected Sertoli cells (Fig. 8C), it is obvious that the knockdown of clathrin blocked the TGF-β3-mediated acceleration of JAM-A endocytosis (Fig. 8C, D). Furthermore, the knockdown of clathrin by RNAi also considerably reduced BTB integral membrane protein (e.g., JAM-A) endocytosis (see Fig. 8C, top panel versus Fig. 4A, second panel) due to the remaining clathrin available to the Sertoli cells in the system. It is also noted that the steady-state protein level of JAM-A in the cell lysates from cultures subjected to clathrin knockdown with or without treatment with TGF-β3 did not alter significantly (Fig. 8C, lower panel, and Fig. 8E).
For more than three decades since the detailed morphological study illustrating the migration of preleptotene and leptotene spermatocytes across the BTB that takes place at stages VIII–IX of the seminiferous epithelial cycle of spermatogenesis in adult rats (Russell, 1977), the biochemical mechanism(s) that regulates this event, however, remains virtually unknown. Even though recent studies have shown that cytokines, such as TGF-β3 and TNFα, regulate the steady-state levels of integral membrane proteins at the BTB (e.g., occludin, ZO-1, claudins) (Hellani et al., 2000; Lui et al., 2001; Lui et al., 2003b; Siu et al., 2003a; Wong et al., 2004; Xia and Cheng, 2005), thereby determining the status of the BTB integrity plausibly via their effects on the transcriptional regulation of specific target genes such as claudins [for a review, see (Lui and Cheng, 2007)], the precise mechanism that is used by the testis involving cytokines to maintain the optimal integral membrane protein levels at the BTB remains obscure. Recent findings based on studies from different epithelia have shown that protein endocytosis is a novel mechanism utilized by a cell epithelium to rapidly alter cell junction dynamics, necessary to facilitate cell movement during embryogenesis, differentiation, and development (Ivanov et al., 2004; Le et al., 1999; Morimoto et al., 2005), it is of interest to determine if the transit of preleptotene/leptotene spermatocytes across the BTB employs similar mechanism involving cytokines, in particular Sertoli and germ cells are known to produce different cytokines in the seminiferous epithelium at relative high concentrations [for reviews, see (Cheng and Mruk, 2002; Mruk and Cheng, 2004; Skinner, 1993)].
In this report, it was shown that TGF-β3 when administered locally to the testis at 0.2 μg/testis, which is the concentration well within the endogenous range of TGF-β3 in normal testes, at 1.2±0.3 μg/testis when assessed by a solid-phase immunoblot-based assay, TGF-β3 was shown to reversibly disrupt the BTB integrity when assessed by a functional in vivo assay. These findings, coupled with the recent studies in which local administration of TNFα was also capable of inducing reversible BTB integrity disruption in vivo (Li et al., 2006), thus suggest that cytokines produced by Sertoli and germ cells locally [for reviews, see (Mruk and Cheng, 2004; Siu and Cheng, 2004; Skinner, 1993)] at the BTB microenvironment can regulate BTB dynamics possibly by regulating the steady-state levels and/or enhancing endocytosis of integral membrane proteins at the BTB (e.g., occludin, JAM-A and N-cadherin), causing a transient dissolution of the TJ-fibrils at the site, thus facilitating the transit of preleptotene/leptotene spermatocytes across the BTB. As shown in this report, the effects of TGF-β3 that enhance protein endocytosis appear to be specific since a partial knock-down of TβRI, the specific receptor for TGF-β, rendered the Sertoli cell cultures with functional BTB incapable of responding to TGF-β3 treatment to accelerate protein endocytosis. The schematic drawing depicted in Fig. 9 illustrating the hypothesis that cytokines (e.g., TGF-β3 and TNFα) contributed by Sertoli and germ cells to the microenvironment at BTB to facilitate spermatocytes in transit is further supported by the findings that TGF-β3 expression at the BTB is stage-specific and its localization at the site of the BTB in the seminiferous epithelium of adult rat testes is relatively high at stages VII–VIII of the epithelial cycle when examined by immunohistochemistry technique (Lui et al., 2003a; Xia et al., 2006). Furthermore, TβR1 was also detected at the BTB by immunohistochemistry at these stages (Xia et al., 2006). The present report thus strengthens the notion that as a preleptotene/leptotene spermatocyte from the basal compartment begins its transit crossing the BTB to enter the adluminal compartment to continue its further development, cytokines are released near its apical region to enhance endocytosis, thereby inducing transient restructuring of the BTB to facilitate cell movement as illustrated in Fig. 9. However, it remains to be determined if the TGF-β3-induced germ cell loss from the seminiferous epithelium as recently reported (Xia et al., 2006) is also mediated by accelerated endocytosis of proteins at the apical ES and/or desmosome-like junctions at the Sertoli-elongating/elongated spermatid and Sertoli-spermatocyte interface. Thus similar investigation should be expanded to the use of Sertoli-germ cell cocultures or similar systems in future studies.
It is of interest to note that by silencing dynamin 2 or both dynamins 2 and 3 via the use of specific dynamin duplexes, it failed to disrupt endocytosis of JAM-A at the BTB in the absence of cytokines. Yet the silencing of dynamins 2 and 3 can effectively block the TGF-β3- and TNFα-induced enhanced endocytosis of integral membrane proteins at the BTB. It seems that while these large GTPases are used by other epithelia to regulate protein endocytosis (McNiven et al., 2000), they are not involved in protein endocytosis under normal conditions. However, a surge of cytokines at the BTB microenvironment may have recruited or activated other adaptors (or kinases, phosphatases) which, in turn, render the physiological involvement of dynamins in this event to accelerate protein endocytosis. This postulate is supported by recent findings that a disruption of germ cell adhesion in the seminiferous epithelium is associated with a transient increase in the binding of dynamin 2 with β-catenin, pulling β-catenin away from the cadherins, thereby destabilizing Sertoli-germ cell adhesion (Lie et al., 2006). This possibility is also supported by recent findings that FAK and Src, both are non-receptor protein tyrosine kinases, are prominently present at the BTB as visualized by immunohistochemistry and fluorescent microscopy (Lee and Cheng, 2005; Siu et al., 2003b). These kinases can thus activate and recruit dynamins to the BTB site to accelerate protein endocytosis to facilitate the transit of preleptotene/leptotene spermatocytes.
In epithelia, protein endocytosis is mediated via either clathrin-dependent, caveolin-dependent, or clathrin and caveolin-independent (e.g., macropinocytosis) mechanism [for reviews, see (Ivanov et al., 2005; Maxfield and McGraw, 2004)]. A recent report using specific inhibitors to clathrin (e.g., PAO) or caveolin (e.g., cholesterol oxidase, CO) have shown that protein endocytosis at the BTB using Sertoli cells cultured in vitro is likely mediated via the clathrin-dependent pathway (Yan et al., 2008). However, many of these classical inhibitors employed to study protein endocytosis are not specific (Ivanov, 2008). For instance, PAO, besides blocking clathrin-mediated endocytosis, also blocked macropinocytosis and phagocytosis, protein tyrosine phosphatase and Rho GTPase activity (Frost et al., 1989; Gerhard et al., 2003; Massol et al., 1998; Retta et al., 1996). In this study, we have used a more specific approach by RNAi using specific siRNA duplexes to knock down clathrin versus scrambled control siRNA duplexes. Using primary Sertoli cell cultures with established BTB including the functional TJ-permeability barrier in vitro, we managed to knock down ~60% of the clathrin protein in these Sertoli cells. While Sertoli cells continued to endocytose BTB proteins (e.g., JAM-A) at a considerably level following the partial knockdown of clathrin, TGF-β3 was no longer capable of enhancing JAM-A endocytosis because of a lack of clathrin in the Sertoli cell epithelium. These findings thus support a recent report (Yan et al., 2008) and results reported herein using inhibitors, illustrating protein endocytosis, in particular TGF-β3-induced accelerated endocytosis, that occurs at the BTB is mediated by a clathrin-dependent mechanism.
Present studies in the field have shown that endocytosed proteins in epithelial cells can either (i) be recycled back to the cell surface via recycling endosomes or undergo de-ubiquitination, or (ii) be recruited to late endosome to be targeted to lysosomes or undergo ubiquitination and targeted to proteasomes for intracellular degradation [for reviews, see (Bright et al., 2005; Katzmann et al., 2002; Lui and Cheng, 2007; Maxfield and McGraw, 2004; Piper and Luzio, 2007). It remains to be determined if the TGF-β3- and TNFα-induced endocytosed proteins would be targeted to lysosomes or proteasomes for intracellular degradation, or the percentage of endocytosed proteins being targeted for intracellular degradation via lysosomes and/or proteasomes versus recycling are differentially regulated, thereby reducing the steady-state levels of integral membrane proteins at the BTB, destabilizing the barrier function at the BTB to facilitate preleptotene/leptotene spermatocyte migration during spermatogenesis. However, using Sertoli cells with established BTB, TGF-β2 and testosterone were shown to enhanced protein endocytosis at the BTB, such as occludin (Yan et al., 2008). But TGF-β2 apparently promoted endocytosed occludin to late endosomes for its intracellular degradation via an increase in association with Rab 9 (a late endosome marker); whereas testosterone stimulated protein recycling (Yan et al., 2008), possibly relocating integral membrane proteins at the BTB near the apical region of a preleptotene spermatocyte in transit to its basal region. This possibility is consistent with the recent findings that testosterone is important to BTB function (Meng et al., 2005; Wang et al., 2006). The present report not only confirms some of these earlier findings, it has unequivocally demonstrated that protein endocytosis at the BTB, such as JAM-A, is mediated via a clathrin-dependent mechanism. Also, besides TGF-β2, TNFα and TGF-β3 can also regulate BTB restructuring via protein endocytosis. In this context, it is of interest to note that the effects of TGF-β3 on the endocytosis of some BTB integral membrane proteins, such as JAM-A, are more potent than TGF-β2 (Yan et al., 2008). This is not entirely unprecedented, since all TGF-βs and their related family members (such as activins, inhibins, bone morphogenetic protein) exert their biological effects via interactions with one of the three TGF-β binding proteins (or receptors) but with different affinities, which, in turn, determines the different biological potencies of different ligands (i.e., TGF-βs or their members) [for reviews, see (Bovd et al., 1990; Massague, 2008)]. These findings also suggest that different cytokines may be expressed temporally and/or spatially at the BTB microenvironment during the epithelial cycle at the time preleptotene spermatocytes are in transit at the BTB by regulating the relocation of proteins at the Sertoli-Sertoli cell interface, such as from the apical region of a migrating spermatocyte to its basal region, via transcytosis. Nonetheless, this possibility must be vigorously investigated in future studies.
It is noted that in the endocytosis assays, there were variations in the recovery of endocytosed biotinylated proteins versus total biotinylated proteins between experiments. This is possibly due to the multi-step processing of samples in these assays: (i) lysates preparation, (ii) the pull-down of biotinylated total proteins by avidin-based beads, (iii) SDS-PAGE, to be followed by (iv) immunoblotting using corresponding antibodies to visualize a specific target protein in the cytosol over an experimental period to detect the endocytosed and biotinylated protein. Indeed, we had minimized intra-experimental variations by processing all samples within an experimental set simultaneously so that any loss of biotinylated proteins would occur uniformly across the samples in a given experiment. Yet, given the number of steps that were involved to process these samples biochemically, it is difficult to conclude that the amount of endocytosed junctional proteins (e.g., JAM-A) is small (such as those shown in Fig. 6E) because of the potential loss of samples during their processing. However, for data reported in Fig. 5B (see g–i versus a–f), these are the 'real-time' JAM-A at the cell-cell interface following TGF-β3 treatment at 30-min, but these results could not yield the time-dependent changes in endocytosis as those depicted in Fig. 6E nor be able to yield semi-quantitative information. But this technique is helpful to assess any changes in protein distribution (such as redistribution or mislocalization) in the Sertoli cells. Again, it is difficult to conclude that there was any loss of JAM-A at the cell-cell interface based on fluorescent staining by confocal microscopy except to conclude that more JAM-A was internalized following treatment with TGF-β3. These observations also expose the limitations of these two powerful and widely used techniques in the field, namely the biochemical-based endocytosis assay and the dual-labeled immunofluorescence analysis by confocal microscopy.
In summary, based on the results reported herein, we have provided a working model possibly used by the testis to regulate the transient 'opening' and 'closing' of the BTB during spermatogenesis involving cytokines and protein endocytosis as depicted in Figure 9. This model will now provide a basis for investigators in the field to design functional experiments to examine the intriguing regulation of junction dynamics at the BTB.
The authors thank Dr. Alison North at The Rockefeller University Bio-Imaging Resource Center for her helpful discussion on the use of confocal microscopy. We are also grateful for the excellent technical assistance of Ms. Eleana Sphicas at The Rockefeller University Bio-Imaging Resource Center for performing electron microscopy.
*This work was supported in part by grants from the National Institutes of Health (NICHD, U01 HD045908; R03 HD051512; U54 HD029990 Project 5 to CYC), and the CONRAD Program (CICCR, C1G 01-72).
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American Chamber of Commerce for Brazil - São Paulo
Belo Horizonte | Brasília | Campinas | Curitiba | Goiânia | Porto Alegre | Recife | Ribeirão Preto | Salvador | São Paulo | Uberlândia
Tel: +188.8.131.523.804
American Chamber of Commerce for Brazil - Rio de Janeiro
Rio de Janeiro | Vitoria
Tel: +55.21.2203.2477
Brazilian National Confederation of Industry - CNI
Brazil Section of BUSBC, Conselho Empresarial Brasil-Estados Unidos - CEBEU
U.S. Department of Commerce
Office of South America
International Trade Administration
14th and Constitution Avenue, NW
Washington, DC 20230
Tel: +1.202.482.0475 or +1.202.482.4157
Fax: +1.202.482.4157
U.S. Department of Commerce Person Finder
Everett Wakai
Commercial Officer (Cônsul Comercial)/Standards Officer for South America
U.S. Commercial Service, U.S. Consulate General
São Paulo, SP - Brazil
Tel +55.11.3250.5402
The Advocacy Center , U.S. Department of Commerce
The Advocacy Center helps to ensure that sales of U.S. products and services have the best possible chance competing abroad. Advocacy assistance is wide and varied but often involves companies that want the U.S. Government to communicate a message to foreign governments or government-owned corporations on behalf of their commercial interest, typically in a competitive bid contest.
Western Hemisphere:
Tathiana Mitchell, Regional Manager: email@example.com
Patrice Williams, Regional Manager: email@example.com
Office of the U.S. Trade Representative
2014 Report on Technical Barriers to Trade (TBT Report)
2014 Report on Sanitary and Phytosanitary Measures (SPS Report)
2014 National Trade Estimate Report on Foreign Trade Barriers (NTE Report)
2014 Special 301 Report
2012 Report on Sanitary and Phytosanitary Measures (SPS Report)
2012 Report on Technical Barriers to Trade (TBT Report)
2012 National Trade Estimate Report on Foreign Trade Barriers
2012 Special 301 Report
Results of the 2012 Section 1377 Review of Telecommunications Trade Agreements
Brazilian Embassy in Washington
Brazilian Consulates in the United States
Brazilian Development Bank (BNDES)
Brazilian Foreign Trade Association
Brazilian Trade and Investment Promotion Agency (APEX)
IBGE-Brazilian Institute of Geography and Statistics
Export-Import Bank of the United States
Inter-American Development Bank (IDB) - booklet
IDB - Procurement FAQs
O Estado de São Paulo (São Paulo)
Folha de São Paulo (São Paulo)
Correio Braziliense (Brasília)


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Thelma, age 88, of Penn Yan, NY, formerly of Corning, NY, passed away on Tuesday, May 7, 2013 at Soldiers & Sailors, Penn Yan, NY.
Born in Riverside, NY on November 5, 1924, she was the daughter of the late James E. and Cora Jane (Briggs) Winfield.
Thelma graduated from Painted Post High School in 1942.
She was predeceased by husbands, William and Joseph; sisters, Neomia Morrow and Betty Maio; and brothers, Edwin G,
James, Jr. and Charles Winfield.
She is survived by sons, Randy Horton of Penn Yan and William (Nan) Horton of Lindley; daughter, Phyllis (Gordon) Casterline of Corning; 8 grandchildren, Scott, Stacy, Diana, Jenny, Jackie, Tony, Jeremy, Jessie; along with several great-grandchildren and nieces and nephews.
All are welcome to join Thelma's family in celebrating her life on Saturday, May 11, 2013 from 12:00PM-2:00PM at Carpenter's Funeral Home, LLC, 14 East Pulteney Street in Corning.
Funeral services will be held immediately following at 2:00PM with Rev. Thomas Wunder officiating.
Thelma will be laid to rest at Coopers Plains Cemetery, Painted Post.
Read Thelma's obituary, share your memories and offer your condolences at www.carpentersfuneralhome.com
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