text:
Saturday Scholar Series presentation available on the Web
by Sara Woolf
R. Scott Appleby, professor of history and the John M. Regan Jr. Director of the Joan B. Kroc Institute for International Peace Studies, John Paul Lederach, professor of international peacebuilding, and A. Rashied Omar, assistant professor of Islamic studies and peacebuilding, delivered the most recent lecture in the University of Notre Dames Saturday Scholar Series,The Role of Religion in Peacebuildingon November 4.
The presentation explored the constructive achievements of religiously inspired peace builders, and what those religious actors have done in the past to stimulate the peacebuilding capacity of people suffering in conflicts.The panel also discussed the role of Notre Dame's Kroc Institute in supporting and promoting peace around the world.
The lecture is available for viewing on the Web at:
(broadband) http://streaming.nd.edu/artsletters/saturday06/peacebuilding.wmv
(modem) http://streaming.nd.edu/artsletters/saturday06/peacebuilding_low.wmv
Sponsored by theCollegeofArtsand Letters, the Saturday Scholar Series presentations begin three and one-half hours before kickoff of each home football game and are held in the Annenberg Auditorium of theSniteMuseum.
The final presentation of 2006 will be November 18 and will feature a musical performance by music department faculty members Georgine Resick (soprano) and John Blacklow (piano).TitledSeeds of Change,the program will feature French musicfrom the first half of the 19th century, including works by Berlioz, Chopin, Liszt, Gounod, and Saint-Säens, as well as spoken commentary drawing parallels between themusic of the period and concurrent trends in literature, the visual arts, social history and politics.
For more information on the Saturday Scholar Series, including a schedule of speakers, visit http://saturdayscholar.nd.edu/ .


text:
See the next phase of the Arch grounds renovation
Posted 5:47 pm, July 12, 2019, by Patrick Clark, Updated at 06:30PM, July 12, 2019
ST. LOUIS, Mo. — It's a goal of the Gateway Arch Park foundation to bring the city to a central point under the Arch. So, how are the Arch grounds holding up after renovations and rediscoveries in downtown? Patrick Clark is reporting on the present and future use of the Arch grounds.
Ryan McClure the new executive director of the Gateway Arch Park Foundation.
"You can see for this time of year the grass is not that bad and it's coming back. That was part of the renovation is replacing the soil and ensuring that the landscape is set up here is set up for success," said Ryan McClure.
McClure is familiar with the nonprofit foundation that works in conjunction with the National Park Service the city of St. Louis and Bi-State. He was there when the City Arch River Project made a push to put a lid on it. The $380 million renovation covered an interstate and re-invigorated this landmark.
"Coming back to Gateway Arch Park Foundation is truly like coming home. I started here in 2011 before the project was even started. So, to see that through this exciting time in the foundation. It's a really exciting new era," said Ryan McClure.
McClure says 221 million of the $380 million dollar renovation project came from private funding. And it is not quite done.
"There's actually one more piece of the project that is yet to be finished. That is in the Old Courthouse. What's happening is really exciting. It's accessibility improvements, making sure more people can enjoy this beautiful historic building that is an integral part of this national park," said Ryan McClure.
Look for more Blues concerts on the north end of the grounds closer to Laclede`S Landing. The stage for concerts will remain temporary. There are no current plans to make that permanent.
Grandson taking grandma to the Arch on last viral national park tour stop
St. Louisan visits all 418 national park sites, ending 2019 at the Gateway Arch
Ohio grandmother visits Gateway Arch on final stop to see every national park
Tim's Travels: Winterfest Ice Rink in Kiener Plaza now open
MLS ownership group says everything on schedule for stadium construction
Tim's Travels: Winterfest Ice Rink All-Star Celebrations at Kiener Plaza
Submissions now open for the "Name The Greenway" Chouteau Greenway project
Arch Madness basketball tournament to remain in St. Louis through 2025
Redo the Lou: Design Downtown STL starts planning for the future
Gingerbread Village now on display at The Chase Park Plaza
Cardinals Care Winter Warm up underway in downtown St. Louis
Gateway Arch offers 'Frights and Heights' a spooky nighttime ride to the top
St. Louis listed as most unsafe city, travelers look past headlines

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: In angular dart, can I find the index in list of strings based on any part of a multi-word criteria In angular dart, I have a list of strings that is filled with available groups and a search string. I want to find the index in the list that contains any whole word of the search string. In this example, I would want it to find index 0 because one of the words in the search string "Client" appears in on of the groups. I know the code below will not work because it is looking for the entire searchtext.
      List<String> groups = [];
      groups .add('Client Information');
      groups .add('User Information');
      groups .add('Activity Information');

      String searchtext = 'Client Customer';

      return groups.indexWhere((item) => item.contains(searchtext));

A: You can dynamically build a RegExp to search for a group of words
final regExp = RegExp('(${searchtext.replaceAll(' ', '|')})');
final index = groups.indexWhere(regExp.hasMatch);

redpajama_set_name: RedPajamaStackExchange


Republican State Assemblyman Dan Logue said he requested an audit of the funds after he heard reports of money spent on activities not clearly connected to mental health.
Democratic State Senator Darrell Steinberg, an author Proposition 63, an income tax on millionaires, replied that tens of thousands of people benefit from the Act, and it has saved lives.
"What may seem odd or frivolous to some, may be the only way, or an effective way, to help people who have not received their share of funding, and have not received care." Steinberg said most of Prop 63 money is going towards services for the people with mental problems, and a smaller portion is being spent on prevention.
The original supporters of Prop. 63, which became the Mental Health Services Act (MHSA), hoped for services designed for prevention and recovery, driven by consumers and families. They wanted better outcomes, reduced homelessness, and less forced treatment and confinement.
They wanted more and better staff training, peer-run programs, family members teaching family members, and more people with "lived experiences" working on the staffs of mental health agencies.
But counties are still not sure what "recovery services" means, or what recovery programs look like. They are paying consultants to tell them, and show them how. New programs also require input from consumers and families before they are lauched. That process has been clumsy and uneven because counties run mental health services in California, and they are all different.
Recovery services are wildly different from county to county. Some of the projects end up being pretty fluffy and "feel-good," like parties or outings.
Here is DJ Jaffe's propaganda piece on the problem. Jaffee speaks for the Treatment Action Center, founded by E. Fuller Torrey, who says the mental health system's job is protecting society from violent people with untreated mental illnesses. TAC favors forced treatment and confinement. Check the comments section on his artical.
A broad coalition of statewide advocacy groups, called Working Well Together (WWT) , is also on the case. Its primary goal is to "ensure public mental health agencies are prepared to recruit, hire, train, support and retain multicultural clients, family members and parents/caregivers as employees.
They are preparing a state peer certification program now under a state contract.
Paid for by the state Mental Health Services Act, the newly formed collaborative of client, family, parent/caregiver, and mental health training and technical assistance organizations consists of the California Network of Mental Health Clients, NAMI California, United Advocates for Children and Families and the California Institute of Mental Health.
Together, these organizations utilize their combined expertise, experience, grassroots networks and mental health system connections to affirm wellness and recovery from mental illness. called s also on the case.
What recovery services would you want this tax to pay for?
WRAP, Mary Ellen Copeland's Wellness Recovery Action Plan has transformed my daughter's life, and that of her two sons. DD was diagnosed with Bipolar 2 two-and-a-half years ago and, yes, she is on medication, but is reducing it with the help and approval of her psychiatrist. She is returning to college in the fall, and attributes 80% of her recovery to the WRAP program she took last year. She has also taken the WRAP facilitator's course and is able to work part-time helping others develop their own road maps to better health. Her new-found stability has also changed the lives of my grandsons, aged 13 and 8, who are recovering from the chaos of their previous existence, including twelve moves in eight years.
Of course, one has to be prepared to do the work involved for WRAP to work. It's a totally individualized program; there are no shortcuts. But for those who are ready to assume personal responsibility for their lives and their illness, it provides a stellar path to re-claiming a productive life.


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text:
Last Ticket to Ride
Letting go of the Centreville carousel.
Winter, 2017–2018 / No. 40
Conan Tobias
In mid-September, as southern Ontario began to experience an unusually late stretch of warm summer weather, Ann Elliott made the one-hour drive from her home, in Ajax, Ontario, to the Toronto Islands ferry dock, accompanied by her daughter, Janet, and granddaughter, LeeAnn. Elliott, who turned eighty-eight at the end of the month, wanted to take a final ride on the Centreville Amusement Park carousel, before it was disassembled and shipped to its new owners, in Carmel, Indiana. The line to ride wasn't especially long, and Elliott soon swung herself onto a horse, with only minor assistance from LeeAnn. Janet stood outside the fence and took photos as her mother smiled and laughed for the ride's three-minute duration. "Mum took me maybe once a year, maybe more, to the islands," Elliott said. "I can't remember any ride but the merry-go-round."
Toronto's islands and mainland waterfront have a long history of amusement, dating back to the nineteenth century, including parks at Sunnyside and Hanlan's Point. The park at Centre Island is the only one remaining. Centreville's carousel was built in 1907, by the G. A. Dentzel Carousel Company, of Philadelphia, and purchased by Centreville in 1966. It features an eclectic mix of fifty-two hand-carved animal mounts, including ostriches, house cats, and, of course, horses. Only a hundred and fifty carousels like it remain in the world.
Severe flooding on the islands this summer forced Centreville to remain closed for most of the season. Representatives of William Beasley Enterprises, the company that runs the park, said selling off assets such as the carousel became necessary to make up the six million dollars in damages and eight million dollars in lost revenue Centreville suffered. As a result, officials from Carmel agreed to purchase the carousel for three million dollars, but, in late September, that city's finance committee voted down the purchase, and the deal was cancelled. Beasley said it planned to look for another buyer.
Elliott, who grew up in Toronto, has made riding the city's carousels a family tradition. In the nineteen-sixties, "I took my daughter, Janet, and her older brother, Bruce, frequently. Then I remember going with my two grandchildren." This was her first visit in nearly two decades. "I'm really sad to see it go," she said. "I have two snapshots, dated '1940, Centre Island,' of my best friend and her mother, and Mum and me and another girlfriend. Just water behind us. I am sure we would have ridden the merry-go-round that day."
The Better Way Christmas, 2004 / No. 13


text:
Starring: Keira Knightley, Matthew MacFadyen, Brenda Blethyn, Donald Sutherland, Judi Dench, Rosamund Pike, Tom Hollander, Simon Woods, Rupert Friend, Claudie Blakley
Directed by: Joe Wright
Windswept moorlands and dishevelled hair, muddy hemlines and walking boots. is this Pride & Prejudice or Wuthering Heights? It's Jane Austen, Jim, but not as we know it.
Director Joe Wright (the man behind the at times unpleasantly oversexed Charles II mini series starring Rufus Sewell) replaces the traditional picture perfect parlours and elegant coiffeurs with sweaty country dance halls and hairstyles that come courtesy of several hedges and a lot of vigorous dragging backwards. The Bennetts' modest Hertfordshire retreat is transformed into a bucolic working farm, ankle deep in mud and flapping geese, complete with rustic labourers and Constable style haywains, more Tom Jones than Pride & Prejudice, whilst Mr Bennett himself (Donald Sutherland) looks positively grizzled, a Captain Ahab in the making.
But wait. In case there's anybody out there who doesn't know the plot of Pride & Prejudice (and if not, where have you been? Have you not see Bridget Jones's Diary?), a quick resumé. It is a truth universally acknowledged, that a single man in possession of a good fortune, must be in want of a wife. Step up Mr and Mrs Bennett and their five daughters: beautiful, placid Jane, spirited, quick witted Elizabeth, serious minded Mary and flirty, flighty and virtually indistinguishable Kitty and Lydia. As Mr Bennett's estate is entailed onto a male relative, Mr Collins, marriage is the Bennett sisters' only chance of escaping poverty. So when the highly eligible Mr Bingley (a gentlemen with £5,000 a year, no less) rents nearby Netherfield Hall, Mrs Bennett soon has him in her sights as a potential husband. And with Mr Bingley comes Mr Darcy, the haughty, stiff-necked snob who somehow ends up falling for Elizabeth.
See, the thing people who've never read Jane Austen don't understand is that her books aren't really about bonnets and balls, soppy love and giggly girl stuff, at all. What Austen writes so brilliantly and perceptively about is money (or, often, the lack of it). Her books deal, pragmatically, with the cold hard economics of the marriage market, the only area of commerce in which women could interact, where beauty and brains could earn one a fortune (£10,000 a year, in Lizzie's case) and one rash gamble could damn you to a lifetime of impecunity (Lydia - whose shotgun wedding to the dashing Orlando Bloom lookalike Mr Wickham can only end in debt and disaster).
And it's this aspect of Austen which Wright's Pride & Prejudice captures brilliantly. For a start, all five daughters actually feature (and they're all young as well - no blushing thirty-year-olds squeezed into shift dresses). Crowded round the dinner table, squeezed onto sofas, tumbling into parlours after eavesdropping at the keyhole - despite the impressive size of the Bennett home, there simply isn't room for so much dishevelled, ultimately useless femininity. They simply have to go, to homes of their own. Plain, slope-shouldered Charlotte Lucas (Claudie Blakley) understands this perfectly. Pretty, sparky Lizzie may turn down the toadying Mr Collins, but for the twenty-seven-year-old spinster Charlotte, too long a burden to her parents, his offer of marriage is a lifeline, a chance to have something of her own.
Is it a coincidence that Lizzie begins to rethink Mr Darcy's marriage proposal after seeing his fabulous estate at Pemberly? Of course not - the place is amazing, who wouldn't be kicking themselves? In Andrew Davies' 1995 TV adaptation, the splendours of Pemberly are accompanied by the, um, splendours of Colin Firth's backside, and we're left to make up our own minds as to which holds more sway. Here the only naked bums on display are in the chilly statue gallery: it's money that's making an impact, not manliness.
Passionless? Well, yes. Whilst Andrew (Tipping the Velvet) Davies' screenplay oozed sex from every pore and was positively heaving with bursting bosoms and taut buttocks, this new version is curiously devoid of sex appeal. Keira Knightley's Elizabeth Bennett is the very antithesis of the busty, arch Lizzie portrayed by Jennifer Ehle, who looked as if she couldn't wait to jump between the sheets with Mr D, £10,000 a year or no. But whilst no-one can deny that Keira is a lovely looking girl, that doesn't stop her spending a large part of the film looking like a skeleton in a wig, all gleaming white teeth and jutting jaw. Meanwhile, Matthew MacFadyen's Mr Darcy is a tasty piece of eye candy but you never really believe that he's in love with Elizabeth. Their relationship is brittle and witty - there's chemistry, certainly, but not of the sexual kind.
Far more engaging than the central characters, however, are those floating around the edges: Brenda Blethyn's marvellously overwrought Mrs Bennett, the hard-nosed marriage broker with a well concealed heart of gold, so garrulously awful we can almost sympathise with Darcy's desire to rescue his friend from her clutches; Simon Wood's charmingly hopeless, ginger- haired Mr Bingley, a nice but dim upper class twit controlled by his glacial bitch of a sister Caroline (Kelly Reilly); Judi Dench's magnificent Lady Catherine De Bourg - she could play the role in her sleep of course, but that doesn't stop her stealing her scenes with gusto.
Best of all, however, is Mr Collins, placed by Tom Hollander (next to be seen in The Libertine and Pirates of the Caribbean 2, alongside a certain Mr Depp). It's easy to overplay the obsequious, self-important parson, but Hollander's performance is judged to perfection: pedantically tactless and toe-curlingly sycophantic, his flat Estuary accent and po face are a cringeworthy delight.
As for the ending, well, everyone knows what happens: there's really no need to drag it out with so much over-explanation. (It's for the Americans, apparently. In case they haven't seen Bridget Jones.) And while opting out of a cheesy double wedding scene is fair enough, denying us one final kiss? Boo!
Like the cold white marble busts in Mr Darcy's home, Pride & Prejudice is fantastic to look at, a real work of art, but somehow lacking in passion and soul. It's captured to perfection the economics of Austen's novel, but lost the heart along the way. As a period drama it's beautiful, full of excellent, well researched and utterly delightful detail, but as a romance it falls a wee bit flat. Refreshingly free of Boobs & Bonnets, this Prim & Practical rewrite of Pride & Prejudice could nevertheless have benefited from a little more Heart & Soul.
The Twilight Saga: Breaking Dawn Part 2
Sun Don't Shine


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text:
typedef struct { // Data stored PER GLYPH
	uint16_t bitmapOffset;     // Pointer into GFXfont->bitmap
	uint8_t  width, height;    // Bitmap dimensions in pixels
	uint8_t  xAdvance;         // Distance to advance cursor (x axis)
	int8_t   xOffset, yOffset; // Dist from cursor pos to UL corner
} GFXglyph;

typedef struct { // Data stored for FONT AS A WHOLE:
	uint8_t  *bitmap;      // Glyph bitmaps, concatenated
	GFXglyph *glyph;       // Glyph array
	uint8_t   first, last; // ASCII extents
	uint8_t   yAdvance;    // Newline distance (y axis)
} GFXfont;

#endif // _GFXFONT_H_

redpajama_set_name: RedPajamaGithub


text:
Quellenamnesie ist ein alltägliches Gedächtnisphänomen. Es beschreibt die Eigenschaft unseres Gedächtnisses, dass wir Fakten unabhängig von ihrer Herkunft speichern. So kann ein Mensch über ein bestimmtes Wissen verfügen (z. B. dass Cäsar von Brutus ermordet wurde) ohne sich daran zu erinnern, wo und wann er die Fakten gelernt hat (z. B. in einer TV-Dokumentation).

Phänomene der Quellenamnesie

Im Zusammenhang mit Quellenamnesien besteht die Möglichkeit, dass sich falsche Quellen in der Erinnerung wiederfinden. Zusammen mit diesem Fehlinformationseffekt bildet die Quellenamnesie den Ursprung vieler falscher Erinnerungen. Auch kann es z. B. Schriftstellern passieren, zu glauben ein Gedanke käme von ihnen, ohne zu wissen, dass die entsprechende Information ursprünglich von außen kam. Ähnliches gilt auch für Zeitzeugen, die bestimmte Ereignisse als eigenes Erleben ansehen ohne sich zu erinnern, dass es aus den Erzählungen anderer Personen stammt oder z. B. aus einem Film.
Ein weiteres Phänomen der Quellenamnesie zeigt sich bei der Erinnerung an Träume: Menschen wissen manchmal nicht, ob ein bestimmtes Ereignis tatsächlich stattfand, oder ob die Erinnerung aus einem Traum stammt.

Gedächtnisstruktur

Die Quelle gehört zu den empfindlichsten Teilen einer Erinnerung. Sie liegt im autobiographischen Gedächtnis, welches nur wenige und bedeutsame Inhalte speichert. Der zentrale Verarbeitungsort hierfür im Gehirn sind die innersten Bereiche des Hippocampus. Dort steht vergleichsweise wenig Platz zur Verfügung.

Das Faktenwissen dagegen zählt zum semantischen Gedächtnis. Der Hippocampus verarbeitet diese Inhalte weiter außen, wobei hierfür mehr Platz vorhanden ist. Daher werden Fakten besser gespeichert als die Quellen.

Literatur

 David G. Myers: Psychologie. 1. Auflage, Springer Medizin Verlag, Heidelberg 2005, ISBN 978-3-540-21358-1.
 Gerhard Roth: Aus Sicht des Gehirns. 2. Auflage, Suhrkamp Verlag, Frankfurt am Main 2009, ISBN 978-3-518-29515-1.
 Gerhard Roth: Persönlichkeit, Entscheidung und Verhalten. 1. Auflage, Klett-Cotta Verlag, Stuttgart 2007, ISBN 978-3-608-94490-7.

Gedächtnis

redpajama_set_name: RedPajamaWikipedia


text:
What Does it Mean, to Heal? - Part II
Issue #7 , November 2018
Issue #15, "Dead and Alive: Being with Ancestors" Part III
Reader Response
Issue #14, "Dead and Alive: Being with Ancestors" Part II
Issue #13, "Dead and Alive: Being with Ancestors" Part I
Issue #12, "How do We Know? Part II"
Issue #11, "A Lesson. A Warning. A Flare: Voices from the Pandemic"
Reader Response: Issue 6-10
Issue #10, "How Do We Know?"
Issue #9, "Extinction Illness: Grave Affliction and Possibility"
Issue #8, "What Does it Mean, to Heal? Part III"
Issue #7, "What Does it Mean, to Heal? Part II"
Issue #6, "What Does it Mean, to Heal?"
Reader Response: Issue 1-5
A Retrospective: Issues #4-5
Issue #5, "Making Kin: Part II"
Issue #4, "Making Kin: Part I"
Issue #3, "Extinction/Devotion"
Issue #2, "Fragile Ongoing"
Issue #1, "Seeing in the Dark"
Issue #7, November 2018
Deena Metzger, Lawrie Hartt, Karen Mutter, Eve Sanders, Andrea Mathieson, Lise Weil, Sharon Simone, Kristin Flyntz
VILLAGE MEDICINE
Deena Metzger
Sharon Simone
Fired Anew
Karen Mutter
Lawrie Hartt
At the Stillpoint of Village Medicine
Eve Rachele Sanders
The View from the Ground
Andrea Mathieson
Listening to Bugs
Christine Holland Cummings
How to Go on When It Keeps Getting Darker
Noelle Imparato
Through Darkness Into Light
Kathy Miriam
Women, Water and Berta Caceras
AFTERMATH: 11/9
Praying Amid the Damage: Dreams, Nightmares, Visions
Kristin Flyntz
The Brown Tide
Befriending the Dragon
Emilee Baum
The Demoness
Kathleen Kesson
After•Word Feverish World, 2018-2068
Lise Weil
After•Word Climate: A New Story and The Book of Joan
1981. I moved to a very small house at the end of a dirt road in a rural canyon without, for a time, running water. Alone, and in such a setting, having uprooted a suburban life, I realized I wanted to be a village healer and so I had to constitute a village. Doing so has been an essential part of my life for the last thirty-seven years.
A village is a field of intelligence, inter-dependence and heart. This village formed around the land where I was living. People gathered and healing gifts arose from the fact of the village's existence. Healing gifts, arising from the fact of the village, called the village into being. Village and healing being entirely reciprocal. Coming to the village doesn't mean, however, that one is part of the village. In order for that to occur, one has to offer oneself, has to include oneself. There is always an invitation – but the task is to accept it.
In one form, the village remains centered on this land in Topanga at the edge of a State Park where the wildlife is protected. We began to see it as a Village Sanctuary for a Viable Future for All Beings. But it is also non-local and therefore invisible, but the bonds are no less real. Those who are part of the village may begin to live elsewhere and form other villages. There is no competition. There are only on-going and repeated interconnections.
Because a village is a field, it is relational, multi-dimensional and complex. Likewise, the healing activities, likewise the nature of healing offered and received; we heal in context of each other. Our lives testify to this. The kind of healing that might occur in a medical office can take place in a village, but village healing cannot be duplicated outside a village. In a village, one's body may heal as one's life is healed. Not life style, life. We say community heals and is healed simultaneously.
Sharon Simone, Karen Mutter and Lawrie Hartt are part of the village and have been for many years. We are also a village unto ourselves and they constitute a village among themselves. Village as field is a state of mind, a nexus of relationships, is constituted in the heart. It has many forms and many possibilities.
These pieces on healing, which they have each written, arise from the village, describe village healing and constitute it at the same time. Our own lives healing from the disconnection intrinsic to modern life. We extend ourselves and break down the ultra- hygienic walls of the medical and corporate establishments. Outside, among the eucalyptus trees or in the oak grove, the old ways of being that sustained each person in relationship to all the others and the natural world, as in an ecosystem, flourish.
Deena Metzger has been writing for fifty years. Story is her medicine. Her latest novel, A Rain of Night Birds, a confrontation between indigenous knowledge and the modern scientific mind, bears witness: climate change arises from the same colonial mind that enacted genocide on the Native people of this country. It was published on Earth Day, April 22, 2017. Her other books include the novels La Negra y Blanca (2012 PEN Oakland Josephine Miles Award for Excellence in Literature), Feral; Ruin and Beauty: New and Selected Poems; Doors: A Fiction for Jazz Horn; Entering the Ghost River: Meditations on the Theory and Practice of Healing and Tree: Essays and Pieces.
To comment on this article, please click here.
Copyright © 2014-2022 Dark Matter: Women Witnessing - All rights reserved to individual authors and artists.
Email: Editor@DarkMatterWomenWitnessing.com


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Attendance at evening meetings, three per annum, including an AGM.
Financial control providing quarterly cash analysis detailing all income and expenditure, with the budget forecast for the rest of the year.
A separate account will be held within the Reel Estates Client Account for service charge funds, and will be administered accordingly.


A great appetizer for Mardi Gras.
Everyone has a favorite Thanksgiving leftovers recipe and this is mine.
Keep this on hand to season everything from eggs to lamb, or give as a holiday gift in little jars.


text:
/**
 * EmailController
 *
 * @description :: Server-side logic for managing emails
 * @help        :: See http://links.sailsjs.org/docs/controllers
 */

module.exports = {

};


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Obama's team split on meeting Putin
Tom Parfitt , Moscow, Michael Evans, Catherine Philp and Rhys Blakely, Washington
September 17 2015, 1:01am, The Times
The White House has not rejected the idea of a one-on-one meeting between President Obama and President Putin in New YorkKevin Lamarque/Reuters
President Obama's top team is split on whether he should meet President Putin when the Russian leader attends the UN General Assembly in New York this month.
Granting Mr Putin an audience would be seen as a victory for the Russian leader, who craves a central role in world affairs. At the same time, Mr Obama's policy of isolating Russia has been attacked as weak in Washington.
Mr Putin's annexation of Crimea, his incursions into Ukraine and his support for President Assad of Syria are issues that the US "can't dance around any longer", Hillary Clinton, the favourite to be the Democratic presidential nominee, has said.
Mr Putin is understood to be pushing for the meeting as a means of boosting his domestic image as…


Carlo must address the Goalkeeping situation now!.
After the last two pre season games I have to say I am worried that when it comes to the crunch Hilario and Turnbull are simply not good enough and we should look to bring in someone else!.
Don't get me wrong, Hilario is a good shot stopper and did well at times yesterday, but you have to agree with me, our defence can be anything but confident heading into the new season without Cech there behind them.
This is one area we are lacking and this could really hurt us this season. I know that more often than not Cech will be there between the sticks and touch wood he will be for the season, but if he isn't then we have problems.
Do you guys agree? Who would you look to bring in as a realistic number 2 and rememeber we are looking for someone who is happy to bide their time on the bench and not someone like Joe Hart who wants first team football.


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Ray Jenkins
OK, so everyone gets one free post for an endorsement. Heh. This one is from the Ray Jenkins camp. Ray is running against Billy Stokes and Kristi Davis for the Circuit Court Judge (Div. I) seat in the May 6 Republican primary.
There are no Democrats, so it's winner take all next month. Early voting started Wednesday and runs through May 1.
Representative Harry Brooks (R – Knoxville) today announced his endorsement for Ray Jenkins for Knox County Circuit Court Judge. Brooks says Jenkins's wide breadth of experience with a proven track record of successes qualify him as a strong candidate for the position.
"Ray Jenkins is the best person for the job," said Harry Brooks, four-term state representative and chairman of the House Education Committee. "He is a constitutional conservative with experience as a lawyer, a community leader and a business leader. We have been long-time friends and he has had impressive successes in every position he has held."
In addition to successful stints in business and politics, Jenkins has a wide variety of legal experience, representing all levels of clients from Fortune 500 companies to families and everything in between.
These experiences have prepared Jenkins to be Circuit Court Judge, as these judges have a heavy role in the general jurisdiction of a variety of legal matters.
Jenkins expressed appreciation for Representative Brooks endorsement and said, "The support of Representative Brooks means a great deal coming from a legislator of his caliber. He has done a great job for Knox County and for Tennessee," he said. "I'm excited for the opportunity to serve Knox County and to apply the law to real people in real situations in our community."
A Knox Co. native, Jenkins is a graduate of Tennessee Tech where he earned his bachelor's degree in Computer Science, Jenkins took an analyst position with the Navy in Virginia after graduation, returning to his home state three years later for a job with the Air Force at Arnold Engineering Development Center where he continued to work while attending law school at the Nashville School of Law.
Check out his website, right smack here.
Posted by Mike Donila at 12:32 PM
Labels: Billy Stokes, Elections, Harry Brooks, Kristi Davis, Ray Jenkins


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Hormones, Growth Factors, and Cell Signaling
Expression and Regulation of Estrogen Receptors in Mesangial Cells: Influence on Matrix Metalloproteinase-9
MYLENE POTIER, SHARON J. ELLIOT, IVAN TACK, OLIVER LENZ, GARY E. STRIKER, LILIANE J. STRIKER and MICHAEL KARL
JASN February 2001, 12 (2) 241-251; DOI: https://doi.org/10.1681/ASN.V122241
MYLENE POTIER
SHARON J. ELLIOT
IVAN TACK
OLIVER LENZ
GARY E. STRIKER
LILIANE J. STRIKER
MICHAEL KARL
Figures & Data Supps
Abstract. Diabetic glomerulosclerosis is characterized by the accumulation of extracellular matrix (ECM) in the mesangium. Estrogens seem to retard whereas estrogen deficiency seems to accelerate progressive glomerulosclerosis. Thus, mesangial cells (MC) may be a target for estrogens. Estrogen action is mediated via estrogen receptor (ER) subtypes ERα and ERβ. Both ER subtypes were expressed in human and mouse MC. Using an estrogen-responsive reporter construct in transfection assays, it also was demonstrated that the nuclear ER were transcriptionally active. In the presence of 17β-estradiol (E2; 10-10 to 10-8 M), there was a progressive increase in the mRNA levels of both ERα (approximately 1.8-fold and approximately 2.7-fold after 24 and 72 h, respectively) and ERβ (approximately 1.3-fold and approximately 2.2-fold after 24 and 72 h, respectively). ERα protein levels increased approximately 2.5-fold after 24 h (10-10 M, E2) and up to approximately 5.4-fold after 72 h (10-9 M, E2). ERβ protein levels increased approximately 2.1-fold in the presence of E2 (10-9 M) after 24 h. Thus, estrogens positively regulate the expression of the ER subtypes, thereby maintaining or increasing MC responsiveness to estrogens. Because diabetic glomerulosclerosis may be due partly to a decrease in ECM degradation, the effects of estrogens on matrix metalloproteinases (MMP) were studied. It was found that E2 (10-10 to 10-8 M) increased both MMP-9 mRNA and MMP-9 activity in MC. This may be an important mechanism by which estrogens influence ECM turnover and protect against progression of diabetic glomerulosclerosis.
The accumulation of extracellular matrix (ECM) in the mesangium in diabetic glomerulosclerosis may result from an imbalance between ECM synthesis and ECM degradation (1). Mesangial cells (MC) play a central role in this process, because they synthesize and secrete both the structural components of the ECM and the enzymes that degrade them (2). Several lines of evidence suggest that there is a decrease in ECM degradation in diabetic nephropathy and that this may be due to decreased matrix metalloproteinase (MMP) synthesis and secretion by MC (3). Thus MC provide a potential therapeutic target to modify progression in diabetic glomerulosclerosis.
The United States Renal Data System data reveal that women in the premenopausal age range have a lower incidence of end-stage renal disease (ESRD) caused by diabetic nephropathy than do men (4). After menopause, there is a sharp increase in the incidence of ESRD caused by diabetic nephropathy, especially in women from ethnic minority groups (4). For instance, the women:men ESRD incident ratio nearly doubles in African Americans (0.8 in the 35- to 39-yr age group compared with 1.5 in the 65- to 69-yr cohort). However, the female preponderance in ESRD caused by diabetic nephropathy after menopause cannot be explained by an increased incidence of diabetes in postmenopausal women compared with age-matched men. A large prospective study that examined the risk to develop type 2 diabetes mellitus in African American and Caucasian adults aged 45 to 64 yr over a 9-yr period showed a nearly equal incidence per 1000 person-years of 25.1 in African American women versus 23.4 in African American men (female:male ratio, 1.07) (5). In Caucasian study participants, there was even a male predominance in developing type 2 diabetes mellitus with a 0.67 female:male incident ratio. Thus, although other mechanisms may be invoked, estrogen status may play an important role in the protection or potentiation of progressive glomerulosclerosis. In fact, estrogens have been shown to inhibit transforming growth factor-β—mediated type IV collagen and to suppress type I collagen expression via activation of activator protein-1 (AP-1) in MC (6,7). Thus, estrogens could decrease the synthesis of ECM components, resulting in decreased accumulation of ECM in the glomerulus. Estrogens could also decrease ECM accumulation by increasing degradation of ECM by MMP. However, this has not been studied in MC.
There are two estrogen receptor (ER) subtypes, ERα and ERβ, that belong to the superfamily of nuclear receptors (8,9,10,11), which mediate the effects of estrogens. Interestingly, both ER subtypes α and β, as well as ER splice variants, have been previously identified in vascular smooth muscle cells including those from the aorta and coronary vessels (12,13,14,15,16,17,18), where the direct actions of estrogens contribute substantially to their cardiovascular protective effects (19). ER subtypes and their regulation and biologic effects have not been reported in MC. Thus, in this study, we investigated ER expression and regulation. We also studied the effect of estrogens on MMP expression and activity in MC. MC expressed both ER subtypes ERα and ERβ, and estrogens regulated their expression. Estrogens and anti-estrogens also regulated the expression of the MMP-9 in MC. These findings may have important pathophysiologic implications for sclerosing glomerular diseases, such as diabetic nephropathy.
Culture media, supplements, and primers were obtained from GIBCO Life Technologies BRL (Grand Island, NY). Charcoalstripped fetal bovine serum (FBS) was purchased from Hyclone (Pittsburgh, PA). 17β-estradiol (E2), tamoxifen, anti—α-smooth muscle actin, and β-galactosidase substrate were purchased from Sigma (St. Louis, MO). ICI 182,780 (ICI) was obtained from Tocris (Ballwin, MO). First Strand cDNA Synthesis Kit for reverse transcription-PCR (RT-PCR) (AMV) and Taq polymerase were purchased from Boehringer Mannheim (Indianapolis, IN). For Western analysis, protein concentrations were measured by using the Pierce BCA Assay from Bio-Rad Laboratories (Hercules, CA). Prestained molecular weight markers were purchased from Bio-Rad Laboratories. ER antibodies and their respective blocking peptides were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The ERα antibody MC-20 is a rabbit polyclonal antibody raised against a peptide mapping at the carboxy terminus of the ERα of mouse origin. The ERα antibody H-184 is a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acid 2 to 185 mapping at the amino terminus of ERα of human origin. The two ERα antibodies recognize both mouse and human ERα protein. N-19, the antibody against human ERα, is a goat polyclonal antibody raised against a peptide mapping at the amino terminus of the ERβ of human origin. Y-19, the antibody against mouse ERβ, is a goat polyclonal antibody raised against a peptide mapping at the amino terminus of the ERβ of mouse origin. Protein A-Agarose and chemiluminescence detection system were purchased from Santa Cruz Biotechnology. ERα and ERβ human recombinant peptide were purchased from Panvera (Madison, WI). Nitrocellulose membranes (Hybond ECL) and films (Hyperfilm ECL) for chemiluminescence detection were purchased from Amersham Pharmacia Biotech (Buckinghamshire, England). For the transfection studies, TransFast and lysis buffer were purchased from Promega (Madison, WI). Zymography gels were purchased from NOVEX (San Diego, CA).
Human MC were obtained from microdissected glomeruli of kidneys not suitable for transplantation. Mesangial-like glomerular outgrowths were patch-cloned, propagated, and grown in Waymouth's medium supplemented with 20% FBS, 1 mM L-glutamine, 100 μg/ml penicillin/streptomycin, and 0.075% Na2HCO3. Human MC were characterized by their stellate morphology and staining pattern, including positivity for α-smooth muscle actin (20). Experiments were performed using cells between passages 3 and 6.
MC from C57BL/6J female mice used for these experiments have been previously described (21). Cells were used between passages 19 and 26. Previous experiments have shown that the cells retain their phenotype at the passages studied. Murine MC were routinely cultured in Dulbecco's modified Eagle's medium/F12 (3/1 vol/vol), supplemented with 20% FBS.
To characterize initially the presence of ER in MC, we performed experiments on human and murine MC maintained in culture medium supplemented with 20% FBS. For the purpose of examining ER regulation, cells were transferred into phenol-red free medium supplemented with charcoal-stripped FBS, because a lipophilic impurity contained in the phenol red has been described as a weak estrogen agonist (22).
Cells were cultured in phenol red-free medium supplemented with 20% charcoal-stripped FBS in the presence of increasing concentrations of E2 (10-10 M to 10-8 M) for 5 d. Cell number was evaluated every 2 d with a Coulter cell counter (Hialeah, FL) and was not affected by the presence of E2 (data not shown).
To examine the regulation of ER mRNA and protein in the absence of estrogens and growth factors, we plated cells in six-well plates and maintained them in phenol red-free medium supplemented with 20% charcoal-stripped FBS. When cells were plated, cell number was adjusted according to the different conditions to obtain a similar high cell density at collection (day 1 and day 3).
To assess the functionality of endogenous ER, we performed transfection experiments. For this purpose, cells were plated, as described previously, and medium was replaced by phenol red-free medium supplemented with 0.1% charcoal-stripped FBS.
To assess the regulation of ER mRNA and protein expression, as well as the regulation of MMP-9 expression and activity by E2, we grew mouse MC initially for 3 d in phenol red-free medium supplemented with 20% charcoal-stripped FBS in six-well plates. The medium was replaced with phenol red-free medium supplemented with 0.1% charcoal-stripped FBS and increasing concentrations of E2 (10-10 M to 10-8 M) for 24 and 72 h. Confluent cells were harvested for RNA and/or protein collection while the supernatants were used to measure MMP-9 activity. All experiments were performed in triplicate.
Isolation of mRNA and RT-PCR
Total RNA was extracted from confluent cell cultures using the guanidium thiocyanate-phenol-chloroform method (Tri-Reagent) (23). RT was performed on 2 μg of total RNA in a total volume of 20 μl. After the total volume was adjusted to 100 μl with diethyl pyrocarbonate water, 2 μl of the cDNA solution was used as a template for the PCR. PCR amplifications were performed in a total volume of 50 μl with 1.5 U of Taq polymerase. The specificity of each reaction was monitored in control reactions, where amplifications were carried out on samples after omission of RT. Amplifications of human ER subtypes α and β in human MC were performed using specific primer pairs previously described by Enmark et al. (10), which resulted in amplicons of 344 bp and 392 bp length, respectively.
To investigate whether murine MC express ERα variants, we used a series of primers to amplify several overlapping segments spanning the entire coding regions of the murine wild-type ERα cDNA, as described previously (24). The forward primers were located in exons 1, 2, 4, and 6, whereas the reverse primers were located in exons 5, 7, and 8.
To assess ERα mRNA expression, we amplified a 408-bp cDNA fragment of the mouse ERα using forward primer TCCTAACTTGCTCCTGGACAGG located at nucleotides 1417 to 1438 and reverse primer CAGGAGCAGGTCATAGAGGGG at nucleotides 1825 to 1805 (nucleotides are numbered according to the sequence published by White et al. (9)). Restriction enzyme analysis was used to confirm the correct sequence of the amplicons (data not shown). To amplify a specific 409-bp amplicon of mouse ERβ, we used to primers located at nucleotide positions 142 to 163 and 551 to 529. Nested PCR was used to confirm the correct sequence (25,26). MMP-9 and glyceraldehyde phosphate dehydrogenase (GAPDH) primer pairs were used as described previously (3), which resulted in PCR products of 414 bp and 561 bp length, respectively. PCR products were separated on 2% agarose gels containing 0.05% ethidium bromide gels and were photographed using an Alpha Innotech Digital Imaging System (San Leandro, CA). Analysis was performed using computer-aided densitometry (NIH image, NCBI, NIH, Bethesda, MD). To determine the PCR assay range, we plotted the number of PCR cycles against the integrated density obtained from the densitometry analysis. GAPDH was used as an internal standard and housekeeping gene. PCR data obtained for ERα, ERβ, and MMP-9 were normalized to GAPDH signals as described previously (3). The samples from at least three different experiments were run in duplicate.
Western Blots
Confluent cell layers were washed once in phosphate-buffered saline (PBS) and collected in the presence of lysis buffer. Cell homogenates were centrifuged 30 min at 15,000 × g at 4°C. Supernatants were collected and protein concentrations were measured. For ERβ protein analysis, samples were immunoprecipitated using the human or mouse ERβ antisera, respectively. Briefly, 150 μg of protein was incubated overnight with the antiserum and protein A-agarose. The immunoprecipitates were washed four times in PBS and resuspended in 40 μl of PBS. To analyze ERα protein, we processed protein homogenates without previous immunoprecipitation. All samples were then diluted in Laemmli buffer and boiled. Equal amounts of protein or immunoprecipitates from each experimental condition were run on a 10% polyacrylamide gel. Prestained markers were electrophoresed in parallel to estimate molecular weight. Electrotransfer of proteins from the gel to the nitrocellulose was performed by electroelution (27). Immunoblotting was performed with either human or mouse ERα and ERβ antisera, and immunoreactive bands were determined by exposing the nitrocellulose blots to a chemiluminescence solution followed by exposure to a Hyperfilm ECL film. Control experiments were performed in the presence of ERα and ERβ human recombinant peptides as positive controls, whereas specificity of the signal was demonstrated by incubating blots with an excess of the corresponding specific immunizing peptide.
Transfection and Luciferase Assays
Before transfection, mouse MC were transferred into 24-well plates and cultured 4 d in phenol-red free medium supplemented with 20% charcoal-stripped FBS. Subsequently, MC were transfected with the reporter construct, pVitA2-ERE-TKLuc (0.25 μg/well) using Trans-Fast, according to the manufacturer's recommendations. VitA2-ERE-TKLuc contains one copy of the Xenopus vitellogenin estrogen responsive element (ERE) proximal to the thymidine kinase promoter, which drives the expression of the luciferase reporter gene in an estrogen-dependent manner. The vector pTKLuc, which does not contain an ERE, served as a control. To adjust for transfection efficiency, MC were cotransfected with pRSV-βgal (0.25 μg/well), a vector that constitutively expresses the β-galactosidase gene. The constructs were a generous gift from Drs. G. Tremblay and V. Giguere (11). One h later, phenol red-free medium supplemented with 10% charcoal-stripped FBS was added to the transfected cells. Cells were incubated for an additional 24 h in the presence of 10-8 M E2 or vehicle (ethanol). The final ethanol concentration was 0.001% in both conditions. For luciferase and galactosidase assays, cells were lysed in 100 μl of Reporter Lysis buffer at room temperature. Light emission was detected using a luminometer (AutoLumat, Wallac, Gaithersburg, MD) after addition of luciferin to 40 μl of cell lysate. Values were expressed as arbitrary light units normalized to the β-galactosidase activity of each sample.
MMP-9 Activity
The cell supernatants were collected 24 and 72 h after treatment. At the time of medium collection, the cells were counted for the purpose of adjusting the volume of the medium to the cell number. MMP-9 activity was assessed using 10% zymogram gels as described previously (28). Briefly, the medium was diluted to normalize for cell number (approximately 25,000 cells/ml), before the addition of 5X Laemmli buffer under nonreducing conditions. After electrophoresis, gels were washed for 1 h in 2.5% Triton X-100 and incubated 40 h in 50 mM Tris buffer. The gels were stained with Coomassie Blue and air-dried. Densitometry, using NIH image 1.6, was used to analyze relative MMP-9 activity.
All experiments were performed at least in triplicate. Data are expressed as percentage of control. Shown are means ± SEM of three or four independent experiments. One-way ANOVA and Dunnett's multiple comparison post hoc test were performed. For transfection experiments, values are expressed as arbitrary light units, normalized to the β-galactosidase activity of each sample.
Expression of ERα and ERβ in Human MC
Total RNA and protein were extracted from MC between passages 3 and 6. Using RT-PCR, we detected both ERα and ERβ transcripts (shown are representative 344-bp and 392-bp amplicons of human ERα and ERβ mRNA; Figure 1A). The expression of ERα and ERβ protein was studied by Western blot analysis. Using ERα and ERβ antisera, we detected signals at approximately 66 and approximately 53 kD, respectively (Figure 1B). The estimated molecular weight of these bands corresponds to the size predicted for the wild-type human ERα and ERβ (8,10). Recombinant human ERα and ERβ peptides served as positive controls (lanes 1 and 4). Preincubation of the ER antisera with their respective immunizing peptides completely abrogated these signals, confirming that the detected bands were ERα and ERβ, respectively (data not shown). Thus, human MC express both ER subtypes ERα and ERβ.
Human mesangial cells (MC) express estrogen receptor (ER). Total RNA and protein were collected from human MC grown in Waymouth's medium supplemented with 20% fetal bovine serum (FBS). (A) ERα and ERβ transcripts were analyzed by reverse transcription-PCR (RT-PCR) using specific primer sequences. Representative 344-bp amplicons of ERα (lane 4) and 392-bp amplicons of ERβ (lane 8), positive controls (lanes 3 and 7), minus (-)RT reaction (lanes 2 and 6), and molecular weight standards (lanes 1 and 5) were run in parallel. (B) Ten μg (lane 2) and 20 μg (lane 3) of human MC homogenates were analyzed by Western blotting using the ERα MC-20 antiserum (left). Ten (lane 5) and 25 μl (lane 6) of ERβ immunoprecipitates from human MC homogenates were analyzed by Western blotting using ERβ N-19 antiserum (right). The 66-kD human recombinant ERα peptide (lane 1, 5 ng) and the 53-kD human recombinant ERβ peptide (lane 4, 0.5 μg) served as positive controls.
Expression of ERα and ERβ in Mouse MC
The expression of ER was examined by RT-PCR and Western blot analysis in MC isolated from the glomeruli of C57BL/6J female mice. Representative amplicons of 408 bp and 409 bp length corresponding to mouse ERα and ERβ transcripts (9,11), respectively, are shown (Figure 2A). To exclude the presence of ERα variants, which originate from alternatively spliced ER transcripts, we amplified the entire coding region of ERα using specific primer pairs. Because all of the PCR products were of the predicted sizes, we concluded that there was no evidence of the expression of alternatively spliced mouse ERα mRNA transcripts (data not shown). The expression of mouse ERα (approximately 67 kD) and ERβ (approximately 55 kD) protein was confirmed by Western blot analysis (Figure 2B). The corresponding signals were abrogated by preincubation with the respective immunizing peptide (data not shown). Human recombinant ERα (66 kD) and ERβ (53 kD) peptides served as positive controls. In summary, human and mouse MC expressed both ER subtypes at the mRNA and protein level.
ER expression in mouse MC. MC isolated from C57BL/6J mouse glomeruli were grown in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) supplemented with 20% FBS. (A) ERα (left) and ERβ (right) transcripts were analyzed by RT-PCR; representative amplicons (ERα, 408 bp; ERβ, 409 bp) are shown. (B) Mouse MC homogenates or immunoprecipitates were analyzed by Western blotting using the ERα MC-20 antiserum (left) and the ERβ Y-19 antiserum (right), respectively. Twenty μg and 10 μg of mouse MC homogenates were loaded in lanes 3 and 4, respectively. In lanes 7 and 8, 25 μl of ERβ immunoprecipitates from mouse MC homogenates were loaded. The 66-kD human recombinant ERα peptide (lane 1, 10 ng; lane 2, 5 ng) and the 53-kD human recombinant ERβ peptide (lane 5, 1 μg; lane 6: 0.5 μg) served as positive controls. Signals that corresponded to the molecular weight of the wild-type mouse ERα of approximately 67 kD (lanes 3 and 4) and the ERβ of approximately 55 kD (lanes 7 and 8) were detected in the immunoblots.
Transcriptional Activity of the ER in Mouse MC
To test the ability of endogenous ER to modulate the transcriptional activity of an ERE-containing promoter, we transfected mouse MC with the reporter construct pVitA2-ERE-TKLuc (a generous gift from Drs. Tremblay and Giguere, Montreal, Canada) (11). In the transfected MC, E2 (10-8 M) induced approximately a twofold increase in luciferase activity (Figure 3). This demonstrates that the endogenous ER maintain their function as ligand-regulated transcription factors in MC.
Transcriptional activity of MC ER. Mouse MC were grown in phenol red-free DMEM/F12 supplemented with 20% charcoalstripped FBS for 4 d. Transfection was performed as described in the Materials and Methods section. After transfection, cells were treated with vehicle or 10-8 M 17β-estradiol (E2) for 24 h in phenol red-free medium containing 20% charcoal stripped FBS. Data are expressed in relative luciferase units. Shown is a representative graph of two independent experiments performed in triplicate.
Regulation of ERα and ERβ mRNA Expression by Culture Conditions in Mouse MC
Estrogens previously have been shown to regulate the levels of both ER subtype mRNA in reproductive tissues (29,30,31); however, little is known about the regulation of ER in vascular smooth muscle cells. We investigated whether E2 modulates ERα and ERβ mRNA in MC. Mouse MC were transferred into phenol red-free medium supplemented with 20% charcoalstripped FBS to minimize the concentrations of compounds that may activate ER (32). After 1 or 3 d in this medium, levels of ERα and ERβ mRNA changed but not in a coordinate manner (ER mRNA were normalized to GAPDH mRNA levels) (Figure 4A). Namely, there was a decrease in ERα mRNA levels, whereas ERβ mRNA levels increased. However, the changes were not of the same magnitude. ERα mRNA decreased to a level of 46.9 ± 8.1% (P < 0.01) after 1 d, and this decrease persisted until day 3. The ERβ mRNA levels were increased at day 1 and continued to increase to reach a maximum at day 3 (268.1 ± 41.3%; P < 0.01; Figure 4B).
Culture conditions change ERα and ERβ mRNA expression. Mouse MC were grown for 1 and 3 d in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. (A) ERα, ERβ, and glyceraldehyde phosphate dehydrogenase (GAPDH) transcripts were analyzed by RT-PCR. Representative amplicons of ERα, ERβ, and GAPDH at day 0 (lanes 3 and 4), day 1 (lanes 5 and 6), and day 3 (lanes 7 and 8) are shown. Molecular weight standards (lane 1) and minus (-)RT reaction (lane 2) were run in parallel. (B) ERα and ERβ mRNA expression, normalized to GAPDH. Data are expressed as percentage of day 0. Shown are means ± SEM of three independent experiments. Statistical significance is indicated by ** (P < 0.01) when compared with baseline (day 0).
In summary, these studies demonstrate that when MC were transferred from regular medium into phenol red-free medium supplemented with charcoal-stripped FBS, ERα and ERβ mRNA levels changed in opposite directions.
Regulation of ERα and ERβ Protein Expression by Culture Conditions in Mouse MC
Estrogens have been shown to accelerate ER protein turnover in a pituitary lactotrope cell line by a proteasome-mediated process, which in turn affects ER protein levels (33). There was approximately a 1.6-fold increase in ERα protein (Figure 5A) and approximately a 3.5-fold increase in ERβ protein levels (Figure 5B) in MC after 3 d of culture in phenol red-free medium supplemented with 20% charcoal-stripped FBS. Thus, the levels of ERα protein and the levels of ERα mRNA are discordant in MC under these culture conditions. Namely, ERα mRNA decreased and ERα protein increased, whereas both ERβ mRNA and ERβ protein increased.
Culture conditions change ERα and ERβ protein expression. Mouse MC were grown for 3 d in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. (A) Mouse MC homogenates collected at 0 (lanes 1 and 2 and lanes 5 and 6) and 3 d (lanes 3 and 4 and lanes 7 and 8) were analyzed by Western blotting using ERα (left) and ERβ (right) MC-20 and Y-19 antisera, respectively. Twenty (lanes 1 and 3) and 30 μg (lanes 2 and 4) of mouse MC homogenates were loaded. In lanes 5 to 8, 25 μ1 of ERβ immunoprecipitates from mouse MC homogenates were loaded. (B) ERα (left) and ERβ (right) protein levels are expressed as percentage of day 0. Shown are means ± SEM of three independent experiments. For ERα, the results shown are from three independent experiments performed with each antiserum, ERα MC-20 and H-184. Statistical significance is indicated by ** and *** (P < 0.01 and P < 0.001, respectively) for comparison with day 0.
Regulation of ERα and ERβ mRNA Expression by Estrogens in Mouse MC
After 3 d in culture, E2 (10-10 to 10-8 M) was added. There was a progressive increase in both ERα and ERβ mRNA levels. ERα mRNA levels were approximately 1.8-fold higher (183.8 ± 27.0%) compared with baseline conditions after 24 h and increased to approximately 2.7-fold (276.4 ± 43.0%; P < 0.01) after 72 h in the presence of 10-9 M E2 (Figure 6A).
E2 increases ERα and ERβ mRNA expression. Mouse MC were grown for 3 d in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. Medium was replaced with phenol red-free DMEM/F12 medium supplemented with 0.1% charcoal-stripped FBS containing E2 (10-10 to 10-8 M) for 24 and 72 h. ERα (A) and ERβ (B) mRNA expression (normalized to GAPDH) in the presence of E2 for 24 h (▪) and 72 h (□). Data are expressed as percentage of control (V, vehicle = 0.001% EtOH) at 24 h, and shown are means ± SEM of three independent experiments. Statistical significance is indicated by ** (P < 0.01) and * (P < 0.05) for comparison with 24-h baseline (V, ▪) and by ▿▿ (P < 0.01) and ▿ (P < 0.05), for comparison with 72-h baseline (V, □).
The increase in ERβ mRNA levels peaked at 10-9 M E2. The ERβ mRNA levels were approximately 1.3-fold higher (127.4 ± 14.5%) at 24 h, and there was approximately a 2.1-fold (211.8 ± 50.0%; P < 0.05) increase at 72 h (Figure 6B).
In summary, in the presence of physiologic concentrations of E2, the mRNA levels of both ER subtypes increased in MC cultured in phenol red-free medium supplemented with charcoal-stripped serum.
Regulation of ERα and ERβ Protein Expression by Estrogens in Mouse MC
E2 (10-10 to 10-8 M) was added to MC after 3 d of culture in phenol red-free medium supplemented with 20% charcoalstripped FBS. There was an increase in the protein level of both ER subtypes. The maximal increase in ERα protein level (approximately 5.4-fold) was seen at 10-9 M E2 (P < 0.05) at 72 h (Figure 7A). ERβ protein levels increased approximately 2.1-fold in the presence of 10-9 M of E2 (P < 0.05) after 24 h (Figure 7B). It is interesting to note that the ERβ protein levels after 72 h were 2.3-fold higher than in the cells at 24 h.
E2 increases ERα and ERβ protein expression. Mouse MC were grown for 3 d in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. The medium was replaced with phenol red-free DMEM/F12 medium supplemented with 0.1% charcoal-stripped FBS and E2 (10-10 to 10-8 M) for 24 and 72 h. ERα (A) and ERβ (B) protein expression in the presence of E2 for 24 h (▪) and 72 h (□). Data are expressed as percentage of control (V = 0.001% EtOH) at 24 h, and shown are means ± SEM of three independent experiments. For ERα, the results shown are from three independent experiments performed with each antiserum, ERα MC-20 and H-184. Statistical significance is indicated by * (P < 0.05) for comparison with 24-h baseline (V, ▪) and by ▿ (P < 0.05), for comparison with 72-h baseline (V, □).
In summary, this demonstrates that estrogens affect the ER protein levels in MC and may maintain or even increase estrogen responsiveness in this cell type.
MMP-9 mRNA Expression and MMP-9 Activity by Estrogens and Antiestrogens in Mouse MC
The levels of MMP-9 mRNA increased after 24 h in the presence of E2 (10-10 to 10-8 M) in cells that had been cultured 3 d in phenol red-free medium supplemented with 20% charcoal-stripped FBS. The maximal MMP-9 mRNA increase was approximately 1.7-fold (175.4 ± 25.0% of control; P < 0.01) in the presence of 10-8 M E2 after 24 h (MMP-9 mRNA was normalized to GAPDH mRNA). No changes in MMP-9 mRNA levels were observed after 72 h (Figure 8A). MMP-9 activity also changed markedly after treatment with E2. There was approximately a 3.3-fold increase (328.4 ± 73.4% of control; P < 0.05) in MMP-9 activity after 24 h of treatment with 10-8 M E2 (Figure 8B). After 72 h, there was approximately a 2.4-fold increase (238.3 ± 37.5% of control; P < 0.05) in MMP-9 activity in the presence of 10-8 M E2 (data not shown). These changes were abolished in the presence of the selective estrogen receptor modulator tamoxifen or the ER antagonist ICI 182,780 (Figure 9). Tamoxifen (10-6 M) and ICI (10-6 M) blocked the E2-induced increase in MMP-9 activity in the mouse MC (60.1 ± 15.4% and 59.9 ± 10.4% of control, respectively) during a 24-h incubation period. Neither of these inhibitors significantly affected baseline MMP-9 activity (126.7 ± 27.45% and 75.7 ± 26.7%, respectively). These studies demonstrate that E2 upregulates MMP-9 expression in mouse MC.
E2 increases matrix metalloproteinase-9 (MMP-9) mRNA expression and activity. Mouse MC were grown for 3 d in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. Medium was replaced with phenol red-free DMEM/F12 medium supplemented with 0.1% charcoal-stripped FBS and E2 (10-10 to 10-8 M) for 24 and 72 h. (A) MMP-9 mRNA expression (normalized to GAPDH) in the presence of E2 for 24 h (▪) and 72 h (□). (B) MMP-9 activity as assessed by zymography 24 h after E2 addition. Data are expressed as percentage of control (V = 0.001% EtOH) at 24 h. Shown are means ± SEM of three independent experiments. Statistical significance is indicated by ** (P < 0.01) and * (P < 0.05) for comparison with 24-h baseline (V, ▪) and by ▿ (P < 0.05), for comparison with 72-h baseline (V, □).
E2 increases MMP-9 activity: An effect abolished by ER antagonists. Mouse MC were grown for 3 d in phenol red-free DMEM/F12 supplemented with 20% charcoal-stripped FBS. Medium was replaced with phenol red-free DMEM/F12 medium supplemented with 0.1% charcoal-stripped FBS with vehicle, 10-8 M E2, 10-6 M tamoxifen (T) alone or in combination with 10-8 M E2, 10-6 M ICI alone or in combination with 10-8 M E2 for 24 h. When T and ICI were used in combination with E2, cells were incubated for 1 h with the antagonists before the addition of E2. (A) A representative zymography assessing MMP-9 activity in the presence of E2 with or without ER antagonists for 24 h (M, markers). (B) Data are expressed as percentage of control (V = 0.001% EtOH). Shown are means ± SEM of three independent experiments. Statistical significance is indicated by ** (P < 0.01) for the comparison, E2 versus V and by ▿▿ (P < 0.01), for comparisons E2 versus T+E2 or ICI+E2.
MC play a central role in the progression of diabetic nephropathy (1,2). Estrogen deficiency in postmenopausal women is associated with a relative increase in the female:male ratio of ESRD caused by diabetic nephropathy (4). This may be due to the effects of estrogens on ECM turnover in the glomerulus, because MC synthesize both ECM components and MMP (2). Thus, estrogen replacement may halt the progression of diabetic glomerulosclerosis in states of estrogen deficiency, i.e., postmenopausal women. The addition of estrogens led to decreased type I collagen synthesis and reduced transforming growth factor-β1—mediated type IV collagen synthesis in mouse MC isolated from male mice (6,7). However, the expression of ER and their subtypes in MC, and their regulation by estrogens, remained to be elucidated. Furthermore, the effects of estrogens on MMP, which play a crucial role in ECM degradation, had not been previously investigated.
In this study, we identified the two known nuclear ER subtypes, ERα and ERβ, in both human and mouse MC (8,9,10,11). We detected the expression of the two ER subtypes at the mRNA level by RT-PCR and confirmed translation into their respective proteins by Western blot analysis. Using an estrogen-responsive reporter construct, we demonstrated further that the nuclear ER were transcriptionally active in MC. Thus, MC expressed functional ER and were able to respond to stimulation by estrogens. This also suggested that MC represent another nonreproductive target cell for estrogens. Furthermore, MC, similar to the findings in arterial vascular smooth muscle cells, expressed both ER subtypes ERα and ERβ (12,13,14,15,16,17,18). It is interesting to note that in experiments with ERα or ERβ knockout mice, the expression of one of the ER subtypes provided protection in a model of arterial vascular injury (34,35). This suggested some degree of redundancy in ERα and ERβ function in arterial vascular smooth muscle cells.
Nonetheless, this functional redundancy may be restricted to specific vascular sites and may not apply to MC.
The modulation of ER by estrogens has been studied extensively in reproductive organs and breast cancer cells. Estrogens either up- or downregulated ER expression, depending on the cell type or cell line (29,31,36). There have been no reports on the regulation of ER subtype expression in MC, and little is known about ER regulation in vascular smooth muscle cells from other vascular beds.
We found that cell culture conditions have a substantial impact on ER levels. The culture conditions that we chose consisted of phenol red-free medium supplemented with charcoal-stripped FBS, a condition generally accepted for studying steroid hormone effects. Phenol red-free medium was selected because phenol red supplements may contain lipophilic impurities, which have weak estrogen agonist activity (22). Charcoal treatment removes steroid hormones and numerous other substances, including growth factors (32,37). After transfer of MC into phenol red-free medium supplemented with charcoal-stripped FBS, there were changes in the ER subtype mRNA levels. ERα mRNA levels decreased after 1 d, and this decrease persisted until day 3, whereas ERβ mRNA increased and reached a maximum after 72 h. Thus, charcoal treatment had discordant effects on the regulation of ER subtype transcription. This observation also suggested that the regulatory regions of the genes for the two ER subtypes may differ in their organization, given that the transcription of the ER subtypes was either decreased (ERα) or increased (ERβ) when phenol red was removed from the basal medium in the presence of charcoal-stripped serum. After incubation in E2-containing medium for 24 or 72 h, we observed an increase in mRNA and protein levels of both ER subtypes at physiologic estrogen concentrations. Thus, the increase in ER protein synthesis seems to be only partially offset by accelerated ER protein turnover, a proteolytic process that occurs rapidly in the presence of E2 (33). These findings provide evidence that the levels of ER protein are sustained or increased in MC in the presence of estrogens. Thus, lack of estrogen for an extended time period, as in menopause, may decrease the capacity to mount an estrogen response. It should be noted that the MC used in these experiments were isolated from young, thus "premenopausal," female C57BL/6J mice (21).
The increased incidence of ESRD caused by diabetic nephropathy in postmenopausal women from ethnic minority groups suggests that the accumulation of ECM is accelerated in estrogen deficiency states (4). Because MMP play an important role in ECM turnover, they may be an important mesangial target gene for estrogens (3). Increased MMP levels contribute to ECM degradation, which could have a protective role in progressive glomerulosclerosis. MMP-2 and MMP-9 belong to a subgroup of matrix-degrading enzymes that exhibit high activity against gelatin and native type IV collagen (3,38). In the present study, we focused on MMP-9. We found that E2 induced MMP-9 mRNA expression and activity in a dose-dependent manner. Tamoxifen, a selective estrogen receptor modulator, and the anti-estrogen ICI 182,780 blocked estrogen-induced MMP-9 activity, suggesting that these effects were ER mediated. The molecular mechanisms by which estrogens regulate MMP-9 transcription in MC are unknown. The MMP-9 promoter does not have a consensus ERE but contains several other important regulatory elements, including three GC boxes, four AP-1 like binding sites, an AP-2 site, and three PEA3 consensus sequences, as well as a nuclear factor-κB (NF-κB) binding site (39). Several of these transcription factors, including the AP-1 complex or NF-κB, have been shown to interact with ER (40). Importantly, at AP-1 binding sites, the two ER subtypes ERα and ERβ signal in opposite directions upon ligation with E2 in vitro (41). Whereas E2 stimulation of ERα increases the transcriptional process, activation of ERβ by its natural ligand downregulates gene transcription at an AP-1 site. The expression of both ER subtypes and the potential of ERα and ERβ to form heterodimers, combined with the presence of four AP-1 binding sites in the 5′-flanking region, add several layers of complexity to the regulation of MMP-9 expression (39,42). In addition, ER have also been shown to interact with NF-κB, a transcription factor that is important in inflammatory processes (43). The complexity of the MMP promoter region may allow precise regulation of MMP-9 expression.
The present data suggest that one mechanism, by which estrogens contribute to the protection from ESRD caused by diabetic nephropathy in premenopausal women, is increased MMP-9 expression in MC. In addition, the data may inject a note of caution in the use of tamoxifen in postmenopausal women with diabetic glomerulosclerosis, because tamoxifen blocked upregulation of MMP-9 expression by estrogens.
In summary, we found that both ER subtypes were expressed in MC and that estrogens positively regulated their transcription and translation. Thus, estrogens maintain or increase the estrogen responsiveness in MC. We found that estrogens upregulated MMP-9 expression and activity in MC. This may be an important mechanism by which estrogens influence ECM turnover and exert their protective effect on the progression of diabetic glomerulosclerosis.
We thank Dr. G. Tremblay and Dr. V. Giguere for the reporter constructs pVitA2-ERE-TKLuc, pTK-Luc, and pRSV-βgal. We are grateful to Christina Romanach for technical assistance.
This work was supported by a grant from the National Institutes of Health (NIH/NIA R01, AG17170-01 to L.J.S.).
M.K. is a recipient of a Career Development Award of the American Diabetes Association (ADA). M.P. is a recipient of a Postdoctoral Fellowship of the American Heart Association (AHA).
© 2001 American Society of Nephrology
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North Carolina Central Athletics | December 21, 2013
NCCU hires Mack as head coach
DURHAM, N.C. -- With 10 years of coaching experience, including stints with five NCAA Division I programs and two conference championship teams, Jerry Mack was announced as North Carolina Central's 22nd head coach on Thursday during a press conference in the Alfonso Elder Student Union on the campus of NCCU.
A native of Memphis, Tenn., Mack, 33, becomes the third-youngest active Division I head football coach. Only Paul Nichols of Davidson (born Jan. 25, 1981) and P.J. Fleck of Western Michigan (born Nov. 29, 1980) are younger than Mack (born Oct. 24, 1980).
Mack comes to NCCU after spending the past two seasons with the South Alabama as wide receivers coach. During that time, three of his receivers finished among the top five on South Alabama's single-season record lists for receptions and receiving yards.
He spent the 2011 campaign in his hometown with Memphis, also as wide receivers coach, where he tutored the receiver with the second-highest single-season receptions in school history.
As offensive coordinator and quarterbacks coach at Arkansas-Pine Bluff in 2010, Mack transformed a unit that ranked 101st in the nation in total offense to 30th nationally and No. 2 in the Southwestern Athletic Conference (SWAC), breaking 10 offensive school records in the process.
Mack served as passing game coordinator and wide receivers coach at Central Arkansas during the 2008-09 seasons, helping to lead the 10-2 Bears to the Southland Conference championship and placing 10th in the FCS in total offense.
In Mack's two seasons (2006-07) as wide receivers and tight ends coach at Jackson State, the Tigers ranked second in the SWAC in scoring offense both years and captured the SWAC championship title in 2007.
Mack started his coaching career as an offensive graduate assistant at Delta State in 2004-05. During that time, he coached running backs, and worked as assistant special teams coordinator and video coordinator, helping the Statesmen to rank third in the NCAA Division II in scoring offense and total offense.
Mack began his collegiate playing career at Jackson State before transferring after one season (1999) to Arkansas State. He lettered three years at Arkansas State (2001-03) before earning his bachelor's degree in management information systems in 2003.
Mack completed his master's degree in physical education from Delta State in 2006 after serving on the Statesmen coaching staff as a graduate assistant.
He has been selected for three internships in the NFL with the New York Jets (2009, 2010) and Buffalo Bills (2008), and participated in the NCAA Coaches Academy program.
Mack's appointment is a five-year term beginning Jan. 6, with an annual salary of $180,000.
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