text:
Tag: New York Times
The Clintons: Back on the campaign trail with the help of the New York Times
This article was published at Salon.com on December 4, 2013 under the headline, "New York Times' blind spot on Clinton and race."
The New York Times published a piece this week in the service of the Democratic Party's campaign for the 2016 elections that reveals a grave misunderstanding of recent history. Reporters Amy Chozick and Jonathan Martin profiled the tactics of former Secretary of State Hillary Clinton and her husband, the 42nd president of the United States, to restore their fragile relationship with African-Americans in anticipation of the former's 2016 presidential run. The Times frames it as an attempt to "to soothe and strengthen their relationship with African-Americans," apparently strained after Bill's 2008 comments about the Obama presidency.
Here is the motivation they assign to the Clintons:
This task [of courting the black vote] has taken on new urgency given the Democratic Party's push to the left, away from the centrist politics with which the Clintons are identified. Strong support from black voters could serve as a bulwark for Mrs. Clinton against a liberal primary challenge should she decide to run for president in 2016.
It would have been illuminating, and accurate as well, to distinguish between Democratic Party functionaries and Democratic voters in their description; I don't see much in the way of Democratic politicos' "push to the left": NDAA 2012/2013, bank bailouts, the ACA, among other laws, don't strike me as overly progressive.
Chozick and Martin assiduously cover the various black leaders with whom the Clintons have consorted since Hillary's resignation as secretary of state earlier this year. Along with that coverage is a telling, if not accurate, description of Bill Clinton's legacy, which Hillary will surely be relying on to vouch for her "progressive" credentials. Here is perhaps the most remarkable paragraph of the article:
Mr. Clinton has a rich, if occasionally fraught, history with African-Americans. He was a New South governor and a progressive on race who would eventually be called "the first black president" by the author Toni Morrison. But he infuriated blacks in 2008 when, after Mr. Obama won a big South Carolina primary victory, he seemed to dismiss the achievement by reminding the press that the Rev. Jesse Jackson had won the state twice and calling Mr. Obama's antiwar position "the biggest fairy tale I've ever seen."
Many African-Americans took Mr. Clinton's fairy tale comment to mean that Mr. Obama's candidacy itself was a hopeless fantasy.
It is true that black Americans were mightily irritated by Bill's comments. But that's hardly the only source of the injury.
(Un)surprisingly, even as Chozick and Martin tritely repeat Toni Morrison's description of Clinton as the first black president, proudly repeated by Bill (and ad nauseam by mainstream media), they don't offer any context for her remarks.
Morrison, writing in the New Yorker in 1998, was reflecting on the Republicans' move to impeach Bill Clinton in the aftermath of revelations of his affair with Monica Lewinsky, his intern at the time. She says:
African-American men seemed to understand it right away. Years ago, in the middle of the Whitewater investigation, one heard the first murmurs: white skin notwithstanding, this is our first black President. Blacker than any actual black person who could ever be elected in our children's lifetime. After all, Clinton displays almost every trope of blackness: single-parent household, born poor, working-class, saxophone-playing, McDonald's-and-junk-food-loving boy from Arkansas. And when virtually all the African-American Clinton appointees began, one by one, to disappear, when the President's body, his privacy, his unpoliced sexuality became the focus of the persecution, when he was metaphorically seized and body-searched, who could gainsay these black men who knew whereof they spoke? The message was clear: "No matter how smart you are, how hard you work, how much coin you earn for us, we will put you in your place or put you out of the place you have somehow, albeit with our permission, achieved. You will be fired from your job, sent away in disgrace, and—who knows?—maybe sentenced and jailed to boot. In short, unless you do as we say (i.e., assimilate at once), your expletives belong to us."
It is clear that Morrison is poetic and pained here. She analogizes the experiences faced by Clinton to those faced all too often by black men. There is much that can be said about this piece. But the cynicism of Clinton and his supporters is such that her phrase was co-opted as an endorsement of his "progressive" politics, rather than what it signaled at the very least; it is a searing insight into the inferior, abject status of black men in the United States at the end of the millennium. And here is Morrison in her own words in 2008.
But Chozick and Martin, in their own perhaps subconscious cynicism merely repeat Morrison's endorsement and omit any discussion of Clinton's policies during his two terms as president, or during his time as governor of Arkansas.
The first "black" president and his partner in devastation proudly designed the prototype of Clinton's famous 1996 welfare reform bill when he was the governor of Arkansas. Women who applied for aid from the state were required, among other indignities, to name the potential fathers of their children. Yes, yes, save your objections: This policy was created to search out "deadbeat dads," and get them to pay child support.
But somehow it never occurred to many — not the press, not white liberals, not liberal feminists, much less the Clintons (if they cared at all) — that such a reform would only be effective in further humiliating already poor women, women who, had they other options, would never have resorted to the state for help. Here's a brilliant letter from a Seattle feminist to N.O.W. back in 2007, which sets out the various assumptions and implications of welfare reform.
The ballast for welfare reform exploited the racial antagonism against black women that was inflated and gained momentum under Ronald Reagan's administration. But as many, from Barbara Ehrenreich to digby to Jason DeParle, point out, the Clintons and their Democratic buddies endorsed the righteous smokescreen that "workfare" was needed to teach the poor how to keep a job rather than asking for money, and to teach poor (black) women "chastity training." Patronizing? Racist? Those words don't even cover half of it, especially as they're accompanied by the convenient selective amnesia about the legacy of slavery and the still-existent practice of institutional discrimination against blacks. We can see this in the history of the drug war, the prison industry, red-lining, not to mention plain old-fashioned racism as seen in our public school system, post-secondary admissions practices, and employment across multiple industries.
Hillary's express support for welfare reform enabled Bill to get the 1996 Personal Responsibility and Work Opportunity Act passed. Peter Edelman, a senior Clinton appointee who resigned in protest of the bill, pointed out that this was the "worst thing Bill Clinton has done." Due to the remarkable efforts of the "first black president" and his wife, and like-minded "liberals" and conservatives who believed that the poor needed to be taught to climb out of a "culture of poverty," welfare was no longer the entitlement that it had been for decades (and should have remained as such). Rather, it was transformed into a sporadic privilege periodically and provisionally bestowed on the poor, all the while leaving millions more in poverty. As Edelman pointed out in 2011, that 1996 bill made things much worse for the poor: "There are now people who cannot find work, and who cannot get welfare."
Needless to say, Democrats and Republicans have managed to augment, enhance, exacerbate the level of nationwide poverty through its support of banking deregulation and absence of serious sanctions for bankers and subprime mortgage companies.
When Chozick and Martin write about Bill Clinton as a "progressive on race," I have to wonder which criteria they use to measure. They use certain famous black politicians' comments (such as those of Democratic Rep. James Clyburn or Rep. Elijah Cummings) or public gestures (such as sitting next to "friend and rival" and former Democratic Virginia Gov. L. Douglas Wilder at Howard University's May 2013 commencement) at face value and out of context. To gauge race progress by which friends a white Democrat sits next to — doesn't this strike anyone as uncomfortably close to the "Some of my best friends" cliché?
Why not consider the effects of NAFTA and WTO, which decimated the manufacturing industry that employed enormous numbers of African-Americans? Many journalists and left economists have detailed the detrimental impact of the offshoring of corporations, the forgiveness of taxes, the eradication of labor protections for foreign nationals who work at formerly American companies. Why does none of this figure into the assessment of "racial progress"? Even one paragraph might have allowed for the possibility that the Times was engaged in some critical questions about the releases and information that they were being fed by the Clinton campaign.
Why not consider the effects of the 1996 Immigration Reform Bill, which was a precursor to the enormous anti-immigration tide that has swept the country, enhanced by the right-wing and neo-patriotic impulses of both Democrats and Republicans in the aftermath of the Sept. 11, 2001, attacks?
Why not consider the effects of the 1994 Crime Bill, which heralded in "three strikes" legislation at the federal level, also signed under the "first black president"? The expansion of the death penalty in the 1996 Anti-Terrorism and Death Penalty Act?
I can hear objections that Hillary should be able to run on her own record. OK, why not examine a few of her votes? Remember, it was Sen. Russ Feingold — not Sen. Clinton, or Sens. Dianne Feinstein and Barbara Boxer, or Secretary of State John Kerry — who stood up against the USA Patriot Act, as a harbinger of a (by now) vengeful, 12-year, racist and arbitrary tide of vitriol against Muslims in the U.S., Iraq, Afghanistan, U.K., Yemen, Pakistan, Somalia and elsewhere in the world. How about on the 2002 authorization to invade Iraq? AUMF 2005? The 2007 surge in Iraq? She voted in favor of them. To her credit, she voted against the 2008 FISA bill, citing checks on presidential authority, even as elsewhere she has been in support of increasing it. How does she feel about WikiLeaks? Edward Snowden? The death penalty (supports it, but not for Iran).
These are hardly left votes. These are hardly liberal votes. These are hardly racially progressive votes.
Let's not judge whether someone is a "race progressive" — especially a politician — by the utterances of his/her friends. Presumably, journalists understand that the notion of an alliance does not confirm the truth of one's race politics; it merely demonstrates that all other concerns have been provisionally subordinated in order to further one particular goal. Sure, we can call it pragmatic, strategic, realpolitik. But regardless of the term used, journalists — of all people — know that citing such alliances does not offer a valuable insight or confirmation about the truth of one's politics.
I tell my students that if they want to write about politics effectively and forcefully, they must major in something other than journalism: history, sociology, ethnic studies, politics — something other than a field that disciplines its students to forget that accurate narratives have a long-seated, deeply buried history that cannot simply be articulated through a repetition of sound bites aired by corporate news media or covering poll results. Facts, those snippets that refer to a certain state of the world, must be assembled and grounded by searching through indirect, long-buried records that have long slipped the public (and corporate media's) memory. Such an excavation requires the skills of an archaeologist and the critical distance of an outsider — not the propitiatory writing skills of someone familiar with the well-worn seat of an election press bus or who lunches with his subjects on a regular basis.
Of course, that assumes that establishment media such as the Times is interested in reportage from a critical perspective. Perhaps that's the most flawed assumption of all.
Author Falguni A. ShethPosted on December 4, 2013 Categories 2013, People of Color, Race, US PoliticsTags Clintons, Hillary Clinton, New York Times, Welfare reform
Heaping Insult on Injury: Bill Keller's Character Assassination of Bradley Manning
On Monday, New York Times columnist and editor Bill Keller made a number of troubling, distracting points in his column about Pfc Bradley Manning's Pre-trial statement, which included some serious morally laden—and ungrounded–condemnations. Keller's position is fairly condemnable itself, given that he works for a newspaper that has been on record as cooperating with maintaining government secrecy on multiple occasions.
1) Keller points out that Manning said he left a voice message for the Times but never heard back. From this, Keller insinuates that Manning was somehow incompetent and wasn't able to get his message to the editors—something that thousands of Times readers do everyday.
It's puzzling to me that a skilled techie capable of managing one of the most monumental leaks ever couldn't figure out how to get an e-mail or phone message to an editor or a reporter at The Times.
First, it's not clear what being a 'skilled techie' has to do with leaving a voicemail. The two, needless to say, are unconnected. Keller's response doesn't squash doubts in my mind that the Bradley may have successfully left a message, only to have it ignored or deleted for any number of reasons: Perhaps because a) the NYT staff found it seemed too outlandish to be true; b) there was incompetence on the part of the staff whose job it was to check the messages; or they decided it was a hoax or unimportant; c) the NYT may have decided to deny receipt of the message in order to stay out of the fray. We know that the Times has had a history of cooperating with the US in protecting state secrets. We saw one glaring example of this under Keller's own watch, in a story admitting that the Times sat on a story for over a year about how the US was illegally wiretapping American citizens.
The White House asked The New York Times not to publish this article, arguing that it could jeopardize continuing investigations and alert would-be terrorists that they might be under scrutiny. After meeting with senior administration officials to hear their concerns, the newspaper delayed publication for a year to conduct additional reporting. Some information that administration officials argued could be useful to terrorists has been omitted.
It is still unclear how much of that story would have endangered national security, and how much of it was a cover-up for a crime conducted under the Bush Administration.
2) That fact again raises doubts about Keller's confidence that if the Times had received information, they would have sifted it carefully and published information that they felt was important for the public to know. We see the repeated collaboration on the part of the NYT with the US government—which often at odds with an American public's interest. The latest example of this was seen in the speculative, often speculative or unfounded story published in last Sunday's NYT about the deaths of Anwar Al-Awlaki, his son Abdulrahman, and his colleague Samir Khan, which several journalists suggest are at odds with known details. Much of that story was confirmed primarily—and only–by government sources, and challenged at many points by detailed investigative independent journalist Marcy Wheeler.
3) Keller assassinates Manning's character by using Manning's self-description as
"emotionally fractured" — a gay man in an institution not hospitable to gays, fragile, lonely, a little pleased with his own cleverness, a little vague about his motives,"
as a vehicle by which to raise doubts about the importance or integrity of Manning's intentions in wanting to share the classified documents with world—given that, as Keller says, Manning's own explanation was inchoate.* I don't see why Keller has to comment on Manning's emotional state which, for someone who most likely knows he's about to get into a massive amount of trouble, isn't surprising. One doesn't need to be an articulate ethics professor in order to know the difference between what is indisputably ethically troubling and what is not. Further, as Nathan Fuller pointed out to Keller in response to his initial column, in fact, Manning had extremely detailed and eloquent objections to the corruption and military practices that he saw.
Regardless, as Keller states, at his sentencing statement—after many years in solitary confinement, during which Manning has much time—if not many hospitable circumstances– to reflect, he makes clear that he is troubled by the dehumanization of the casualties in one military attack captured on video, where the US soldiers who perpetuated the attack cheered at the large number of casualities that they managed to engender. That statement is consistent with his earlier actions and perspective, to say the least.
4) Finally, in order to show that the NYT has no obligation to with support–either a whistleblower or– treasonous 'enemy' of the state, Keller refers to Max Frankel, who was The Times's Washington bureau chief during the Daniel Ellsberg's leaking of the Pentagon Papers. According to Frankel:
"[Ellsberg] was committing an act of civil disobedience and presumably knew that required accepting the punishment. We were privately pleased that the prosecution overreached and failed, but we did not consider ourselves his partner in any way.
But isn't that precisely the ethical and deceptively neutral stance that journalists such as Nathan Fuller, Kevin Gosztola, Glenn Greenwald, and others are challenging? If you're a journalist—and that means anything to you—it means reporting to the public—even, especially, at risk of pissing off the state. And that means standing with your sources and ensuring that they are not targeted or persecuted for sharing important information with the Fourth Estate. That requires public support for information-sharing.
This should be the rule for journalists, especially when there may be concerns about criminal activities on the part of the state. Protect your sources, support them, and report on harassment, corruption and wrongdoing. Such a stance would be 'truly' neutral, because it would ensure allegiances to its public readership, and promote trust by showing its adversarial, watchdog–not lapdog–stance toward the state.
5) Keller suggests that Manning 'pilfered' documents. That suggests that Manning was stealing property that is owned by the government. In fact, this is precisely the issue that is at stake regarding Manning's actions. As such, Keller is begging the question (i.e., assuming the very thing that is being questioned).
Did Manning steal? Or did he release documents that the state wanted to be kept secret for "national security" reasons–which are unconvincing to many, many people, including former whistleblowers, journalists, and a segment of the American population. In many people's understanding of a liberal democratic state—a government is accountable to its people. If this is the case, then a government's actions must be made known to it people.
If we accept the latter explanation which, barring an actual state of emergency, is the only Constitutional one, another interpretation of Manning's actions is NOT that he was stealing, but rather attempting to share evidence of state corruption and wrongdoing with Americans—those people to whom the US government is accountable. That would make him by most accounts except for those who are concerned about having wrongdoing exposed—a whistleblower.
6) Finally, as whistleblower Daniel Ellsberg stated yesterday, there is a misconception about the kind of material that Bradley Manning leaked. In his letter, Ellsberg clarifies Manning WAS selective in the information he leaked. From Ellsberg's statement on the Manning recorded statement, released today:
"MANNING WAS DISCRIMINATING
Critics have alleged that a major difference between my case and Manning's is that I was discriminating in what I leaked, while Manning wasn't. He just dumped some material that doesn't need to be out, they say. This is simply false.
First, it's important to point out most of the material he put out was unclassified. The rest was classified 'secret,' which is relatively low level. All of the Pentagon Papers was classified top secret.
But in a fact no one seems to observe from his statement, Manning was working within a "SCIF," which stands for Sensitive Compartmented Information Facility. To get into a SCIF, a soldier needs a clearance higher than top secret. This means he had access to the highest classified material, such as communications and signals intelligence. This means he could've put out information top secret and higher, and purposely chose not to do so."
Especially in light of Ellsberg's point, Keller's statement seems disingenuous at best, and self (and employer-) serving as well. But the set of terms that Keller deploys to refer to Manning–a 'geek,' 'fragile gay man' (how is his sexuality relevant?), "nervous, troubled, angry young Army private," and in effect, a thief, suggests a character assassination more than a clarifying explanation of the NYT role in this affair. Neither courageous nor morally upstanding on the part of Keller. And not surprising.
*Since I first wrote this piece, independent journalists such as Nathan Fuller, Kevin Gosztola, and Greg Mitchell have published challenges to Kellers' assessment of Manning's motivations. I have revised to include some of their points; my assessement overlaps at points with theirs.
Author Falguni A. ShethPosted on March 13, 2013 March 13, 2013 Categories 2013Tags Bill Keller, Bradley Manning, New York Times, Whistleblowers2 Comments on Heaping Insult on Injury: Bill Keller's Character Assassination of Bradley Manning


text:
"""This file contains a unit test for the EventObject.

This is an implementation of an unit test for EventObject storage mechanism for
plaso.

The test consists of creating six EventObjects.

Error handling. The following tests are performed for error handling:
 + Access attributes that are not set.
"""

import unittest

from plaso.dfwinreg import fake as dfwinreg_fake
from plaso.events import text_events
from plaso.events import windows_events
from plaso.lib import event
from plaso.lib import timelib

class TestEvent(event.EventObject):
  """A test event object."""
  DATA_TYPE = 'test:event'

  def __init__(self, timestamp, attributes):
    """Initializes the test event object."""
    super(TestEvent, self).__init__()
    self.timestamp = timestamp
    self.timestamp_desc = u'Some time in the future'
    for attribute, value in attributes.iteritems():
      setattr(self, attribute, value)

class FailEvent(event.EventObject):
  """An test event object without the minimal required initialization."""

def GetEventObjects():
  """Returns a list of test event objects."""
  event_objects = []
  hostname = u'MYHOSTNAME'
  data_type = 'test:event'

  event_object = event.EventObject()
  event_object.username = u'joesmith'
  event_object.filename = u'c:/Users/joesmith/NTUSER.DAT'
  event_object.hostname = hostname
  event_object.timestamp = 0
  event_object.data_type = data_type
  event_object.text = u''
  event_objects.append(event_object)

  filetime = dfwinreg_fake.Filetime()

  # TODO: move this to a WindowsRegistryEvent unit test.
  filetime.CopyFromString(u'2012-04-20 22:38:46.929596')
  values_dict = {u'Run': u'c:/Temp/evil.exe'}
  event_object = windows_events.WindowsRegistryEvent(
      filetime.timestamp, u'MY AutoRun key', values_dict)
  event_object.hostname = hostname
  event_objects.append(event_object)

  filetime.CopyFromString(u'2012-04-20 23:56:46.929596')
  values_dict = {u'Value': u'send all the exes to the other world'}
  event_object = windows_events.WindowsRegistryEvent(
      filetime.timestamp, u'//HKCU/Secret/EvilEmpire/Malicious_key',
      values_dict)
  event_object.hostname = hostname
  event_objects.append(event_object)

  filetime.CopyFromString(u'2012-04-20 16:44:46.000000')
  values_dict = {u'Value': u'run all the benign stuff'}
  event_object = windows_events.WindowsRegistryEvent(
      filetime.timestamp, u'//HKCU/Windows/Normal', values_dict)
  event_object.hostname = hostname
  event_objects.append(event_object)

  timestamp = timelib.Timestamp.CopyFromString(u'2012-04-30 10:29:47.929596')
  filename = u'c:/Temp/evil.exe'
  attributes = {
      u'text': u'This log line reads ohh so much.'}
  event_object = TestEvent(timestamp, attributes)
  event_object.filename = filename
  event_object.hostname = hostname

  event_objects.append(event_object)

  timestamp = timelib.Timestamp.CopyFromString(u'2012-04-30 10:29:47.929596')
  attributes = {
      u'text': u'Nothing of interest here, move on.'}
  event_object = TestEvent(timestamp, attributes)
  event_object.filename = filename
  event_object.hostname = hostname

  event_objects.append(event_object)

  timestamp = timelib.Timestamp.CopyFromString(u'2012-04-30 13:06:47.939596')
  attributes = {
      u'text': u'Mr. Evil just logged into the machine and got root.'}
  event_object = TestEvent(timestamp, attributes)
  event_object.filename = filename
  event_object.hostname = hostname

  event_objects.append(event_object)

  text_dict = {u'body': (
      u'This is a line by someone not reading the log line properly. And '
      u'since this log line exceeds the accepted 80 chars it will be '
      u'shortened.'), u'hostname': u'nomachine', u'username': u'johndoe'}

  # TODO: move this to a TextEvent unit test.
  timestamp = timelib.Timestamp.CopyFromString(u'2012-06-05 22:14:19.000000')
  event_object = text_events.TextEvent(timestamp, 12, text_dict)
  event_object.text = event_object.body
  event_object.hostname = hostname
  event_object.filename = filename

  event_objects.append(event_object)

  return event_objects

class EventObjectTest(unittest.TestCase):
  """Tests for the event object."""

  def testSameEvent(self):
    """Test the EventObject comparison."""
    event_a = event.EventObject()
    event_b = event.EventObject()
    event_c = event.EventObject()
    event_d = event.EventObject()
    event_e = event.EventObject()

    event_a.timestamp = 123
    event_a.timestamp_desc = u'LAST WRITTEN'
    event_a.data_type = 'mock:nothing'
    event_a.inode = 124
    event_a.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_a.another_attribute = False
    event_a.metadata = {
        u'author': u'Some Random Dude',
        u'version': 1245L,
        u'last_changed': u'Long time ago'}
    event_a.strings = [
        u'This ', u'is a ', u'long string']

    event_b.timestamp = 123
    event_b.timestamp_desc = 'LAST WRITTEN'
    event_b.data_type = 'mock:nothing'
    event_b.inode = 124
    event_b.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_b.another_attribute = False
    event_b.metadata = {
        u'author': u'Some Random Dude',
        u'version': 1245L,
        u'last_changed': u'Long time ago'}
    event_b.strings = [
        u'This ', u'is a ', u'long string']

    event_c.timestamp = 123
    event_c.timestamp_desc = u'LAST UPDATED'
    event_c.data_type = 'mock:nothing'
    event_c.inode = 124
    event_c.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_c.another_attribute = False

    event_d.timestamp = 14523
    event_d.timestamp_desc = u'LAST WRITTEN'
    event_d.data_type = 'mock:nothing'
    event_d.inode = 124
    event_d.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_d.another_attribute = False

    event_e.timestamp = 123
    event_e.timestamp_desc = u'LAST WRITTEN'
    event_e.data_type = 'mock:nothing'
    event_e.inode = 623423
    event_e.filename = u'c:/afrit/onnurskra.txt'
    event_e.another_attribute = False
    event_e.metadata = {
        u'author': u'Some Random Dude',
        u'version': 1245,
        u'last_changed': u'Long time ago'}
    event_e.strings = [
        u'This ', u'is a ', u'long string']

    self.assertEqual(event_a, event_b)
    self.assertNotEqual(event_a, event_c)
    self.assertEqual(event_a, event_e)
    self.assertNotEqual(event_c, event_d)

  def testEqualityString(self):
    """Test the EventObject EqualityString."""
    event_a = event.EventObject()
    event_b = event.EventObject()
    event_c = event.EventObject()
    event_d = event.EventObject()
    event_e = event.EventObject()
    event_f = event.EventObject()

    event_a.timestamp = 123
    event_a.timestamp_desc = u'LAST WRITTEN'
    event_a.data_type = 'mock:nothing'
    event_a.inode = 124
    event_a.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_a.another_attribute = False

    event_b.timestamp = 123
    event_b.timestamp_desc = u'LAST WRITTEN'
    event_b.data_type = 'mock:nothing'
    event_b.inode = 124
    event_b.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_b.another_attribute = False

    event_c.timestamp = 123
    event_c.timestamp_desc = u'LAST UPDATED'
    event_c.data_type = 'mock:nothing'
    event_c.inode = 124
    event_c.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_c.another_attribute = False

    event_d.timestamp = 14523
    event_d.timestamp_desc = u'LAST WRITTEN'
    event_d.data_type = 'mock:nothing'
    event_d.inode = 124
    event_d.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_d.another_attribute = False

    event_e.timestamp = 123
    event_e.timestamp_desc = u'LAST WRITTEN'
    event_e.data_type = 'mock:nothing'
    event_e.inode = 623423
    event_e.filename = u'c:/afrit/öñṅûŗ₅ḱŖūα.txt'
    event_e.another_attribute = False

    event_f.timestamp = 14523
    event_f.timestamp_desc = u'LAST WRITTEN'
    event_f.data_type = 'mock:nothing'
    event_f.inode = 124
    event_f.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_f.another_attribute = False
    event_f.weirdness = u'I am a potato'

    self.assertEqual(event_a.EqualityString(), event_b.EqualityString())
    self.assertNotEqual(event_a.EqualityString(), event_c.EqualityString())
    self.assertEqual(event_a.EqualityString(), event_e.EqualityString())
    self.assertNotEqual(event_c.EqualityString(), event_d.EqualityString())
    self.assertNotEqual(event_d.EqualityString(), event_f.EqualityString())

  def testEqualityFileStatParserMissingInode(self):
    """Test that FileStatParser files with missing inodes are distinct"""
    event_a = event.EventObject()
    event_b = event.EventObject()

    event_a.timestamp = 123
    event_a.timestamp_desc = u'LAST WRITTEN'
    event_a.data_type = 'mock:nothing'
    event_a.parser = u'filestat'
    event_a.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_a.another_attribute = False

    event_b.timestamp = 123
    event_b.timestamp_desc = u'LAST WRITTEN'
    event_b.data_type = 'mock:nothing'
    event_b.parser = u'filestat'
    event_b.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_b.another_attribute = False

    self.assertNotEqual(event_a, event_b)

  def testEqualityStringFileStatParserMissingInode(self):
    """Test that FileStatParser files with missing inodes are distinct"""
    event_a = event.EventObject()
    event_b = event.EventObject()

    event_a.timestamp = 123
    event_a.timestamp_desc = u'LAST WRITTEN'
    event_a.data_type = 'mock:nothing'
    event_a.parser = u'filestat'
    event_a.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_a.another_attribute = False

    event_b.timestamp = 123
    event_b.timestamp_desc = u'LAST WRITTEN'
    event_b.data_type = 'mock:nothing'
    event_b.parser = u'filestat'
    event_b.filename = u'c:/bull/skrytinmappa/skra.txt'
    event_b.another_attribute = False

    self.assertNotEqual(event_a.EqualityString(), event_b.EqualityString())

  def testNotInEventAndNoParent(self):
    """Call to an attribute that does not exist."""
    event_object = TestEvent(0, {})

    with self.assertRaises(AttributeError):
      getattr(event_object, u'doesnotexist')

  def testFailEvent(self):
    """Calls to format_string_short that has not been defined."""
    e = FailEvent()

    with self.assertRaises(AttributeError):
      getattr(e, u'format_string_short')

if __name__ == '__main__':
  unittest.main()

redpajama_set_name: RedPajamaGithub


text:
El municipio de Mound (en inglés: Mound Township) es un municipio ubicado en el condado de Miami en el estado estadounidense de Kansas. En el año 2010 tenía una población de 729 habitantes y una densidad poblacional de 8,75 personas por km².

Geografía
El municipio de Mound se encuentra ubicado en las coordenadas . Según la Oficina del Censo de los Estados Unidos, el municipio tiene una superficie total de 83.33 km², de la cual 82,86 km² corresponden a tierra firme y (0,56 %) 0,47 km² es agua.

Demografía
Según el censo de 2010, había 729 personas residiendo en el municipio de Mound. La densidad de población era de 8,75 hab./km². De los 729 habitantes, el municipio de Mound estaba compuesto por el 98,22 % blancos, el 0,82 % eran afroamericanos, el 0,27 % eran amerindios, el 0,14 % eran isleños del Pacífico, el 0,27 % eran de otras razas y el 0,27 % eran de una mezcla de razas. Del total de la población el 1,1 % eran hispanos o latinos de cualquier raza.

Referencias

Enlaces externos

Municipios de Kansas
Localidades del condado de Miami (Kansas)

redpajama_set_name: RedPajamaWikipedia


text:
Policies & Permissions
jem Home » 2000 Archive » 4 December » 192 (11): 1625
Short Telomeres Result in Organismal Hypersensitivity to Ionizing Radiation in Mammals
Fermín A. Goytisolo, Enrique Samper, Juan Martín-Caballero, Paul Finnon, Eloísa Herrera, Juana M. Flores, Simon D. Bouffler, María A. Blasco
Fermín A. Goytisolo
Enrique Samper
Juan Martín-Caballero
Paul Finnon
Eloísa Herrera
Juana M. Flores
Simon D. Bouffler
María A. Blasco
DOI: 10.1084/jem.192.11.1625 | Published December 4, 2000
Here we show a correlation between telomere length and organismal sensitivity to ionizing radiation (IR) in mammals. In particular, fifth generation (G5) mouse telomerase RNA (mTR)−/− mice, with telomeres 40% shorter than in wild-type mice, are hypersensitive to cumulative doses of gamma rays. 60% of the irradiated G5 mTR−/− mice die of acute radiation toxicity in the gastrointestinal tract, lymphoid organs, and kidney. The affected G5 mTR−/− mice show higher chromosomal damage and greater apoptosis than similarly irradiated wild-type controls. Furthermore, we show that G5 mTR−/− mice show normal frequencies of sister chromatid exchange and normal V(D)J recombination, suggesting that short telomeres do not significantly affect the efficiency of DNA double strand break repair in mammals. The IR-sensitive phenotype of G5 mTR−/− mice suggests that telomere function is one of the determinants of radiation sensitivity of whole animals.
radiosensitivity
DNA DSB repair
Ionizing radiation (IR) is a potent inducer of DNA double strand breaks (DSBs), which, if unrepaired, may either lead to cell death or trigger neoplastic transformation (1). Studies in mammals and yeast have shown that mutations in genes responsible for DNA DSB repair result in increased IR sensitivity (2)(3). Two major mechanisms are involved in repairing DSB in mammalian cells: nonhomologous end joining (NHEJ) and homologous recombination (HR) (4)(5).
Telomeres are specialized protective structures at the ends of chromosomes (6). There is increasing evidence that proteins involved in DSB DNA repair may have a role at the telomeres. Yeast telomeres serve as storage sites for DNA repair proteins such as yKu80 and Sir3p, which, upon induction of DSB, are mobilized from the telomeres to the sites of damage (7)(8). In mammals, Ku proteins bind to telomeric sequences (9)(10) and protect telomeres from end–end fusions (11), and the DNA repair complex RAD50/MRE11/NBS1 associates with telomeric protein TRF2 (12). Telomere length can also influence IR sensitivity, in particular, mutations in DNA repair proteins yKu80, yKu70, Mre11, Rad50, Xrs2, Sir2, and Sir3 result in yeast strains with altered telomere length and altered IR sensitivities (13). In Caenorhabditis elegans, DNA repair mtr-2 mutant shows telomere shortening and IR hypersensitivity (14). An association between telomere length and IR has not been established yet in mammals. Furthermore, it is not clear how short telomeres influence IR sensitivity in mammals. Here, we study the radiation sensitivity of mice lacking the RNA component of telomerase (mouse telomerase RNA [mTR]−/−) (15). These mice lack telomerase activity and show telomere shortening at a rate of 4–5 kb per mouse generation (15). Telomere shortening in these mice is accompanied by an increase in the number of chromosome ends without detectable telomeres and in the frequency of chromosome fusions (15)(16). Our results indicate that short telomeres result in organismal hypersensitivity to IR and in an increased DNA damage after irradiation. Furthermore, we show that this increased radiation-induced DNA damage in late generation mTR−/− mice does not seem to be the direct consequence of a defect in a known DNA DSB repair pathway.
Wild-type (wt) and different generations of mTR−/− mice were generated as described (15). The genetic background was 60% C57BL6, 37.5% 129Sv, and 2.5% SJL (15)(16)(17).
Statistical Analysis.
Sample means, standard deviations, chi-squares, and Student's t tests were calculated using Microsoft Excel.
Gamma Irradiation.
Single doses of 1.75 Gy were administered to age-matched 5-mo-old wt and mTR−/− mice weekly using a 137Cs source (MARK 1-30 Irradiator; Shepherd & Associates) at a rate of 1.14 Gy/min.
Histopathological Analysis.
Sections from different organs from nonirradiated and irradiated wt and mTR−/− mice were fixed in 10% buffered formalin and stained in parallel with hematoxylin–eosin.
Telomere Length.
To measure telomere length, FLOW-FISH (fluorescence in situ hybridization) was carried out on fresh bone marrow (BM) cells (a total of 1,500–4,000 nuclei for each sample were analyzed) as described (17). For FLOW-FISH on blood cells, blood (100–200 μl) was obtained before and after irradiation from the retroorbital plexus. Red blood cells were lysed by osmotic shock. Lymphocytes were resuspended in FCS, and an equal volume of FCS plus 20% DMSO was added dropwise. The cells were frozen gradually at −80°C and kept in liquid nitrogen. After collection of all blood samples, cells were thawed at 37°C, and an equal volume of 8% dextran (10% Rheomacrodex; Pharmacia Antibioticos Farma, S.A.) and 5% human albumin was added. Cells were centrifuged at 900 g for 20 min at 4°C and were resuspended in RPMI 1640 containing 10% FBS and 0.55 mM β-mercaptoethanol. Approximately 0.5 × 106 cells were used per sample, and FLOW-FISH was carried out as described (17).
Cytogenetic Determination of Radiation-induced Chromosomal Damage in wt and mTR−/− Mice.
Direct BM chromosome preparations were made from unirradiated control and gamma-irradiated mice as described (18) and stained with concentrated paint probes for chromosomes 1, 2, and 3 (Cambio) simultaneously. Slides were pretreated, denatured (19), and hybridized with a total of 10 μl of hybridization mix containing 2 μl of biotin-labeled chromosome 3 probe, 2 μl of FITC-labeled chromosome 2 probe, 1 μl each of biotin- and FITC-labeled chromosome 1 probe, 0.5 μg of mouse CoT 1 DNA (Life Technologies), and 10 μg of human total genomic DNA. After 48-h hybridization at 42°C, slides were washed in 2× SSC, followed by two 15-min washes in 0.5× SSC/50% formamide at 42°C and a final wash in 2× SSC. Biotin-labeled probes were detected with alternating layers of avidin–Texas Red and biotinylated goat anti–avidin D antibody (Vector Laboratories), and FITC-labeled probes were detected with alternating layers of rabbit anti-FITC–IgG (Dako) and FITC-labeled goat anti–rabbit IgG (Sigma-Aldrich) (20). Three layers of each fluorescent detecting reagent were applied. Slides were mounted in Vectashield (Vector Laboratories) containing 400 ng/ml DAPI (4,6-diamino-2-phenylindole) counterstain and scored using a Nikon epifluorescence microscope equipped with appropriate filter sets. Aberrations involving painted chromosomes were scored. Events that were clearly clonal in origin were counted only once in the determination of aberration frequencies. Quantitative comparison of all chromosome-type aberrations taken together (chromosome-type aberration score) employed a modified version (19) of the PAINT classification system (21) in which the number of color junctions arising from translocations, dicentrics, etc. and the number of deletions are summed and expressed per 100 cells.
Annexin V Labeling of Fresh Splenocytes to Determine In Vivo Apoptosis upon Irradiation in wt and G2 and G5 mTR−/− Mice.
The following reagents were used: FITC-conjugated annexin V (Immunotech), biotinylated rat anti–mouse B220 (Southern Biotechnology Associates), PE-conjugated hamster anti–mouse CD3 (Southern Biotechnology Associates), and spectral red label (SPRD)-conjugated streptavidin (Southern Biotechnology Associates). 24 h after irradiation, mouse splenocytes were incubated with biotinylated anti-B220 and SPRD-conjugated streptavidin, followed by incubation with FITC-conjugated annexin V and TRI-COLOR flow cytometry analysis.
Apoptosis and Cell Cycle Profile in Proliferating Splenocytes.
24 h after irradiation at the indicated gamma ray dose, splenocytes were isolated, and a total of 0.8 × 106 splenocytes were incubated in RPMI 1640 containing 10% FCS, 0.55 mM β-mercaptoethanol, and 10 mg/ml LPS (Sigma-Aldrich). After 48 h of culture, 0.250 × 106 cells were incubated with rat FITC-conjugated anti–mouse CD45R/B220 (BD PharMingen) and 20 mg/ml propidium iodide and analyzed by flow cytometry.
Sister Chromatid Exchange Determination.
Sister chromatid exchange (SCE) frequencies were determined in splenocytes. Spleens were removed, disaggregated in RPMI 1640/20% FCS, and stored on ice for 24 h. 5-ml cultures at 2 × 106 cells/ml in RPMI 1640/20% FBS were stimulated with 2 μg/ml Con A and 25 μg/ml LPS and grown in the presence of 10 μM bromodeoxyuridine for 48 h at 37°C. Cultures were irradiated with a 60Co gamma source (0.75 Gy/min) after 24 h of culture. Cells were harvested by hypotonic KCl/methanol–acetic acid fixation after colcemid treatment (0.004 μg/ml) for the final 2 h of culture. Slides were prepared and stained (22), and 25 metaphases were scored per treatment.
V(D)J Recombination Experiments.
PCR primers used were DHall: 5′-GGGA/CTTTTTGTC/GAAGGG/TATCTACTACTGTG-3′; JH4: 5′-AAAGACCTGCAGAGGCCATTCTTACC-3′; VH7183: 5′-CGCGAAGCTTCGTGGAGTCTGGGGGA-3′; DR218: 5′- ACCCCAGTAGTCCATAGCATAGTAAT-3′; Vβ8.1, 2, 3: 5′-GAGGAAAGGTGACATTGAGC-3′; Jβ2.6: 5′-GCCTGGTGCCGGGACCGAAGTA-3′; Vβ8 probe: 5′-GGGCTGAGGCTGATCCATTA-3′; 5′-intervening sequence (IVS): 5′-gtaagaatggcctctccaggt-3′; and 3′-IVS: 5′-GACTCAATCACTAAGACAGCT-3′.
Single-cell suspensions from lymphoid organs and BM from 6-wk-old mice were obtained. DNA was prepared for PCR as described (23). Semiquantitative PCR was done as described (23). PCR reactions (20 μl) contained 2–8 μl of template (∼10,000–40,000 genomes, which correspond to 50–200 ng of DNA), 160 ng of each primer, 1 U of Taq polymerase (PerkinElmer), and the reaction buffer provided by the manufacturer containing 1.5 mM MgCl2. Amplification (30 cycles) was performed at an annealing temperature of 65°C. The reaction was analyzed in 2.5% agarose gel electrophoresis and was transferred onto a nylon membrane (Hybond-N+). Oligo probes were labeled with T4 polynucleotide kinase and γ-[32P] (Amersham Pharmacia Biotech), and the probe was separated from unincorporated γ-[32P]ATP in a NAP™ 5 column (Amersham Pharmacia Biotech). For analysis of Ig coding joints, two sets of primers were used. To evaluate the D-JH rearrangement, the pair included the degenerate primer DHall and JH4 (23); for V-DJH rearrangement, the JH4 primer was combined with VH7183 (24). A JH4-specific probe (DR218) was used for the detection of Ig coding joint PCR products as described elsewhere (24). The TCR coding joints Vβ8-DJβ2.6 were detected using the primers Vβ8.1, 2, 3 and Jβ2.6 (25) and probed with the Vβ8 probe (25). A sequence from the heavy chain locus that is not affected by Ig rearrangement was amplified as a control (see IVS in Fig. 6 A) using 5′- and 3′-IVS primers (26).
Western Blots.
Nuclear extracts were prepared from passage two wt, Ku86−/−, or different generation mTR−/− mouse embryonic fibroblasts as described (27). Nuclear extracts were separated on SDS/7.5% polyacrylamide gels for detection of Ku70 and RNA pol II and on SDS/5% polyacrylamide gels for detection of poly(ADP-ribose)polymerase (PARP) and catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). PARP: mAb from Labgen; DNA-PKcs: rabbit anti–human DNA-PKcs polyclonal antibody from Oncogene Research Products; Ku70 mAb from Kamiya Biomedical Company; RNA polymerase II: rabbit anti–mouse polyclonal antibody from Santa Cruz Biotechnology, Inc.
mTR−/− Mice with Short Telomeres Are Hypersensitive to IR.
To assess the role of telomere length in organismal sensitivity to IR, wt, second generation (G2), and fifth generation (G5) mTR−/− mice were irradiated with cumulative weekly doses of gamma rays (1.75 Gy each dose). We purposely used G5 rather than G6 mTR−/− mice because G5 mTR−/− mice have 40% shorter telomeres than wt mice but do not show proliferative defects or pathologies, except for increased infertility (15)(16)(28). Thus, pathologies appearing in irradiated G5 mTR−/− mice will be a direct consequence of radiation. wt and G2 mTR−/− mice did not show radiation-induced loss of viability after six doses of 1.75 Gy gamma rays (Fig. 1), and these mice survived up to 2 mo after the sixth radiation dose, showing no signs of disease. In contrast, irradiated G5 mTR−/− mice showed mortality after the third dose of radiation (Fig. 1), with 60% of the irradiated G5 mTR−/− mice dead after the sixth irradiation. None of the control nonirradiated age-matched wt and G5 mTR−/− mice showed mortality during the course of the irradiation experiment or in the following 2 mo (Fig. 1). Similarly, none of the irradiated G5 mTR−/− that survived the six radiation doses showed signs of disease during the subsequent 2 mo (not shown). A Student's t test value of P = 0.0487 indicates that the mortality curve of the irradiated G5 mTR−/− mice is significantly different from that of wt irradiated, wt nonirradiated, G2 mTR−/− irradiated, G2 mTR−/− nonirradiated, and G5 mTR−/− nonirradiated mice. A recent study using G6 mTR−/− instead of G5 mTR−/− mice also described IR hypersensitivity of these mice as compared with wt (29).
Viability of wt (17 nonirradiated and 34 irradiated) and G2 (12 nonirradiated and 16 irradiated) and G5 mTR−/− (11 nonirradiated and 25 irradiated) mice after administration of cumulative doses of gamma rays (1.75, 3.50, 5.25, 7.00, 8.75, and 10.50 Gy). The graph shows survival until 1 wk after the administration of the sixth dose. The wt and G2 and G5 mTR−/− survivors survived at least up to 2 mo after the sixth irradiation dose, at which point the mice were killed.
To determine the cause of death, irradiated G5 mTR−/− mice showing signs of poor health (moribund) were killed and examined in parallel with age-matched nonirradiated G5 mTR−/− mice and with similarly irradiated wt and G5 mTR−/− mice showing no signs of disease (survivors). Heart, liver, bladder, salivary glands, and lungs were histologically normal (Table 1). Spleen and BM were depleted of cells in moribund irradiated G5 mTR−/− mice but not in similarly irradiated wt and G5 mTR−/− survivors or in nonirradiated G5 mTR−/− mice (Table 1). Fig. 2 shows normal histology of BM, stomach, small intestine, and kidney from irradiated wt and G5 mTR−/− survivors as well as in the nonirradiated controls. In contrast, these organs showed severe defects in the irradiated G5 mTR−/− moribunds. In particular, irradiated G5 mTR−/− moribunds showed severe BM aplasia (Fig. 2, arrows), parietal cell degeneration, and cystic formation in the stomach mucosa (Fig. 2, arrows), degeneration of villi and crypts in the small intestine (Fig. 2, arrows), and acute atrophy of kidney tubules (Fig. 2, arrows), a hallmark of radiation nephropathy (30). Similar radiation toxicity has been described mice deficient in ATM (ataxia telangiectasia mutated) and PARP, both proteins that are important in the DNA damage response (31)(32).
Summary of Pathologies
Histopathological analysis of nonirradiated and irradiated wt and G5 mTR−/− mice. Bone, stomach, small intestine, and kidney histological sections corresponding to nonirradiated and irradiated wt and G5-mTR−/− mice are shown. Only the irradiated moribund G5 mTR−/− mice showed radiation-induced pathologies such as dramatic BM aplasia, degeneration of parietal cells, and cystic formation in the stomach, acute atrophy of the villi with crypt degeneration in the small intestine, and acute degeneration of kidney tubules (see arrows indicating abnormalities). Differences in staining are due to slight differences in the thicknesses of the sections. All images were taken at the same magnification (99×) except BM (150×). Sections were fixed in 10% buffered formalin and stained with hematoxylin–eosin.
Increased Apoptosis and Normal Cell Cycle Profile in Irradiated G5 mTR−/− Mice.
Annexin V labels cells undergoing apoptosis in vivo. We measured the percentage of annexin V–labeled spleen B cells (B220+ cells) isolated from nonirradiated and irradiated wt and G2 and G5 mTR−/− mice. In the absence of irradiation, there were no differences in annexin V staining between genotypes: 18.4, 12.7, and 12.9 arbitrary fluorescence units for wt, G2, and G5 mTR−/− mice, respectively (Table 2). After irradiation at different doses, G5 mTR−/− spleens contained more annexin V–positive B cells than similarly irradiated wt mice, 28.8 and 137.9 arbitrary units for 1.75 and 4 Gy, respectively, in the case of the G5 mTR−/− mice as compared with 12.5 and 16.8 arbitrary units in the case of wt (Table 2). To determine if IR hypersensitivity of G5 mTR−/− mice could also be the consequence of a cell cycle defect associated with short telomeres, we analyzed by flow cytometry the cell cycle profile of spleen B cells (B220+/CR45R+) that were induced to enter the cell cycle upon stimulation with LPS (a B cell–specific mitogen). In the absence of irradiation, there were no statistically significant differences in cell cycle profile between genotypes (Table 3). Upon irradiation with a single gamma dose of 4 Gy, G5 and G6 mTR−/− splenocytes showed higher apoptosis than those from similarly irradiated wt mice; the percentages of apoptotic cells were 63.9 ± 8.9 and 63.8 ± 2.1 for G5 and G6 mTR−/− splenocytes, respectively, as compared with 36.0 ± 6.2 for similarly irradiated wt. All together, these results indicate increased IR-induced apoptosis in the G5 and G6 mTR−/− mice (Table 3). This increase in apoptosis in G5 and G6 mTR−/− mice was statistically significant, as indicated by Student's t test values of P = 0.0007 and 0.0003, respectively. Importantly, G0-G1 and S-G2 values were similar in all genotypes after irradiation (Table 3), indicating no defects in proliferation or cell cycle checkpoints in late generation mTR−/− cells.
Increased Cell Death in Irradiated G5 mTR−/− Splenocytes (B Cells)
Apoptosis and Cell Cycle Profile in LPS-stimulated Unirradiated and Irradiated wt and G2, G5, and G6 mTR−/− B Splenocytes
Inverse Correlation between Telomere Length and Organismal IR Sensitivity.
The fact that G5 but not G2 mTR−/− mice showed decreased viability and increased apoptosis after gamma irradiation suggests an inverse correlation between telomere length and organismal sensitivity to IR in mammals. We measured telomere length in fresh BM cells in groups of nonirradiated and irradiated wt and G5 mTR−/− cohorts using FLOW-FISH (reference 7; Fig. 3 A). Telomere fluorescence was significantly higher in BM cells from unirradiated wt mice than in those from unirradiated G5 mTR−/− cohorts (3.351 ± 0.548 and 2.236 ± 0.420 arbitrary fluorescence units, respectively; Fig. 3 A; Student's t test, P = 9.07659 × 10−5), in agreement with telomere loss occurring with increasing mTR−/− generations, as previously described (15)(16)(28). Importantly, telomere fluorescence was lower in the group of irradiated G5 mTR−/− mice that showed signs of radiosensitivity (Fig. 3 A, asterisk, moribunds) than in the irradiated surviving G5 mTR−/− group (survivors; 1.636 ± 0.119 and 2.088 ± 0.233 arbitrary fluorescence units, respectively; Fig. 3 A). A Student's t test value of P = 0.00173146 indicates that the difference in telomere length between both groups of mice is statistically significant. The telomere fluorescence intensity was also lower in the irradiated moribund G5 mTR−/− group than in the group of nonirradiated G5 mTR−/− mice (1.636 ± 0.119 and 2.236 ± 0.420 arbitrary fluorescence units, respectively; Fig. 3 A). A Student's t test value of P = 0.00136244 indicates that it is a statistically significant difference. It is important to point out that there is no statistically significant difference in telomere length between nonirradiated G5 mTR−/− mice and the irradiated G5 mTR−/− survivors (Student's t test, P = 0.34908841), indicating that the apparent elongation of telomeres in the surviving irradiated G5 mTR−/− mice (Fig. 3 A) is not statistically significant.
(A) Telomere length analysis using FLOW-FISH in fresh BM cells from nonirradiated (N) and irradiated (I) wt (n = 10 of each) and G5 mTR−/− (N, n = 10; and I, n = 14) cohorts. FLOW-FISH was carried out as described (17). Telomere length is represented as arbitrary units of fluorescence. Average telomere fluorescence values and their corresponding SDs are indicated in the text. Mice that died upon irradiation are indicated with an asterisk (moribund). (B) Telomere length analysis using FLOW-FISH in blood cells from the same mouse before (B) or after (A) irradiation. wt (n = 6) and G2 (n = 6) and G5 mTR−/− (n = 9) cohorts were used. Each nonirradiated or irradiated mouse is indicated by the same symbol before (B) and after (A) the completion of the irradiation protocol. TFU, arbitrary telomere fluorescence units.
To address whether loss of viability in irradiated G5 mTR−/− mice could be due to an additional radiation-induced telomere loss, we used FLOW-FISH to measure telomere length in blood cells taken from the same animal before and after whole body irradiation. Fig. 3 B shows that in individual wt and G2 and G5 mTR−/− mice, telomeres did not significantly change as a consequence of irradiation. Student's t test analysis showed that telomeres were not significantly different in blood cells extracted from the same animal before or after it was irradiated. Thus, irradiation does not trigger significant changes in telomere length: wt mice before versus after irradiation, P = 0.49800000 (no statistically significant difference in telomere length); G2 mTR−/− mice before versus after irradiation, P = 0.95686187 (no statistically significant difference in telomere length); and G5 mTR−/− mice before versus after treatment, P = 0.06622803 (no statistically significant difference in telomere length).
Increased Radiation-induced Chromosomal Damage in IR-sensitive G5 mTR−/− Mice.
A reduction in, or lack of, recovery after split/fractionated doses of radiation, as observed here in 60% of the G5 mTR−/− mice, may be indicative of a higher radiation-induced DNA damage. To address this indication, we performed a cytogenetic analysis of unirradiated and gamma irradiated wt and G2 and G5 mTR−/− mice. For this, fresh BM chromosome preparations were made and stained with concentrated paint probes for chromosomes 1, 2, and 3 simultaneously (Fig. 4). The frequency of aberrations involving chromosomes 1, 2, and 3 was determined by chromosome painting. As previously described, we detected an spontaneous frequency of end–end fusions in unirradiated G5 mTR−/− animals (Table 4) (15)(16). Gamma radiation exposure elevated the frequency of aberrations in all genotypes (Table 4). Importantly, whereas wt and G2 mTR−/− mice did not show any end–end fusions or deletions upon irradiation (zero Robertsonian fusions, dicentrics, and deletions per 100 metaphases; Table 4), the frequency of these chromosomal aberrations was significantly increased in the group of irradiated G5 mTR−/− mice as compared with nonirradiated controls. For statistical analysis, a chi-squared test was performed: dicentrics, none induced in wt or G2 mTR−/− mice; G5 mTR−/− (unirradiated, 1/347; irradiated, 9/259), chi-squared = 9.11, 1 degree of freedom (df), P < 0.01, statistically significant; deletions, significant only in G5 mTR−/− mice (unirradiated, 0/347; irradiated, 6/259), chi-squared = 8.06, 1 df, P < 0.01, statistically significant; Robertsonian-like fusions (p-arm events), none scored in wt or G2 mTR−/− mice; G5 mTR−/− (unirradiated, 2/347; irradiated, 28/259), chi-squared = 31.39, 1 df, P < 0.001, statistically significant; telomeric associations + Robertsonian-like fusions (p-arm events), wt and G2 mTR−/− none scored, and G5 mTR−/− (unirradiated, 27/347; irradiated, 49/259), chi-squared = 14.67, 1 df, P < 0.001, statistically significant. In contrast, the frequency of chromatid-type aberrations such as chromatid breaks, isochromatid breaks, and chromatid exchanges was not significantly elevated in irradiated G5 mTR−/− mice (2.4 per 100 metaphases) as compared with similarly irradiated wt (0.5 per 100 metaphases) or G2 mTR−/− mice (2.2 per 100 metaphases) (Table 4).
Normalized Frequencies of Chromosomal Aberrations in BM Cells of wt and G2 and G5 mTR−/− Mice
Representative chromosomal aberrations in irradiated G5 mTR−/− BM cells. (A) Control, unirradiated wt cell with no aberrations in chromosomes 3 (red), 2 (green), or 1 (red/green). (B–E) Aberrations observed in irradiated G5 mTR−/− cells: interhomologue Robertsonian fusion of chromosome 1 (B), interchromosome Robertsonian fusion involving chromosome 3 and an unidentified chromosome (C), translocation between chromosome 2 (green) and chromosome 3 (red) (D), and q-q arm dicentric between chromosome 3 and an unidentified chromosome (E). See Table 4 for complete cytogenetic analysis of irradiated control and mTR−/− mice.
DNA DSB Repair in wt and Different Generation mTR−/− Mice.
IR is a potent inducer of DSB in the DNA, and many radiosensitive phenotypes are associated with defects in DSB repair. There are two major mechanisms responsible for DSB repair in mammalian cells, NHEJ and HR. To investigate if any of these pathways is affected in late generation mTR−/− mice, we determined: (a) SCE frequencies in wt and G6 mTR−/− primary splenocytes as an indication of HR frequencies (33) and (b) V(D)J recombination in thymus and BM from wt and G6 mTR−/− mice (34) as an indication of end-joining activities.
SCE Frequencies.
Baseline SCE frequency in unirradiated G6 mTR−/− splenocytes was slightly elevated compared with wt (8.4 ± 0.73 and 6.6 ± 0.39, respectively). Student's t test P < 0.05 indicates a statistically significant difference; Fig. 5 shows representative examples and quantification of SCE frequencies at different doses. Radiation exposure elevated SCE frequencies in both genotypes in a dose-dependent fashion (10.4 ± 2.4 and 12.2 ± 1.4 for 0.5 and 1.0 Gys, respectively, in the case of wt cells and 11.0 ± 2.8 and 14.1 ± 4.8 for 0.5 and 1.0 Gys, respectively, in the case of G6 mTR−/− cells; Fig. 5). The absolute frequencies of SCE attained in G6 mTR−/− cells were higher than in wt, but the differences were not statistically significant (Student's t test, P > 0.05). The numbers of SCEs induced over baseline were comparable (Fig. 5). As SCEs arise largely as a consequence of HR (33), these data indicate that HR is active in G6 mTR−/− cells. The slight elevation of baseline SCE may indicate an elevated HR activity in G6 mTR−/− cells; similar elevations have been noted in cells deficient in components of the NHEJ pathway, such as in Ku86−/− cells (34).
Examples of spleen cell metaphases stained to visualize sister chromatid exchanges. wt metaphase, unirradiated. G6 mTR−/− metaphase, unirradiated. SCEs can be seen at points at which dark and light staining exchanges between chromatids. Frequencies of baseline and gamma radiation–induced SCE in wt and G6 mTR−/− splenocytes are also shown. 25 metaphases scored per treatment.
V(D)J Recombination.
The variable region genes of Igs and TCR molecules are assembled from germline segments during development of B and T lymphocytes by a DNA rearrangement process known as V(D)J recombination (35). The V(D)J recombination process involves a step of cleavage or introduction of DSB at specific sequences and a joining step. Several mutations that affect V(D)J recombination also affect NHEJ DSB repair, such as Ku70−/−, Ku86−/−, and DNA-PKcs (35), indicating that a defective V(D)J recombination could be indicative of a DSB NHEJ repair defect. To determine whether G6 mTR−/− mice showed a defective V(D)J recombination, we first studied the formation of Ig heavy chain rearrangements in the B cell lineage using a semiquantitative PCR assay on genomic DNA from BM cells. Two different wt and two G6 mTR−/− mice showed similar formation of DH-JH4 and VH7183-DJH coding joints (Fig. 6 A). As expected, Ku86−/− mice did not show any of the two rearrangements (Fig. 6 A). A positive control for input DNA is also shown (Fig. 6 A). To detect coding joints formed by TCR rearrangement, semiquantitative PCR analysis of thymocyte DNA preparations was performed to detect Vβ8-DJβ2.6 joints. Similar amounts of PCR products of the appropriate sizes were detected in two independent wt and G6 mTR−/− mice but not in the Ku86−/− mice, as expected (Fig. 6 A). These results clearly show that late generation mTR−/− mice do not have a defect in V(D)J recombination, indicating that the DSB DNA repair activities involved in this process are fully functional in the late generation mTR−/− mice (Fig. 6 A). To rule out that a defect in NHEJ-mediated DSB repair in late generation mTR−/− mice could be due to degradation of some of the major DNA repair proteins, we studied DNA-PKcs, Ku70, and PARP status in wt and G6 mTR−/− nuclear extracts. As shown in Fig. 6 B, all of these DNA repair proteins were present in both wt and G6 mTR−/− extracts to similar levels (Fig. 6 B).
(A) Joint formation in two wt (1 and 2), one Ku86−/− (1), and two G6 mTR−/− mice (1 and 2). For analysis of Ig coding joins, two sets of primers were used: D-JH rearrangement, primers DHall and JH4; V-DJH rearrangement, primers JH4 and VH7183. The JH4-specific probe (DR218) was used for the detection of Ig coding joints. The TCR coding joins Vβ8-DJβ2.6 were detected using the primers Vβ8.1, 2, 3 and Jβ2.6 for PCR and the Vβ8 probe for detection. An IVS from the heavy chain locus that is not affected by Ig rearrangement was amplified as a control for PCR reaction (IVS) using 5′-IVS and 3′-IVS primers. (B) Western blots to detect endogenous levels of DNAPKcs (∼470 kD), Ku70 (∼70 kD), and PARP (∼110 kD). Western blots were carried out as described in Materials and Methods. The positions of the different specific bands are indicated by arrows. As loading control, RNA pol II levels were detected in the different extracts.
Conclusions.
The study of telomerase-deficient mice with different telomere lengths has provided strong evidence that telomere length and hypersensitivity to IR are connected in mammalian cells. The absence of telomerase activity per se does not affect radiation sensitivity when telomeres are sufficiently long (G2 mTR−/− mice). Importantly, gamma IR per se does not result in significant telomere shortening in telomerase-deficient mTR−/− mice, suggesting that exhaustion of proliferative potential of cells is not likely to be the cause of the phenotype. Indeed, we show that fractionated gamma radiation induces a high level of chromosomal damage in the G5 mTR−/− mice as compared with wt or G2 mTR−/− mice. Importantly, similar SCE frequencies and V(D)J recombination efficiency of wt and late generation mTR−/− mice suggest that decreased HR or NHEJ efficiency is not the direct cause of higher DNA damage present in irradiated G5 mTR−/− mice. Thus, short telomeres do not seem to be affecting the efficiency of the DSB repair machinery as we would have predicted from studies in yeast (7)(8). Indeed, the radiation-sensitive phenotype of G5 mTR−/− mice is consistent with a direct role of telomeres in the DNA damage response. It is likely that the DNA repair machinery regards short telomeres as DNA breaks, triggering end–end fusions such as the Robersonian fusions and dicentrics found in irradiated G5 mTR−/− mice. We believe that the results presented here could have important implications for both radiotherapy and radiation protection. Telomerase inhibition in tumors (i.e., chemotherapy with telomerase inhibitors) could result in telomere loss, which in turn is likely to render the tumor more sensitive to radiotherapy. Similarly, telomere length may be one of the factors contributing to interindividual variation in radiation sensitivity.
We are indebted to G. Taccioli for V(D)J primers, P. Bonner and R. Hidalgo for gamma irradiations, E. Santos for mice genotyping, and M. Serrano for discussions.
F.A. Goytisolo and E. Samper have fellowships from the regional government of Madrid (Comunidad Autónoma de Madrid). Research at the laboratory of M.A. Blasco is funded by The Swiss Bridge Foundation and by grant PM97-0133 from the Ministry of Science and Technology, Spain, grant 08.1/0030/98 from CAM, grants EURATOM/991/0201 and FIGH-CT-1999-00002 from the European Union, and by the Department of Immunology and Oncology, Centro Nacional de Biotecnología-CSIC. This work was supported by EU Concerted Action F14PCT90066.
Abbreviations used in this paper: BM, bone marrow; DSBs, double strand breaks; FISH, fluorescence in situ hybridization; HR, homologous recombination; IR, ionizing radiation; IVS, intervening sequence; mTR, mouse telomerase RNA; NHEJ, nonhomologous end joining; PARP, poly(ADP-ribose)polymerase; SCE, sister chromatid exchange; wt, wild-type.
Submitted: 4 August 2000
Revision requested 9 October 2000
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© 2000 The Rockefeller University Press
Journal of Experimental Medicine Dec 2000, 192 (11) 1625-1636; DOI: 10.1084/jem.192.11.1625
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