On FlashPaper
A Format for Sharing Documents on the Web
An image embedded in FlashPaper. (Coffee with a drop of milk)
One of our goals at Scribd is to create "the best way to publish a documentonline." For short and simple documents, it is generally best to display the text inHTML format. However, for any document that is more than a few pages long or has complex formatting, HTML doesn't work very well. We could allow people to just attach their document as a PDF or Microsoft Word file, but then the sitewouldn't be any fun to browse, because users would have to download eachdocument they want to view. We saw the difficulty with sharing formatteddocuments as a real problem, and we looked for a way to solve it.What we found was a product called Macromedia FlashPaper, which we hadactually never heard of. The document you’re reading now, by the way, is inFlashPaper format. FlashPaper documents are Flash SWF files that look andfunction a lot like PDF files.
T
FlashPaper works great for technical articles, faithfully preserving formatting.
A
role
for
the
anaphase-promoting
complex
inhibitor
Emi2 XErp1,
a
homolog
of
early
mitotic
inhibitor
1,
in
cytostatic
factor
arrest
of
Xenopus
eggs
Jeffrey
J.
Tung*
†
, David V.
Hansen*
†
, Kenneth H.
Ban*
†
, Alexander V.
Loktev*,
Matthew
K.
Summers*,
John
R.
Adler
III*,
and
Peter
K.
Jackson*
†‡
*Department
of
Pathology
and
†
Program
in
Cancer
Biology,
Stanford
University
School
of
Medicine,
300 Pasteur
Drive,
Stanford,
CA
94305
Communicated
by
Marc
W.
Kirschner,
Harvard
Medical
School,
Boston,
MA,
February
9, 2005
(received
for
review
November
30, 2004)
Unfertilized
vertebrate
eggs
are
arrested
in
metaphase
of
meiosis
II
with
high
cyclin
B
Cdc2
activity
to
prevent
parthenogenesis.
Until fertilization,
exit
from
metaphase
is
blocked
by an activity
called cytostatic
factor
(CSF),
which
stabilizes
cyclin
B
by
inhibiting
the
anaphase-promoting
complex
(APC)
ubiquitin
ligase.
The
APC
in-
hibitor
early mitotic
inhibitor
1
(Emi1)
wasrecently
found
to
be required
for
maintenance
of
CSF
arrest.
We
show here
that
exog-
enous
Emi1
is
unstable
in
CSF-arrested
Xenopus
eggs
and
is
destroyed
by
the
SCF
TrCP
ubiquitin
ligase,suggesting
that
endog-
enous
Emi1,
an
apparent
44-kDa
protein,
requires
a
stabilizing
factor.
However,
anti-Emi1antibodies
crossreact
with
native
Emi2Erp1
FBXO43,
a homolog
of
Emi1
and
conserved
APC
inhibitor.
Emi2
is
stable
in
CSF-arrested
eggs,
is
sufficient
to
prevent
CSF
release,
and
is
rapidly degraded in a
Polo-like
kinase
1-dependent
manner in
response
to
calcium-mediated
eggactivation.
These results
identify
Emi2
as
a
candidate
CSF
maintenance
protein.
cyclin
B
meiosis
maturation-promoting
factor
oocyte
maturation
o
prevent
parthenogene
sis,
unfertilized
eggs
f
rom
many
an
imals
arre
st
in
met
aphase
of
meiosis
II
(MII).
Sperm
penetration
triggers
the
release
f
rom
met
aphase
arre
st
and
the
c
ommencement
of
alternating
cycles
o
f
DNA
replication
and
cell
div
ision
in
the
embr
yo.
The
regulator
y
basis
for
met
aphase
II
arre
st
was
first
characterized
in
f
rog
eggs
30
years
ago
and
ter
med
c
y
tost
atic
factor
(CSF)
(1).
CSF
is
operationallydefined as
an
activ
it
y,
rather
than
a
single
molecule,
pre
sentin
unfer- tilized
eggs
that
blocks
cleavage
of
div
iding
blastomere
s
upon
injection
(rev
iewed
in
ref.
2).
Mos,
an
activator
of
the
mitogen- activated
protein
k
inase
Rsk
pathway,
is
a
key
c
omponent
of
CSF
that
appears
at
the
onset
of
meiosis
I
(MI)
and
activate
s
CSF
to
block
cleavage
of
blastomere
s
(3).
The
anaphase-promoting
c
omplex
(A
PC)
is
an
E3
ubiquitin
ligase
that
triggers
M-phase
ex
it
by
directing proteasome-
dependent
cyclin
B
destr
uction
(4),
re
sulting
in
the
sw
if
t
inac-
tivation
of
the
cyclin
B
Cdc2
k
inase,
or maturation-
promoting factor
(MPF)
(5,
6).
A
rise
inintracellular
calcium
af
ter
fertil- ization
induce
s
met
aphase
II
release
by
relieving
the
A
PC
f
rom
repre
ssion.
Early
mitoticinhibitor
1
(Emi1),
originally
cloned
f
rom
a
Xenopus
ooc
y
t
e
cDNA
librar
y,
blocks
the
cleavage
of
injected
blastomere
s
similar
to
CSF
(7)
and
ef
ficiently
inhibits
the
A
PC
in
vitro
(8).Recently,
Emi1
was
shown
to
be
requiredUpon
fertilization
of
Xenopus
eggs,
calcium
signaling
i
nactivate
s
CSF
arre
st,
which
require
s
the
Xenopus
Polo-like
k
inase
1
(Plx1).
The
t
a
rget
of
Plx1
in
this
pathway
remains
unk
nown
(13).
I
n
humansomatic
cells,
MPF
and
human
Polo-like
k
inase
1
(
Plk1)
t
arget
Emi1
f
or
deg
radation
by
the
Skpl
Cullin
F-box
protein
(SCF)
TrCP
ubiquitin
ligase
(14
–17).
Specifically,
Plk1
phosphor
ylate
s
Emi1
on
its
DSGxxS
sequence,
creating
a
c
onsensus
deg
ron
recognized
by
TrCP
(17).
Thus,
Xenopus
Emi1(xEmi1)
c
ould
b
e
a
Plx1
t
arget
downstream
of
calcium
signaling.
A
n
apparent
paradox
is
how
Emi1
levels
are
sustained
in
the
CSF-arrested
egg
amid
h
igh
MPF
and
Plx1
activ
i
tie
s
.
I
n
line
w
iththis
paradox,
a
recent
report
suggests
that
Emi1
is
unstable
andundetect
able
i
n
Xenopus
eggs
(18).
On
the
other
hand,
Emi1
appears
to
be
pre
sent
in
mouse
eggs
(10).
In
this
study,
we
want
to
clarif
y
our
underst
anding
of
Emi1
regulation
in
Xenopus
eggs
and
find
t
hat
Emi2,
an
Emi1
homolog,
may
c
ontribute
to
CSF
arre
st.
Methods
Reagents.
Sera
f
rom
four
rabbits
immunized
w
i
th
maltose
binding
protein
(MBP)-Emi1
fusion
protein
were
af
fin
it
y-purified
b
y
f low-
ing
over
a
c
olumn
of
GST-Emi1
i
mmobilized
on
CNBr-Sepharose
re
sin
w
ith
acid
e
lution.
Other
antibodie
s
used
wereagainst
-cate-
n
i
n,
cyclin
B2,
Plx1,
Plk1
(Zymed),
myc
epitope,
and
actin
(Sant
a Cr
uz
Biotechnolog
y).
xEmi2
was
PCR-cloned
f
r
om
an
ooc
y
t
e
cDNA
librar
y,
and
a
human
Emi2
(
hEmi2)clone
was
purchased
f
rom
Inv
itrogen.
pCS2-cDNA
c
onstr
uctswere
linearized
and
in
vitro
-transcribed
to
generate
mRNA
by
using
a
mMe
ssage
Machine
k
i
t
(Ambion,
Austin,
TX).
pCS2-cDNA
c
onstr
ucts
were
in
vitro
-
translated
(I
VT)
in
rabbitreticuloc
y
t
e
l
ysate
(TNT,
Promega)
and
labeled
w
i
th
35
S-methionine.
A
ll
Emi1
and
Emi2
e
x
periments
used
Xenopus
sequence
s
unle
ss
other
w
i
se
noted
as
hEmi1
and
hEmi2
f
or
humansequence
s.
MBP-fusion
proteins
and
GST-Plk1
were
ex-
pre
ssed
in
Escher
ichia
coli
and
purified
b
y
batch
binding
bacterial
protein
l
ysate
to
af
fin
it
y
r
e
s
in
and
elution
w
ith
maltose
or
gluta-thione,
then
dialyzed
into
XB
buf fer
(
20
mM
Hepe
s,
pH
7.7 100
mM
KCl).
Point
mut
ations
were
engineered
w
i
th
a
QuikChange
k
i
t
(Strat
agene).
Handling
of
Xenopus
Oocytes.
Ooc
y
t
e
s
were
obt
ained
and
proce
ssed
for
H1
k
i
nase
activ
it
y
and
immunoblot
as
de
scribed(19).
Ooc
y
t
e
s
were
injected
w
i
th
30
ng
of
MBP-Emi1
f
usion
protein
o
r
10
n
g
o
f
various
mRNA
in
tot
al
volume
s
not
exceeding
50
nl.
Maturation
for
maintenance
of
CSF
arre
st
in
f
rog
and
mouse
eggs.
Immu-
nodepletion
of
Emi1
f
rom
Xenopus
CSF
egg
extract
cause
s
rapid
cyclin
B
proteolysis
and
ex
it
f
rom
met
aphase
arre
st
independent
of
calcium
mobilization,
and
ablation
of
Emi1
by
small
inter
fer-
ing
RNA
in
mouse
ooc
y
t
e
s
induce
s
parthenogene
sis
(9,
10).
Recent
work
has
shown
that
the
Mos
mitogen-activated
protein
k
inase
Rsk
pathway
e
s
t
ablishe
s,
but
is
not
required
to
maintain,CSF
arre
st
(11,
12).
Therefore,
CSF
arre
st
is
a
c
omplex
proce
ss
e
s
t
ablished
by
the
mitogen-activated
protein
k
inase
pathway
and
maintained
through
inhibition
of
the
A
PC.
Freely
available
online
through
the
PNAS
open
access
option.
Abbreviations:
APC,
anaphase-promoting
complex;
CHX,
cycloheximide;
CSF,
cytostatic
factor;
Emi,
early
mitotic
inhibitor;
hEmi,
human
Emi; xEmi,
Xenopus
Emi;
GVBD,
germinal
vesicle
breakdown;
IVT,
in
vitro-
translated;
MI,
m
eiosis
I;
MII,
meiosisII;
MBP,
Maltose
binding
protein;
MPF,
mitosis-promoting
factor;
Plk1,
human
Polo-like
kinase
1;
Plx1,
Xenopus
Polo-like
kinase
1;
SCF,
Skpl
Cullin
F-box
protein.
Data
deposition:
The
sequence
reported
in
this
paper
has
been
deposited
in
the
GenBank
database
(accession
no.
AY928267).
‡
To
whom
correspondence
should
be
addressed.
E-mail:
pjackson@stanford.edu.
©
2005
by
The
National
Academy
o
f
Sciences
of
the
USA
4318
–
4323
PNAS
March 22, 2005
vol.
102
no.
12
www.pnas.org
cgi
doi
10.1073
pnas.0501108102
Search History:
Result 00 of 00
00 results for result for
p.
Notes
Load more
