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Journal of Histochemistry
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BIOCHEMICAL AND HISTOCHEMICAL IDENTIFICATION OF SIALIC ACID CONTAINING MUCINS OF
RODENT VAGINA AND SALIVARY GLANDS
LEONARD WARREN and SAMUEL S. SPICER
J Histochem Cytochem 1961 9: 400
DOI: 10.1177/9.4.400
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BIOCHEMICAL
ACID
AND
HISTOCHEMICAL
CONTAINING
MUCINS
SALIVARY
Institutes
Received
The
mucification
thehium
reaction
of the
gated
mouse
extensivehy
the
mucus
this
filled
cells
oestrous
anti
phases
pregnancy
process
16).
flicting
studies
has
have
involved.
dioestrous
more
been
stuolietl
from
carried
prusent
histochemical
(23) methods
sulfated
siahic
which
in
vagnma
and
thie
establishing
mucification
is
that
sialomucimi
of the vagina
also
shown
sialomucins
with
of
that
differ
respect
cheniical
to
form
is
the
hiistochemically
accoroling
to
enzymatic
of siahic
and
auicl
and
before
organ
and
rats
killing
each
the
For
dry,
by
was
acid,
and
then
homnogenized
was
were
heated
acids.
thiobarbituric
of
at
acid
homogenate
mg
of
Norit
N
80#{176}
C for
were
method
chromatography
in
0.5
the
1 hour
to
was
of
10 g
of sialic
the
* 1 filter
paper
by
ascending
the
N
of
then
Aliquots
which
and
separate
HC1,
: Solvent
1:2:1
ammonia,
(21).
80:20:1.
chromatograms
spray
reagent
each
tissue
and
also
by
(24).
For
extract
with
was
added
Sialidase
fluid
by
M.
was
an
embedding
Deparaffinized,
for 30 minutes
rehydrated
in 1%
acid
sohmtion
followed
stain
according
to
Mowry
and
acid
thuis
pH
Winkler
puirplish
red
method
a 30
1.5-3.0
stained
minute
(8)
prior
to
sectioning
sections
blue
at
in
a periodic
combined
were
3%
acid
procedure
This
or purple
method
blue
colors
and
neutral
(magenta).
of
staining
exposure
phosphate-citrate
appear
obtained
imfor 24 hours
in
alcian
by
the
(11).
mmiucins blue
second
involved
in
method
J. S. Anderson,
and
Nutrition,
and
Schiff
release
and
Tissues
were fixed
formalin
methods:
mortem
acetate
acetic
by
obtained
perfringens
of Nebraska.2
paraffin
ns
method
were
unpublished
B. Rhodes
Biochemistry
of
also
Clostridium
of
25-fold
roumtine
A
the
Vibrio
cholerac’
by the
(1). The best preparations
culture
post
calcium
muici
of
homog-
assayed
was
acid
on the
alone
mediately
neuitral
homogenates
and
located
3 ml
Norit
of
water:
purified.
the
many
ml
both
acid
purifleti
of
blotted
somewhat
with
to
azure
0.02%
buffer
more
azure
(8).
A
A
Mucins
metachro-
(23).
time hydrolysate
A was
of
analysis
were
to the
0.1
Aliquots
Part
hours
Ethanol:
medium
of
and Fremichi
5
L.
stained
hours
examination.
tissues
added
concentration
sialic
24
chemnical
histologic
bound
tissue
for
for
acid
fasted
anaesthesia.
used
sialic
enates
5-10
were
of
Sulfuiric
a final
mice
Whatman
solvents,
Histochemical
the
present.
chloroform
remainder
weighed
For
and
studied
analysis
water.
to
NIH
the
ethanol.
identification,
University
EXPERIMENTAL
Stock
70%
on
were
acid
and
species
digestibility
on
and
with
approximately
two
of H. E. Feeney,
Departmnent
rat.
identified
site
of
thiobarbituric
fromn
and
ml
washed
fromn N-acetylneuraminic
n-propanol:
0.1
1800-fold
the
in the
mouse
acid
24
2.
culture
of Ada
presented
is elaborated
in
sialic
obtained
decanted
authentic
N-acetylneuraminic
acid isolated
from
human
bloooi serum
(22, 26).
Siahidase
(neuraminidase)
was purified
from the
em-
of
the
solution
was
A was
chromatographed
non-
mucins
Eviolence
in
sialic
identify
been
Norit
placed
acids
the
devel-
from
The
0.5
were
Sialic
biochemical
sulfated
supernatant
centrifuging
containing
Solvent
mucins
recently
and
specifically
numcimis,
have
glamitls.
10,
con-
time
with
method
sulfated
few chemi-
aiiol
characterization
salivary
(5,
eluted
N-glycolyl
1. n-Butanol:
of
on
19, 20)
distinguish
acid
mucimis,
acid-containing
ployed
ouit
report
(17,
if
in detail
and
The
and
chromatographed
of hormones
preliminary
water
acid
and
abundant
influence
aside
been
the
In
opeol
It
lxcome
The
However,
the
1960
(14).
stirring
eluiates
during
data
suggesting
the presence
and nonsulfated
acid mucus
(19)
( 25)
cal
to
supervenes.
this
13,
in
of
fluctuate
disappearimig
reappearing
pro-oestrous
on
epithehium
11,
discarded.
that
layer
14, Maryland
October
after
investi-
It is known
S. SPICER
Bethesda
solution
epi-
been
superficial
cycle,
Health,
publication
vaginal
has
imi the
squamous
the
oestrus
rat
(7, 9, 12, 15).
stratified
with
the
in
and
for
SAMUEL
AND
of
OF SIALIC
AND
GLANDS
WARREN
LEONARD
National
IDENTIFICATION
RODENT
VAGINA
OF
centrifuged
adided
to
of
and
the
1
each
for
about
2
supernatant
Miss
We should
like to thank
Dr. John
Hornibrook
growing
Vibrio
cholerae.
Pumrification
of siahidase
was
carried
out
C. Spearing.
400
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by
IDENTIFICATION
if the
matic
in place
section
of the
alcohols
and
resinous
dehydration
xylol
medium
prior
such
to
as
RAT
mounting
cellulose
a
Combined
staining
with Halmi’s
aldehyde
fuchsimi
(4) followed
by alcian
blue or with
alcian
blue
followed
by aldehyde
fuchsin
was carried
out as
described
previously
(19).
N,N
- Dimethyl
p - phenylenediamine
monohydrochloride
was employed
as a reagent
for the
histological
demonstration
of
periodate
engendered
the
aldehydes
periodic
enediamine
(18).
of
In
procedure
brief
either
sections
24
to
at
tissue
sections
dried
by
section
This
of
in
0.1
ml
buffer
protein
in
and
as
37#{176}Cin
tissue
sections
enzyme-buffer
contaimied
ions.
case
35 g
It
of
of
also
con-
the
Vibrio
protein
with
in ucin
of the
TABLE
Staining
properties
of vaginal
and
Mouse
Alcian
A
Azure
A pH
alcian
++++
2.5
fuchsin
+++
then
sialic
fluids
and
were
sections
distilled
were
water.
The
acids.
++++
characterization
mouse
of
vaginal
mucus:
and
blue
rat
in
untreated,
nmethilated,
sections
Mild
purple
1)11 1.5
Aldehyde
digested
No Treatment
blue-PAS4
Azure
of
occa-
I
pregnant
sialidase
Stain
Species
nil
on
solution.
In alcian
blue periodic
acid Schiff stained
sections,
the surface
mucus
of the pregnant
mouse
or rat
vagina
reveals
the purple
blue color characteristic
of many
mucopolyasccharides
(Table
I, Fig.
1).
The lack of reactivity
with the 4 hour dimethylp-phenylenediamine
exposure
after
11104 and the
greyish
purple
coloration
with
time prolonged
diamine
exposure
also point
to the presence
of
acid mucopolysaccharide
(Table
I, Fig. 5). Like
nonsulfated
acid mucins
with strongly
dissociated
carboxyl
groups
or sulfated
mucins
with partially
masked
sulfate
esters,
this material
colors
meta-
by
at
solution
the
total
Histochemical
deparaffinized,
of
or
RESULTS
(0.01
N).
0.04 M calcium
preparation
cholerae
for
basophilia,
the
0.1
of
sections
buffer,
supernatant
pipette
phenyl-
-
citrate
4 hours
mucins
5.25
and
5 g
to
accomplished
(0.1
layering
with
pH
M acetate
tained
of
out
solution.
for
HCI
digestion
carried
p
of
was
Comitrol
enzyme-buffer
fine
39#{176}C,
layer
oxidized
M)
blockade
(17),
with
at
a
combined
supernatant
and rinse
solutions
were
assayed
for sialic
acid by the thiobarbituric
acid
procedure
(23).
I)igested
and
control
sectioiis
were then
stained,
or in sonie experiments
were
scraped
into test tubes,
hydrolyzed
and assayed
6.5.
pH
twice
histologic
hours
above
with
the
a
18
dishes.
inactivated
with
The
to
close
Petri
digestion,
rinsed
elsewhere
-
(0.03
After
rods
similarly
heat
aspirated
an exposure
periodate
N,N-dimethyl
methanolic
Sialidase
of
covered
with
3
glass
incubated
sion
phenyl-
-
described
a phosphate
described
immersing
0.1
according
p
involves
hours
methylation”
preheated
be
method
48
0.1%
previously
air
or
in
“Mild
sections
-
to
new
this
6,
enediamine
M) buffer
was
in tissue
acid-N,N-dimethyl
in
were
incubated
on
water
enzyme.
perfringens
were
supported
(8a).
401
SIALOMUCINS
Clostridium
sections
with
caprate
MOUSE
AND
dry with filter paper
through
graded
is blotted
usual
OF
Methylation
++++
Blockade
red
Sialidase
++++
Digested
Sections
red
unstained
unstained
unstained
purple
unstained
uinstai
ned
blue
blue
Alcian
hyde
blue
then
fuichsin
PAD**
4 h
alde-
++
blue
&
purple
unstained
++
brown
+++
orange
brown
+++
brown
brown
PAD 48 h
Alcian
blue-PAS
Rat
Azure
A pH
+++
++++
1.5
alcian
blue
blue
hyde
*
Periodic
Periodic
acid
then
+++
then
ahde-
++++
Schiff
+++
++++
red
++
purple
blue
blue
unstained
&
purple
procedure.
N-dimethyh-p-phenylenediamine
++++
unstained
blue
fuchsin
acid-N,
purple
blue
unstained
Azure
A pH 2.5
Aldehyde
fuchsin
Alcian
grey
purple
procedure.
Downloaded from jhc.sagepub.com by guest on June 11, 2014
+++
purple
blue
unstained
purple
blue
402
L.
chironiaticmtlly
Fig.
3)
at
hut
sulfated
staimi
with
blue
tinily
the
w-ith
color
I)
method
blockade
(19).
(Table
forniation
iii
miomisuilfated
A an(i
I)
(17)
are
for the
sulfated
niuicins.
as
of
as
of alcian
vaginal
mnucus
in
the
had
with
alcian
75%
with
acid
siahiolase,
frommi
which
mucins
comparisomi,
siniila
accomplished
by
selectively
(Table
r
I,
cleaves
Figs.
2,
histochemnical
sialidase
4,
6).
changes
digestion
of
the
mouse
sublimigual
glitnol are shown
in Figs.
7-10.
Here
also
the
emizymne
specifically
remnoves
exposed
sialic
acid groups
of the polymiier
responsible
for
alcian
blue
affinity,
for mnetachromasia
at pH
2.5, ammo!, as observed
in experiniemits
not illuist rated
here,
for the
ation
slow
the
in
diamnine
reaction
post
and
H104
l)ro(edltmre
lack
of brown
color-
10%
alcian
a
positive
from
the
section
supernatant
fluid
Iii Table
niemit. designed
FIG.
staimi.
Fma.
staimiing
FIG.
of
vaginal
epitheliumii
in 16 hours
(Table
II).
results
of an
III are seen
to
1. Mouse
X200.
2. Section
assess
vagina
Mouse
the
in
adjacent
characteristic
3.
the
iii
acid
To
1,
which
digested
16
showing
staimiing
sialic
acid
sialic
staining
still
had
of
the
section
after
released.
of the
When
section
remiiained
detectable.
hr
be
sialic
evemi
been
was
6
could
tissuie
observed,
acid
cleavage
of
noted
are
Siniilarly
dimnethyl-p-phemiylene-
approximately
particularly
digested
to
sections
alone
stained
appear
half
is the
appear
alcian
liberated
tions
of
blue
by
the
of
the
observation
by
from
there
blue
hours
the
tissue
Sialic
acid
with
Alcian
metachromnatic
solution
mnucin.
by
cholerae
acids,
residue.
acid
upon
Azure
measurable
hydrolyzing
periodic
A
tissue
acid
Note
Schiff
the
stain.
stain
to
deterMore-
treated
acid-Schiff
mucin.
measured
thiobarbituric
in contrast
sialidase.
blue-periodic
sialic
prepara-
been
of sialidase
blue
free
fragments,
siahic
of the
assay
Alcian
Vibrio
only
has
in siahic
acid
ant
acid
Schiff
muet hod
sectiomis.
by the
procedure,
is no increase
ex-
digested
sections
acid
of measuring
unbound
forni
thiobarbituric
suipernat
after
whereas
periodic
acid
as umidigested
supernatant
amid this
methods
only
the
the
the
samne
unstained
alone,
is the fact that
sialic acid-containing
sialidase.
in the section
acid method,
mucopolysaccharide.
pregnancy
of the
released,
Alt hough
.
26%
i)luie
posure
the
purple
i micreased
about
was
of
dimet.hyl-p-
only
orange
be
over
experi-
showing
Fig.
the
u)f alcian
pregnancy
to
sialic
into
dependency
of neuitral
vagina
the
HIO4
that
sialidase
changes
the reaction
of the niucins
toward
the alcian
blue periodic
acid Schiff
stain
from the puirple
blue of acid mucopolysaccharide
to the magenta
or red of neutral
mucopolysaccharide,
indicating
that the siahic acid end groups
are hydrolyzed
without
appreciable
degradation
of tue periodic
acid
reactive
polymer.
Sections
other
mimics
of
post
affinity
blue
blue
characteristics
acid.
arations
all
ng
from
alcian
blue
of the
after
is
essentially
released
by
alcian
Also noteworthy
acid, rather
than
ciiniethyl-p-phenylene-
(18).
acid
acid.
digested
intervals
diamine
reaction
was discernable
when 20% of the
sialic acid had been removed,
amid reached
a maxi-
Assay
of the siahic
acidi
released
into the supermiatamit fluid amid that remnainirig
in the tissue
sections
after
emizymiiatic
digestion
substantiates
the
idemitification
of sialomucimis.
The sialidase
prepreleased
the
no
sialic
siahic
the
sialic
in
beemi
of
staimii
l)rocedtire
after
tissue
gland
were
for various
staining
by
decrease
acid
mum
of alcian
tiomi
of
of siahic
t he
miiucimis
some
blue stainimig
and
azurophilic
miietachromiiasia,
and
the alteration
of post 11104
dimnethyl-p-phienylenediamnine
staimming
by diges-
For
from
amid
neutral
cie-
structiomi
derived
amount
increased,
reased
only
acids
methylatimig
tue
a sialomumcimi
di-
interpreted
blockade
dec
detected
HIO4
of carboxylic
As
sections
phenylemiethiamiiine
miiethylation
Stronger
iclemitification
moumse
in
acid
post
be
esters
J)ar-
reactivity
mild
mnay
required
in
the
a
level
the
the
previously
of acidic
fromii
niticins.
affinity
Specific
loss
azure
of miiethyl
conditions
ies
nonsulfated
omi the
of tinie.
fuchsiri
of
staining
mucin
least
pru)pert
blue
SPICER
achro-
met
at
aldlehvde
The
resulting
the
bluie
with
are
alciutmi blue,
amnimie
and
S.
mouse
suhlimigual
of Vibrio
cholerae
mmmialdehvde
iii
S.
Sections
of
by siahidase
where
of this
blue
I,
1.5,
show
sequence,
AND
(Table
pH
tendency
characteristic
(Table
A
at
alcian
sequence
as
toward
the
purple
reverse
recognized
muicins
noted
blue
tti
azuire
rides
formuerly
fuchsimi-alciami
the
2.5
uiiistutined
mu ticopolvsacchia
The
miiasiit.
to
pH
remnains
WARREN
at
red
X200.
pH
2.5.
X200.
FIG.
4. Section
adjacent
to Fig. 3, digested
16 hiours
with
Vibrio
cholerae
sialidase.
Note
nietachromatic
azurophihia
from epithehial
mucin
and stained
mnetachromasia
in mast cells in the
propria.
Azure
A at pH 2.5. X200.
FIG.
5. Moumse vagina
in pregnancy
showing
greyish
purple
acid mucimi. Periodic
acid-diamine
48
hours.
X200.
Fmtu. 6. Section
staimiing
FIGS.
lingumal
to Fig. 5, digested
16 hours with Vibrio
of neutral
mucopolysaccharide.
Periodic
7, 8, 9 amid 10 resemble
Figs. 1, 2, 3 and 4, respectively
adjacemit
characteristic
gland
cholerae
sialidase.
acid-diamine
except
that
staining.
Downloaded from jhc.sagepub.com by guest on June 11, 2014
Note
reddish
loss of
lamina
stain,
brown
stain,
48 hours.
X200.
they illustrate
mouse
sub-
t
..S..
1
li.
1#{149}
.1.;’
:
r
.,‘c
.I.
&
...
,,
.
_,.1
_%
-
lms.
l--1()
Downloaded from jhc.sagepub.com by guest on June 11, 2014
S
..i
Downloaded from jhc.sagepub.com by guest on June 11, 2014
Il)ENTIFICATION
sections
acid.
With
hydrolysis
release
it
all
of the
be
concluded
miiay
tions
Flu
rality
to
)rolonged
sialiolase.
Rat
that
and
of sections
sialic
over
the
also
been
observed
acids
are
in
different
ample
a
of
of
preparatiomi
the
the
of
the
tissues
the
muicosa
40
41
l)uirple
70
(ii
PuirPle
red
whether
sialidase
homogenates
zyme
action
fixation
releases
that
siahic
on
sialic
the
substrate.
acids
from
mouse
tissues,
were
cleaved
from
were
In
while
rat
brow-mi
red
+++
omamige
brow-mi
varies
420
100
red
+ + +
oramige
brown
exSections
of
to
one
24
Digestion
of
tissue
vaginal
anioumnts
con-
II
sections
pregnant
mouse
by sialidase
Sialic
Acid
Alcian
Reaction
ditions.
of Section
useol.
in the
measuired
two
met
was
ever,
in
not
the
sections
be
enzyme
amount
did
of
the
not
sialic
acid
be
of serumm proteins.
acid of rat serum
Howtissue
mouse
acid
time
have
The
from
found
tissues
their
comitemit
that
is rapidly
of
small
rat
by
re-
amount
amoumnt
tissues.
for
proteins
was
the
released
We
hydrol-
of
sialic
rat
I.
completion.
increase
accounted
were
Table
Increasing
froni
of two
sections
enzymatic
to
III).
released
possibly
that
of
and
5-fold
acid
solutions
hydrolysis
all
II
conindicated
PAI)--See
go
enzymatic
(Tables
times
the
PAS,
rioted
is
from
sections
the
amid
to
virtually
leased
At
hools.
of
5 IL sections
sumpernatant
allowed
sialidase
procedumre
Umitreated
aciol.
acids
It should
ysis
might
Blue.PAS
Adjacent
were
by
the
text.
Mg of siahic
siahic
by
experimental
were
5.7
stained
is
the
block
tained
sialic
of
epithelium
in
sections
it
digesteui
The
cholerae.
dlescribed
fresh
similar
were
Vibrio
of
TABLE
brow-n
100
IV
small
tinder
browmi
300
extensively
only
tissues
browmi
oramige
which
lack of enbe dume to
liberated
oramige
+++
orange
from
Table
+++
orange
20
purple
brown
+++
determine
acids
to
ptmrple
orange
+++
16 hours,
to
of rat tissues,
since the
on tissue
sections
might
effects
seen
sought
+
red
of diges-
after
unstained
blume
red
the
experiments
Purple
75
1’ibrio
4 hours
12 to
purple
bluie
to Puirple
92
reaction
only
0
17
95
hours.
Further
Reaction
120
For
from
after
gland
at
section
1 to
0
resuper-
mouse.
staining
after
submaxillary
rate
sialidase
acid
gland
vaginal
proper-
the
the
sublingual
PAD
6 Hour
l5
diges-
suhlinguial
into
the
of
mouse
sialidase
AicisBIue-PAS
a high
of rat
from
of
by
“in
of
resist
miiouse
that
tissues
sublingual
unaltered
contain
acid
III
sections
leased
sections.
removed
eliminated
cholerae
of
Stic
t he
histochemical
1)igestion
widely
tion,
their
those
tissue
of
acid-
preparations
they
of
histochemical
also
that
TABLE
Digestion
Thaeo
remiiains
glands
405
SIALOMUCINS
gland
miiolecules.
to
MOUSE
preparasialic
its
resemble
sialic
and
miiouise,
flumid
has
mouse
the
measurable
HO
It
despite
fact
acid
(20).
natant
the
to
Suirprisingly,
rat,
AND
mucins
sialidase
exposure
of
of siahic
leases
of
various
mucin
sublingual
imi spite
glands
of
knowmi
soluble
sjalomucins:
following
closely
the
HAT
of such
are
from
liberate
in the
simiiilarity
ties
not
of
mucus
level
acids
fragments
vaginal
conditiomis
H2504)
that
do
containing
the
‘s
0.1
sialic
employed
tion
Since
(80#{176}Ciii
OF
the
sialic
liberated
by
sialidase.
Resistance
pg
1.
Undigested
control
2.
Supernatant
3.
Section
section
of
Section
blue
However,
by
rat
digestion...
after
enzyme
0.2
++++
red
0.2
++-F+
red
cholera
digestion...
of 3
1.1
that
6.
of
1.4
tissue
16
hours
procedure
PAS
digestion
is
=
4
described
periodic
39#{176}C. The
at
in
acid
the
Schiff.
text.
experimental
rat
mucimis
to
we
have
well
tissue
found
from
in
that
mouse
the
presence
siahic
in
and
sialidase-sensitive
is released
homogenates
in
the
absence
of
after
purification
on
of
to
mice
free
the
with
paper
Table
Downloaded from jhc.sagepub.com by guest on June 11, 2014
the resistance
the chemical
differs
froni
mucins.
homogenates
hydrolyzed
standards.
actiomi
homogenates.
cally
graphed
the
acid
tissue
Another
possible
explanation
for
of rat mucins
to sialidase
is that
form
of sialic
acid
in these
mucins
5. Supernatant
Supernatant
certain
might
be accounted
for by the presence
inhibitory
to sialidase
in rat tissues.
sialidase
equally
Cbs-
perfringens
enzyme
4.
purple
0.0
1
after
tridium
++++
1.5
of
of sialidase
of materials
buffer
in
V
and
sialic
Norit
two
shows
A
solvents
that
Accordingly
chemi-
rats were
acids
and
were
these
chromatowith
a sialic
known
acid
in-
406
L.
ANI)
WARREN
S.
TABLE
Sialidase
digestion
of
rat
and
S.
SPICEIt
IV
mouse
mucins
in
tissue
homogenates
Release
Amount
of
Sialic
Acid
in Tissue
C1,,slridi,i,,i
of
Sialic
perfringens
enzyme
A.
pg
pg
5.5
0.5
17.0
0.6
Acids
-____________________
Vibric
clilorerae
enzyme
pg
%
9.1
0.1
3.5
0.4
1.8
2.4
0
0
0
0
1.6
3.0
1.2
2.2
Rat
Vaginal
epithehium
(non-pregnant)
Vaginal
epitlielium
(pregnant)
Stibmaxillary
B.
8.6
glandi
Stiblingumal
54.1
gland
Mouse
Vaginal
epithelium
Sublingual
Submaxillary
Each
vessel
ILmoles;
drolysis
gland4
contaimiecl
The
acids
was
tubes
the
*
were
carried
out
directly
were
aci(ls
paper
the
vessels
51.1
3.4
75.6
60.2
36.2
62.5
41.4
14.9
36.0
21.7
52.2
sodium
acetate
sialic
vessels
buffer,
37#{176}C.The
inctubation.
thiobarbituric
Homogenates
to l)revent
the
for
omily
and
mouse
acid.
served
tissues
has
only
Rat
not
Mouse
been
neuraminic
acid
Type
neurammnic
of
acid
Tissue
Fast
component
N-acetyi
I
N
epi+
+
+
+
+
+
+
states:
+
mouse.
fairly
acid
Sublingual
gland
+
N-Acetylneuraminic
Solvent
2
Solvent
1 RF
material:
acid:
RF
0.35.
0.20,
Solvent
1 RF
0.30,
N-Ulycolvlneuraminic
Solvent
Solvent
1 HF
2
HF
0.53,
acid:
0.27.
Solvent
Fast
2 RF
moving
0.47.
level
alcian
from
N-acetylneuraminic
N ,O-diacetylneuraminic
acid,
chromiiatographically
tylneuramninic
lingual
gIant!
acid)
gland
mouse.
anti
the
is present
iii
and
It
is
miiouse
which
vagimia
gland
that
also
(or
rat
contain
of
the
submaxillary
N-glycolyl-
There
level
rat
of
and
The
trolled
sialidase
sialidase
to
the
the
it
moiety
to which
acid
content
physiological
in the
the
non-pregnant
be seen
that
between
the
there
sialic
mucin
appear
to be
an
as
followed
sialic
is a
acid
visualized
index
mucification
reaction
which
pattern
of chamige
throughout
Both
of
ascribed
resistance
of i.e. attachment
a differ-
variation
of
can
amount
be
and
sialic
different
vagina
miiate-
suisceptible
mode
mucin,
in
of
rat
moving
that
or sugar
V)
forms
resistant
cannot
the
rat
ob-
(Table
the
suggest
histology
vagina
with
of the
underthe
cycle.
mucification
and
by
injection
the
of
reaction,
biochemical
estradiol
results
imi a marked
of the
staining.
methods,
administration
comitent
bluie
fast
in
is considerable
the
blue.
the
results
In Table
VI
good
correlation
chemical
behaves
samiie w-ay as N-acein the vagina,
sub-
submaxillary
of interest
acid
in
mouse
extent
of the
goes a regular
oestrus
distinguishable
and
to
The
material
sialidase
those
resistance
factor.
Variations
rat and
of
Submaxillary
glamid
sialic
70#{176}Cfor
interfered
gland
in
for
as
to sialidase
of
sialic
acidsis due
to
N -e-
moving
Since
found
except
same
mucins,
fast
submaxillary
ent bond configuration,
sialic acids are attached.
theliuim
free
at
which
characterized.
are,
the
to this
The
acid
mucins
rial,
Vaginal
for
20
acid
hy35 pg of
enzyme,
of material
in rat
neuraminic
comym
CaCl2,
by
acid
assay
were
incubated
release
neuramiiinic
rat
chromatography
N-gmy-
50 pmoles;
2 hours.
V
in
of
5.25,
above
(determined
or clostridial
assay.
incubated
Type
pH
acid
listed
1 Mg of protein,
emizyme,
at
after
to experimemital
identified
by
2.3
:34.9
of
cholera
for 3 hours
on
TABLE
Sialic
ml;
amount
homogenates);
acid
vessels
of 0.3
the
incubated
thiobarbittiric
These
volume
containing
adolitiomi
3 miiinutes before
with
imi a fimial
tissue
homogenate
of an ahiquot
of the
protein.
4.5
58.0
(pregmiant)
ghamid*
vagina
However,
Downloaded from jhc.sagepub.com by guest on June 11, 2014
of
into
lowering
and
by
histobe
may
hormones.
The
pre-ovuhatory
of
the
the
elimination
the
injection
conmice
sialic
acid
of alcian
of
proges-
IDENTIFICATION
TABLE
Correlation
of
reactive
sialic
macin
RAT
VI
acid
in
OF
of sialic
level
the
and
vagina
alcian
of
the
iaic
act
Species
PAS
0
Re-
action
21
mg/g
wet
tissue
1-2 month
old..
Mouse
1
1.10
Normal
1-2 month
old..
Mouse
1
1.05
Normal
1-2
old..
Mouse
1
1.24
mnonth
++
the
carboxyl
tions
over
the
sialic
acid
such
mucins.
The
Normal
1-2 nionth
old..
Mouse
1
1.00
Normal
1-2 month
old..
Mouse
1
1.68
+++
Norlnal
Normal
1-2 month
1-2 month
old..
old..
Mouse
1.36
++
0.77
+
Normal
1-2 month
old..
Mouse
1
1
1
0.84
+
Normal
1-2
old..
Mouse
1
0.63
0-+.
Normal
1-2
month
old..
Mouse
1
0.67
0
reagent
Normal
1-2
month
old..
Mouse
1
0
as demonstrating
Normal
1-2
month
old.
Mouse
1
O.66
0.33
Normal
1-2
month
old..
Mouse
1
0.67
Normal
1-2
month
old.
Mouse
1
0.40
Pregnant
Mouse
1
Pregnant
Mouse
1
Pregnant
Mouse
1
1.54+++
Estradiol
Mouse
6
0.45
Estradiol
month
Mouse
taming
well
sides
tam.
0
Mouse
6
0.96
++
Rat
4
0.43
0-+
Pregnant
Rat
4
2. 22, + + +
of
4
old
in
corn
and
female
injected
oil
day
estradiol
days
terone
after
staining
content
of
the
animal.
at which
vagina
The
for
(Table
VI).
back
in
in
sialic
acid
alcian
salivary
ties
levels
of
the
consistently
blue
is
the
high
alcian
blue
sialic
acid
be
correspondingly
The
gation
methods
employed
localize
in epithehial
certain
tissue.
Since
philia
from
with
purified
sialic
acid
by
of
the
great
in
the
present
the
enzymatic
removal
tile
a
orcimiol
lie
imiterprets
in ganglio-
siahidase
which
as
thd’v
of at
least
hormonal
consome
control.
take
in
staining
hiistochemically
preparations
potent
as
proper-
identified
as sialo-
elimination
by
sialidase
the
to
vagina
have
been
the
of
specific
baso-
digestion
the
removal
is not
periodic
in the
fails
source
like
of
accompanied
acid
Schiff
of
staimiing
the
polymer
place.
rich,
and
re-
mouse,
mucopolysacchiaride.
degradation
alters
alter
mucificatiomi
anti
However,
by
extensive
quickly
We
of
sialidase.
vagina
gland.
a
have
the
rat
hiistochemical
sections
slowly
in the
submaxillary
as
thie
basis
effected
not
the
acid
tissue
hence
does
investispecifically
show
ami alteration
glanol
sialomucins
developed
lw
acid
in
of
these
on
mucins
vaginas
DISCUSSION
as
l)resent
BIAL
synthesis
vagina
mouse
Siahidase
pregnant
and
ac-
there
are several
types
origin
which
vary
in
elaborated
the
mucins,
In the
animal,
in
the
has
which
is un(ler
a nonsulfated
mucins
killed.
may
non-pregnant
by
ohigestion
the
mucins
of
and
reaction
the
been
reaction,
residues
of sialic
addition,
of
for
VI).
prominent
are
the
The
old
were
to
form
sialomucins
action
day
and
then
vegetable
the
rise
mucification
animals
affinity
they
50 g
daily
21
in
In
these
con-
tissue.
be noted
that
of epithehial
the
in
sul-
acid
SUMMARY
Schiff.
(Table
is always
pregnant
time
brings
absent
mucification
Eight
attendant
the
or
given
subcutaneously
killed.
acid
an
were
treated
similarly
of progesterone
estradiol
with
Although
present
were
1 mg
periodic
=
mice
oil,
then
5 days
for
PAS
female
were
mice
with
daily
as
other
sialic
the
acid
other
has
3)
sectiomis
and
fromn
and
orcinol
(2,
sialic
of
also
acid
sections
with
susceptibility
well
these
described
tissue
esters
procedure
of the
Diezel
of nervous
their
2. 26 + + +
Non-pregnant
21
in
methyl
of
tissue
reaction
It shoulol
of sialomucins
+
Eight
20).
chromogenic
(6)
of
By
methods
(17,
More-
hvaluronic
miieans
IW the
authors
of
progesterone
in
by
as
Cbs-
virus.3
localization
materials
complished
cholerae,
distinguished
as
removal
prepara-
Vibrio
staining.
are
with
The
methvlation
sialoniucins
it is
combine
influenza
formatiomi
mild
basophilic
fated
+
and
probable
mucins
dyes
ehimidyes,
by sialidase
sources,
the
basic
of sialomnucins.
three
by
the
be effecteol
perfringens,
acid
++
tan
sections
with
that
groups
from
tridiu,n
imi tissue
staimiing
of basophilia
criteria
+
nitlcifls
to assume
blocks
Normal
from
characteristic
reasonable
Basophilia
with
Alcian
Blue-
407
SIALOMUCINS
acid
nates
blue
rodent
0
Condition
MOUSE
AND
used
of
are
cholera
Downloaded from jhc.sagepub.com by guest on June 11, 2014
staining
of
sublingual
amiol even
I)igestion
more
with
reactions
similar
of the
in
niucin
pregnant
commercial
sialo-
relatively
slowly
in the
the enzyme
the
siahic
acid-
imi tho salivary
rat,
ant!
fails
influenza
vaccine
However,
these
neuraminidase.
relatively
and
clostridial
mouse
gland,
crude
and
not
preparations.
as
408
L.
to) rtluase
sialit
or
tid)Iis
thit
variatn)mls
l3othi
orgamis.
in
mutitin
is
ai
Iiiiut,
)rogesttr(
ill
thiuse
of
the
(.hirOn1:mtOgrImJ)hi
\agimia
glan(ls
all
that
niotse
IliSt)
tt)iitaiii
amid!
low
pmd-
with
sialic
acid
liv
thiiit
:tnut
sumbsu’-
amiul
mitt
imi two)
solvemits
acid
is
in
Au
.
with
presemit
a 11(1
gIant!
ati(l
of neurainimiic
of
auidl
sul)mnaxillary
‘-glvcolvlmicuramiiimiiu
form
nieans
thie rat
a mit!
sIml)miiaXillary
N-auetylneumamnimiiu
vagimia
maxillary
is
truatt(l
imicitase
ruveals
sul)lingual
contaiii
identified
in
cu)ml(emitmatu)n
u)eStflI(liu)l
)StIlt
huit
hiisto-
ulevateol
)mle t r(atnient.
lulentificatiomi
mno)umse
aciul
vagina
cyclic
t)l)Sum’e(l
d()nsistemlt.ly
sialiu
see-
thic
the
vu ra llul
of rnuuin
are
‘l’hie
ovumlatory
l)ility
rat
level
uyule
AND
histologic
acid
t)f tIme allit)tlmlt
prtgmiamity.
fromn
these
sialiu
otstrouls
tlieiiiitally.
l)1PtI
of
imi thc
durimig
Oltl(mlt
tither
hiomiiogumiatus
(‘hianges
ami(I
Itui(l
WARREN
uinmo-
rapid
thit
rat
sub-
glamitl.
ACKNOWLEDGMENTS
I’hit
i\Iarsh
ance
authors
antI
are
imi this
grateful
J. U.
\Irs.
to
Hcnson
\Ir.
for
BIaIl(leiS
assist-
valtiecl
U. L.
E. L. Purification
FRENCH,
AND
2.
1)mEzEr,
P.
ehungemi
i nfammtelemi
an
B.
den
Histochemnische
diffuisemi
4.
5.
Virchows
I)mEzEL,
P. B. In:
Blackwell,
Oxford,
of ovulatiomi
vomii
206-228,
and
associated
webe).
270:
der
Substamiz
imn Nervenge-
Hoppe-Seylers
Z.
Physiol.
Chem.
185-193,
1941.
7. LATASTE,
F.
Transformatiomi
periodique
de
l’epithelium
dum vagimi
des rongeumrs
(rythme
vaginal).
(‘.
H. soc.
biol.
44: 765-769,
1892.
8. LmmImE,
H.
I).
Histopathologic
Technic
and
Practical
Histochemistry,
Blakiston
Co.,
New
York,
1954.
8a. LILLIE,
R. I). Stain
Technol.
30:
133,
1954.
9. Loxa, J. A. AND EVANS,
H. MeL.
The oestrous
imi
t lie
rat
and
its
associated
pile-
)olysacchiari(les
. J.
Histochem
. (ytochem.
8: 18-35, 1960.
SemcER,
S. S. AND JARREL5,
M. Histochernical
reaction
of ami muroniatic
clianiimie
with
acid
groups
and
periodate
engendered
aldehyoles
i Ii
niucopolysacchanides
.
J.
Histochem.
(‘ytochem.
9: 368-379,
1961.
Sm’ICER,
S. S. AND MEYER,
1). B. Histochemiiical
different
Typus
histological
changes.
Proc.
Soc.
Exp.
Biol.
Med.
25: 754-756,
1928.
KLENK,
E. AND
LANGERBEINS,
H. tber die
Verteilung
der
Neumraminshure
im
Gehirn
(Mit
einer
Mikromiiethode
zur quamititativen
Bestimmung
19.
famniliaren
Arch.
327:
1955.
The
(‘erebral Lipidoses,
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