Hedial Research Society
8P
the association of the glycolytic enzyme glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) with the cell membrane were studied.
Erythrocytes were untreated (UT) or thiol groups were
alkylated at pH7.4, 2°C with either NEM or IAamide. GAPDH
pretreated with NEM or IAamide was also added to ghosted
erythrocytes. Ghost membranes were prepared, separated by
polyacrylamide gel electrophoresis and stained with Coomassie blue
GAPDH was identified by sequencing and quantified using the ratio of
staining density to actin.
NEM increased GAPDH association with the cell membrane
This only occurred with the intact cell and could not be achieved by
NEM pretreated GAPDH. IAamide also increased GAPDH association
with the cell membrane but this could be achieved with IAamide
pretreated GAPDH.
UT
Mean GAPDWACTM ratio
Mamide GAPDH
GAPDH
+NEM
NEM
0.340 1.383*
P < 0.001 v UT
1.362*
0.238
GAPDR
+IAamide
I .085*
0.100
A membrane protein that is specifically very reactive to NEM
modulates GAPDH association with the cell membrane. Associations
between enzymes and cell membrane proteins are important in
metabolic control and an abnormality in the function of such a
membrane protein, indicated by N a L i CT could have important
metabolic effects.
28 ENDOGENOUSLY ELICITED ANTIBODIES TO
HUMAN PLATELET CYTOSOLIC PROTEIN INHIBIT
ATHEROSCLEROSIS IN THE CHOLESTEROL-FED RABBIT
DJ LAMB and GAA FERNS
Centre for Clinical Science and Measurement, School of Biological
Sciences, University of Surrey, Guildford, GU2 5XH, UK
Our previous studies and those from other workers have shown that
several growth factors may be important in atherogenesis. These
growth factors may be released from platelets or be expressed by
cells within the arterial wall. In order to investigate their role in
atherogenesis, New Zealand White rabbits were immunised against
cytosolic protein from human platelets (PCP) or saline until high
antibody titres were obtained. Rabbits were then fed a 0.25-1%
cholesterol-containing diet for 10 weeks in order to induce
atherosclerotic lesions. Weekly blood samples were assayed for
antibody titres and cholesterol levels. Rabbits were killed by
anaesthetic overdose and perfusion fixed with 4% paraformaldehyde
in isotonic PBS. Aortae were removed and the extent of
atherosclerosis assessed by en face image analysis of aortae stained
with oil red 0. Histological sections were taken at the level of the first
intercostal branch and the intirnal and medial areas quantitated by
image analysis. Anti-PCP antibody titres increased rapidly 2 weeks
following immunisation against PCP but not saline, and reached a
plateau after 8 weeks. Integrated plasma cholesterol levels were
similar in both the PCP (109.W5.9mMweeks) and saline (124.5k6.0
mMweeks) groups. Compared to non-immune rabbits (n=10),
animals immunised against PCP (n=lO) had a significantly smaller
area of the aorta containing atherosclerotic lesions (18.7i4.2% vs
34.4i4.3% ( ~ ~ 0 . 0 1 )This
) . was associated with a smaller
intimalmedial area ratio in aortae taken from PCP imunised rabbits
(0.03*0.02) than from non-immune animals (0.16kO.07) ( ~ ~ 0 . 0 5 ) .
These data suggest that growth factors released from platelets may
be involved in cholesterol-induced atherosclerosis.
We would like to thank the British Heart Foundation for their support.
29
URINE PTERlN LEVELS IN PATIENTS WITH PRE-CANCEROUS
LESIONS OF THE UTERINE CERVIX.
27 FUNCTIONAL CELL MEMBRANE ABNORMALITY IN
AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE
(ADPKD)
SL WATKINS', IC WEST*, R WILKMSON* and TH THOMAS
('Introduced)
Department of Medicine. University of Newcastle-upon-Tyne, UK
Associations of enzymes with cell membranes are imponant in
metabolism The glycolytic enzyme glyceraldehyde-)-phosphate
dehydrogenase (GAPDH) binds to the integral band 3 membrane
protein. GAPDH association with the cell membrane and the effects of
a thiol protein that is abnormal in ADPKD were studied in erythrocytes
from normal controls (NC) and ADPKD patients.
Erythrocytes were untreated (UT) or thiol groups were
alkylated at pH7.4. 2°C with either N-ethylmaleimide (NEM) or
iodoacetamide (IAamide) Ghost membranes were prepared, separated
by polyacrylamide gel electrophoresis and stained with Coomassie
blue GAPDH was identified by sequencing and quantified using the
ratio of staining density to actin.
The association of GAPDH with the cell membrane was greatly
increased by treatment ofcells from NC with NEM The effect of
NEM on cells from ADPKD was much less In contrast, IAamide
increased the level ofassociation strongly in both NC and ADPKD
GAPOWACTM ratio
UT
NEM
ADPKD
Nc ADPKD
Mean 0340 0278
SD
(0090) (0 106)
P<OOOIvNEMNC
I383
OS69'
(0264) (0 189)
IAamide
Nc
JJ RIPPIN", TC HARDMA", RCLIFFORD', DL EUSTACE2*('lntmdumd)
'Dept of Cardiovascular Medicine, Imperial Colle e School of Medicine,
Charing Cross Hospital, London W6 8RF and Dept of Obstetrics &
H
Gynaecology, Queen Mary's Hospital, Kent DA14 6LT.
Early detection and hearment of invasive carcinoma of the cervix impmves pient
prognosis. Markm of immunological cell adivation may pndia diwau.Cell arltlnes
stimulated with mitogens produce pterins as a marker of immune activation in selected
disease states. In vivo, Urinary neOpterin levels have been shown to be predictive of
survival time in patients with invasive cervical carcinoma prior to therapy. T k present
study investigates whether there is evidence of cellular immune activation m patients with
cervical inhaepithelial neoplasia (CM); a generic term defining a specbum of
inmepithelialchanges representing a pre-cancerous state preceding invasiw minoma of
the cervix.
We compared urinary excretion of pterins in 51 patients with abnormal smear ~ u h to
s
excretion in 30 healthy female volunteers. Subjects provided a urine sample prior fa
assessment by coloscopy. Routine coloscopy was performed and biopsies taken according
to the normal procedure of identification of any suspicious areas afler application of 5%
acetic acid. Patients were grouped on the basis of severity of CM (incming in severity
through grades I, 2, and 3). Smears and colposcopic biopsies were processed routinely and
urine neoptrin and matinine levels measured using standard methods.
16 patients demonstrated @ I CIN, 15 gmde 2. and 7 gra& 3. 13 of the total of 51
patients had nonnal colposcopy on repeat cytology. 2 patients denwnmted urinary
neoptrin above the upper limit of the reference range. These two CM 2 patients had SLE,
explainingthe elevated neopterin; both patients were excluded from the final data analysis.
Differencesbenvgn the groups were tested by hkall-Wallis -is,
no differenm in
pterin excretion emerged benwenthe four study groups (Table).
I
controls
I
CMI
I
CMZ
I
CM~
ADPKD
I362
1.40')
(0228) (0295)
Renrlrs exprwed as median [range]
A thiol protein with specific high reactivity to NEM normally
modulates GAPDH association with the cell membrane. This protein
has impaired function in ADPKD. This may have important effects in
metabolism and could have a role in the insulin resistance seen in
ADPKD.
There was no evidence of i n c d pterin excretion in patients with CM compared to
.healthy controls, precluding any diagnostic or prognostic value of measluing Urinary
pterins. The apparent difference in the predictive nature of neopterin betweeninvasive and
non-invasive disease may be due to lack of mscularization of the cetvical mernhane
above the baremen! membrane in CM. This would impair lymphqte trafficking and
monocyte activation limiting pterin production.