Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102, Bl-B30 B1

REVUE TRIMESTRIELLE ('), OCTOBRE 1993

DRIEMAANDELIJKS TIJDSCHRIFT (I), OKTOBER 1993

SOCIETE BELGE DE BIOCHIMIE ET DE BIOLOGIE MOLECULAIRE

BELGISCHE VERENIGING VOOR BIOCHEMIE EN MOLECULAIRE BIOLOGIE
l5SC&UNION, UCL, 20 NOWMBRE 1993
~ ~ ~ ~

6 e Congr&sMultidisciplinaire
Transplantation et Molecular mechanisms of tumor rejection
1. C O N F ~ E N C E S A. AMAR-COSTESBC,D. GODELAINB, B. VAN DEN EYNDB(I), V. VER-
LANT and H. BEAUPAY[International Instihue of Cellular and Molecular
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15

B. VANDEN E ~ (LICR,
E Brussels) Pathologv and (I) Ludwig Instilute for Cancer Research, B w h ]
Genes coding for tumor rejection antigens. Characterization of the muriae tumor-specific protein P l A p
A.B. R I C ~ S Q (Birmingham,
N U.K.) Mouse mastocytomaP8lS expresses several antigens which are targets
Molecular analysis of cytosolic T lymphocytes recogni- of a T cell-mediated rejection response in syngeneic animals.Two of these
tion of EBV malignancies. antigens, which were demonstrated in vitro with cytolytic T lymphocytes,
are encoded by gene PlA (VANDEN EYNDEet al., 1991). This gene is
MEMBRES PROTECTEURS expressed in different tumors, but is silent in most normal tissues, with
BESCHERMENDE LEDEN the exception of testis and placenta. Activation of gene PlA is linked
to tumoral transformation in mast cells. Our current knowledge about
AMERSHAM Belgium the gene product (PlAp) is scarce :the putative protein (224 amino-acid
ANALIS residues) is acidic (PI = 3.5). The assoCiation of PlAp with the transformed
APPLIED BIOSYSTEMS phenotype suggestsa function in cell growth and a possible involvement
BEUN-DE RONDE in oncogenesis.
BIO-RAD LABORATORIES In order to identify protein PlAp, an oligopeptide based on the
BOEHRINGER INGELHEIM BIOWHITTAKER predicted C-terminal sequence of the protein was synthesized, coupled
BOEHRINGER-MANNHEIM (Belgium) to ovalbumin and used for rabbit immunization. The antisera were then
used to test cell lysates by immunoblotting after SDS-PAGE. A compo-
CANBERRA PACKARD nent migrating at 42 kd was detected in lysates from P1.HTR (PlA-
CARL ZEISS N.V./S.A.
For personal use only.

positive) cells, or from DAP-3L fibroblasts transfected with a cosmid

CERESTAR gruppo Ferruui containing the entire PlA gene. This band did not show up in antigen-
CURRENT BIOLOGY loss variants (Pl.ist A-B-, P0.HTR and P 1 . k A-B-) derived from PI .HTR
DENLEY Europe cells and in mock-transfe-cted DAP3L fibroblasts. Thus, the 42-kd com-
DEVOS-FRANCOIS ponent may be identified as PlAp.
DU PONT DE NEMOURS Rabbit reticulocyte lysates and wheat-germ extracts were programmed
FISONS INSTRUMENTS for protein synthesis with in vitro transcripts of a PlA cDNA. The pro-
minent translation product migrated at 42 kd and was specifically ad-
HEWLETT PACKARD sorbed onto protein A-Sepharosebeads in the presence of the antiserum.
JANSSEN PHARMACEUTICA This further identifies the immunoreactive 42-kd polypeptide as PlAp,
LABAZ-SANOFI in spite of an unexpectedly high apparent Mrvalue which may be due
LAMECO to the acidic regions of the molecule.
LAND& & GLINDERMAN N.V. The antipeptide antiserum was used to extract the immunoreactive
LIFE TECHNOLOGIES component from P1.HTR cells and transfected DAP3L fibroblasts, after
MEDATOM EUROPA metabolic labeling with [35S]-methionine,or [31P]-orthophosphateand
MEDECINE/SCIENCE-JOHN LIBBEY EUROTEXT lysis in Nonidet NP-40. In each case two %-or "P-labeled polypeptides,
migrating at 42 and 65 kd in SDS-PAGE, were specifically removed from
MERCK-BELGOLABO the cell lysates. Thus PlAp is probably d a t e d with another polypep
METTLER-TOLEDO tide, forming either a stable heteromeric functionalunit, or a dissociable
NEW BRUNSWICK SCIENTIFIC multiprotein complex. In addition, both PlAp and the associated 65-kd
OMNILABO polypeptide occur, at least partly, in a phosphorylated form.
PHARMACIA LKB In subcellular fractionation of P1 .HTR cells or transfected DAPJL
PLEUGER fibroblasts by differential centrifugation, the bulk of the 42-kd protein
PROMEGA demonstrated by immunoblotting was bound to sedimentable material.
SIGMA-ALDRICH The distribution showed a peak in the light mitochondrial fraction and
ruled-out a preferential association with nuclei, or mitochondria. In
SMITHKLINE BEECHAM BIOLOGICALS sucrose gradient the density distribution of PlAp was broad, differed
UCB from those of the plasma membrane and lysosomal markers, and par-
VAN HOPPLYNUS tially correlated with that of mannosyltransferase.Our interpretation is
WATERS Chromatography Division/MILLIPORE that PlAp is bound to the endoplasmic reticulum, or more broadly to
membranes of the secretory pathway. PlAp was not released by washing
the cytoplasmic organeUes with 1 M KCI. However, it was digested by
2. COMMUNICATIONS trypsin in the absence of a detergent, leaving an immunoreactive frag-
ment (20 kd) bound to the membranes. Thus, PlAp is exposed at the
cytoplasmic surface; it is either a type-I1 transmembrane protein, or a
peripheral protein bound to a membrane constituent which could be the
65-kd molecule.
(I) Publication subsidike par le Minist&e de I'Education Natio-
nale et de la Culture. Reference
Publikatie gesubsidieerd door het Ministerie van Nationale VAN DEN EYNDE, B., L d , B, VAN PEL, A., DB PIABN, E. & BOON, T (1991) J. &.
Opvoeding en Cultuur. Med. 173, 1373-1384.

Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)

 B2
Ph. BASTJN(1. 2), I. COPPENS (1. 2), J.-M. SAINT-
REMY P. BAUDHUIN
(3), (I), F.R. OPPERDOES (z)
c)
and P.J. COURTOY [(I) Cell Biology, (*)Research Unit
for TropicalDkases and (7 Allergy and ClinicalImmunology
Unit, Catholic University of Louvain and International Institute
of Cellular and Molecular Pathology, Brussels, Belgium.
Identification of a specific epitope on the ex-
tracellular domain of the LDL-receptor of
Trypanosoma brucei brucei
We have previously shown that an antiserum rai-
sed against the Mr 86 0o0 fragment of the LDL-
receptor of bloodstream Trypanosoma brucei brucei
shows extensive cross-reactivity with the mammalian
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
LDL-receptor (COPPENS et al., 1992). Here we report
on the production and characterization of 30
monoclonal antibodies (MAbs) raised against the same
Mr 86 OOO fragment of the T. b. brucei LDL-receptor.
Of these, only 8 MAbs recognize in an ELISA test the
purified (presumably intact M,145 OOO LDL-receptor.
Seven of them also recognize the LDL-receptors
isolated from rat and rabbit, whereas one MAb (1A9)
is specific for the trypanosome LDL-receptor.
A p001 of several MAbs inhibits by 90% the specific
binding of T - L D L to trypanosomes at 4”C, but does
not interfere with binding of lZ51-LDLto rat fibroblasts.
”’I-MAb 1A9 is efficiently taken up by T. b. brucei
at 30°C and its uptake is inhibited by an excess of non-
labelled LDL particles, indicating that MAb 1A9
For personal use only.
follows the LDL-receptor pathway. Uptake of IZ5I-
MAb 1A9 by rat fibroblasts is less efficient and is not
significantlyreduced by an excess of non-labelled LDL.
Following internalization by T. b. brucei, 1z51-MAb1A9
appears to recycle back to the surface. MAb 1A9 as
well as other pooled MAbs activate rabbit complement,
leading to lysis of trypanosomes in vitro.
We conclude that the T. b. brucei LDL-receptor
contains at least one specific epitope that is accessible
on live cells to antibodies and can mediate activation
of the complement system.
Reference
I., BASTIN,
COPPBNS, P., OPPERWBS,
Ph., BAUDHUIN, F.R. & C o n -
TOY, P.J. (1992) &-.Parasitol. 14, 77-86.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
socnk3 BELOE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
M. BERBEROF(I), L. V
- (’), A. PAYS(‘),
P. TEBABI(I), D. JEFFERIES
(z) and E. PAYS (I)
[(I) Department of Molecular Biology, University of Brussels,
67, rue des Chevaux, 8-1640 Rhode Saint Gentire and
( l ) Present address :Department of Genetics, University of
Glasgow, Church Street GI1 SJS, Glasgow, Scotland]
A short sequence at the 3’ end of the VSG mRNA
is responsible for its stage-regulated stability
African trypanosomes have to survive in two rather
different environments :the bloodstream of their mam-
malian hosts and the midgut of the tsetse fly.
The surface of the bloodstream form of T. brucei
is covered with a coat of a glycoprotein termed the VSG
(Variant Surface Glycoprotein). The continuous varia-
tion of antigenic determinants of this VSG allow the
parasite to escape the host immune response.
In the fly, the VSG coat is replaced by another ma-
jor surface glycoprotein called procyclin.
While the VSG mRNA is the most abundant
messenger of the bloodstream form, it has been shown
to be preferentially degraded upon differentiation of
the parasite into the procyclic form (EHLERSet al.,
1987). Due to its high level of sequence conservation,
we have speculated that the 3’ UTR (UnTranslated
Region) of the VSG mRNA is the target for
mechanisms controlling its differential stability during
the life cycle of the parasite.
In order to test this hypothesis, we have cloned the
3’ UTR of the VSG gene downstream from a reporter
gene (CAT, for Chloramphenicol Acetyl Transferase)
whose activity has been measured by transient and
stable assays in both the bloodstream and the procyclic
form. The presence of this sequence led to a marked
differential behaviour; when compared with a control
construct, the CAT activity was found to be respectively
increased in bloodstream forms and reduced in pro-
cyclic forms. Site-directed mutagenesis enabled us to
conclude that two particularly conserved stretches,
termed the 8-mer and 14-mer (Amm et al., 1989), are
involved in these processes.
By measuring the amount of CAT mRNA in
trypanosomes transformed by constructs differing in
their 3’ UTR sequence, we could conclude that the dif-
ferential gene expression conferred by the 3’ UTR of
the VSG mRNA occurs at least partly at the level of
RNA stability.
This work was supported by a Wellcome Trust Training
Fellowship in TropicalMedicineto D.J. an IRSIA fellowship to M.B.
and a FNRS fellowship to L.V. Support was also received from the
Belgian ‘<Fondsde la Recherche Scientsque Mddicale”, the ‘%bnds
Sp4cial de la Recherche de I’Universitd Libre de Bruxelles“ and
through a research contract with the “Communautd Francaise de
Belgique” (ARC 89194-134). It was also funded by the Agreement
for Collaborative Research between ILRAD (Nairobi) and Belgian
Research Centres.
References
ALINE,R. & STUART, K. (1989) Exp. Parasitol. 68, 57-66.
EHLERS,B., CUCHOS,J. & OVBRATH, P.(1987) Mol. Cell. Biol. 7,
1242- 1249.
 SOCJkl'l? BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
F.Boucm, F. PVIAINFERME, R. WATTIAUX
and S. WAT-
TIAUX-DE
CONINCK (Laboratoire de Chimie Physiologi-
que, Facult& Universitaires Notre-Dame de la Pair, rue de
Bnurelles, 61, 8-5000 Namur, Belgium)
Cloning of a cDNA of a rat liver lysosomal mem-
brane protein, LGPlODlO
A rat liver cDNA expression library in X g t l 1 was
screened with a specific monoclonal antibody, 10Dl0,
raised in our laboratory against LGPlOD10, a lyso-
somal membrane glycoprotein of rat liver (WATTIAUX-
DE CONINCKet al., 1991). One putative clone was
isolated. The bacteriophage DNA was amplified by the
Polymerase Chain Reaction (PCR) technique and
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
digested with EcoRI. The EcoRI-excised cDNA insert
of 1.2 kb was subcloned into the plasmid vector
+
pGEM-3Zf( ) and sequenced by the dideoxynucleo-
tide chain-termination method. As the estimated M.W.
of the polypeptide is 48 OOO, this insert cannot repre-
sent the full-length cDNA, which should contain at least
1.5 kb. To obtain the rest of full-length cDNA of
LGPlODlO, we used the 5' RACE system (Rapid Am-
plification of cDNA Ends). This procedure exploits the
advantage of in vitro amplication offered by the PCR
technique to facilitate the isolation of cDNA for which
only limited sequence information is available. Two
anti-sense primers specific for LGPlOD10, called RT
and AMP 5', designed from the known sequence of the
cloned 1.2-kb cDNA were prepared. First strand
For personal use only.
cDNA, primed with RT, was synthesised, using rat-liver
total RNA isolated by the guanidine isothiocyanate/
acid-phenol method. Afterwards, the original mRNA
template was destroyed with RNAse H and an homo-
polymeric tail (oligo-dC anchor sequence) was added
to the 3' end of the first strand cDNA using terminal
deoxynucleotidyltransferase and dCTP. PCR amplifi-
cation of dC-tailed cDNA was carried out using two
primers :one is the oligo-dG ANCHOR primer which
anneals to the homopolymeric tail and contains at the
5' end (anchor) restriction sites allowing further clon-
ing of the synthesised cDNA and the other is AMP 5 ' .
Specific amplication of the dC-tailed first strand cDNA
product resulted in a prominent 650-pb fragment. This
second fragment of the LGPlODlO cDNA was also
cloned in the pGEM3Zf( +) vector. Sequencing of this
fragment would complete the total sequence of
LGPlODlO cDNA.
This work was supported by the Fonds National de la Recher-
che Scientifique and the Fonds de la Recherche Scienti3queMedicale
(contract 11'3.4523.91).
Reference
WATTIAUX-DECONINCK,S., GONZE, M.M.,MAINFERME,F.,
V m DER SMISSEN,P., COURTOY, P.J., DE WAELE,L.,
THIRION,J., LETESSON, J.J. & WATTIAUX, R. (1991)Current
Topics in Endocytosk (COURTOY, P.J. ed.) Springer Verlag,
pp. 231-238.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
B3
J. BOUCKAERT,
R. LORISand L. WYNS(Vrue Universiteit
BrusSe, Imtituut voor Moleculaire Biologie. Paardemtraat 65.
8-1640 Sint-Genesiw-Rode, Belgium)
High resolution crystallographic studies of the
demetallised concanavalin A lectin
Like all legume lectins, concanavalin A contains a
Mn2+ion and a Ca2+ion that are essential for its specific
carbohydrate binding and biological activities. In order
to study the structural differences in the metal-binding
region between the native -MAN et al., 1982) and the
demetallised lectin (apo-Con A) and to understand their
effect on the residues involved in the specific binding of
saccharides (DEREWENDA et af., 1989), an improvement
of the structure of demetallised concanavalin A (SHOHAM
et al., 1979; REmE et ul., 1978) is required. Kinetic nuclear
magnetic resonance dispersion (NMRD) studies (BREWER
et al., 1983) indicate a slow transition between the native
and the demetallised form, characterised by a 22 kcal mol-'
energy barrier. The latter may be associated by a cis into
trans conversion of the peptide bond between Ala207 and
Asp208 upon demetallisation. The two independent
crystallographic studies of apo-Con A disagree at this
point, probably due to un.clarity in the electron density
map at the resolution (3 A).
Apo-Con A crystals, reaching the size of size
0.7 x 0.25 x 0.25 mm3, were obtained in sitting drop in
2 M (NH,),SO, at pH 5.0 in presence of Chelex 100. Data
were collected on a FAST area detector in combination
with a rotating anode source, to 2.2 A resolution with an
overall completeness of 87%. The P2,2'2 space group of
apo-Con A is related to the I222 space group of the native
concanavalin A crystals by loss of crystallographic sym-
metry in the native tetramer towards a local diad axis in
the dimer of apo-Con A. Least-squares restrained refine-
ment was performed with RESTRAIN and PROLSQ,
making use of the non-crystallographicsymmetry. Initially
the metal-binding loop and the non-proline cis-peptide
region were omitted from the model. In the present struc-
ture the peptide bond between Ma207 and Asp208 is clear-
ly in the trans configuration. This has important
implications in terms of rearrangement of the residues in-
volved in the saccharide binding, and correlated with this,
the chelation of the calcium ion. The manganese site of
the demetallised concanavalin A remains rather unaf-
fected, but the calcium-binding site is destroyed. The
ligands of the calcium become almost invisible in the elec-
tron density, because the metal-binding loop extends in
the solvent. Soaking experiments on the apo-Con A
crystals showed that the crystals convert to space group
I222 upon occupation of the calcium binding site, while
no such conversion is observed if only one transition metal
ion is bound. This change in space group coincides with
a significant improvement in diffraction quality of the
crystals.
This work was supported by the VLAB project of the Flemish
government. J. BOUCKABRTreceived a gront from the IXONL.
R. LORISis a Research Assistent of the NFWO.
References
BREWER et al. (1983)J. Biomol. Struct. Dyn. 1, %1-997.
HARDMAN et al. (1982)J. Mol. Biol. 157, 69-86.
DEREWENDA, Z.et al. (1989)EMBO J. 8, 2189-2193.
REEKE et al. (1978)Proc. Natl. Acad. Sci. 75, 2286-2290.
SHOHAM et al. (1979)J. Mol. Biol. 131, 137-155.
 B4
D. CASTEELS
and J. MERREQAERT
(University of Antwerp,
Department of Biochemistry, Laboratory of Molecular
Biotechnology, Universiteitsplein I , B-2610 Wilrok)
The primary structure of the mouse fau gene and
two retropseudogenes
The fau gene is the cellular homolog of the fox se-
quence of the Finkel-Biskis-Reilly-Murine Sarcoma Virus
(FBR-MuSV). It encodes the S30 ribosomal protein fus-
ed in frame to a ubiquitine-like protein (FUBI). Thefau
gene is expressed in a wide range of tissues and is con-
served in different species (MICHIELS et al., 1993).
Hybridization studies of genomic DNA of Balb/c mice
with a fau cDNA probe suggests that there are 10 to 12
copies of thefau gene in the genome. Many other mam-
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
malian ribosomal proteins genes are present in multiple
copies but up to now it seems that there is only one func-
tional gene; the others are nonfunctional
retropseudogenes. Apparently, this is also true for thefau
gene of the mouse.
The mouse fau gene contains 5 exons, the first exon
contains an untranslated leader sequence. The following
2 exons are coding for the FUBI-part and the last 2 exons
encode the S30-part of the gene. This exon/intron con-
figuration is identical to the primary structure of the
humanfau gene (KAS et al., 1992) and to the UbCEP pro-
teins, fusion proteins of a ribosomal protein and ubi-
quitine (FINLEY et al., 1989).
The promoter regions of the mouse and the human
gene are similar. They contain a degenerated TATA-box
For personal use only.
(TACAAA) flanked by GC-rich sequepces with 2 recogni-
tion sites for the Sp-1 transcription factor. A recognition
site for the &factor, typical for ribosomal proteins is also
found in mouse and human (HARIIIARAN et al., 1991).
Two fau retropseudogenes (MMpFAUl and
MMpFAU2) have been identified. They have almost all
known properties of retropseudogenes,sequencesformed
by reverse transcription of the mRNA and insertion in the
genome (WEINERet al., 1986). They have a poly-A tail
at the 3’ end and a polyadenylationsignal which is mutated
in MMpFAU2. MMpFAU2 also has a 6-bp repeat at both
ends of the fau specific sequence. Both pseudogenes
are f 75% identical to the fau cDNA but both are shorter
due to a deletion at the 5’ end and do not encode for a
functional protein because of point mutations, deletions
and insertions in the fau-encoding region. The main dif-
ference between these sequences is at a AG-rich region
which encodes 5 lysines in the cDNA. In MMpFAUl this
region is 350-bp long while in MMpFAU2 it is only 10-bp
long. The mapping of the mouse fau gene and the 2
pseudogenes is currently under investigation.
is a bursar of the IWONL.
D. CASTEELS
References
FPILHY, D., BARTEL,
B. & VARSHAVSKY,A. (1989) Nature 338,
394-401.
Hmmmm, N.,KELLEY,D.E. & PERRY,R.P. (1991) Proc. Natl.
Acad. Sci. USA 88, 9799-9803.
Kns, K., Mc-, L. & MERREOAERT, J. (1992) Biochem. Biophys.
Res. Comm. 187, 927-933.
MCEIIBLS, L., VANDBR RAUWELAHRT, E., VAN HASSELT, F., KAS, K.
& MERREOAERT, J. (1993) Oncogene 3, 2537-2546.
WEINER,A.M., DEININOER, P.L. & EPSTRATIADIS, A. (1986) Annu.
Rev. Biochem. 55, 631-661.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
S O C ~ T EBELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
L. ( I 9 2), Z m o , J. ( I 9 2), GEUSENS,P. (1.2, 3),
CELLS,
DEQUEKER, J. (3) and UUS, J. 2) [(I) Deportment
(19
of Immunology, Dr. Willems Instituut; (=)Limburgs Univer-
sitair Centrum, Diepenbeek. Belgium and (7Department of
Rheumatology, K.U.Leuven. Belgium]
Characteristics of synovial inflammatory T cells
in patients with rheumatoid arthritis
Rheumatoid Arthitis (RA) is a chronic inflam-
matory disease of the joints, characterized by infiltra-
tion of activated T cells in the synovium. Autoreactive
T cells and yS T cells are implicated in the diseases’
pathogenesis (PANAYIet al., 1992). One of the can-
didate antigens potentially involved in the autoimmune
process is mycobacterial 65-kd heat-shock protein
(HSP) (GASTONet al., 1990).
In this study we examined the frequency of IL-2
responsive cells and yS T cells in synovial fluid (SF) as
compared to paired peripheral blood (PB) of seven RA
patients with different clinical characteristics. Cells ob-
tained from both compartments were first analysed for
TcRarO or yS and CD4 or CD8 phenotypic expression.
No significant differences could be observed between
SF- and PB-derived cells as to their phenotypic pro-
file. The frequency of IL-2 responsive cells and $ T
cells was analysed by stimulation with IL-2 and PHA
respectively. Our data revealed that the IL-Zresponsive
cells occurred at a higher frequency of yS T cells was
slightly higer in SF as compared to paired PB.
However, one patient with acute untreated RA had a
substantiallyhigher frequency of 76 T cells in SF. More
work needs to be done in order to draw a significant
conclusion as to their precursor frequency and func-
tional characteristics.
In addition, we started with the isolation and
characterization of T cell lines reactive t o
Mycobacteriurn. From paired SF and PB of one pa-
tient we derived T cells responding to Mycobacteriurn
bovis 65 kD HSP or Mycobacterium tuberculosis-70 kd
HSP. All these HSP-specific T cell clones express the
arb TCR and the CD4 phenotype with one exception
that stained substantially with an anti-CD8 antibody.
Analysis of the TcR variable regions of these HSP-
reactive T cells may provide further insights into the
autoimmune mechanisms underlying the diseases’
pathogenesis.
This work is supported by a grant rom the NFWO and by the
Sociale Investe~ngsmaatschappuvoor Limburg (SIM). Recombinant
Mycobacterium bovis 65 kD HSP and Mycobacterium tuberculosis
70 kD HSP were a kind gift of Dr. J. VAN EMBDEN(National in-
stitute of Public Health and Environmental Protection) supported
by the UNDP/ World Bank/WHO Special programme for research
and training in tropical diseases (TDR).
References
GASTON,J.S.H., LIFE, P.F., JENNER, P.J., COLSTON, M.J. &
BACON,P.A. (1990) J. Exp. Med. 171, 831-841.
PANAYI,
G.S.. LANCHBURY, J.S. & KINOSLEY,G.H. (1992)Arthritis
Rheum. 35, 729-735.
 S O C ~ T I ?BELQE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
F. CHAUMONT,
J.J. BLUM c) and F.R. OPPERDOES Nathalie CHEVALIER,
[Research Unit for Tropical Diseases, International Institute
of Cellular and Molecular Pathology, 8-1200, Belgium and
( I ) Division of Physiology, Department of Cell Biology, Duke
University Medical Center, NC, USA]
Aerobic and anaerobic glucose metabolism of
Phytomonas sp. isolated from Euphorbia
characias
Trypanosomatids of the genus Phytomonas are
etiological agents of diseases affecting economical im-
portant crop but they also parasitize many plants and
fruits without any apparent pathogenicity. Metabolic
studies of Phytomonas sp. isolated from Euphorbia
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
churacias indicated that glucose, fructose and mannose
serve as major energy substrates and that metabolites
excreted into the growth medium were acetate, ethanol,
succinate, glycine, pyruvate and glycerol (SANCHEZet
ul., 1992, 1993).
In order to characterize further the carbohydrate
metabolism of Phytomonas sp., we conducted ex-
periments using washed cells. Cells were collected from
SDM-79 culture medium by centrifugation, washed
twice and resuspended in Hanks’ balanced salt solu-
tion. Cells were incubated in a shaker bath at 26°C in
the same buffer containing 4 mM glucose at a fmal pro-
tein concentration of about 1 mg protein d-’. Before
incubation and after different times, samples were im-
mersed in a boiling water for 90 s, centrifuged and
For personal use only.
supernatants assayed for metabolites using enzymatic
assays. By this method, we observed glucose was the
preferred energy and carbon substrate during
logarithmic growth. In stationary phase cells glucose
consumption was dramatically reduced.
Glucose consumption and metabolite production
were measured on logarithmically growing cells, both
under aerobic (air and 95% 0,/5%CO,) and anaerobic
(95% NJS% CO, and 100% N,) conditions. The
glucose consumption was nearly similar under the four
conditions suggesting that Phytomonas sp. lacks both
a “Pasteur effect’’ and a “reverse Pasteur effect”. The
main end-products formed in aerobiosis were acetate
and CO, and in anaerobiosis ethanol, CO, and glycerol.
Smaller amounts of pyruvate, succinate, L-malate and
L-lactate were also detected. However, a quantitative
analysis of glucose fermentation revealed that known
end-products are not sufficient to account for all the
glucose used during the logarithmic phase of
Phytomonas.
References
SANCHEZ-MORENO, M., LASZTITY,D., COPPENS,L. & OPPER-
DOES, F.R. (1992) Mol. Biochem. Parasitol. 54, 185-200.
SANCHEZ-MORENO, M., MONBSTJRR, A., BECERRA,C.F., LOPEZ-
GAY.J., OPPERDOES, F.R. & Osma, A. (1993) J. Protozool.,
in press.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
B5
Mia CALLENS
and P.A.M. MICHELS
(International Institute of Cellular and Molecular Pathology,
Research Unit for Tropical Diseases, Bnmeb)
High-level expression of Trypanosoma bnrcei
fructose-1,Cbisphosphate aldolase in Escherichia
calk purification and characterizationof the recom-
binant protein
Glycolytic enzymes of African trypanosomes are con-
sidered promising targets for the design of new trypanocidal
drugs, because of their vital role in the parasite’s energy
metabolism. Moreover, most of these enzymes have a number
of unique structural and kinetic features which, at least in
part, can be attributed to their unusual compartmentation
in microbody-like organelles called glycosomes. We have em-
barked upon a project to determine essential differences in
structure between the glycolytic enzymes of the trypanosome
and those of its mammalian host.
The purification of all glycosomal enzymes involved in
glycolysis from the bloodstream from Trypanosoma brucei
has been achieved previously (MISSETet al., 1986). Most of
the purified enzymes, including fructose-bisphosphate
aldolase, have been used for detailed kinetic analysis and
crystallization trials. However, resolution of the crystal struc-
ture of most enzymes has been hampered by the low amounts
of proteins that can be obtained from trypanosomes grown
in laboratory animals or in culture. The availability of the
gene for aldolase (MARCHANDet al., 1988) enabled us to
develop a procedure for high-level production of the enzyme
in a heterologous expression system. Therefore, a specific
restriction site was introduced by mutagenesis at the front
of the gene, enabling ligation of the gene to the vector pET3A,
immediately downstream of the phage ’I7 promoter and
ribosomebindingregion (STUDIER et al., 1990). This construct
was introduced in E. coli BL21 (DE3)pLys, a strain that har-
bours the T7 RNA polymerase gene in its chromosome under
the control of the LacUVS promoter. It also contains the
plasmid pLys, supplying low levels of T7 lysozyme, to reduce
basal activity of the polymerase prior to induction.
High levels of aldolase expression were achieved by growth
of the E. coli cells at 37OC in LB medium till OD- = 0.6,
with subsequent induction of T7 RNA polymerase with IFTG
for 2 hours. After harvesting and rupture of the bacteria in
a French press, 50% of aldolase activity was found as active
protein in the soluble cell fraction. Nucleic acids and some
other cellular components were eliminated by treatment with
nuclease and by precipitation with protamine sulphate (0.5
mg/ml). Aldolase was further purified to near-homogeneity
by ammonium-sulphateprecipition (6OVo) and passage over
a CM-cellulose column. The procedure appears reproduci-
ble. From 1 litre of E. coli culture 60-120 mg protein was
obtained, with a specific activity of 20-27 U/ml. The recom-
binant aldolase is essentially indistinguishable from the en-
zyme previously purified from the bloodstream form T.brucei
(CALLENS et al., 1991), with respect to its subunit Mr (41 kd),
isoelectric point (9.3), pH-activity profile, stability and kinetic
properties. The recombinant aldolase is currently being used
in crystallization experiments.
References
CWNS, M. el al. (1991) Mol. Biochem. Parasitol. 47. 1-10.
MARCHAND, M. et al. (1988) Mol. Biochem. Parasitol. 29, 65-16.
MLSSBT,0. ef ai. (1986) Eur. J. Bioehem. 157, 441-453.
S m m t , R. et al. (1990) Methods in Enwrnology 185, 60-89.
 B6
J. KUMMERTand P. LEPOIVRE
D. COLINET, (Luboratoire
de Puthologie Vpgptole, Fucultk des Sciences Agronomiques,
8-5030 Gembloux, Belgium)
Sequence relationship between the N-terminal part
of the coat protein of two strains of sweet-potato
latent virus.
Coat-protein sequence data have been used to
distinguish between viruses and between strains of
viruses in the potyvirus group (SHUKLA & WARD,1989).
Coat protein of distinct viruses are generally 38 to 71'To
identical, and differ markedly in the length and se-
quence of their amino N-termini, whereas coat proteins
of different strains of the same virus are more than 90%
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
identical and have very similar amino-terminal se-
quences (SHUICLA & WARD,1989).
In a previous work, a combined assay of reverse
transcription and polymerase chain reaction (RT-PCR)
utilizing degenerate primers allowed to identify three
distrinct potyviruses in a sweet potato clone originated
from China (COLINET et al., 1993). Dot-blot assays in-
dicated that one of these three potyviruses was closely
related to a sweet-potato latent virus isolate from
Taiwan (SPLV-T) (COLINET et al., 1993).
In order to corroborate this relationship, a 1.30-kb
fragment was amplified from SPLV-T-infected Nico-
tiana benthamiana using the same degenerate primers.
The partial nucleotide and deduced amino-acid se-
quences of the SPLV-T 1.30-kb fragment were com-
For personal use only.
pared to those previously determined for the 1.30-kb
fragment amplified from the SPLV-like virus from
China (SPLV-CH) (COLINET et al., 1993). This com-
parison showed 96.4Vo and 100% homology at the
nucleotide and amino-acid level, respectively, in part
of the C-terminal region of the putative RNA
polymerase, and 93.0% and 87.9% homology at the
nucleotide and amino-acid level, respectively, in the N-
terminal region of the coat protein, indicating that the
two viruses should be considered as two strains of
SPLV.
One important difference between the coat protein
amino-acid sequence of the two strains concerns the
DAG box involved in aphid transmission of potyviruses
(ATREYA et al., 1991). The lack of aphid-transmissi-
bility of SPLV-T (MOYER& SALAZAR, 1989) could be
explained by the mutation of a A to a T in its DAG
box. This mutation is not present in the DAG box of
SPLV-CH, for which the aphid-transmissibilityhas not
been determined yet.
This work was financially supported by the EEC (Project STD3
N" TS3-CT91-0013).
References
ATREYA,P.L., ATREYA, C.D. & PIRONB, T.P. (1991)Proc. Nutl.
Acud. Sci. USA 88, 7887-7891.
COWT, D., KUMMERT, J., LBPOIVRE,P. & SEW, J. (1993)
Phytoputhology (in press).
MOYER,J.W.& SAWWR, L.F. (1989) Plant Bis. 73, 451455.
SHUIILA,
D.D. &WARD,C.W. (1989)Arch. Virol. 106. 171-200.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994. 102 (1)
SOCI&& BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
I. COPPENS(I9 2), Ph. BASTIN(1*2), P. BAUDHUIN v),
F.R. OPPERDOES (z) and P.J. COURTOY
(') I(')
Cell
Biologv Unit and ( l ) Tropicul Diseuses Unit, ICP & UCL, 75
uv. Hippocrate. 8-1200 Brussels]
Activity and inhibition of hydroxymethylglutaryl-
CoA reductase in Trypanosoma brucei brucei
The rapid growth of Trypanosoma brucei brucei,
the causative agent of the African sleeping sickness in
livestock, shows a crucial requirement for low-density
lipoproteins (LDL), probably because of the cholesterol
that these particles carry. Import of cholesterol from
the host plasma LDL after receptor-mediated en-
docytosis and protein degradation has been
demonstrated (COPPENS et al., 1988, 1993; GJLLETT&
OWEN,1987). In mammalian cells, sterols are synthesiz-
ed by the mevalonate pathway and some intermediates
of this pathway have been recently detected in these
parasites (Ldw et al., 1991). We examined the contribu-
tion of a de novo sterol synthesis in the bloodstream
forms and culture-adapted procyclic forms of T. b.
brucei. We also asked whether a deprivation of ex-
ogenous supply of cholesterol together with a phar-
macological inhibition of the endogenous synthesis of
sterols could have a synergistic effect on the inhibition
of parasite growth.
An activity of hydroxymethylglutaryl-CoA(HMG-
CoA) reductase, the rate-controlling enzyme in the
biosynthesis of steroids and polyisoprenoids in mam-
malian cells, is detected in both forms of trypanosomes
and is enriched in microsomal preparations. In vitro,
the multiplication of both forms is arrested by the com-
petitive inhibitor of HMG-CoA reductase, synvinolin
(simvastatin) at an IC,, of 25 pM. Concentrationsabove
50 pM produce severe cytopathic effects leading to
parasite death in vitro, but fail to cause any detectable
toxic effects on mammalian cells. Growth inhibition
by synvinolin is partially overcome by the supplemen-
tation of late products of the mevalonate pathway, e.g.
cholesterol or squalene, and by LDL. Conversely, this
growth inhibition is potentiated by agents interfering
with the exogenous supply of cholesterol, such as an-
tibodies against the LDL receptor, or chloroquine that
impair the LDL degradation. Low concentrations of
synvinolin that are not cytotoxic and produce only
marginal inhibition of proliferation (1.25 to 12.5 pM),
-
induce a 2-fold increase of HMG-CoA reductase ac-
tivity and result in an enhanced capacity for binding
and internalization of LDL particles. The latter results
suggest that HMG-CoA reductase and LDL receptor
expression could be regulated by the cytosolic concen-
tration of cholesterol in T. brucei, like in mammalian
cells.
References
COPPENS, I., BAUDHUIN,P., OPPERDOES, F.R. & COURTOY, P.J.
(1988)Roc. Natl. Acud. Sci. USA 85, 6753-6151.
COPPENS,I., BAUDHUIN, P., OPPERDOES,F.R. & COURTOY, P.J.
(1993)Mol. Biochem. Parusitol. 58, 223-232.
G I L E ' ~ ,M.P.T. & OWEN,J.S. (1987)Biochem. SOC. Trum. 15,
258-259.
LOW, P., DALLNER, G . , ~ Y O R S.,
, COHEN,S., C m , B.T. &
MENON,A.K. (1991)J. Biol. Chem. 266, 19250-19257.
 SOCI&Tg BELOE DE BIOCHIMJE, UCL, 20 NOVEMBRE 1993
0. CULIc, R., LEMMENS,L. VANDUPFEL
and H. MCHY P. CUPERS,A. VEITHEN, P. BAUDWINand P.J. C o n -
(Limburgs Universitair Centrum, Deprrrlement M.B. W.,
Universitaire Campus, B-3590 Diepenbeek)
A novel method for the cytochemical detection of
phosphohydrolases at the cellular level
A general principle of cytochemical o r
histochemical detection of cell phosphohydrolases is in
situ precipitation of the phosphate released by the en-
zyme action. Various ions have been tested for that pur-
pose and among them Pb2+and Ce3+have been most
commonly used (FIRTEI, 1978). However, Pb” has been
shown to inhibit some of the phosphohydrolases and
also to form nonspecific deposits, while the use of CeJ+
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
is limited to electron-microscopy-based studies since the
CePO, precipitate is not visible in optical microscopy
(ROBINSON BE KARNOVSKY,1983).
The aim of this study was t o develop a reliable and
sensitive method for the light microscopy detection of
ectonucleotidases(and other phosphohydrolases)at the
cellular level.
The new method is a combination of the classical
cerium-precipitation approach and the sensitivity of
autoradiography. It is based on the use of radioactive-
ly labeled enzyme substrates C’P) or radioactively label-
ed capturing agent (I4*Ce),which is more universal since
it can be used for just any kind of phosphohydrolase
activity.
Slides with cells carrying precipitated radioactive
For personal use only.
CePO,, built up during enzyme reaction are coated with
autoradiographic emulsion (Kodak NTB2) and are
developed after a sufficiently long exposure time.
We evaluated this method for cellular visualization
of the ecto-ATPase, ecto-ADPase, 5’-nucleotidase and
alkaline phosphatase. It can also be used for monitor-
ing more complex cellular events, such as adenosine
production from extracellular ATP ( C m c et al., 1990).
For this purpose we used different cell lines as model
systems in order to validate a correlation between
biochemical measurements of the enzyme activity and
cytochemical findings. When applying this method,
conditions like concentration of Ce3+, fixation pro-
cedure, possible interferences of capturing agent with
other components of the reaction mixture (substrate,
for example), and interference of the other
phosphohydrolases have to be carefully checked.
This method is suitable for studies of
phosphohydrolase expression and its regulation,
monitoring of transfection efficiency, and functional
expression cloning of phophohydrolases.
References
CULIc, O., Smonc, I. & ZANIC-GRUBISIC, T. (1990) Biochim.
Biophys. Acta. 1030, 143-151.
FIRTH,J.A. (1978) Histochem. J. 10, 253-269.
ROBINSON,J.M. &KARNOVSKY, M.J. (1983) J. Histochem. cytochem.
10, 1197-1208.
Archives Internationales de Physiologie, de Biochimie et de Biophysique. 1994. 102 (1)
B7
TOY (Cell Biology Unit, University of Louvain and Internatinal
Institute of Cellular and Molecular Pathology, Avenue Hippocrate,
75, B-1200 Bru~elle~)
Fluid and membrane endocytosis in rat foetal
fibroblasts proceed normally in the absence of
clathrin coated pits.
In primary cultures of rat foetal fibroblasts, en-
docytosis of transferrin is inhibited by -90 to 95% by
intracellular potassium depletion, a procedure that arrests
coated-pit formation. In these conditions, fluid-phase en-
docytosis of peroxidase is not affected (CUPERS et al.,
1993). We have now focussed on effects on endocytic
membrane traffic. Membrane endocytosis was estimated
by the internalizationand recycling of two fluorescent pro-
bes : [N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hex-
anoylglucosylsphingosine (C6-NBD-CICer) and
1-[4-(trimethylamino)phenyl]d-phenylhexa-l,3,5-triene
(TMA-DPH). C6-NBD-GICer inserts in plasma mem-
brane and can be removed by a back-exchangeprocedure
with BSA (KOKet al., 1992). TMA-DPH is a probe
fluorescing when inserted into lipid membranes; it can be
removed simply by phase partition in an aqueous medium
(ILLINOER et al., 1990).
Membrane labelling was achieved with C6-NBD-GlCer
or TMA-DPH at 4°C and cells were then rewarmed at
37°C to allow internalization. After various internaliza-
tion intervals, cells were cooled agains to 4OC and sur-
face tracers were removed by methods described above.
Intracellular membrane tracer accumulation was a non-
linear process. In potassium-depletedcells, kinetics of en-
try was indistinguishable from that of control cells and
a steady-state between internalization and recycling was
reached after 60 min, corresponding with both markers
to 20% of internalization an 80% remaining on surface
plasma membrane.
Membrane tracer recycling was further analysed with
C6-NBD-GlCer. Whereas potassium depletion accelerates
2-fold fluid regurgitation (Cupms et al., 1993), no signifi-
cant difference between control and potassium-depleted
cells was observed in the recycling kinetics of C6-NBD-
GlCer and in the recyclable fraction (- 64% in control
cells and 55% in potassium-depleted cells).
In conclusion, both fluid and membrane endocytosis
in rat foetal fibroblasts proceed with a normal rate in the
absence of clathrin-coated pits. A first explanation may
be that clathrin-independent endocytic pathway is
stimulated or induced to the level that compensates for
the loss of the clathrin-dependent pathway, i.e. is sub-
jected to regulation. A simpler explanation may be that
clathrin is not an essential component of the molecular
machinery involved in the formation of pits and their bud-
ding into vesicles, but is only required to allow selective
internalization of receptor. In both cases, the size and
abundance of endocytic pits and vesicles must be iden-
tical in control and treated-cells.
We want to thank Ms.F. N’KuLI, T. h c and M. Lea- for their
excellent technical assistance. A. V B ~ isNan IRSIA fellow.
References
Cupms, P.,Krss, A.K.. BAUD-, P.& Covaro~.P.J. (1993)NATOASI
Series, Vol. H74 : Molecular Mechanisms of Membrane TrcGf/ic.
M o d , HOWELL& BBROERONeds, Springer-Vslag.
Iumain, D.,POINDRON, P.,Gussaa, N., MODOLLHL, M.& K ~ ~ R YJ .,4 .
(1990)Biochim. Biophys. Acta 1030, 73-81.
KOK, J. W.,HOBKSTRA. K., ESKBLINBN,S. & HOBKSTRA,D.(1992)J. Cell sfi.
103. 1139-1152.
 B8
D. DETAIIU, Ph. JACQUES and Ph. TEONART
( h t r e Wallon
de Biologie Industrielle, Universitk de Liege, B-4OOO Liege. Sart
Tilrnan)
Biodegradation of the polycyclic aromatic hydrocar-
bons phenanthrene, naphthalene and biphenyl by
bacteria isolated from a contaminated soil
Polyaromatic hydrocarbons (PAH) are widespread en-
vironmental pollutants which are of great concern because
of their toxicity, carcinogenicity and resistance to biodegrada-
tion (ATLAS,1981; LEAKY& COLWELL, 1990). However,
numerous microbial species are known to degrade these PAH
and there appears to be several metabolic pathways available,
depending upon the species, strain and even the culture con-
ditions (CBRNIOLIA, 1982,1984). Therefore it is important to
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
characterize the degradation parameters on a laboratory scale.
This work deals with the characterization of phenan-
threne, napthalene and biphenyl degradationunder different
conditions by pure cultures of microorganisms belonging to
three different species (Arthrobacter, Pseudomonas and
Alcaligenes).
In this study, we have shown that each selected
microorganisms is able to degrade easily its own substrate
used as sole source of carbon and energy. In experiments in
which 1 g hydrocarbon per litre is added to 100 ml mineral
salt medium supplemented with a vitamined solution, it was
shown that after incubation periods of 24 h (for
Pseudomonas), 40 h (for Alcaligenes) and 72 h (for Ar-
throbacter),levels of degradation between 90% and 100%
have been measured. Moreover, increasing hydrocarbons con-
centrations in the culture medium (up to 10 gllitre) resulted
in a decrease of biomass, while the amount of hydrocarbon
For personal use only.
really degraded is more important. This result suggests that
the abilities of bacteria to degrade these compounds, even
in non-limiting but toxic concentrations, are restricted.
In order to reduce the toxicity of these xenobiotic
substances, we have achieved a fed-batch experiment on a
culture of Pseudomonas. After successive addition of
naphthalene (2 x 1 gllitre) in the culture medium, we note
after 72 h of incubation,that absorbance doesn't reach a three
times value of the one reached after a 24 h incubation in the
same medium. Moreover, the percentage of naphthalene
degradation collapses after the second fed-batch. We observed
the same results when we achieved a complete fed-batch.This
experiment shows indirectly that the growth of bacteria is in-
hibited, not by a limitant factor but probably by a metabolite
produced by the oxidative metabolism of naphthalene.
On the other hand, we observed that supplementationof
the culture medium with glucose (2 g/litre) had a positive in-
fluence on PAH degradation, probably because it stimulated
metabolism of the cells and facilitated growth. This result
is supported by the observations of h u m & REHM(1991)
on phenanthrene biodegradation by Arthrobacter
polychromogenes.
We have also noted that each strain is unable to degrade
completely a mixture of PAH at low amounts (500 mgll) and
that biodegradation of each hydrocarbon leads to produc-
tion and accumulation of metabolites.
The authors thank Dr. MEROEAY (SCK-VITO B-2400 Mol, Belgium)
for kind gifts of the bacterial strains.
References
ATWS, R.M. (1981) Microbiol. Rev. 45, 180-209.
C~RNIOLIA, C.E. (1982) Rev. Biochem. Toxicol. 3, 321-361.
CERNIOLIA, C.E. (1984) Adv. Appl. Microbiol. 30, 31-71.
KBvla, S . &REEM, H.-J. (199l)Appl.Microbiol. Biotechnol. 34.804-808.
LRAEY,J.G. & COLWLL,R.R. (1990) Microbiol. Rev. 54, 305-315.
Archives Internationales de Physiologie, de Biochimie et de Biophysigue, 1994, 102 ( 1 )
socnb~3BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
M. DE WOLFand E. DAMS(RUCA-Laboratory for Human
Biochemistry, University of Antwerp, Groenenborgerlaan 171,
8-2020 Antwerp)
Regeneration of receptor recognition domains on
the B subunit of cholera toxh by formation of
hybrids from inactive mutants.
A Trp88Phe inactive mutant and a Lys34Glu,
Lys91Glu inactive double mutant of the B subunite of
cholera toxin (CTB) were prepared by site-directed
mutagenesis as previously described (DAMS et al., 1992).
Mutants were released from the periplasmic space of
E. coli JM83 host cells with polymixin B and purified
by affinity chromatography over an anti-CTB antibody
Sepharose 4B column.
Submitting a mixture of equal amounts of these in-
active mutants to a denaturation-renaturation cycle
resulted in the formation of hybrid CTB pentamers
(hCTB) which were able to bind GM,. The pentameric
nature of these hybrids was revealed by gel-filtration
chromatography and by SDS-polyacrylamide gel-
electrophoresis after crosslinking with disuccinimidyl
tartrate. The affinity of hCTB for GM,, as measured
indirectly by a competitive solid-phase radiobinding
assay, was unexpectedly high and only 2.5-fold lower
than that of its native counterpart. The average number
of active binding sites on hCTB was estimated from
(i) titration with the oligosaccharide moiety of GM,
(oligo-GM,) and monitoring the reversal of the Trp
fluorescence quenching by iodide ions (DE WOLFet al.,
1981), and (ii) rapid gel-filtration over a Superdex HR
column of a mixture of hybrid CTB and an excess of
3H-labelled oligo-GM,. The data indicated the forma-
tion of one active binding site per four reconstituted
binding sites on hCTB, which is consistent with a ran-
dom association of CTB monomers during the
denaturation-renaturation cycle.
Assuming a binomial distribution, 1/16 of hCTB
molecules should have a composition similar to that
of the parental mutants, 10/16 should have one active
binding site and 5/16 two active binding sites. This
calculated fraction of divalent hCTB molecules in the
hCTB preparation is consistent with its reduced capaci-
ty (30 percent of that of native CTB) to agglutinate
GM1-containing phospholipid vesicles.
In conclusion, the binding characteristics of hCTB
prepared from Trp88Phe and Lys91Glu inactive
mutants are identical with those previousIy reported
(DE WOLFet al., 1992) for hybrids prepared from in-
active chemical derivatives and further support the view
that receptor recognition domains are shared between
adjacent @-chainsof CTB.
This work was supported by FGWO grant no. 3.0005.91.
References
DEWOLF,M.J.S.,FRIDIUN,M.&KOHN,L.D.(~~~~)J.B~~~.
256, 5489-5496.
DB WOW, M., DAMS, E. & DmRICK. W. (1992) Bacterial Protein
Toxins, Zbl. Bakt. Suppl. 23 (WITEIOLT,B., h m , J.E.,
B o u ~ ~ uG.J.,
s , COSSART, P., DIIKSTRA,
B.W., FALMAONE, P.,
~ C H F.J., ,FRBBR. J. & NIA~A". H.,
eds) pp. 201-202.
 socnM BELOE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
M. DE WOLFand E. DAMS (RUCA-Laboratory for Human
Biochemktv, Universityof Antwerp, Groenenborgerlaan I 71,
8-2020 Antwerp)
Biologic activity of cholera toxin containing a
hybrid B subunit formed from inactive mutants
The mechanism by which the initial interaction bet-
ween cholera toxin (CT) and its receptor, the
monosialoganglioside GM,, triggers A,-peptide inser-
tion into the membrane is still poorly understood. It
has been suggested that pentavalent binding of CT plays
an important role in this process (FISHMAN, 1982).
To further explore the role of pentavalent binding
in CT action, we studied the interaction of a CT
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
derivative containing a reduced number of binding sites
with intact cells in culture. This CT derivative was con-
structed from a hybrid B subunit (hCTB), which was
prepared from an inactive Trp88Phe mutant on the one
hand and an inactive Lys34Glu, Lys91Glu double mu-
tant on the other hand.
When submitted to a denaturation-renaturationcy-
cle, the parental mutants and hCTB were able to
reassociate with CTA and form the corresponding
holotoxins (Phe88CT, Glu34Glu91CT and hCT) as
measured by gel-filtration chromatography on a Biogel
PlOO column. The GM,-binding characteristics of hCT
were identical to those observed for hCTB and consis-
tent with the presence of only one or two functional
binding sites per hCT molecule. In contrast to Phe88CT
For personal use only.
and Glu34,Glu91CT, hCT was able to increase the in-
tracellular CAMPlevel in intact Vero cells and human
skin fibroblasts. At concentrations as low as a few
nanograms of hCT per ml, a significant increase in the
CAMPlevel could be observed, whereas at a concen-
tration of 1 pg of hCT per ml accumulation of CAMP
reached a maximum. Activation kinetics were identical
to those previously reported for hCT containing a
hybrid B subunit prepared from inactive chemical
derivatives of CTB (DE WOLFet al., 1992).
The present results further support the view that
pentavalent binding and clustering of GM1 molecules
is not essential for CT action.
This work was supported by FGWO grant no. 3.0005.91.
References
FISHMAN,P.H. (1982) J. Membrane Biol. 69, 85-97.
DB WOLF, M., DAMS,E. & DIBRICK. W. (1992) Bacterial Protein
Toxins, Zbl. Bakt. Suppl. 23 (WITHOLT, B., ALOUF,J.E.,
Boullus, G.J., COSSART.P., D ~ T R AB.W.,
, FALMAONB. P.,
FBBRBNBAcH, F.J.. FREER.
J. & NIBMA",H. eds), pp. 201-202.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994. 102 (1)
B9
L. DROOGMANS (l), I. CLUDTS(l), Y. CLEUTER (l),
A. Van den Broeke (l), L. WILLEMS(2), R. KETT-
MA" (') and A. BURNY 2) [(')Luboratoirede Chimie
(19
Biologique. Universitk Libre de Bruxelles, 67, rue des Chevaux,
81640 Rhode-St-GenPse et (') Facultk des Sciences Agronomi-
ques de Gembloux, UER Biologie Molkculaire et Physiologie
Animale, 13, avenue Markchal Juin. 8-5030 Gemblow,
Belgium]
The expression of interleukin4 receptors and
interleukin-6 mRNA by bovine-leukemia-virus-
induced tumor cells is independent of virus ex-
pression
Bovine leukemia virus (BLV) is the etiologic agent
of bovine leucosis. The virus induces malignancies of
the B-cell lineage (1eukemiaAymphoma). Since
interleukin-6 (IL-6) has been shown to be involved in
B-cell malignancies such as multiple myeloma or
plasmocytoma, the role played by this cytokine in the
BLV-induced leukemogenesis process was evaluated.
We derived six cell lines from BLV-induced tumors
(LB155, LB159, BL167, YR1, YR2 and M51). In situ
hybridization indicated that BLV expression occurs in
a small proportion of the cells of five cell lines (LB155,
LB159, LB167, YRl and M51). No expression was
found in the YR2 cell line. The six cell lines were tested
for the expression of IL-6 receptors by measuring the
specific binding of 1251-labelledrecombinant human
IL-6 to the cells. The results indicate that two cell lines
(LB155 and YR2) display 250-300 receptors per cell
(&= 10-'OM) whereas the other four (LB159, LB167,
YR1 and M51) do not display detectable amounts of
receptors. Despite the presence of IL-6 receptors on the
surface of LB155 and YR2 cells, no influence of ex-
ogenous IL-6 on their growth has been observed, even
when the serum concentration in the culture medium
was lowered to 0.5%.
Northern analyses indicated the presence of IL-6
transcripts only in the case of mRNA isolated from
LB155 cells. Since this cell line also expresses receptors
for the cytokine, an autocrine loop may exist in these
cells. Experiments in which different cell lines (OVK,
MDBK, BL' or BL-) were transfected with a plasmid
containing the bovine IL-6 promoter controlling the ex-
pression of the reporter cat gene failed to indicate any
influence of BLV expression on the activity of this pro-
moter, even when the cells were cotransfected with a
plasmid allowing the expression of the viral transac-
tivator p34m.
Taken together, these results indicate that the ex-
pression of IL-6 and its receptor can occur in some
BLV-induced tumors but is independant of BLV infec-
tion. The expression of the cytokine and of its recep-
tor could represent one of the possible secondary events
leading to full malignancy.
R.K. is Directeur de Recherches and L.D. and L.W.are Cher-
cheur Qualifi6 of the FNRS (Fonds National Belge de la Recherche
Scientifque). We thank the C a k e Genkraled'Epargne et de Retraite
(C.G.E.R.), the Fonds de la Recherche Fondamentale Collective
(F.R.F.C.) and the Bekales Foundation for financial support.
 B10
M.-C. DUBOIS,I. MARTIN,E. GOORMAGHTIGH and
J .-M. RWSSCHAERT(Free University of Brussels, Cam-
pus PIaine CP 206/2. B-1050 Bruxella)
Fusion in vitro of gastric tubulovesicles : a
fluorescence study
The parietal cells of gastric mucosa secrete
hydrochloric acid. In the resting state, these cells con-
tain a large number of vesicular membranes. The H+K+-
ATPase, the hydrogen-ion pump of the stomach is in-
serted in those membranes. Upon stimulation, the so-
called tubulovesicles fuse with the apical plasma mem-
brane to form secretory canaliculi. This fusion process
leads insertion of the ATPase with the proper orienta-
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
tion of the pump in the plasma membrane. The
molecular mechanism of this fusion is still unknown.
In order to study this fusion process by an in vitro
fluorescent method, we first inserted the oc-
tadecylrhodamine B fluorescent probe (HOEKSTRA et
a/., 1984) in the membrane of isolated tubulovesicles,
and followed the fluorescence dequenching resulting
from fusion between labelled tubulovesicles and
unlabelled ones. We noticed in control experimentsthat
fluorescence dequenching is observed even in the
absence of unlabelled vesicles. We therefore develop-
ped a new approach to follow the lipid mixing between
biological membranes, based on fluorescence energy
transfer between a donor (NBD-PE) and an acceptor
(Rhodamine-PE) fluorophore (STRUCK et al. , 1981).
For personal use only.
The experimental design of this method normally
precludes its application in studies involving the fusion
between native biological membranes, because
reconstitution techniques are required for proper mem-
brane insertion of the probes. However, we develop-
ped a new approach to insert the probes in biological
membranes :NBD-PE and Rhodamine-PE were incor-
porated into the vesicular membranes in the presence
of a small concentration of detergent (C12E8),which
is later removed by ultracentrifugation. We have used
this new method to identify components that may pro-
mote fusion between isolated tubulovesicles. We show
here that calmodulin and calcium incubated for 30 min,
at pH 7.4 and 37°C promote about 25% of fusion bet-
ween tubulovesicles. Light-scattering assays confirm-
ed this result :tubulovesiclesincubated with calmodulin
are larger than non-treated vesicles, Our results indicate
that it is possible to follow the fusion in vitro of
tubulovesicles by a fluorescent method, and that
calmodulin could stimulate this process. This calcium-
binding protein has also been implicated in
neurotransmitter release by chromaffin cells, resulting
from fusion between secretory granules and the plasma
membrane (CREUTZ,1981).
M.-C.D. is an IRSIA fellow.
References
CRBUTZ,C.E. (1981) J. Cell Biol. 91, 247-256.
HOEKSTRA,D., DE BOER,T., KLAPPE, K. & WILSCHUT,J. (1984)
Biochemisfry23, 5675-5681.
STRUCK,D.K.. HOEKSTRA. D. & PAGANO, R. (1981) Biochernisfry
20,4093-4098.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
S O C D ~ TBELGE
~ DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
I. FERRAO(I), M. CRABEEL(’), GHISLAIN,M. (9,M. MI-
NET (‘) and M. JACOBS (3) [(’) UniversifdLibre de Bnurelles,
( l ) Vriie Universiteit 8-1. diemt Erfelijkheidsleer en Microbiologie,
c/o COOVI, E. Gtysonlaan. 1. 8-1070 Brussel, (3) Vrue Universiteit
Brussel, dienst Plantengenetico. 1640 St-Genesius-Rodeand (*) Centre
de G4ndtique rnoldculaire du CNRS,91198 Gif-sur-Yvette]
Isolation of a A . thuliuna cDNA complementing the
aminotriazole sensitivity of a S. cerevisiue strain bear-
ing a gcn4 deletion
An A. thutiunu gene encoding an aspartate kinase-homoserine
dehydrogenase bifunctional enzyme (ak-hsdh) has been recent-
ly cloned (M. GHISLAJN, PhD. Thesis, 1992). Its promotor was
shown to contain repeats of the TGACTC sequence, which in
yeast is known to mediate the “General Amino-Acid Control”
that increases the transcription of several genes encoding amino-
acid biosynthetic enzymes under conditions of amino-acid star-
vation. Gnc4p is the transactivator in that process. TGACTC
elements are also present in other A. thulium genes involved in
amino-acid biosynthesis. Moreover, preliminary results indicated
the dependence on TGACTC and on Gnc4p of the function of
the ak-hsdh promoter in yeast (M. CHISLAIN, PhD. Thesis, 1992).
These data suggesting the existence of a possible general
amino-acid control in this plant prompted us to try to isolate
a plant homolog of GCN4. By analogy with the procedure used
to isolate the yeast GCNQgene, we have selected aminotriazole-
resistant transformants of a gcn4-deleted yeast strain, using a
A. thuliuna cDNA expression library (AT is an inhibitor of the
HIS3 gene product whose effect can be overcome by overexpres-
sion of HIS3).
We have isolated one plasmid conferring resistance to
aminotriazole and also to aminoethylcysteine, a second analog
to which gcn4- strains are sensitive and that is used as an her-
bicide. We confirmed the A. thaliana origin of the 1.93-kb Not1
insert by Southern blot hybridization and determined the DNA
sequence. The strand under the control of the PGK expression
promoter of the plasmid encodes a potential protein of 586 aa
which seems to be responsible for the complementation, as no
suppression of the aminotriazole sensitivity is observed when the
insert is recloned in the opposite orientation.
A search for homology in the SwissProt database revealed
no similar proteins and the Prosite program detected no obvious
domain signature, in particular no bZIP motif characteristicof
the GCN4/APl family of DNA-binding proteins. Moreover the
phenotypes expected from a functional equivalent of Gcn4p (such
as resistance to other analogs and increased expression of specific
genes) were not observed. Thus, te isolated gene does not seem
to encode a TGACTC-dependent transcriptional activator. The
absence of complementation of his3 and his1 mutants indicate
that we have not isolated the functional equivalent of HIS3 or
HISl (increase in quantity of Hislp, a feedback-regulated en-
zyme, might also lead to an ATR phenotype). The hydropathic
profde of the A. thulianu protein being incompatible with its loca-
tion in a membrane, it is also not the equivalent of Atrlp, a yeast
pump active in the specific efflux of aminotriazole. Other
hypotheses, such as the plant protein being a transcription fac-
tor with a uncharacterized DNA-binding domain regulating
HIS3, HISl or ATRI are still to be tested.
The structure of the isolated cDNA is very peculiar, the
“sense” strand (the strand equivalent to the poly A mRNA that
generated the cDNA) bearing no significant O W .The long ORF
with complementing activity is encoded by the opposite strand.
The latter is presumably also transcribed in the plant because
there is a TATA box consensus 105 nucleotides upstream of the
initiating ATG and 2 AATAAA polyadenylationsignals 256 and
280 nucleotides downstream of the stop codon; the insert is in-
terrupted a few nucleotides after the second AATAAA.
Confiiation of the existence of two overlapping messengers
in the plant should rise the question of the role of the antisense
mRNA.
 BELOE DE BIOCHIMIE,
SOCD~TI~ UCL, 20 NOVEMBRE 1993
C. GIBLENS,Natalie DE GEEST,Xue-Qin Xm and
Gis&leP h U X (Laboratorium voor Biochemie, Katholieke
Universiteit Leuven. Dekenstraat 6, 8-3000 Leuven)
Identification of a thioether bond and of disulphide
bridges in functional units d and g of Jc-
haemocyanin of Helix pomatia
The 8 functional units (a-h) of the subunits
(Mr 450 OOO) of the 8,-haemocyanin @?Hc) of Helix
pomatia can be liberated by limited proteolysis. The se-
quence of d (DREXELet al., 1987) and of g (XIN et al.,
1990) revealed respectively 5 (positions 50,59, 232, 321,
332) and 6 cysteinyl residues (positions 56,65, 166,233,
320,326) while amino-acid analyses, after performic-acid
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
oxidation, pointed to 7 of them in both. As, moreover,
no thiol group could be titrated it was decided to localize
the free thiol group(s), if any, and the disulphide groups
in d and g.
The pepsinolysates at pH 2.1 were fractionated by
Sephadex G-25 sf chromatography and further by
reversed-phase HPLC. The Cys-rich peptides were iden-
tified by amino-acid analyses and the expected positions
in the primary structure confirmed by sequencing. They
corresponded for d to residues 44-67, 157-168 + 230-236,
and 319-339 and for g to 50-72, 161-167 + 218-234, and
318-328 with in d(44-67) and g(50-72)a third Cys, respec-
tively identified as Sersoand Sera, on sequencing, and in
d(157-168) an unexpected Cys instead of
Disulphide bridges thus undoubtedly link C y s 165 and 232
and 321 and 332 in d and Cys 166 and 233 and 320 and
For personal use only.
326 in g and further also C y s 50 and 59 in d, as deduced
from the sequencing of the subpeptides 44-56 and 57-67
obtained on reduction, pyridylethylation, and trypsin-
olysis of d(44-67), and by analogy also Cys 56 and 65 in g.
Another peculiarity of d(44-67) and g(50-71) is their
unusual strong absorption at 255 nm, like found for a pep-
tide of tyrosinase of Neurospora crassa containing a
thioether bridge between a C y s and a His (LERCH,1982).
Such a bond fits well with the resistance of the third Cys
to thiol reagents, with its oxidation by performic acid, with
its detection as Ser on sequencing and with the amino-acid
analyses, which revealed for both peptides some
2-thiolhistidineand another unusual component. The lat-
ter most likely corresponds to S-(2-histidinyl)cysteine as
may be expected from a thioether bond between a C y s and
His. Sequencing of the subfragment 457-67) yielded, next
to Serso,2-thiolhistidine in position 62.
The thioether bridge may be a general feature for func-
tional units of molluscan Hc as, when the sequences were
reached via cDNA, a Cys and His were always deduced
at the corresponding position (LONTIEet al., 1990).
We wish to thank the Fund for Joint Basic Research for Research
grants and TheKatholieke Universiteit te Leuven for graduate fellowships
(X.-Q.X.).
References
-1, R., SIB~MUND,S., SCEINEJDKR,H.-J., LINZEN,B., GIEIBNS, C.,
Pmhux, G., LONTIE, R., Kmmnm”, J. & LOT-I‘SPEICH, F. (1987)
Biol. Chem. Hoppe-Seyler 368, 617-635.
LERCH,K. (1982) J. Biol. Chem. 257, 6414-6419.
LONTIE, R., W m ,R., GIHLWS,C. & FWhux, G. (1990)in Invertebrate
Dioxygen Carriers (PRb~ux,G. & LONTIB, R., eds). pp. 141-146,
Leuven University Press, Louvain.
XIN,X.Q.,GIBLWS,C., Wm,R.&Pmhux,G.(199O)hInvertebmte
Dioxygen Carriers (PR~Aux, 0. & LONTIE, R. eds), pp. 113-117,
Leuven University Press, Louvain.
Archives Internationales de Physiologie, de Biochimie et de Biophysique. 1994, 102 ( 1 )
B11
F. GOETHALS,J.M. BOCQUET,L. XENITOSand
M. ROBERFROID(Unit4 de Biochimie Toxicologique et
Canc&ologique, Ddpartement des Sciences Pharmaceutiques,
Universitt! Catholique de Louvain, UCL 7369, B-1200Bruxelles)
Use of histological and biochemical criteria for
assessing nephrotoxicity using rat kidney slices
The toxicity of several acute nephrotoxicants was
evaluated in vitro using renal slices prepared from male
Wistar rats with the Krumdieck slicer (KRUMDIECKet
al., 1980). Slices were incubated in serum-free DMEM/
Ham F12 medium gassed with O,/CO, (95/5) for up
to 8 h with the test compounds. ATP and GSH con-
tent, protein synthesis and histopathology (hematox-
ylin/eosin, Brachet, PAS staining) were monitored
during the incubation time.
In addition, incubation media were assessed for the
activity of lactate dehydrogenase, alkaline phosphatase,
y-glutamyl transpeptidase and N-acetyl-8-glucosamini-
dase.
Mercuric chloride (HgCl,) modified all the para-
meters tested at a concentration of M within 2 h
of incubation, whereas at lW5M, only protein synthesis
was inhibited after 4 h of incubation.
Sodium chromate (NazCr04)decreased ATP con-
tent and inhibited protein synthesis at lo+ M, without
inducing any enzyme release. At lo4 M, all the func-
tional parameters tested were modified after 2 h of in-
cubation; these alterations preceeded the increase in
enzymes leakage or the alterations in morphology.
The N-acetylcysteine conjugate of hexachlorobuta-
diene was tested at 5 x M and 5 x lom5M.
Significant increase of enzymes leakage was observed
at 2 h for 5 x M and at 4 h for 5 x 1W6 M and
preceded the inhibition of a-methylglucose uptake and
protein synthesis.
Cis-platin decreased the ATP content at lW4 M,
moreover RNA and protein synthesis were inhibited
from 2 h of incubation. Histological analysis demon-
strate proximal tubular alterations whereas glomeruli
and medulla were not damaged. The effect of trans-
platin and carboplatin were also evaluated. As com-
pared to cis-platin, carboplatin was less nephrotoxic,
confirming the situation in vivo.
Reference
KRUMDIECK,C.L., Dos SANTOS.J.E. & Ho, K.J. (1980) Anal.
Biochem. 104, 118-123.
 B12
A. GOOSSBNS,R. GEREMIA,M. VAN MONTAGUand
.
G ANQBNON (Laboratorium voor Genetica, Universiteit Gent)
Characterizationof arcelin 5, a seed storage protein
of wild beans
Resistance to the insect pest Zabrotes subfasciatus (Mex-
ican bean weevil), an important cause of post-harvest losses
in cultivated common beans from tropical countries, was
found in some wild Phaseolus vulgaris accessions. Arcelin,
a seed storage protein which is only present in these wild ac-
cessions is thought to be the factor responsible for this
resistance. Six different arcelin alleles have been identified
of which arcelin 1 and arcelin 5 seem the most promising in
conferring resistance towards insects (OSBORN et al., 1986;
LIOI& BOLLINI,1989; CARDONA et al., 1990; S m m o et al.,
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
1991). The arcelin 5 protein and the corresponding gene are
characterized with the aim of (i) determining the nature and
biochemical properties of arcelin 5 and homology and rela-
tionships with other arcelins and lectins, and (ii) establishing
the role of arcelin in resistance towards Z. subfasciatus.
The arcelin 5 protein was extracted from bean flour of
the wild P. vulgaris accession GO2771 with 10 mM NaCl, pH
2.4. Upon dialysis of the extract against double distilled water
and subsequent chromatofocussing of the precipitate from
the dialysis, three polypeptides with identical isoelectric points
and identical amino-terminal sequences were present : two
major polypeptides of 33 and 32 kd which are both
glycosylated and one minor polypeptide of 31 kd which was
not further characterized.
Reverse transcriptase polymerase chain reactions were per-
formed using total RNA of immature seeds of the GO2771
accession as a template and degenerate oligonucleotide
For personal use only.
primers designed on the basis of internal peptide sequences
and the amino-terminal sequence from the 33-kd protein. In
this way, overlapping cDNA clones were obtained, Correspon-
ding to the 5’ end, an internal fragment, and the 3’ end of
the arcelin 5 mRNA. Sequencing of the cDNA clones reveal-
ed the presence of two classes of clones, both encoding pro-
teins of 261 amino acids with signal peptides of 21 amino
acids. The first class corresponds to the 33-kd protein and
the second one most probably to the 32-kd protein. The iden-
tity between the two is 99% at the DNA level and 97% at
the level of the deduced amino-acid sequences. The arcelin-5
amino acid-sequence is 63% identical with arcelin 1 and
arcelin 2, 53% with phytohemagglutinin-E, 55Vo with
phytohemagglutinin-L, and 57% with the bean a-amylase in-
hibitor. These comparisons show very clearly that arcelin-5
is related to the other arcelin variants and lectins of P. vulgaris
but they also point out that arcelin 5 represents a new type
of arcelin. Southern-blot analysis of the DNA from the
GO2771 accession indicated the presence of two different
fragments hybridizing with an arcelin 5 cDNA probe.
Further research will focus on the isolation of the cor-
responding genomic clones and the involvement of the
arcelin-5 proteins in the resistance towards Z. subfasciatus.
References
C.,KORNEGAY,J., Posso, C.E.,MOPALES,F.& RAMIREz, H
CARDONA,
(1990) Entomol. Exp. Appl. 56, 197-206.
LIOI, L. & B o w l , R. (1989) Bean improvement Cooperative 32, 28.
OSBOiw, T.C., BLAKE,T.,GEPTS,R. & BLISS,F.A.(1986) Theor. Appl.
Genet. 71, 847-855.
A.,VAISASINA,B., Lior, L., VITALE,A. & BOLLINI,R. (1991)
SANTINO,
Plant Physiol. (Life Sci. Adv.) 10, 7-11.
Archives Internationales de Physiologie. de Biochimie et de Biophysique, 1994, 102 (1)
SOCI~TEBELGE DE BIOCHJMIE, UCL, 20 NOVEMBRE 1993
J.P. GIUTU (Loboratoire de Microbiologie, ULB - Facult6 de Mtdecine,
CP. 614, B-1070B m k )
Stable and unstable noncomplementing diploidy in
Escherichia coU and in E. coli : Serratia hybrids
Cell hybridization has always attracted cell an molecular biologists
as it concerns the fundamental question of tolerance between cell
constituants from genetically unrelated organisms. Protoplast fusion
in two genetically labelled Bacillus subtilk strains produces com-
plementing diploids persisting as such until segregation of parental
or recombinant haploid bacteria occurs. But this haploidy is
sometimes phenotypical, diploidy being maintained in some clones
where “rejection” consists in the switching off of one persistent
genome in a cell expressing the other parental genome. The totally
inactive chromosome of a stable non-complementing diploid (Ncd)
can be activated after polyethylene-glycol-inducedcell fusion. The
silent chromosome is therefore replicated but not transcribed (HmIi-
KISS & GABOR,1980; GUILLEN et al., 1985).
Analogous observations concern a highly reversible double mu-
tant of E. coli K12 strains previously interpreted as resulting either
from a phase variation involvingtwo distinct genes (GRATIA, 1989a)
or from an interaction between their products in the outer membrane
(GRATIA,1989b).The conclusion that such double mutants were rather
non complementing diploids, characterizd by the alternate expres-
sion of two genomes whose one was carryingtwo mutations, has been
finally reached on the basis of the following observations. (1) Such
bacteria though initially non-fertile (F-) can promote the formation
of complete heterozygotesupon contact with other polyauxotrophic
E. coli cells even F and/or RecA-. Segregants of complementing
diploids selected by their transient prototrophic character were found,
after re-isolation on non-selectivemedium, to be able to grow in the
presence of nutrients required by either parent but not anymore in
their absence (“biparental clones”). (2) Markers implied in the
segregation of parental types were located throughout the
chromosome (thr, leu, lac, pro, gal, att A, trp, att80, fig, his, r e d ,
met C,argG. mal, metB). ( 3 ) Stable parental types behaved in the
same way and were, like the initial double mutant, able to pass from
one type to the other, especially upon contact with other bacteria
as visualized by the use of genetically labelled partners. (4) Lysogeniza-
tion behaved as any other marker including immunity (prophages
A, 680 and +T were used). A zygotic-induction-likephenomenon was
observed when the lysogenic state was triggered in a clone where the
chromosomeCanying the prophage was previously switched off with
as a result the loss of the corresponding immunity repressor.
Any Ncd clone A/B issuing from successive crosses behaved as
if heterozygosis was limited to the newly acquired chromosome (b)
and only one of the previous genomes (a), suggesting that a regulatory
mechanism is responsible for a joint replication of two but only two
chromosomesat each cycle. This was also observed in heterospecific
crosses, e.g. between E. coli A/B and Serratia marcexens SMGIK)
(let say partner C). Upon re-isolation of selected prototrophic lac+
complementing types (E. coli was auxotrophic but lac+),stable E.
coli like Ncd types were isolated which proved to yield Serratia-like
clones, even after reisolation of the E. coii-like colonies, showing
the hybrid nature of the genetic constitution of the latter. Then, the
E. coli phenotype was restricted to either A or B, the A-B variation
being substituted by variation either from A to C (the reverse could
not be observed) or from B to C. Stability of Ncd E. coli-Serratia
A/C or B/C hybrids expressingthe E. coli genes was compromised
by continuous subcloning on account of a possible incompatibility-
like mechanism. As well as in B. subtilis, recombination between E.
coli chromosomes in stable Ncd cells, and a fortiori between E. coli
and Serratio, was inhibited. Quite informative was the isolation of
a hybrid clone where the phage sensitivity pattern of the E. coli p e n -
tal type B was altered until the clone was cured of the variation.
Therefore, it is being tried to find out whether the co-existence of
two unrelated genomes, expressed transiently, generates the forma-
tion of hybrid molecules.
References
GRATIA,J.P. (1989a) Arch. In?. Physiol, Biochem. 97,B32.
GRATIA,J.P. (1989b) Ibid. 97. 894.
G-N, N., AYAR, M. & HIRSCHBBIN, L. (1985) EMBO J. 4. 1333-1338.
Horc~luss,R . D . & G A B O R , M . H . ( I ~ ~ O )Nutl.
P ~ ~Acad.
~. Sci. USA 77,3553-3557.
 soc1131-13
BELGE DE BIOCHIMIE, UCL. 20 NOVEMBRE 1993
J.P. G u m (I), D. DBKEGEL (,), G . VANHEULE
(,), M. GALLENI (9,
R. DESCTNEER (I) .and I. TwadM (3) I(') hboratoire de
Microbiologie, Facult6 de MMminc, ULB. Brusseiq (3 Institut Pustar du Bra-
bcmr, BrusVts and (9 Centre d'Ing6nieurie des Prottins. Llniversit6 de Lipge]
Dissodrtionbewtcen Lolingand morphological alterationsupon
exposure of Serratia marcescens SMG40 to meclllinam
The work of LUND & TYBRINO (1972) and of SPRATT(1975) on 8-
lactam mecillinam has opened a considerable investigation field concer-
ning cell regulation and morphogenesis (cf D ' m & Bovloc, 1990).
Serratia marcexem is known to be insensitive to the lytic activity of
mecillinam in most cases. However, at low doses far from being growth-
inhibitory, it can cause the formation of round ceUs (GUNKEL et al., 1991;
this work). This fmding and the isolation of lov mutants in E. coli K12
(BOW et al., 1989; VINELLAet al., 19!92), which are also induced to
spherical morphology by mecillinam without being killed, shows that mor-
phological alterations and killing can be dissociated. The analysisof factors
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
deciding whether bacteria will keep on dividing as round cells or will die
is particularly attractive. The S. marcescens strain SMG40 which is sen-
sitive to cephalosporins of 3rd generation and to mecillinam (GRATIA & the trans-acting proteins involved in the breast-specific
CRENIER, 1992) seems to be appropriatein such studies. Indeed, subclones
were isolated with various levels of sensitivity to mecillinam (MIC bet-
ween 4 and loo0 pg ml-l) though being converted to spherical forms with
2-4 Fg ml-' mecillinam.
The substrainsconsidered here derived from the most CTX-sensitive
pigmented variant which, in spite of variation in pigmentation, kept the
same sensitivityto cefotaxime (MIC 0,2 pg ml-l) and the same 8-lactamase
activity [assayed in envelopes before complete neutralization by 1 pM
cloxacillin, using nitrocefm as substrate according to JORISet al. (1986)I.
No difference was detected in the penicillin-binding proteins of high
molecular weight (PBPlA,B,C, PBP2) among which PBPlA was
prevalent (58.2%), as in the strains studied by GUNKEL et al. (1991), and
the one which was most inhibited by cefotaxime. The PBPZ content was
uniform (20.5%) and completely inhibited by mecillinam (1 pg per 20
pg of total protein) in spite of varying activities of the drug in vivo. The
recent technique of GALLBNI et al. (1993) using fluorescent ampicillin pro-
For personal use only.
ved very convenient in this study.
Electron microscopy revealed that the most sensitive cells evolved to
large spheres before dying, while in more resistant populations, most cells
were smaller (1.5 to 2.5 pm instead of 4-6 pm in diameter). But, after
exposure of the sensitive cells to high doses of mecillinam (100-1OOOpg
m1-I) followed by a ten-times dilution in distilled water, cells lysed long
before they rounded and enlarged. Therefore, killing may arise at two
different stages :after rounding and enlargement on the one hand, and
through a lesion at the tip of the still rod-shaped cells treated at high
concentration on the other hand.
In all subclones showing high or low sensitivity to mecillinam in terms
of lysis, the addition of CaCI, did not interfere with rounding of the cell
but inhibited enlargementthrough regulation of the cell volume and divi-
sion (GRATIA,1993) and protected cells from lysis when treated under
drastic conditions. The ratio between actively dividing Cocci in the presence
of Caa+and colony-forming cells surviving to mecillinam in the absence
of this cation increased with time following a coefficient strictly depen-
dent on the concentrationof added Ca2+(from 50 pM to 25 mM) whatever
the lytic rersponse to mecillinatn. Results of experiments with EDTA in-
dicated that differences between substrains were not due to variation in
the ability to take up Ca**(or Mg*+)ions from the medium (Mueller-
Hinton Broth).
In conclusion, killing by mecillinam in Serratia possessing PBPZ and
responding to that drug by cell rounding depends on several mechanisms.
One of them is related with the presence and activity of Ca2+ions and
is common to the various substrains studied here. In addition, one may
suggest the role of a regulator, e.g. ppGpp as in E. coli mutants (VINELLA
et ul., 1992), to explain the quantitativedifferences in the level of resistance
to mecillinam between these substrains.
One of us (J.P.G.) gratefully acknowledged Prof. J.M.GHWSBNfor hospitality and
interst in the present work.
References
B o r n . Ph.. J m , A. & D'ARI.R. (1989) EMBO J. 1. 317-323.
D ' w . R. & Bouroc. Ph. (1990) TIES IS. 191-194.
o m k , M. el a/. (i993) ilioerem. J. 29i. 19-21.
GRAM, J.P. (1993) Arch. Inl. Physiol. Biochim. Biophys. 101, BIZ.
GRAM, J.P. & CPHNrmt. L. (1992) Zb. Baktenol. 216, 340-346.
G-, A.G.. HBcma. U. & hlmm. H.H.(1991) J. gen. Microbiol. 131, 243-252.
Jous. B. el al.. (1986) Biochem. J. 23% 581-586.
L m . F. & Tysamo. L. (1972) Nature 236. 135-137.
SPFSTT.B.G. (1975) Boc. Notl. Acad. Sci. USA 12, 2999-3003.
Vnraun, D., D'ARI,R. & B o r n , Ph. (1992) EMBO J. 11. 1493-1501
Archives Internationales de Physiologie. de Biochimie et de Biophysique, 1994, 102 (1)
B13
F. PASLEAU,
M. GROOTECLAES, P. BERZIand R. GOL-
WINKLER (Laboratory of Molecular Oncology, University
of Liege, Tour de Pathologie, 823, 84000 Liege, Belgium)
Breast-specific expression of erbB2-LUC reporter
genes
The product of the c-erbB2 oncogene is an EGF-
R-related receptor present in low quantities in normal
breast (PRESS et ul., 1990). The proto-oncogene is ac-
tivated by overexpression in at least 30% of primary
breast adenocarcinoma (SLAMON et ul., 1989). This
overexpression correlates with aggressive behavior of
the tumor and poor prognosis for the patient (PERREN
et al., 1991).
Our aims are to identify the DNA sequences and
control of c-erbB2 gene expression and to characterize
the alterations responsible for the overexpression of the
gene in primary breast cancers.
A 6-kb fragment, containing the c-erbB2 upstream
regulatory region, was isolated from a normal human
lymphocyte genomic DNA library and sequenced
(PASLEAU et ul., 1990). A functional assay has been used
to compare the transcriptional activity of this normal
c-erbB2 promoter in different mammary cell lines
overexpressing or not the c-erbB2 mRNA. Reporter
vectors containing fragments of increasing size (255 to
6 047 bp) of the c-erbB2 promoter linked to the
luciferase gene have been transfected in the low express-
ing HBL-100, T-47D and HeLa cells and in the high
expressing MDA-MB-453 and BT-474 cells. This ap-
proach dlows the delimitation of activator and
repressor sequences of the cerbB2 gene. Two transcrip-
tional activators, particularly active in the BT-474, were
located between -0.5 and -0.75 kb and between -2 and
-4 kb. A repressor sequence located in the -0.7 to -2 kb
fragment was shown to be active in all cell lines studied.
Nuclear proteins extracted from the different cell lines
were compared, in a gel retardation assay, for binding
to the 250-bp PvuII-SmaI fragment containing the c-
erbB2 activator. A specific pattern of retarded com-
plexes were observed in the tumor BT-474 cells com-
pared to the other cell lines.
Together, these results suggest that extended
fragments of the c-erbB2 regulatory region must be
considered in order to find the elements involved in
tissue-specific control or deregulated expression. By
footprinting assays, the important regulatory sequences
will be precisely delimited and possible mutations of
this sequences will be searched in the tumor cells.
References
PRSSS,M.F. d at. (1990) Oncogene 5, 953-962.
SLAMON,D.J. et al. (1989) Science 244, 707-712.
PERREN,T.J. (1991) Br. J. Cancer 63, 328-332.
PASLEAU,F. et al. (1990) Arch. int. Physiol. Biochem. 98, B40.
 B14
E. HANNECART-POKORNI, L. SONCK,F. DEPUYDT,
N. GERLACHE
and R. VANHOOF (Antibiotic Research
Unit. Pasteur Institute, 1180 Brussels)
Study of aminoglycoside-resistance mechanisms in
Xanthomonas (Pseudomonas)maltophilia
Literature on the aminoglycoside-resistance
mechanisms in X. maltophilia is scanty. However general
resistance behaviours are clinically observed, maybe as a
species-linked phenomenon.
We studied this resistance, in six strains isolated in a
Brussels general hospital (1984-1985). Two major
mechanisms were investigated, i.e. : - resistance by en-
zymatic modification of -OH or -NH, groups in the
aminoglycoside molecule; - resistance due to decreased
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
permeability of the cell wall for aminoglycosides. A third
resistance mechanism due to ribosomal mutation has been
discarded since it is only rarely encountered.
Using the radiolabeled cofactors method, enzymatic
modification could not be detected in the six strains. Con-
sequently resistance was supposed to be due to a lack of
penetration of the antibiotics. This has been confirmed
by kinetic uptake studies, using [3H]gentamicin.
Uptake of aminoglycosides into the Gram-negative
bacterium involves an initial interaction with negatively
charged residues of the lipopolysaccharide (LPS), the ma-
jor component of the outer membrane. The result of this
interaction is the displacement of divalent cations by the
antibioticand the subsequentdisruption of the outer mem-
brane which becomes permeable (HANCOCK et al., 1991).
For personal use only.
The following step of the antibiotic uptake involves the
penetration across the energized cytoplasmic membrane.
Concerning the first step, the lipopolysaccharides of
the six strains have been extracted and characterized. The
following findings can be forwarded :
- the presence of polysaccharide O-chains has been
confirmed by electrophoresis. Those O-chains provide the
hydrophilic environment necessary for the binding of
aminoglycosides on the bacterial surface (BRYANet al.,
1984);
- anionic charges (carboxyl and phosphate groups)
responsible for the interaction with the aminoglycosides
have been chemically detected.
These observations have been confirmed by the possi-
ble interaction of dansyl-polymyxin with both isolated
LPS and bacterial cells. As a matter of fact fluorescent
data have shown that polymyxin B binds to multiple LPS
anionic sites comparably to the polycationic
aminoglycoside (MOORE et al., 1986).
It can be concludedtherefore that the observed interac-
tions of LPS to aminoglycosidesdo not account for the
resistance phenomenon observed in the strains studied.
Consequentlythe subsequent steps of aminoglycosideup-
take through the cytoplasmic membrane are probably in-
volved in the impairment of the penetration process in X .
maltophilia.
References
BRYAN,L.E., O'HARA,K. & WONG,S. (1984) Antimicrob. Agents.
Chemother. 26, 250-255.
HANCOCK,R.E.W., FARMER,S.W., Lr, Z. & POOLE, K. (1991) An-
timicrob. Agents. Chemother. 35, 1309-1314.
MOORE,R.A., BATES,N.C. & HANCOCK,R.E.W. (1986) Antimicrob.
Agents. Chemother. 29, 496-500.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
soc16~6BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
and G.M. PETIAU-DE
A. HERMAN VRIES(Chimie Gbnd-
rale I, CP l60/18, UniversitbLibre de Bnucelles, Avenue F.D.
Roosevelt, 50, 8-1050 Bruxelles, Belgium)
Characterization of J-galactosidase from a murine
kidney tumor induced by leukemia cells
The activity and isoenzyme patterns of
glycosyltransferases and glycosidases are frequently
altered in cancer diseases.
This study examines the properties of 0-
galactosidase in normal kidney and in experimental
kidney tumors induced by an injection of murine cells,
line BL/RLlZ-NP derived from a C57 BL/Ka thymic
lymphosarcoma radiation-induced line (LJEBERMAN et
al., 1979), in normal C57 BL mice. A crude extract was
obtained after extraction in the presence of 1070Triton
X-100 and centrifugation; the activity was estimated
at pH 5 in the presence of 4-methylumbelliferyl-0-D-
galactopyranoside as substrate. The enzymatic proper-
ties were similar : the optimal pH was 4 and the op-
timal temperature, 55OC.
The KM of the tumor 6-Galactosidase was inferior
to that of the control (0.195 mM versus 0.286 mM).
Both were inhibited by D-galactono-1,Clactone. When
compared, the chromatofocusingpatterns of 0-galacto-
sidase in normal kidney and tumor revealed the pre-
sence of one major basic form in both extracts. The PI
of the second major form was about 7 in the normal
kidney and about 6.8 in the tumor. After treatment
with neuraminidase, the PI of some acidic isoforms was
raised. For comparison, the chromatofocusing pattern
of the cultured BL/RLlZNP cells revealed the presence
of one major form (PI = 6.7).
In conclusion, the results presented here indicate
that the 8-galactosidases of normal and tumor mouse
kidney have similar properties in spite of a higher
degree of sialylation of tumor isoenzymes.
This work was supported by grants from Galephar S.A. and by
the Banque Nationale de Belgique. The authors thank Dr. R. Hooc;~e
(V.I.T.O., Mol) for the tumors.
Reference
LIEBERMAN,M., DECL~WE, A . , RICCIARDI-CASTAQNOLI, P .,
BONIVER,J., FINN, 0. & KAPLAN, H.S. (1979) Int. J. Cancer
24, 168-177.
 SOCII~TB BELGE DE BIOCHIMIE,
UCL, 20 NOVEMBRE 1993
D. HODZIC(l), S. LAMBERT
(z) and R. GOL-WINKLER
(‘)
Laboratoire d‘Oncologie Molkculaire, Centre Hospitalier
[(I)
Universitaire. Universitk de Li&ge, Belgique and (’) Present
adress: Unitk INSERM 353. Hdpital St Louis, Institut
d’hkmatologie, 75475 Paris C e d e 10. France]
Use of inverse PCR in genomic library screening
Recent studies suggest that Insulin-like-Growth Fac-
tor I1 (IGF-11) may play an important role in tumor
development.
In fact, many researchers have demonstrated
overexpression of IGF-I1 mRNA in different types of
tumors such as Wilm’s tumor, pheochromocytoma,
hepatocarcinoma or mesothelioma (GELATO&
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
V m o n (1990); ELBADRYet al., 1991;HASELBACHER
et al., 1987).
We have shown a 2 8Wfold overexpression of IGF-
I1 mRNA in colon carcinomas over the levels detected
in normal colonic tissues resected from the same pa-
tients.
Hybridization in situ experiments have localized the
IGF-I1 mRNA overexpression in the carcinomatous
cells (LAMBERT et al., 1990). Interestingly, tumors
found to overexpress IGF-I1 mRNA carry a rearran-
ged allele of the IGF-I1 gene. This appears as extra
hybridization bands in tumor DNA digested with
several restriction enzymes. Hybridization with probes
from the ninth exon have localized the rearrangement
at the 3’ end of the IGF-I1 gene.
For personal use only.
To further investigate the role of the rearrangement
in IFG-I1 overexpression, a genomic library was con-
structed in Lambda FIX I1 (Stratagene)with DNA ex-
tracted from a tumor overexpresing 200 fold IGF-I1
mRNA. A first screening of this library with probe “C”
gave 3 positive clones. Their characterization by restric-
tion mapping showed that they contain different
fragments of the normal IGF-I1 allele.
The library is now being differentially rescreened
with two probes, “C” and “B”. In order to avoid isola-
tion of previously analysed clones, only those positive
with probe “C” and negative with probe “B” will be
lifted. Simultaneously, the breakpoint corresponding
to the beginning of the rearrangement will be cloned
directly using INVERSE-PCR.
References
GELATO,M.C. & V m m n , J. (1990) J. Clin.Endocrin. Metab. 71,
1168.
G.K. et al. (1987) Proc. Natl. Acad. Sci. USA 84,
HASELBACHBR,
1104.
LAMBERT,S. et al. (1990) Int. J. Cuncer 46, 405.
O m ,M. EL-BADRYet al. (1991) J. Clin. Invest. 87, 648.
Archives Internationales de Physiologie, de Biochimie et de Biophysique. 1994, 102 (1)
B15
K. KAS, D.J. STICKENS
and J. MERREGAERT
(university
of Antwerp. Dept. Biochemistry, Lab. of Molecular
Biotechnology, Unversiteitsplein 1. 8-2610 Wilrijk. Belgium)
FAUlP, a processed pseudogene of human FAU1,
contains an expanded trinudeotide repeat
A member of the human FAU gene subfamily, en-
coding the ribosomal protein S30 fused to a ubiquitin-
like protein, was cloned, sequenced and analysed. This
clone has all the characteristics of a processed (reverse-
transcribed) pseudogene, and is called FAUlP. It lacks
all four of the FAU introns, carries a 12-bp poly (A)
tail at the 3’ end and is flanked by a direct repeat of
13 nucleotides in the 5’ and 3’ regions. The pseudogene
sequence does not contain mutations that would lead
to a frameshift or a premature termination codon.
Hence, the completely intact open reading frame en-
codes a putative protein with 92% identity to the
FAUl-encoded protein (KAS et al. , 1992). Transcribed
retropseudogenes, although a rare occurence, have been
described before (MCCARREY& THOMAS,1987).
Whether FAUlP is transcribed/translated, needs fur-
ther investigation.
Noteworthy, this FAUl pseudogene shows an ex-
pansion of the (AAG) triplet repeat present in the
coding part of the FAUl gene. Recently, a new form
of human mutation - expansion of trinucleotide repeats
- has been found to cause the diseases of Fragile X syn-
drome, Myotonic dystrophy, Kennedy’s disease, Hun-
tington disease and spinocerebellar ataxia type 1 (Ross
et al., 1993). Here, we show the first example of an
(AAG) triplet repeat which has the capacity to expand.
Since the described triplet repeat expansion is in a pro-
cessed pseudogene of FAU1, this implies that FAUl
itself might be a target for expansion. FAUl localizes
to chromosome llq13, a region containing a large
number of disease loci (KAS et al., 1993). However,
there is no evidence yet that FAUl is associated with
a disease. Whether the triplet repeat expansion seen in
FAUlP has any significance, remains to be proven.
K. is an Aspirant of the NFWO.
References
KAS, K., WC~ELS, L. & MERREGAERT, J. (1992) Biochem. Biophys.
Res. Comm. 187, 927-933.
KAS, K., SCHOENMAKERS, E., VAN V m ,W., WEBER, G., Nomm-
SKJOLD, M., MERREGAERT, J. & LARSSON, C. (1993) Genomics
17, 387-392.
MCCARRE,J.R. & THOMAS, K. (1987) Nature 326, 501-505.
Ross, C.A., MCINNES,M.G., MARGOLIS,R.L. & LI, S.-H. (1993)
TINS 16, 254-260.
 B16
Z. KHOUITI and J.P. SmON (Unite de biotechnologie, CERIA,
Avenue Emile Gryson 1. 81070 Bruxellm)
Carnocin KZ213,a new bacteriocin produced in the
genus Carnobacterium spp.
Lactic-Acid Bacteria (LAB) became more and more im-
portant in food technology because they produce several an-
tibacterial factors including organic acids, hydrogen peroxyde
and bacteriocins (PIARD& DESMAZEAUD, 1992). LAB are
predominant on vacuum-packed meat ( A m & STILES, 1990).
A new genus Carnobacterium, proposed by COLLIN et al.
(1987), groups some species of non aciduric lactobacilli, in-
cluding Carnobacteriumpkcicola, Carnobacterium divergem,
Carnobacterium mobile and Carnobacterium gallinarum,
especially isolated from poultry and meat. The genus Car-
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
nobacterium spp. has the ability to produce bacteriocins. The
latter are proteinaceous compounds that generally inhibit
growth of closely related species (TA~oet al., 1976). Some
of them have an antagonistic activity against pathogenic
micro-organisms and food spoilage. These may be of interest
in food preservation. In this report we describe a new
bacteriocin called Carnocin KZ213, produced by Car-
nobacteriumpiscicola 213 isolated from meat by MONTEL et
al. (1992).
Several strains of Carnobacterium spp. from ATCC
(Americain Type Culture Collection), and from INRA (In-
stitut National de la Recherche Agronomique) have been
screened for bacteriocin production. The agar-well diffusion
method was used for detection of the antagonistic activity.
One strain, Carnobacterium piscicola 213, has proved to
possess an antagonistic activity. The cell-free supernatant of
an 24-h culture of the producer strain grown on modified
For personal use only.
MRS broth, (MRS broth perpared without ammonium citrate
or sodium acetate), has been tested for inhibition zone on
an indicator lawn.
Carnocin KZ213 was active at neutral pH, its activity was
not modified by the catalase (68 U/ml), so that inhibition
by organic acids and hydrogen peroxyde was not involved.
The antagonisticactivity was stable after heat treatement du-
ring 20 min at 121°C. But, it was completely destroyed by
the proteolytic enzymes tested (pepsin and pronase, 1 mg/mI).
Carnobacterium piscicola 213 produced a proteinaceous
bacteriocin-like substance with a preferential antagonistic ac-
tivity against Carnobacterium spp. Carnocin KZ213 did not
inhibit a wide variety of lactic-acid bacteria, so it’s different
from the bacteriocin produced by the known strains Car-
nobacteriumpiscicolaLV17 and U149 (AHN & STILES, 1990;
STOFFELS et al., 1992). Carnocin KZ213 was active after
autoclaving at 121°C for 20 min, contrary to Carnocin CP5
(MATHIEUet al., 1993).
Carnocin KZ213 could be true bacteriocin and was
secreted at a high level.
References
AHN, C. & STILES,M.E. (1990) J. Appl. Bacteriol. 69, 302-310.
AHN,C. &STILES,M.E. (1990) Appl. Environ. Microbiol. 56,2503-2510.
M.D., FARROW,
COLLINS, J.A.E., €H
’ ELPS, B.A., Fwvsv,S. & JONES,D.
(1987) Int. J. Syst. Bacteriol. 31, 310-316.
MATHIEU,F., MICHEL,M. & LEPEBVBE, G. (1993) Biotechnol. Lett. 15,
587-590.
MONTEL, M.C., TALON,R.,FOURNAUD, J. & CHM~POMIGR,M.C. (1 992)
J. Appl. Bacteriol. 10, 469-472.
PIARD,J.C. & DESMAZEAUD, M. (1992) Lait 72, 113-142.
STOFFELS,
G., NISSEN-MEYER. J., GUDMUNDSDOTTKR, A., SLETTEN,K.,
HOLO,H. & NES, I.F. (1992) Appl. Environ. Microbiol. 58,
1417-1422.
TAGG,J.R., DAIANI,A S . & WANNAMAKER,L.W. (19767 Bacteriol, Rev.
40, 722-756.
Archives Internationales de Physiologie, de Biochimie et de Biophysigue, 1994, 102 (1)
socnh? BELOE DB BIOCHIMIE, UCL, 20 NOVEMBRE 1993
L. KOHL, C.D. Do THI, F.R. OPPERDOES and
P.A.M. MICHELS(Research Unit f o r Tropical Diseases,
International Institute of Cellular and Molecular Pathology,
Brussels, Belgium)
Trypanosoma brucei contains a unique
glycerol-3-phosphate dehydrogenase in its
glycosome
In trypanosomes the majority of the enzymes of the
glycolytic pathway and two enzymes of the glycerol
metabolism are localized in a microbody-like organelle,
the glycosome. Glycerol-3-phosphate dehydrogenase
(GDH) plays an essential role in the metabolism of the
trypanosome, because it is responsible for maintain-
ing a redox balance within the organelle. A preliminary
analysis of the structural and kinetic properties of the
native enzyme showed some peculiarities of
trypanosomal GDH :subunit mass 2-3 kd larger than
its mammalian counterpart, highly charged (PI 10) and
unique kinetic properties. We have screened a Xgtl 1 ex-
pression library, prepared from poly A+ RNA from
bloodstream form Trypanosomabrucei, with a rabbit
polyclonal antiserum raised against purified T. brucei
GDH. After rescreening, 8 positive clones were
isolated. The inserts of the recombinant phage genomes
were amplified by PCR. All phages contained an iden-
tical insert with a size of 1.2 kb. Four different clones
were chosen for further analysis. Sequence analysis
showed that all clones contain a full length cDNA. The
5’ end of the cDNA codes for an amino-acid sequence
that is identical to the amino-terminal sequence of
GDH ,determined by microsequencing of the purified
protein.
The cloned cDNA encodes a polypeptide of 350
amino acids, which corresponds to a calculated M, of
37 518. The calculated net charge of the protein is + 14.
These results are in good agreement with those
previously obtained for the purified protein (MISSETet
al. , 1986). A characteristic motif of NAD+-binding do-
mains could be distinguished, but the remainder of the
sequence shows only a very low percentage of identity
(18-20%) with the other GDH sequences.
Its unique primary structure, its essential metabolic
role and its peculiar kinetic properties render
glycosomal GDH a promising target for the design of
new drugs against trypanosomes.
L. KOHLis a recipient of a fellowship Formation-Recherche by
the Government of Luxemburg.
Reference
MJSWT,O., Bos, 0. F.R. (1 986) Eur. J. Biochem.
J.M. & OPPERDOES,
157, 441-453.
 SOCIJ~TE BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
J. KUMMBRT,J.L. LEMAJRE,
G. RUFFLARD,
D. COLINET
and
P. LEPO~VRB
(Laboratoire de Puthologie v~gdtule,Facult6 des
Sciencer ugronomiques. 55030 Gembloux)
Detection and charactehtion of a barley yellow mosaic
virus isolate susceptible to infect the resistant barley
cultivar express
Barley yellow mosaic diseases result from the infection by
one or both bymoviruses : Barley yellow mosaic virus (BaYMV)
and Barley mild mosaic virus (BaMMV).
Since 1990, mosaic symptoms have been observed in one field
in Huccorgne (Belgium) on several barley cultivars considered
so far as resistant to BaYMV and BaMMV. Extracts from these
symptomatic plants did not present serological reactions in the
presence of a set of monoclonal antibodies currently used for
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
the identification of BaYMV and BaMMV in field plants.
The objectiveof the present work was to clarify the etiology
of these mosaic symptoms and to identify the causal viral agent
by using a combined assay of reverse transcription and
polymerase chain reaction (RT-PCRR) utilizing degenerate
primers.
The material used for this characterizationconsisted in total
RNA preparations from barley plants of the cultivar Express
recolted in the field at Huccorgne.
Sequence data existing for the RNAl of BaYMV
(KASFIIWAZAKJ et ul., 1990; PEERENBOOM e? al. , 1992) and of
BaMMV (KASHIWW et al., 1992) have been compared
together and with the sequences of several potyviruses in order
to determine degenerate primers to be used in RT-PCR reactions.
Three sets of primers were selected allowing amplification
of sequences of either different bymo- and potyviruses, or of
BaYMV or BaMMV alone.
A 1130-bp fragment was amplified from infected barley plants
For personal use only.
of the cultivar Express, with the specific primers for BaYMV,
whereas no amplification occurred with the specific primers for
BaMMV.
The non specific third set of primers, allowing the amplifica-
tion of a 840-bp fragment for BaMMV and of a 960-pb frag-
ment for BaYMV, give a single 960-bp fragment from infected
barley plants of the cultivar Express, whereas both fragments
(840 bp and 960 bp) are consistently amplified in varying pro-
portions from infected barley plants of sensitive cultivars.
These results suggest that the viral agent infecting the cultivar
Express is an isolate of BaYMV. Partial sequencing of the
amplified 960-bp fragment which corresponds to the 5’ end of
the coat-protein gene showed more than 94% and 97%
homology, at the nucleotide level, with the sequences published
respectively for a japanese (KASEIWAZAKI et al., 1990) or a ger-
man (PEERENBOOM et al., 1992) strain of BaYMV isolated from
infected sensitive barley plants. None of these differences was
reflected at the amino-acid level of the putative translated pro-
duct, whereas less than 30% homology was observed with the
corresponding sequences of BaMMV.
The amplified fragments obtained from RT-PCR with the
three sets of primers for total RNA extracts from sensitive barley
cultivars and cv. Express from Huccorgne were cloned with the
p-Bluescript in E. coli in order to develop non radioactive pro-
bes for the detection and characterization of viruses infecting
barley by dot blot hybridization assays.
This work was supported by the IRSIA, Brussels.
References
KAWWAZAU, S . , Mmom, Y.,O m ,T.& Hmmo, H. (1990) J. Gen. Virol.
71, 2781-2790.
KASWVAZAU, S., NOMUM,T.,KURODA, H., 110,K. & Hmmo, H.(1992)
J. Gen. Virol. 73, 2173-2181.
P ~ ~ R ~ N B OE.,
O PROLS,
M. A.D.
M., Scmu, J., S~amrss,H.H. & DAVIDSON,
(1992) J. Gen. Virol. 72, 1303-1308.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
B17
Vtronique LEFEBVRE
and P. BUC-CALDERON
(unite de
Biochimie Toxicologique et Cancerologique, Departement des
Sciences Pharmaceutiques, UniversitkCatholiquede Louvain,
1200 Bruxelles, Belgium)
Proposals for a potential clinical use of fructose
during liver transplantation
Since 1963, liver transplantation surgery is becom-
ing an accepted therapy for patients with severe hepatic
failure (ST- et ul., 1963). For the transplantation
procedure to be successful, the preservation of the
organ during storage and transportation is vital. At pre-
sent, simple cold storage of liver in artificial in-
tracellular medium is used, and preservation time is
limited (TODOet al., 1989). With regard to hypoxic
damage during preservation, the development of
strategies that would improve organs storage could be
important clinically.
Our results show that elevated fructose concentra-
tions allow a better cellular resistance against hypoxia-
mediated cell lysis. Paradoxically, this better cell sur-
vival, which is associated with the high lactate forma-
tion from high concentration of fructose, is observed
at the lower ATP levels. Although fructose allowed a
maintenance of low but constant intracellular ATP
levels, the depressed protein synthesis induced by
hypoxia was not restored (LEFEBVRE et al., 1993). In-
deed, measurement of protein-synthesis rates could be
used as a parameter to predict whether the hepatic tissue
would be metabolically active once transplanted
(NAKAGOHRIet ul., 1989). In addition, preservation
methods which maintain protein synthesis in the liver
may be shown to improve the overall quality of liver
preservation (VREUGDENHIL et al., 1992). Since the
reoxygenation of hypoxic hepatocytes previously in-
cubated in the presence of fructose allow a faster
protein-synthesis recovery than the reoxygenation of
hypoxic cells preincubated in the absence of fructose,
we suggest that liver must be perfused with a fructose-
containing solution before its removal from liver
donors and further hypoxic preservation. In this way,
during such an aerobic perfusion liver tissues can ac-
cumulate a sufficient amount of fructose 1-phosphate
and ATP, produced by the anaerobic glycolysis, will
be mainly used for the maintenance of critical
functions.
V.L. is Research Assistant of the National Fonds for Scientific
Research (FNRS, Belgium).
References
LEPBBVRE,
V., BECKWS,
V., VAN STBBNBRUOOE, M., ROBEXFROID,M.
& BUC-CALDERON,P. (1993) Arch. Biochem. Biophys. 304,
322-331.
NAKAGOHRI, T.,Aslwo, T., GOTO, T., KENMOCHI.T., SAKAMOTO,K.,
O c m , T.& ISONO, K. (1989) Tramplant. Proc. 21,2292-2293.
STARZL,T.E.,MARCHIORO, T.L., VON KAULLA,K.N., HKRMA”,G..
B m m , R.S.& W A D D ~W.R. , (1963)Surg. Gynecol. Obstet.
117, 659-676.
Tono, S., NERY,J., YANAOA.K.. PODESTA, L., GORDON, R.D. &
STARZL, T.E. (1989) J. Am. Med. Assoc. 261, 711-714.
J.H.
VREUGDENHIL,P.K.,MARSE,D.C., BELZER,F.O.&SOUTHARD.
(1992) Hepatology 16, 241-246.
 B18
S.M.DURVIAUX
F.P. LEMAIGRE, and G.G. ROUSSEAU
(Hormone and Metabolic Research Unit, Universitt! Catholi-
que de Louvain and International Institute of Cellular and
Molecular Pathotop, Avenue Hippocrate 75, B-I2(KIBnurella)
Liver-specificfactor binding to the liver promoter
of a 6-phosphofructo-2-kinase/fructose-2,6-
bisphosphatase gene
6-Phosphofructo-2-kinase/fructose-2,6-bisphosp-
hatase (PFK-Z/FBPase-2) catalyzes the synthesis and
degradation of fructose-2,6-bisphosphate,a potent
stimulator of glycolysis. One gene codes for the liver,
muscle and hepatoma isozymes of PFK-2/FBPase-2 by
the alternative use of three promoters (DARVILLE et af.,
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
1989; DUPRIEZet al., 1993). We have delineated earlier
several sites of protein-DNA interactions on the liver
promoter (LEMAIGREet af., 1991) and are now identi-
fying the proteins involved in these interactions.
Two of these sites, called sites I11 (-112 to -138) and
IV (-196 to -216), bind liver-specific factors and con-
tribute to the activity of the promoter in transfection
experiments. Site I11 binds in a mutually exclusive way
octamer factor-1 and the liver-specificHNF3a, /3 and
y. Site IV binds two classes of liver-enriched proteins,
namely CCAAT/enhancer binding protein (C/EBP)-
related factors and a factor that we call LP4.
LP4 can bind to a HNF-3 site. It differs from
HNF-3 a,B and y as shown by use of antibodies and
methylation interference analyis. DNase-I footprinting
For personal use only.
and electrophoretic mobility shift assays, in the
presence of competing oligonucleotides corresponding
to binding sites for known liver-specific factors, in-
dicate that LP4 behaves as an undescribed liver-specific
DNA-binding protein. We have purified LP4 to near
homogeneity by chromatography through heparin-
agarose, wheat-germ agglutinin-agarose and DNA-
affinity columns. The Mr of LP4 is 55 O00, which is
compatible with the Mr of 65 OOO calculated earlier in
UV-crosslinking experiments.
This work was supported in part by the FRSM,the “P6les &At-
traction Interuniversitaire” (PAI) and the “Association contre le
Cancer”.
References
M.I., CREPIN,K.M.,HUE,
DARVILLE, L. & ROUSSEAU. G.G. (1989)
Proc. Natl. Acad. Sci. USA 86, 65434547.
DUPRIEZ,V.J., DARVILLE, M.I., ANTON, I.V., GEOONNE, A.,
GHYSDABL. J. & ROUSSEAU,G.G. (1993)Proc. Natl. Acad. Sci.
USA 90, 8224-8228.
LEMAIGRE,F.P., DURVIAUX, S.M.& ROUSSEAU, G.G. (1991)Mol.
Cell. Biol. 11, 1099-1106.
Archives Internationales de Physiologie. de Biochimie et de Biophysique, 1994, 102 (1)
SOCD~TI~
BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
I. LEONARD(’), J.F. BECKERS(9. J. ZANEN(I), D. NON-
(I), J.A. STIENNON-HEUSON
CLERCQ (I), G. TOUBEAU (I),
c),
P. SCHAUDIES G. LAIJRENT(I) and P. FALMAGNE r)
Service d’Histologie et (3 Service de Chimie Biologique. LMwrtement
[(l)
de Biologie er de Pathologie Cellulaires, UniversitkdeMom-Hainaut; (>)Ser-
vice de Physiologie de la Reproduction, Universitt?de Liege and (9 Depart-
ment of Nephrology, W.R.A. I.R.. Washington, D. C., U.S.A. 1
Modification of epidermal growth factor (EGF) level in rat
kidney tissue after tubular necrosis induced by tobramycin
or cisplatin
Several lines of evidence indicate that EGF, a polypeptide
growth factor synthesized in the submaxillary gland and in the
kidney, might act as a positive regulator during renal tubular
regeneration after nephrotoxic or hypoxic injury (NORMAN et al.,
1990;COIMBIU ef af., 1990;M o m et af., 1992). However, the
precise mechanism of action of EGF in the kidney remains to
be established, inasmuch as the growth factor mostly occurs in
renal tissue as a high molecular weight, membrane-bound precur-
SOT (BREYER & COHBN, 1990).
In this study, rat kidney tissue was examined with respect
to EGF level and distribution, proliferation rate and renal
dysfunction during an episode of tubular necrosishegeneration
caused by two nephrotoxins exhibiting different mechanisms of
renal toxicity. Nephrotoxic injury was induced by a four-day
treatment with tobramycin or cispaltin at daily doses of 200
mg/kg or 2 mg/kg respectively. The animals were sacrificed 1,
4,7,14,21 and 60 days after the end of treatment. Immunoreac-
tive EGF was evaluated by RIA in the supernatant of renal cor-
tex and OSOM homogenates after high speed centrifugation
(soluble EGF) or in renal homogenates pretreated with Triton
X-100 (total tissue EGF, mainly membrane-bound EGF)
(LBONARD et af.,1993). These measurements were supported by
the immunohistochemical demonstration of EGF immunoreac-
tivity on kidney paraffin sections. Proliferation rate was
estimated by immunolocalization of S-phase cells pulse-labelled
with BrdU. Renal glomerular filtration impairment was
monitored by serum creatinine and BUN levels.
Tubular necrosis was followed by regenerative hyperplasia
reflected by an elevation of proliferation rate in renal tissue of
treated animals during the first week after treatment. In parallel
with tubular necrosis and regeneration, total EGF immunoreac-
tivity markedly decreased at day 1. In tobramycin-treated group,
this decrease was transient since EGF level returned to control
value at day 21. In contrast, in cisplatin-treated rats, the decrease
was still present two months after the end of treatment. These
results were confirmed by immunohistochemical studies. Level
of soluble EGF remained unchanged during the whole period
of observation. Similarly, renal dysfunction was transient in
tobramycin-treated groups but persisted in cisplatin-treated
animals.
These results are in favour of an involvement of EGF dur-
ing tubular regeneration. We suggest that the membrane-bound
EGF level decreases as a consequence of an enzymatic proces-
sing into soluble EGF. However, the pool of soluble EGF might
be governed by complex kinetics and so remains apparently con-
stant. Moreover, cisplatin, which is known to interact with DNA,
induces cystic tubular degeneration possibly related to defective
tissue repair (DOBYAN et al., 1981). As indicated here, this might
be due to an impairment of EGF activity in renal tissue.
This work was supported by grants from the Belgian FNRS and FRSM. D.
NONCLBRCQ and G. LAWRBNT are Charge de Recherche and Chercheur Quaiifit?,
respectively. of the FNRS. I. LEONARD is a recipient of an IRSIA fellowship.
References
BREYBR,J.A. & COEEN,S. (1990) J. Biol. Chem. 265, 16564-16570.
Conrslu, T.M.,Cmsmrsm, D.A. & H w s , H.D. (1990) Am. J. Physiol. 259,
F438-F443.
D~BYAN, D.C., HILL,D., LEWIS,T. & Bumma, R.E. (1961) Lab. Invest. 45,
260268.
U O NI.,~M. o w , S., BHcraas, J.F., LAURRNT,0.. NONCLE.RCQ,D., ZA”, J.,
HEWSON-SWNNON, J.A. & F A L M AP.
~ ,(1993) Atrh. Int. Physiol. Biochim.
Biophys. 101, B22.
Mom, N.J., LAURBNT, G . , NONCLBRCQ, D., T O ~ A UG., , HEUSON-STIBN-
NON, J.A., BEROBUON, M.G.&BBAUC-, D. (1992)Am. J. Physiol. 263,
F806-F811.
NORMAN, J., Tuu, Y.K., BACAY, A. & FINE,L.G. (1990) Clin. Sci. (London) 78,
445-450.
 SOCdTI? BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
E. MARBAIX (7,
( 1 s 2), I. KOKORINE(11 3), P. HENRIET
J .DONNEZ c)and Y. EECKIIOUT
(4), P .J. COURTOY (7
I(')Cell Biology Unit and (7 Connective Tissue Group. ICE ( I )
Deportment of Pathology and (') Lkpariment of Gynecology, Saint
Luc University Clinics, University of Louvain Medical School,
Belgium]
Influence of the menstrual cycle, of progesterone and
of estradiol on the expression of collagenase in
human endometrial explants
Progesterone has been shown to inhibit the release of in-
terstitial collagenase (Matrix Metalloproteinase-1) from
human endometrial explants (MARBAM et al., 1992). We now
report the effects of the sampling period during the menstrual
cycle and of estradiol on the extent of collagenase release,
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
together with the influence of progesterone on the abundance
of collagenase release, together with the influence of pro-
gesterone on the abundance of collagenase mRNA. Col-
lagenase was activated and assayed as previously described
(MARBAM et al., 1992). Total RNA was extracted from ex-
plants after 2 or 3 days of culture and analysed by Northern
blotting using a 1.7-kb "P-labelled cDNA probe, derived
from the human collagenase cDNA (gift from H. NAGASE,
University of Kansas, USA) inserted in pBluescripP .
In the absence of sex hormone, media conditioned dur-
ing the first day of culture contained low or undetectable col-
lagenase activities (0-0.4 U/ml) for all endometria sampled
during the proliferative, early and mid-secretory phases of
the menstrual cycle (n= 21). The release of collagenase in-
creased during the second and the third day of culture. In
contrast, high collagenase activities (0.8-2.5 U/ml) were
released during the first day of culture of 2 out of 3 late-
For personal use only.
secretory endometria and of the 3 menstrual ones.
Progesterone (50-100 nM) inhibited the release of col-
lagenase from explants of all endometria (n= 8), but had less
or no effect on late-secretory and menstrual endometria
(n= 4). Estradiol alone (1 nM)also inhibited the release of
collagenase but to a lesser extent than progesterone. On the
other hand, estradiol in combination with progesterone
enhanced the effect of progesterone. Priming by estradiol dur-
ing the first day of culture also enhanced the inhibitory ef-
fect of progesterone added during the following days.
Collagenase mRNA was detected in all endometrial ex-
tracts tested (n= 6). Preliminary experiments indicated that
progesterone induces a 30 to 90% decrease of collagenase
mRNA in explants from early and mid-secretory endometria
(n= 3) but has no effect on explants from late-secretory or
menstrual endometria (n= 3).
In conclusion, the correlation between the dating of the
menstrual cycle and the extent of initial collagenase release
in culture provides additional support to our hypothesis that
collagenase plays a key role in the remodeling of human en-
dometrium. The decrease or loss of inhibition of collagenase
release by progesterone in perimenstrual endometria could
result from a decline in the abundance of progesterone recep-
tors. Conversely, the moderate effect of estradiol alone and
its enhancement of the inhibition produced by progesterone
could result from an induction of the expression of pro-
gesterone receptors.
This work was supported by grant 3.4566.91 of the Belgian Fonds
National de la Recherche Scientfique and by a pant from Ipsen-Biotech,
France, P.H. is a Research Fellow of the Imtitutpour I'Encouragement
de la Recherche Scientflque dam I'lndustrie et I'Agriculture.
Reference
MARBNX, E., D o m . J., COURTOY,P. J. & EECKHOUT,
Y.(1992) Roc.
Natl. Acad. Sci. USA 89, 11789-11793.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
B19
A. MARON (I), T. GUSTIN
(2), R. DEMEURE (3), I. MOT-
TET C), J.F. GOUDEMANT (3) and J.N. OCTAVE p)
e)
[(I) Laboratoire de Neurochimie; Service de Neumhirurgie
and (7 Dppartement de Radiologie, U.C.L.. Bruxelles]
A first step in the development of gene therapy for
brain tumors :expression of Herpes s i m p k virus
thymidine kinase in rat C6 glioma cells
In humans, gliomas are malignant and invasive
tumors which are often inoperable and resistant to
chimio- and radiotherapy. We have developed an ap-
proach using gene transfer technology for the treatment
of brain tumors, using the experimental model of the
rat C6 glioma.
The suicide gene that we have used is the thymidine
kinase gene from Herpes simplex virus (HSV-tk)
(CULVERet al., 1992). In contrast with cellular
thymidine kinase, the HSV-tk phosphorylates a
nucleoside analogue called gancyclovir. The
phosphorylated gancyclovir is incorporated into DNA,
leading to DNA polymerase inhibition and cell death.
The HSV-tk gene was transfected into rat C6 glioma
cells, and their sensitivity to gancyclovir was measured,
using an MTT test. The transfected cells were sensitive
to low gancyclovir concentrations (lo-' M), and all the
cells died in a medium containing 3 x M gan-
cyclovir.
These transfected cells were injected by stereotaxic
guidance in the right caudate nucleus of Wistar rats.
First, the tumor growth of non-treated rats was
measured in High Resolution Magnetic Resonance Im-
aging (HR-MRI) (DEMELJRE et al., 1993). This allowed
to measure the tumor volume and thickness. Untreated
rats died about 25 days after cells injection. Histology
showed the proliferating tumoral cells in the rat brain.
Furthermore, the tumor evolution of a rat treated
with gancyclovir was followed. During gancyclovir
treatment, HR-MRI showed changes in the nature and
the size of the tumor tissue. Gancyclovir treatment
resulted in the disappearance of the tumoral cells pro-
gressively replaced by a cystic cavity. Histology con-
firmed the regression of the tumor.
In conclusion, an animal model of rat glioma was
established using HSV-tk + C6 cells sensitive to gan-
cyclovir. A rat glioma induced with these cells com-
pletely regressed under gancyclovir treatment. These
preliminary data constitute the first step of a gene
therapy for brain tumors.
This work was supported by a grant FNRS Televieto A. MARON.
J.N. OCTAVE is Chercheur Qualifit of the FNRS.
References
K.W.,RAM,Z., WALLBREGE,S., ISHI, H., OLDFIELD,
CULVER, E.H.
& BLAESE,R.M. (1992) Science 256, 1150-1152.
DEMBURB,R., MOTET, I., GOUDEMANT, J.F., WON, A., Gus-
TIN, A. & OCTAVE,J.N. (1993) 2ndInternational Conference
on Magnetic Resonance Microscopy (Heidelberg).
 B20
I. b m , M.-C. DUBOISand J.-M. RWSSCHAERT N. MERTENS
(Laboratoire de Chimie-Physiquedes Macromoldcules aux In- and J. b u s (192) [(‘) Dr. L. Willems Instituut, Diepenbeek
terfaces CP206/2, Universitd Libre de Bruxeiles, 8-1050
Brussek, Belgium)
The mode of insertion of the NHZ-terminal SIV
fusion peptide into the membrane is mediated by
the lipid bilayer composition
Although a great deal of effort has been made to
understand the genetic and immunological aspects of
HIV infection, the molecular mechanism of fusion bet-
ween the virus and its host cell remains poorly
understood. Most viral fusogenic proteins contain a
short amino-terminal hydrophobic segment which has
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
been proposed to interact with the lipid membrane dur-
ing the fusion event. The HIV gp41 N-terminal pep-
tide has been shown to induce lipid mixing of lipid
vesicles ( m m et af., 1993). We report here on the
interaction of a synthetic 12-residuespeptide correspon-
ding to the NH,-terminal sequence of the gp32 from
SIV with phospholipid bilayers. This peptide has been
shown to induce lipid mixing of PC/PE/SM/Chol Luv
(Large Unilamellar Vesicles) at pH 7.4 and 37°C (m-
TIN et al., 1991). In the present study, this fusion pro-
cess was inhibited by addition of lysophosphatidylcho-
line (lysoPC) to the lipid bilayer of PC/PE/SM/Chol
LUV. This inhibition can be interpreted in terms of
molecular “shape”. Lysophosphatidylcholine adopts
a conical shape complementary to that of PE and in
For personal use only.
an equimolar mixture of lysoPC and unsaturated PE,
lysoPC stabilizes the bilayer organization. Moreover,
the results suggest that the overall PC to PE ratio
modulates the fusogenic activity. Fourier Transform
Infrared Spectroscopy (FTIR) (GOORMAGHTIGH et af.,
1990; GOORGMAGHTIGH & RUYSSCHAERT, 1993) reveals
that the orientation of the SIV fusion peptide with
respect to the lipid acyl chains depends on the presence
of lysoPC in the lipid bilayer but that the peptide secon-
dary structure and the amount of lipid-associatedpep-
tides do not depend on the lipid composition. The
peptide is obliquely inserted into the lipid bilayer of
vesicles without lysoPC, whereas it is oriented parallel
to the lipid-water interface in the vesicles containing
lysoPC. The data suggest that the insertion of the SIV
fusion peptide into the host cell lipid membrane plays
a crucial role in vesicle fusion and in virus-cell mem-
brane fusion. It has been proposed that this penetra-
tion favors the formation of transient lipid species such
as inverted micelles which are involved in the fusion
process.
References
G~~RMAGHTTGE, E., C m u x , V. & RWSSCHAERT, J.-M. (1990) Eur.
J. Biochem. 193, 409-420.
GOORMACXITXX~. E. & RWSSCHAERT,J.-M. (1993) Subcellular
Biochem., in press.
hlARTIN, I., DEFRISE-QUERTAIN, F., MANDIMU, V., NIBISEN,N.M.,
A RB~ ,m , A.. BRASSEUR,
S A B R ~ ~T., R., R w s s c ~ u e a J.-M.
~,
& VANDIMBRANDBN, M. (1991) Biochem. Biophys. Res. Comm.
175, 872-879.
Mmm, I., DEFRISE-QUERTAIN, F., DECROLY, E., S m m , T.,
BURNY, A., BRASSEIJR,R . , VANDENBRANDEN, M. &
RWSSCEARRT, J.-M. (1993) Biochim. Biophys. Acta 1145,
124-133.
Archives Internationales de Physiologie. de Biochimie et de Biophysique, 1994, 102 (1)
socnid BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
(19 2), C. VANDEVYVER(l), Z. JINGWY (l)
and ( l ) Limburgs UniversitairCentmm. Diepenbwk, Belgium]
Charasterization of the T cell receptor (TCR) in
multiple sclerosis
Autoimmune mechanism mediated by T cells, which
specificallyrecognize a myelin protein, may play an im-
portant role in Multiple Sclerosis (MS).
A panel of 25 (myelin basic protein; MBP)-specific
T lymphocyte clones were generated from the blood
of 8 MS-patients and one normal individual. These
clones, which are all CD4+,show a helper function and
are HLA-DR MHC-restricted. Most of the clones
recognize immunodominant regions of MBP although
several displayed distinct antigen reactivities.
We investigated the V(D)J-regions used among
these T-cells by determining their sequence. This V(D)J-
region is believed to play a major role in antigen
recognition.
The strategy used for this sequence analysis from
a- and @-chainis the reamplification of previously
amplified products (for V gene usage determination)
with the same V-primer and a biotinylated constant
primer which anneals within a sequence just behind the
V(D)J-region. Low melting point gel purification from
the amplification products obtained was required prior
to direct sequencing. The purified material was bound
to magnetic beads (DYNAL), dissolved in a 0.05%
Nonidet P-40 aqueous solution and manually sequen-
ced using the standard Sanger dideoxy-chain-
termination method.
Each of the V a and V@ sequences obtained con-
sisted of one of the known V sequences, except that
two amino-acid substitutions from the known Vp20.1
sequence and one amino-acid substitution from the
VP17.1 sequencewere found. This is ascribableto either
an allelic variation or a new member of this V sub-
family.
Repeated a- and @-sequenceswere found when
analysing different clones according to one individual
indicating that MBP-specific T cells undergo clonal ex-
pansion in the blood. This clonal expansion was also
observed in the normal subject, presumably due to ex-
pansion by a superantigen.
Furthermore no common motifs were identified in
the a- or @-chainCDRs of the clones sequenced.
This work was supported by a grant of the IWONL to N. MER-
TENS,the “Sociale Investeringsmaatschappij Limburg” (SIM), the
‘WationaleLater$', and the NFWO “LevenslijnMulriple Sclermis”.
 K. MOTMANS
(19 2), C. VANDEVYVER(l) and J. RAUS(1,2)
Dr. L. Wilfem-Instituut,Department of Biotechnology/Im-
[(I)
munolow, Universitaire Campus, 8-3590 Diepenbeek and
Lintburgs UniversitairCentrum, Universitaire Campus, 8-3590
Diepenbeek]
Construction of chimeric T-cell receptor genes
using PCR
There are numerous experiments where two DNA
fragments from different origin have to be joined to
generate hybrid genes. Standard methods for generating
recombinant DNA constructs involve ligating different
restriction fragments together, which is a tedious and
time-consuming process. The join between the two
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
fragments is sequence-dependent and governed by the
availability of restriction sites, which is not always
ideal.
In our results, we want to increase the tumor
specificity of Tumor Infiltrating Lymphocytes (TIL) by
teh expression of a chimeric T-cell receptor (cTCR) in
which the antigen-binding domain is replaced by an
analogous domain of a breast-cancer-specific
monoclonal antibody (MoAB). The resulting cTCR can
recognize and bind the antigen in a non-Major-Histo-
compatibility-Compex (MHC)-restricted manner
(GROSSet al., 1989).
Here we discribe the construction of the cTCR-
genes using three PCR-reactions. The genes encoding
the antigen binding parts of the tumor-specific MoABs
For personal use only.
are isolated, amplified by PCR and cloned for sequence
analysis. These cloned genes are used as template in a
first series of PCRs. In a second series of PCRs, the
TCR constant a- and @-chainsare isolated, starting
from Jurkat-RNA. The primers that are used in these
amplification reactions, at the ends that are to be fus-
ed, are made complementary to one another by in-
cluding nucleotides at their 5’ ends that are
complementary to the 3’ portion of the other primer.
This makes the 3’ part of the cDNA, encoding for the
MoAB-part identical to the 5’ part of the cDNA for
the TCR constant parts.
Both PCR products are used as template in a third
series of PCRs, where the chimeric genes are synthesiz-
ed. The strands that contained the overlap sequences
at their 3’ end serve as primer for each other. Exten-
tion of this overlap yields a cTCR-chain. As soon as
the recombinant product is synthesized, it gets exponen-
tially amplified by two external primers. These exter-
nal primers contain unique restriction sites which make
direct cloning of the chimeric genes in an expression
vector possible.
This work was supported by a fellowship (Aspirant Navorser)
from the Belgian Nationaal Fonds voor WetenschappelukOnder-
zoek to K. MOTMANS.the Vereniging voor Kankerbestruding, the
Limburgs Universitair Centrum and the Sociale Invester-
ingsmaatschappij voor Limburg (SIM).
Reference
GROSS,G., W a s , T. & ESSHAR,
Z. (1989) Proc. Natl. Acad. USA
86, 10024-10028.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
M. PRAET,S. PIRON, F. HOMBLE,
P. STRYCKMANS (l),
M. LOOS(’) and J.M. RWSSCHAERT[Luboratoirede
Chimie-Physique des Macromol&Ies aux Interfaces, Universitk
libre de Bwelles, Boulevurd du Triomphe, B-10.50Bmeiles
and (I) Laboratoire d’Hdmatologie. Institut Bordet, 8-loo0
Bruxelles]
Comparative cellular uptake of anthracyclines in
human multidrug-resistant leukemia cells
Multidrug resistance (MDR) is observed in tumor
cells after exposure to antitumoral agents such as an-
thracyclines and vinka aldoids. In numerous cell lines,
MDR is associated with the presence of an = 170-kd
plasma membrane-associated glycoprotein termed P-
glycoprotein (Pgp) that may act as a drug-efflux pump
and decreases the intracellular antitumor drug concen-
tration (KARTNER et al., 1983). Little is still known
about the molecular mechanism underlying the drug
efflux and the way Pgp handles drug structurally very
different. The understanding of this mechanism would
help to design on a rational basis, new agents able to
overcome multidrug resistance or which would not be
rejected out of the cells displaying the MDR phenotype.
We present here a preliminary study designed to clarify
the importance on the efflux process of physico-
chemical parameters associated to antitumor agents,
such as steric hindrance, hydrophobicity and the charge
at physiological pH. We measured the uptake of dif-
ferent anthracyclinesinto sensitive human myelogenous
leukemia K562 cells and into K562/DNR cells, a subline
resistant to daunorubicin (DNR). It was shown that the
K562/DNR subline expresses Pgp at high rate at the
membrane surface (HAMADA& Tsmuo, 1988). We in-
cubated 106 cells in 1 ml of RPMI 1640 medium dur-
ing 3 hours in the presence of M of DNR,
4-demethoxyDNR (idarubicin, IDA), 4’-
deoxyadriamycin (4’-deoxyADM) and 4’-deoxy-4’-
iodoADM respectively. Laser flow cytometry was us-
ed to quantitate intracellular anthracycline content. Up-
take of DNR and 4’-deoxyADM was minimal in the
K562/DNR resistant sublines as compared to the up-
take in the sensitive cells. On the opposite 4’-deoxy-4’-
iodoADM and IDA, were taken up nearly to the same
extent into the sensitive and resistant K562 cell lines.
It is presently unclear why IDA and 4’-deoxy-4’-
iodoADM are able to overcome the MDR phenotype,
since their molecular structure is nearly similar to that
of DNR and 4’-deoxyADM. A crucial feature shared
by the two compounds is their highly lipophilic coeffi-
cient which could facilitate their uptake into resistant
cells and eventually counterbalance the Pgp-mediated
efflux. It will be of importance to determine whether
subtle structural differences could prevent the interac-
tion with Pgp. From a clinical point of view, these
results suggest that 4’-deoxy-4’-iodoADM and IDA
could be efficient against leukemia cells displaying the
MDR phenotype.
References
HAMADA,H. & Tsmuo, T . (1988) J. Biol. Chem. 263, 1454-1458.
KARTNER, N., RIORDAN,J.R. & LING, V. (1983) Science 221,
1285-1287.
 B22
and J.M. RUYSSCHAERT
V. RAUSSENS,E. GOORMAGHTIGH
(Free University of Brussels, Campus plaine CP206/2,B-1050
Brussek)
Contribution to the study of the structure of the
gastric H + ,I(+ -ATPase in the tubulovesicles
The acid secretion by the parietal cells of the stomach
is mediated by an H+,K+-ATPaselocated in the apical
membrane of the stimulated cells.
The parietal cells contain tubulovesicles,which can be
easily purified by fractionedisopycnic centrifugationswith
the cytoplasmic side oriented outside. The H+,K+-ATPase
represents 85% of the tubdovesicular membrane proteins.
The gastric H+,K+-ATPaseis an C Y heterodimer.
, ~ The
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
hydropathy profiles predict 8 or 10 membrane-spanning
CY helices for the CY sub-unit and only one for the p sub-
unit. Most of the rest of the protein is predicted to be
located on the cytoplasmic face.
In order to evaluate the validity of the predicted
topological models :
1. proteinase K and pronase were used as aspecificpro-
teases in order to cleave the H+,K+-ATPasedomains pro-
tuding into the aqueous phase. The activity of these
proteases is limited to the cytoplasmic face of the vesicles.
After digestion, the proteases and the peptides were
separated from the tubulovesiclemembrane by gel filtra-
tion. The vesicles containing the membrane-associated
peptides of the ATPase were analysed for protein and lipid
content. The low amount (35%) of digested protein does
not fit with the H+,K+-ATPasemodels predicting that 60%
For personal use only.
of the protein is accessible to proteases.
2. IR-attenued total reflexion spectroscopy was used
to determine the secondary structures (GOORMAGTIOH et
al., 1990) of the H+,K+-ATPase and of its transmembrane
regions as isolated by proteolysis. In both cases, the (11helix
content (30%), the 0 sheet content (30Vo), the random
fraction (1’7%) and the turn fraction (23%) do not fit
with the H’,K+-ATPase models which predict an increase
of the ar helix content in the transmembrane region. Linear
dichroism measurements(GOORMAGTIGH & RUYSSCHAERT,
1990) indicated that the CY helices are mainly perpen-
dicular~oriented with respect to the lipid bilayer plane.
3. The accessibilityof the protein and of its transmem-
brane region to the solvent was analyzed by H/D
(hydrogen/deuterium)exchange kinetics followed by IR
spectroscopy. The exchange rates for the entire protein
and for the transmembraneregion were shown to be very
similar, 65% of the peptidic bonds were exchanged after
1 hour. Such a high accessibility of the transmembrane
region to the solvent has been found for the human
erythrocyteglucose exchanger. In this case, the hypothesis
of an aqueous channel formed by the assembly of
transmembraneCY helices has been proposed (ALVAREZ et
al., 1987). Similarly, our H/D exchange measurements on
the H+,K+-ATPasecould suggest the presence of an
aqueous channel.
References
ALVAREZ, J., LEE,D.C., BALDWW,S.A. & CHAPMAN,D. ( 1 987) J. Biol.
Chem. 262, 3502-3509.
GWRMAGHTIQH, E.,CABIAUX, V. & RLJYSSCHAERT, J.M. (1 990) Eur. J.
Biochem. 193, 409420.
G o o ~ ~ ~ a mE.o&~ RWSSCHAERT,
i, J.M.(1990) in Molecular descrip-
tion of biological membrane components by computer-aidedcon-
formational analysis, CRC press.
Archives Internationales de Physiologie. de Biochimie et de Biophysique. 1994, 102 (1)
SOCU~TEBELGE DE BIOCHLMIE, UCL, 20 NOVEMBRE 1993
J. ROBBENS,E. REMAUTand W. FIERS(Laboratorium voor
Moleculaire Biologie, Universiteit Gent, K.L.Ledeganckstraat
35, B-9ooo Gent, Belgium)
Periplasmic secretion in Escherichia coli of murine
interleukin-2 fused to the ompA signal peptide :
influence of amino-acid sequence at the cleavage
site
The overexpression of heterologous proteins in
Escherichia coli (E. coli) has become an “established
technique”. Expression levels up to 20% of total
cellular protein are not exceptional. Unfortunately,
many overexpressed proteins are found in inclusion
bodies, insoluble aggregates, in which the protein is pre-
sent in a biologically inactive form (SCHEIN, 1989). To
avoid this problem, several approaches can be follow-
ed. We and others reported already about the co-
expression of chaperones and heterologous proteins
(ROBBENS,1993a, 1993b; BLUM,1992). Another ap-
proach that can be explored is secretion of the
heterologous proteins into the periplasm of E. coli.
Besides avoiding intracellular precipitation, penplasmic
secretion may be advantageous in that the protein of
interest is present in a cellular compartment where
relatively few endogenous proteins are found. The lat-
ter aspect may contribute significantly to the ease of
purification of the desired protein.
Secretion of proteins into the periplasm requires an
amino-terminal signal peptide fused to a mature pro-
tein. Although, the signal peptide is a prerequisite for
secretion, the precise requirements regarding the se-
quence at the cleavage site have not been unambiguous-
ly established. In the present study, we report on the
dramatic improvement in the secretion of murine
interleukin-2 (mIL2) by a single amino-acid change at
the signal peptide cleavage site. A fusion comprising
the naturally occuring outer membrane protein A (om-
PA) signal peptide precisely fused to mature mIL2
secreted about 200 units/ml in the periplasm. Insertion
of a serine residue between the ompA signal peptide
and the mature protein increased the secretionto a level
of 8 x 106 units/ml. After purification, the specific
biological activity was determined to be 5 x lo7
unitslmg, which is equal to the specific biological ac-
tivity of natural mIL2 and about 10 times higher than
mIL2, purified from inclusion bodies (GUISEZ,1993).
References
BLUM,P., VRUIW, M.,Lm, N. & MATIN,A. (1992)BioNeChnology
10, 301-304.
GUISEZ, Y., DEMOLDER,J.. MERTENS,N., RAEYMAEKERS, A.,
PLAETINCK, G., ROBBENS,J.. VANDEJCERCKHOVE, J., RE-
MAUT, E. & FIRM,W. (1993) Protein expression andpurifica-
tion 4, 240-246.
ROBBENS,J., REMAUT,E. & Fmns, W. (1993) Arch. Int. Physiol.
Biochem. Biophys. 101. B35.
ROBBENS,J., REMAUT,E. & FmRs, W. (1993) submitted.
SCHEIN.C.H. (1989) Bio/Technology 7, 1141-1 149.
 SOCdTg BELOE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
N. SONVEAUX (I), C. INGELS (I), D. THINES(z) and
J.M. RUYSSCHAERT (I) [(I) hboratoirede Chimie-Physique
des Macromolkcules aux Interfaces, CP 206/2. Universitk Libre
de Bruxelltx, Brussels, Belgium and (') hboratoire de Biologie
Cellulaire, Universitk Catholique de Louvain, I place Croix-du-
Sud, Louvain-la-Neuve. Belgium]
Mode of organization of lipids in HBsAg particles
Infection by Hepatitis B Virus is associated with the syn-
thesis of empty viral envelopes into the blood stream of pa-
tients (PATZBR et al., 1984; DUB~IS et al., 1980). These
spherical particles, called HBsAg particles, are composed of
the viral envelope proteins embedded into host-derived lipids
WERMANN et al., 1984; UEDAet al., 1991). Similar particles
have been produced in yeast by genetic engineering and are
widely used as a vaccine ~ T R etE al., Valemela et al., 1982).
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
Little is known about the lipidic organization of such par-
ticles. The only available information concerns their lipid
composition;phospholipids constitute the major component
(f 80%) as well for the plasma-derived particles as for the
yeast-derived particles (P~TRE et al., 1987; GALWANES et ul.,
1982). Several arguments suggest a bilayer organization of
the lipids within HBsAg particles : the HBsAg protein is a
major component of the viral envelope which is organized
as a bilayer and the length of several predicted hydrophobic
stretches of the HBsAg protein corresponds to the thickness
of a lipid bilayer. Nevertheless, the high radius of curvature
of the particles and their low lipid content (approximately
25% in weight of lipids in hepatitis particles) (GUBRRBRO et
al., 1988) do not allow to exclude that it could be surround-
ed with a monolayer of lipids, as demonstrated for the LDL
(Low Density Lipoprotein) particles (YEAGLEet al., 1978).
Phospholipase D was used to investigate the mode of
For personal use only.
organization of phospholipids in the envelope of yeast-HBsAg
particles. This enzyme cleaves only the phospholipidsexpos-
ed on the outer surface of the particle and allows therefore
to determine whether the envelope surrounding the particle
is made of a bilayer or a monolayer. The percentages of cleav-
ed HBsAg phospholipids are in agreement with a bilayer
organization. Incubation of the native HBsAg particles with
phospholipase D did not alter their morphology or their den-
sity as demonstrated by electron microscopy and migration
on a sucrose gradient. Our data strongly suggest that HBsAg
particles envelope is organized as a discontinuous bilayer of
lipids surrounding protein aggregates.
N.S. is an IRSIA fellow (InstitutpourI'encouragement de la recherche
scientifique duns I'industrie et I'agriculture). We thank SmithKline
Beecham Biologicals (Rixensart, Belgium) for providing us the yeast-
HBsAg particles.
References
Drrso~s,M.F., POURCEL, C., ROUSSET,S., CHANY,C. & TIOUAIS,P.
(1980) Proc. Natl. Acad. Sci. USA 7l,4549-4553.
GAIJVANES, F., GONZALES-ROS, J.M. & PETERSON, D.L. (1982) J. Biol.
Chem. 257, 1110-1111.
GUERRERO, E., GALIVANES, F. & PETERSON, D. (1988) Viral Hepatitis
and Liver Disease, 1094-1101.
HBERMA", K.H., GOLDMA", V., SIWARTZ, W., SEYFFARTFI, T.,
BAUMCURTEN, H. & GEIUICH,W.H . (1 984)J. Virology55,396402.
PATZER,E.J., NAKAMURA, G.R.& Y m , A. (1984) J. Virology 51,
346-353.
P%TRE,J., VAN WUNENDAELE, F., DE NEYS,B., CONRATH, K., VAN
OPSTAL., 0.. P., RUTOERS,T.,CAEEZON,T., CAPIAU,C.,
HAUSER,
HARFOW,N., DE WILDE, M., STEFTIENNE, J., CARR.S., HEM-
LING, H. & SWADESH,
J. (1987) PostgraduateMedicul Journal63.
13-81.
UEDA,K., TSURIMOTO,
T. & MATWEAM, K. (1991) J. Virology 65,
3521-3529.
VALENZIJEU, P., MEDINA, A., RWER, W.J., A M ~ ~ E R E RG.
, &
HALL,B.D. (1982) Nature 298, 347-350.
YEAOLE,P.L., MARTIN, R.B., POTTENGER, R.G.(1918)
L., LANODON,
Biochemistry 17, 2101-2710.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994. 102 (1)
B23
C. TANS,F. DUBOIS,Z.D. ZHONG,M. JAWT, R. WAT-
TIAUX and s. WATTUUX-DE CONINCK(Laboratoire
de Chimie Physiologique, Facult& UniversitairerNotre Dame
de la Pa&, 61, rue de Bwelles, B-5ooO Namur)
Uptake by rat liver of bovine growth hormone free
(Gh) or bound to a monoclonal antibody (Ghab)
Several works have shown that the binding of a
monoclonal antibody to growth hormone enhances its
biological activity in vivo (HOLDER et ul., 1985; BOM-
FORD t ASTON, 1990). Until now, the mechanism of
this phenomenom is unknown. The liver is a major site
of action of Gh. After binding to receptors located in
hepatocytes plasma membrane, Gh is internalized and
to a large extent reaches lysosomes where it is degrad-
ed (POSTEL-VINAY et al., 1982). Nothing has been
described concerning the fate of Ghab when it is an im-
portant question with respect to its hormonal action;
indeed Ghab may be considered as a molecule different
than Gh that could be targeted to other cells with con-
sequences for its biological activity. In the work
reported here, we have compared by centrifugation
methods, the uptake by rat liver and the intracellular
distribution of Gh and Ghab; the molecules were label-
ed with lZ5lor with IZ5ltyramine cellobiose. Results
show that : 1) the elimination of Gh from the blood
is considerably more rapid than that of Ghab; 2) both
molecules are quickly taken up by the liver; 3) probably
after travelling through endosomes, Gh and Ghab get
to lysosomes where they are degraded. However, by
using a method we have recently described (WATTIAUX
et ul., 1993) allowing to distinguish between hepatocyte
lysosomes and sinusoidal cell lysosomes, we have found
that Gh mostly ends up in hepatocyte lysosomes when
Ghab is recovered in hepatocyte and in sinusoidal cell
organelles; 4) binding and internalization by isolated
hepatocytes and clone 9 hepatocytes are markedly less
efficient for Ghab than for Gh. Therefore, it is pro-
bable that in the liver, an important proportion of Ghab
is targeted towards another kind of cell than Gh, what
obviously sets a problem with respect to its activity in
vivo.
This work was supported by the Fonds de la Recherche Scien-
tifique. the Fonds de la Recherche Scientifique Mtdicalc (contract
no 3.4523.91) and the Fonds de la Recherche FondamentaleCollec-
tive (contract no 2.45592).
References
B~MPORD, R. & ASTON.A. (1990) J. Endocrinol. 125, 31-38.
HOLDER, A.T., ASTON,R., PRECCE, M.A. & IVANYI, J. (1985)
J. Endocrinol. 107. 9-12.
POSTEL-VINAY, M.C., KAYSER, C. & DESBUQUOIS, B. (1982)
Endocrinol. 111. 244-251.
WATTIAUX,R., GENTINNE, F., JADOT. M., Dvaors, F. & WATTIAUX-
DE CONINCK, S. (1993) Biochem. Biophys. Res. Comm. 190.
808-8 13.
 B24
Sandrine A. TINTON c),S.C. CHOW (l), P.M. BUC-CAIDERON (‘),
G.E. Ioss (3 and s. ORRBNIUs (’) I(’) Unit6 de Biochimie
Can&roIogique et Tomkologique, LMpartement des Sciences Pharmaceuti-
q u a 8-7369. Universitd Catholique de Louvain. S l 2 W Bruxelles, Belgium
and )(‘ InstifUte of EnvironrnentalMedicine, Division of T o d c o o ~Kamlin-
,
ska Instituter, Box 210. S-171 77 Stockholm. Sweden]
Change in eplcium homeostasis is not involved in the in-
hibition of protein synthesis induced by extracellular
adenosine and ATP in isolated rat hepatocytes
Extracellular ATP and adenosine regulate many physiological
functions by interacting with purinoreceptors P, and P, (EL
MOATASSIMet al.. 1992; DAVALet al., 1991). However, purine
ribonucleosidessuch as adenosine also inhibit the growth of several
cell lines (HENDERSON & Scon, 1981)and extracellular ATP is lytic
for hepatocytes (ZOETEW et a/., 1993).
We have previously shown that incubation of isolated hepatocytes
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
with extracellular adenosineor ATP inhibit the synthesisof proteins
by mechanism(s) still poorly understood (TINTON et a/., 1993).
The requirement of a calcium sequestred pool rather than of an
increasein free cytowlic calciumconcentrationhas been demonstrated
for the maintenance of an optimal rate of protein synthesis in
eukaryotic cells (BROSTROM & BROSTROM, 1990). Therefore we in-
vestigated the relationship between change in free cytosolic calcium
concentration [Ca2+Iiand protein-synthesis inhibition induced by ex-
tracellular adenosine and ATP in isolated rat hepatocytes.
Addition of adenosine(0.5 mM) to fura-2-loaded cells resuspended
in buffer containing CP’ produces a small and sustained [Ca2+Iiin-
crease as compared to ATP (0.5 mM), vasopressin (10 nM) and thap-
sigargin (1 pM). The nucleoside mobilizes a small calcium pool in
hepatmes suspended in nominally CaKfree medium. Upon restora-
tion of calcium into the medium, adenosine stimulates a Ca” increase,
suggesting an influx pathway. Similar [Ca2+Iichanges were observ-
ed with APPCP and APCPP, two methylene analogs of ATP.
For personal use only.
However, only adenosine and APPCP inhibit the protein synthesis
after two hours of incubation by about 45% while APCPP does not.
In contrast to adenosine, ATP (0.5 mM) produces a rapid [Ca1+Ii
increase similar to vasopressin and mobilizes the same hormone
IPS-sensitiveCa” pool. GTP or UTP (0.5 mM) produce a similar
[CaJ+Iiincrease as ATP but only ATP induces a significant inhibi-
tion of protein synthesis (6OVo after 2 h of incubation); in such con-
ditions, treatment of cells with vasopressin, GTP and UTP inhibit
the synthesis of proteins by less than 20%. The inhibitor effect of
adenosine and ATP on protein synthesis is not potentiated by the
addition of vasopressin. Addition of thapsigargin (1 pM) to
fura-24oaded hepatocytes causes a rapid [Ca2+Iiincrease in cells
resuspended in buffer containing calcium, mobilizes the reticulum
endoplasmic Ca” pool when fura-2-loaded cells are incubated in a
nominally calcium-free medium and stimulates Cal+ influx after
restoration of calcium in the extracellular medium (LLOPJS et al.,
1992). Although incubation 0 s isolated hepatocytes with 1 p M thap-
sigargin for 150 min results in a strong inhibition of the incorpora-
tion of labelled leucine into proteins, our results show a significant
additive effect of adenosine and ATP on the protein-synthesis in-
hibition induced by thapsigargin. AU these present data rule out
change in [Ca2+Iias a mechanism involved in the inhibition of pro-
tein synthesis induced by extracellular adenosine and ATP in isolated
rat hepatocytes but rather suggest a metabolic effect triggered by the
uptake of adenine compounds after their previous hydrolysis into
9-8-D ribofuranosyladenine.
This work was supported by grant RF/93/9fT (1993) from the European Science
Foundation (ESF Research fellowship in Toxicolog~).
References
BROSTTKOX. C.O. & BROSTROM, M.A.(1990) Ann. Rev. Physiol. 52, 577-590.
DAVAL,J.-L.,NHHLIO,A. & Nrcous, F. (1991) Life Sciences 49, 1435-1453.
C.,
E,LMOATA.%~~, ~ ~ ,&MANI,J.-L. (1992)Biochim.Biaphys. Acta
D o n b i ~J.-L.
1134, 31-45.
HBNDERSON, J.F. & SCOTT,F.W. (1981) Pharmac. Ther. 8, 539-571.
LLOPIS,J.. Knss, 0..G w ,A. & Onasms, S. (1992) Biochem. J. 284,243-247.
TINTON, s., LEmBVRE. V., COUSIN, 0. & BuC-CALDERON,P . (1993) Biochim.
Biophys. A d a 1176, 1-6.
ZOBTEWII. J.P., VAN DB WATER,B., D8 BONT,H., MULDBR,G . & NAOELKERKE, J.
(1993) J. Biol. Chem. 268, 3384-3388.
Archive Internationales de Physiologie. de Biochimie el de Biophysique, 1994, 102 (1)
SOCI6Tfi BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
A. PAYS,P. TEBABI
L. VANHAMME, and E. PAYS(Vniver-
site Libre de Bruxelles, Ddpartement de Biologie Moldculaire,
Lrrboratoire de Parmitologie Mol&uIaire, 67, rue des C h e v m ,
B-1640, Rhode-St-GenPse)
A mutational analysis of the variant surface
glycoprotein (VSG) gene promoter in
Trypanosoma brucei
African trypanosomes are the agents of parasitic
diseases, sleeping sickness in humans and nagana in cat-
tle. Trypanosoma brucei is transmitted to its mam-
malian host by an insect vector, the tse-tse fly.
Bloodstream (mammalian)and procyclic (insect) forms
are covered by a stage-specific dense coat of variant
surface glycoprotein (VSG) and procyclin respective-
ly. Bloodstream forms escape the immune system of
their host through periodic changes of the VSG express-
ed, selected among a repertoire of a thousand genes
(PAYS& STEINERT, 1988).
We have analysed the function and structure of the
VSG promoter. Although the expression of the VSG is
stage-specific, the promoter is active in both procyclic
and bloodstream forms. Instead the expression of the
VSG is modulated, at least partly, by the 3’-untrans-
lated region of its mRNA (BERBEROF et ul., 1994). Dele-
tional analysis of the AnTat 1.3A VSG promoter
defines a minimal 80-bp region sufficient for basal pro-
moter activity. Fine deletions inside this region seem
to indicate that the spacing between different elements
of the promoter is critical. Mutational analysis enabl-
ed us to pinpoint 2 short stretches of 4 bp essential for
promoter activity and centered at positions -62 and -36.
Mutation at position + 1 also destroys promoter ac-
tivity. The VSG promoter does not show any homology
with known eukaryotic promoter consensus sequences,
although 2 zones of restricted homology with
trypanosome procyclin and rDNA promoters can be
found. Whether this observation can be related to the
current proposal that the VSG gene is transcribed by
a ribosomal-type (pol I) polymerase will be discussed.
L.V. is Char& de Recherches at the FNRS. This work was sup-
ported by grants from the Fonds de la Recherche Scientifique
Mddicale, the Fonds SMciaI de la Recherche de f‘llniversitd Libre
de Bruxelles and through a research contract with the Communautd
Francake de Belgique (ARC89/94-134).
References
M.,VANEAMME,L., PAYS,A., TEBABI.P., JEFFENES, D.
BERBEROF,
& PAYS,E. (1994) Arch. int. Physiol. Biochim. Biophys. 102,
B000.
PAYS,E. & STEINBRT,
M. (1988) Ann. Rev. Genet. 22, 107-126.
 SOC&'I% BELOE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
M. VAN HELVOIRT, C. VANDEWVER, G. MASSAand
J. RAUS (Dr. L. Willemsinstituut, Department of
Biotechnology and the Limburgs UniversitairCentrum, Univer-
sitaire campus Bldg. A , B-3590 Diepenbeek)
Mutation analysis in congenital adrenal
hyperplasia : genotype-phenotype correlation in
the belgian CAH patients
Congenital Adrenal Hyperplasia (CAH) is an
autosomal recessive trait due to a defect in the synthesis
of cortisol. In 95% of the patients this is a result of a defi-
ciency of the enzyme 21-hydroxylase, which is necessary
for the conversion of 17-OH progesterone t o
11-deoxycortisol.
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
Clinically, there are 3 different forms of CAH : in the
most severe Salt-Wasting form (SW), cortisol- and
aldosterone production is impaired; those children have
severe urinary salt loosing besides the virilization of the
external genitalia. In the Simple Virilizing form (SV), the
aldosterone synthesis remains intact and in the milder,
non-classic forms (NC), symptoms are only seen during
childhood (late onset) or not at all (cryptic).
The enzyme 21-hydroxylaseis encoded by the CYP2IB
gene. This gene is located, together with its pseudogene
CYPZIA, in the Class-111 region of the human Major
Histocompatibility Complex on the short arm of
chromosome 6, near the genes coding for the fourth com-
plement factor. The genes CYP21B and CYP2ZA are both
about 3.1-kb long and each gene contains 10 exons. Their
nucleotide sequences are 98% identical in the exons and
For personal use only.
96% identical in the introns. CYP2ZA is inactive due to
a 8-bp deletion and several point mutations.
All mutations causing 21-hydroxylase deficiency are
the result of either unequal crossing-over during meiosis,
resulting in a complete deletion of CYP21B, or of gene
conversion events resulting in the transfer of deleterious
mutations present in the pseudogene to the active gene.
By use of Restriction Fragment-Lenght Polymor-
phism~(RFLP) analysis using the restriction enzymes Taql
and BglII in combination with a genomic probe one can
detect those deletions and duplications in the CYP21B
locus. Furhterrnore, these RFLPs give rise to five
haplotypes and when using also a C4 probe, 11 haplotypes
can be defined. By associating CAH with the haplotype
of the affected child (index case), one can locate the
segregation within a famyli, making genetic counseling
possible.
Identification of point mutations, responsible for
21-OH deficiency, is possible by use of the Polymerase
Chain Reaction (PCR) of the CYP21B gene, in combina-
tion with allele-specific oligonucleotidehybridisation. To-
day, about 9 point mutations have been characterized and
recently, a correlation was reported between some com-
binations of mutant CYP21B alleles and the clinical
phenotype. Therefore, the determination of the genotype
of CAH patients can, beside the genetic counseling, be
helpful in predicting the severity of the disease.
So far, we screened 1I patients and their relatives (35
samples). Our results will be discussed.
This work was supported by the Belgian Study Group for
Pediatric Endocrinology to Dr. G . MASSA.
Archives Internationales de Physiologie, d e Biochimie et de Biophysique, 1994, 102 (1)
B25
Karen VANHOORELBEKE,Annick GOOSSENS, C. GIELENS
and GistIe PRgAUX (Laf~oratorium voor Biochemie,
Katholieke Universiteit Leuven. Dekenstraat 6, 8-3000 Leuven)
An a2-macroglobulin-uke proteinase inhibitor in the
haemolymph of the Decabrachia cephalopod Sepia
officiinalir
The haemolymph of Sepia officinalis contains, next to the
haemojranin (Hc), a second protein component (PR&AUX et
al., 1979) corresponding to a glycosylated h e r (Mr350 OOO)
of subunits linked by disulphide bridge(s), which, when not
removed, apparently led to a slow-down of the limited pro-
teolysis of the Hc (VANHOORBLBBKE el al., 1992). This promp-
ted us to identify it as a possible proteinase inhibitor (I).
Apart preincubation for 30 min of several types of pro-
teinases (Es) with I in a molar ration E/I of 1/4 inhibited
their proteolytic activity on Hc and on casein ( E / S , 1/1OOO,
g/g, borate-HC1 buffer, pH 8.2, I 50 mM) but not the
hydrolytic activity of trypsin on N-a-benzoyl-DL-arginine
nitroanilide even when soybean trypsin inhibitor had been
added. The inhibitory activity of I thus seems to result in a
trapping of the enzyme like first described for the human
az-macroglobulin (azM) (BARRET et al., 1979).
The azM character of I was further corroborated at other
levels. On denaturation with 2% SDS two thiol groups
became detectable. Storage overnight in the presence of 1 M
(NH.),SO, or for months in Tris-HC1 buffer, pH 8.2, I 50
mM, led to an inactivation and to a transformation into an
electrophoreticallyfast form at pH 8.8. The proteinases, on
interaction, cleaved the subunits into 2 fragments of about
equal size, heat treatment (9S°C, 30 min) split them into a
1/3 and a 2/3 part but only when I had not first been inac-
tivated.
Partial DNA sequencing of a 1.2-kb fragment, im-
munologically selected from the cDNA library of DECLERCQ
et al. (1989), led to the sequence KTNFWEVPSSEMMIP-
TASIVISCITTSTKLMVDASTVKISIAFKNDVVGAFTES-
EGKPGTNVDFXVKTLPGSAVFVMAVDKNLHLLQDA-
KYITEDMIYGAL, homologous to residues 498-599 of
human a2M (SOITRIJP-JENSEN et al., 1984); partial amino-
acid sequence of a CNBr-fragment yielded KNFAPNLYX-
H",homologous to residues 955-%7, and even to 954-%7
on including the methionyl residue expected at the left and
present there in human a2M.
Proteins of a similar azM type were also isolated from
the haemolymph of arthropods, among which from Astacus
(ST&KERet al., 1991), and from the haemolymph of the
mollusc Octopus vulgaris (WERSEN et al., 1992).
We wish to thank the Fund for Joint Basic Research for research grants.
References
(1979) Biochem. J. 181.
BARRBTT,A.G..BROWN,M.A.&SAYERS,C.A.
401-418.
DECLERCQ, L., VANDAMME,A.-M. & P ~ A I J XG,. (1989) Arch. Intern.
Physiol. Biochim. 91, B193.
Pmt~ux,G., GIELENS, C. & LONTIE,R. (1979) In Metalloproteins. Struc-
ture, Molecular Function and Clinical Aspects (WESER, U., ed.),
pp. 73-80, Georg Thieme Verlag, Stuttgart & New York.
SOTTRIJP-JENSEN, L., STRPANIK,T.M., KRISTENSEN, T., WIBRZ~IC-
KI,D.M., JONES,C.M., UNBLAP.B., D, MAONUSSUN, S. & PETER-
SEN, T.E. (1984) . I Biol.
. Chem. 259, 8318-8327.
STOCKER, w., BREIT, s., SOlTRUP-JENSEN, L. & Z R " 0 , R. (1991)
Comp. Biochem. Physiol. 98B,501-509.
T ~ Q R R S EI.B.,
N , SALVESEN, G . , BRUCATO,F.H., Pmo, S.V. & ENO-
HILD, J.J. (1992) Biochem. J. 285, 521-527.
VANHOO-, K., BOWAN,A., GIEISNS, C., LONTIE, R. &pRBAux, G .
(1992) International Congress on Invertebrate Dioxygen Carriers,
April 12-17, 1992, Lunteren, The Netherlands, Abstract book,
11-PO1.
 B26
and M.DE LEY(Laboratorium voor
J. VAN WEYENBERGH
Biochemie. KULeuven, Dekenstraat 6, 8-3000 Leuven)
Zinc induction of femtin heavy chain mRNA in
human monocytes
The interaction of zinc and the immune system has
been the subject of extensive investigation, mainly
focussing on its clinical aspects. While the underlying
mechanisms at the molecular level remain unclear, Zinc
has been shown to induce a number of cytokines in
mononuclear cells.
Using human monocytes as an in vitro model
system, we examined the effects of zinc-
supplementation of the cell culture medium at the
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
mRNA level.
cDNA derived from zinc-supplemented cells was
radiolabelled and mixed with a 20-fold excess of
unlabelled cDNA from unsupplemented cells. On dot
blot, this mixture was shown to detect metallothionein
cDNA, but not actin cDNA, a zinc-induced and a non-
induced house-keeping gene, respectively. Screening of
a human peripheral blood monocyte cDNA library with
this mixture yielded eleven positive clones, four of
which coded for ferritin (Ft) heavy (H) chain, but none
for Ft light (L) chain. This induction of H-Ft was con-
f m e d by Northern-blot analysis of poly-A+RNA from
unsupplemented and zinc-supplemented cells.
Besides its function in iron metabolism, Ft has been
suggested to play a role in immune response (KUMAGAI
For personal use only.
et al., 1992). Although the influence of zinc on L-Ft
mRNA needs to be further investigated, it is not sur-
prising that only H-Ft should be induced by zinc, since
it can be considered as a regulatory cytokine that down-
regulates cell proliferation (BROXMEYER, 1992). This
cytokine activity is most probably exerted through an
H-specific Ft-receptor on hematopoietic and hepatoma
cells, different from the one on normal hepatic cells,
which has similar binding capacities for both H and
L chains, and is only involved in iron metabolism (Moss
et al., 1992).
In conclusion, the induction of H-ferritin mRNA
in human monocytes by zinc can offer more evidence
on the immunological importance of this trace metal.
This work was supported by a research grant o f the National
Lottery (Grant nr. N1364-1A) and of the Belgian NFWO (Grant nr.
3.0078.91). We are grateful to Dr. M A ~ and U the staff of the
Belgian Red Cross Transfusion Centre of Aalst for providing fresh
buffy coats.
References
BROXMEYER, H.E. (1992) J. Lab. Clin. Med. 120, 367-370.
KWGAI, T., AWN, M. & OKADA,S. (1992) Pathol. Res. Pract. 188,
93 1-941.
Moss, D., FARGION, S., ~ C A N Z A N I , A.L., LEVI, S., CAPPEUI-
NI,M.D.,AROSIO,P.,POWEU, L.W.&HALLDAY, J.W.(1992)
J. Inorg. Biochem. 47, 219-227.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
SOCI~TE BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
and P. J. COUR-
A. VEITHEN,P. CUPERS,P. BAUDHUJN
TOY (Unite de Biologie, Cellulaire, University of Louvain and
InternationaJInstitute of Cellular and Molecular Pathology,
Av. Hippocrate 75. B-12W Brwcelles)
Endocytosis in v-Src transformed fibroblasts
We have investigated the effects of v-src on en-
docytosis in the Rat-VtsLA29 fibroblasts cell line ex-
pressing a thermosensitive form of the v-Src protein.
When isolated, this protein is inactive at 40"C, a per-
missive temperatue for cell transformation (VAN DER
VALK et al., 1987). At permissive temperature, in-
tracellular accumulation of the fluid-phase tracer
horseradish peroxidase in Rat- l/tsLA29 was two-fold
higher than in non-transforming conditions. This
stimulation cannot be accounted for by a decrease of
fluid regurgitation, that was actually slightly enhanc-
ed, and corresponds thus to a two-fold increase of fluid-
phase endocytosis. In contrast, receptor-mediated ac-
cumulation of transferrin was only marginally enhanc-
ed (20% higher than the control), indicating that
clathrin-coated pits are not implicated in the stimula-
tion of fluid-phase endocytosis. The stimulatory effect
of v-Src was rapidly suppressed by amiloride, a
macropinocytosis inhibitor (WESTet al., 1989), sug-
gesting that v-Src, like several growth factors, induces
macropinocytosis.
Temporal relation of v-Src protein activation with
the simulation of endocytosis was further investigated.
We found that fluid-phase endocytosis stimulation oc-
curs more than 8 h after transfer of the cells to the per-
missive temperature, whereas transfer to the
non-permissive temperature induced within 2 h a par-
tial reversal of the stimulation. Full reversal was observ-
ed after 3 days at 40°C. Finally, the protein-synthesis
inhibitor cycloheximide produced after 6 h a 40%
decrease of fluid-phase endocytosis rate in transforming
conditions without appreciable effect in control cells.
Two hypotheses can explain those results. Firstly,
v-Src could induce the synthesis of proteins involved
in the stimulation of fluid-phase endocytosis. Second-
ly, v-Src could simply potentiate those factors. In this
case, enhanced stimulation of v-Src synthesis, as
described in chick embryo fibroblasts (HAMAGUCHI et
al., 1993), would also be required.
References
HAMAGUCHI, M., Xuo, H., UBHARA, Y., OHNISHI.Y. & NAOAI,Y.
(1993) Oncogene 8, 559-564.
VAN DBRVALIC, J., VERLMN, I., DE LAAT,S.W. & MOOLENAAR, W.H.
(1987) J. Biol. Chem. 262. 2431-2434.
WEST,M.A.. BRETSCHER, M.S. & WATTS, C. (1989) J. Cell Biol. 109,
273 1-2739.
 S O C d ~BELOE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993 B27

I. WERY,B. DEMOULIN,
E. ROSATOet G. DANDRIFOSSE
(Service de Biochimie et de Physioiogie gknkraie, Institut de
Chimie, Universitk de Lipge, Sart Tiiman. 40 L e e , Beigique)
Evolution de param&tresintestinam suite h la prise
orale de spermine chez les rats non sevrds
Nous avons ttabli la cinktique des variations de dif-
ftrentes caracttristiques de l’intestin, a p r b administra-
tion de spermine (8 pmol/50 pl d’eau) per 0 s A des rats
ggts de 11jours. Des changementsimportants apparais-
sent d&sla deuxihme heure qui suit le traitement et 30-40
h aprhs l’ingestion de spermine. L’ttude montre des
phtnom&nes semblables dans la partie proximale et
dans la partie distale de l’intestin.
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15

Quelques heures a p r b le traitement, on observe

principalement une diminution du poids par cm, une
baisse de l’activitt enzymatique spkcifique de la lactase
et de la maltase ainsi qu’une augmentation t r b impor-
tante de la teneur en spermine. Trente heures aprks l’in-
gestion de spermine, on constate une augmentation
importante du poids d’intestin par cm, une apparition
d’activitt de saccharase, une augmentation de l’activitt
enzymatique sptcifique de la maltase, une hausse de
la teneur en spermidineet un retour de la teneur en sper-
mine A une valeur proche de celle mesurte chez les tt-
moins. L’activitt enzymatique sptcifique de la lactase
reste basse jusqu’au terme de I’exptrience (70 h aprhs
le traitement).
On a isolt des enttrocytes d’intestin proximal de rats
For personal use only.

gg6s de 11 jours, traitts par la spermine 2 h ou 4 h avant

le sacrifice. On a mesurt les activitts enzymatiques
sptcifiques de la sucrase, de la lactase et de la maltase
ainsi que les teneurs en putrescine, en spermidine et en
spermine dans difftrents groupes d’enttrocytes selon
leur position sur l’axe crypto-villositaire. L’activitt de
intact ATPase 40.9% 38.5% 20.5% la sucrase reste non-dttectable dans les deux ex-
treated ATPase 42.2% 37.5% 17.4% ptriences. Deux heures apres le traitement, I’activitt
sptcifique de la lactase augmente fortement dans les
cellules de la partie haute des villositts et diminue dans
les cellules du bas des villositts; I’activitC sptcifique de
la maltase est doublk dans tous les enttrocytes de l’axe
crypto-villositaireexcept6 dans ceux du bas des cryptes;
la teneur en putrescine augmente dans les enttrocytes
du dessus des villositts et dans les cellules proches de
la frontikre villositt-crypte; les teneurs en spermidine
et en spermine augmentent dans toutes les cattgories
d’enttrocytes ttudits. Quatre heures a p r b le traitement,
l’activitt sptcifique de la lactase diminue dans les
enttrocytes inftrieurs des villositts; I’activitt de la
maltase diminue dans les enttrocytes villositaires; la
teneur en putrescine augmentet dans les enttrocytes
villositaires alors que la teneur en spermidine diminue
dans ces mi3mes cellules; la teneur en spermine reste
tlevte dans tous les enttrocytes ttudiks mais n’est
significativement plus tlevte, par rapport aux valeurs
mesurtes chez les ttmoins, que dans les enttrocytes de
la partie haute des villositts et de la partie basse des
cryptes.
Subsidit par le SPPS (contrat HH/006).

Archives Internationales de Physioiogie, de Biochimie et de Biophysiqtie, 1994, 102 (1)

 B28
and B. JORIS(centred'hgdnierie
Sabine WILLEM,Ph. LEDENT
des Proteines, B6, ULg. B4OOO Liege)
Role of conserved residues in class D J-lactamase
studied by site-directed mutagenesis.
8-Lactamases efficiently hydrolyse penicillins, and
constitute the major cause of bacterial resistance to these
antibiotics. The interaction between the 8-lactamase (E)
E +S - -ES E-S* -
and the substrate (S) is described by the model :
E + P, where ES is the non-
covalent Michaelis complex and E-S* the covalent acyl-
enzyme.
The OXA-2 enzyme, isolated on a multiresistance
plasmid of Salmonelly typhimurium, is a serine 8-
lactamase belonging to the class D (JORIS,1988). It ex-
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
hibits original catalytic properties :
- oxacillin is the most sensitive substrate to hydrolysis,
in contrast with other classes of 8-lactamases (DALE,
1971);
- halogenides induce a partial inactivation of the en-
zyme (DALE,1972);
- complex kinetics (burst or delay), explained by bran-
ched pathways, were observed with most substrates.
The OXA-2 primary structure shows no significant
similarity with 8-lactamases of other classes. However,
the four structural elements ("boxes") conserved in ac-
tive site of all penicillin-recognizing enzymes are found
in the OXA-2 sequence (JORIS,1991).
Box 1 is constituted by a S-X-X-K sequence contain-
ing the active serine involved in the acyl-enzyme in-
termediate.
For personal use only.
Box 2 is composed of a S(Y)-D(A, G)-N stretch.
Box 3 is an acidic residue, D or E, which plays the role
of a general base catalyst in class A 0-lactamases.
A triad K(H)-T(S)-G forms the box 4. A positive
charge on the first residue is important for the interac-
tion with the free carboxylate of the substrate.
In order to prove the presence of this four putative
elements in the catalytic cavity of the OXA-2 enzyme, a
site-directed mutagenesis approach was initiated. Here,
we report four mutations located in two of the four
putative boxes : the Glu140 was replaced by Asp or Ala
in box 3, and the Lys189 by His or Asn in box 4.
The different mutated proteins were purifed using
either hydrophobic interaction chromatography (HIC) or
affinity chromatography (CARTWRICHT, 1984).
The kinetic studies were initiated to precise the in-
fluence of the mutated residues on the substrate profile
and the partial inactivation induced by substrates and
halogenides.
Preliminary results clearly demonstrate that the Glu140
residue is not involved in the catalytic mechanism.
On the opposite, the replacement of Lys189 by His or
Asn results in dramatic changes in the kinetic parameters,
showing that this residue plays a role in the hydrolysis of
@-lactams.
References
~ , & WALEY,S.G. (1984)Biochem. J. 221, 505-512.
C A R T W I US.J.
DALE,J.W. & SMITH,J.T. (1971)Biochem. J. 123, 493-512.
DALE,J.W. (1972)Biochem. J. 128, 173-174.
JORIS, B., GHUYSEN,J.-M., DIVE,G., RENARD, A., DIDEBERG, O.,
CHARLIER, P., FRERE,J.-M., KELLY,J.A., BOYINQTON,J.C.,
M o ~ w s P.C.
, & &ox, J.R. (1988)Biochem. J. 250, 313-324.
JORIS, B., LGDENT, Ph., DIDEBERG,O., FONZE, E., LAMOTTE-
BRASSEUR,J., KELLY,J.A., GHWSEN,J.-M., FRERE,J.-M. (1991)
Antim. Ag. and Chemoth. 35, n"l1, 2294-2301.
Archives Internationales de Physiologie. de Biochimie et de Biophysique, 1994, 102 (1)
SOCII?TE BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
S. WOUTERS (l), M. VANDENBRANDEN (I), J.M. R w s -
(z) and E. DECROLY
SCHAERT (l), P.J. COURTOY (l)
[(I) Laboratoire de Chimie Physique des Macrornolkcules aux
interfaces, CP206/2, Universitk Libre de Bruxelles, B-I050
Brussels and (') Unite de Biologie Cellulaire, Louvain Univer-
sity Medical School and International Institute of Cellular and
Molecular Pathology, 8-1200 Brussek]
Idenfication of endoprotease activity against the
precursor of HIY fusion glycoprotein (gp160) in
microsomal fractions
Fusion between HIV-1 virus and the host-cell mem-
brane requires the proteolytic cleavage of the envelope
protein (gp160) into two subunits gp120 and gp41 (Mc
CUNEet ul., 1988). The site of cleavage is located im-
mediately after a sequence rich in basic amino acids.
Comparison of the cleavage sites of some thirty
enveloped viruses indicates that R-X-K/R-R is the
minimal sequence required and that mechanisms im-
plied in viral maturation by cleavage seem to be very
close. MOULARDand collaborators (1992) also
demonstrated that the proteolytic cleavage of the gp160
is Ca2+-dependent.Cleavage occurs in an intracellular
compartiment tentatively assigned to the Golgi com-
plex (PAL et ul., 1989). Recently, SAKAGUCHI and
coworkers (1991) reported that an endoprotease activity
against the fusion glycoprotein precursor of virulent
Newcastle Disease Virus was indeed associated with the
trans-Golgi membranes. The aim of our work is to
localize and to characterize proteases potentially able
to specifically cleave the gp160 into gp120 and gp41.
Crude microsomal fractions were isolated from rat
liver by differential centrifugation. The microsomal
components were separated in discontinuous sucrose
gradients. The light fractions : 8/33 (collected at the
interface of 8% and 33% sucrose) and 36 (collected in
the 36% sucrose layer), which are enriched in galac-
tosyltransfkrase, cleaved gp160 into gp120 and gp41,
although non specific degradation of gp41 was also
observed at high enzyme/substrate ratio. The en-
doprotease activity of the 8/33 fraction was further
characterized with fluorogenic peptides (peptides-7-
amino-4-methyl coumarine) mimiking the cleavage site
of the gp160. The endoprotease in light microsomal
particles showed a maximum activity at pH = 7.5-8 and
was totally inhibited by EDTA (20 mM). Our data are
compatible with the Golgi localization of an en-
doprotease whose activity depends on CaZ+or other
metallic ions.
References
Mc CUNE, J.M., RABIN, L.B., FEINEERC,M.B., REYES,G.R. &
WEISMAN,I.L. (1988)Cell 53, 55-67.
MOULARD, M. (1992)Thkse de doctorat. Laboratoire de Biochimie,
CNRS URA 1455, Facultt de Mkdecine Secteur.
PAL, R., HOKE,G. & SARNOADHAN, M. (1989)Proc. Natl. Acad.
Sci, USA 86, 3384-3388.
SAKAGUCHI, T., MATSUDA,Y., KNOKAGE, R., KAWAHARA, N.,
KIYOTANI, K.,KATUNUMA,N., NACAI,Y.& YOSHIDA,T.(1991)
Virology 184, 504-512.
 SOCdTE BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993
W. WWTS, P. TYLZANOWSKI
and J. MERREGAERT
(Lkpt Biochem.. Lab of Mol. Biotech., University of Antwerp,
Universiteitsplein I , 8-2610 Wilrdk, Belgium)
Sequence of mouse bone sialoprotein I1 (BSP)
cDNA
We have sequenced a cDNA encoding the bone
sialoprotein I1 of the mouse. The entire clone of 1 968
bases has been sequenced from both directions. The largest
open reading frame is located between positions 83 and
1055 and encodes a protein of 324 amino acids. A
polyadenylation signal (AATAAA) is found between
nucleotide positions 1549 and 1554.
Three ATG codons were found in the same open
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15
reading frame and in the proximity of each other. Follow-
ing the first ATG is a possible hydrophobic signal pep-
tide of 16 amino acids, analogous to human BSP (FISHER
et al., 1990). The second one (nucleotide position 119) Lies
within a consensus sequence characteristic for the
eukaryotic initiation codon.
The mouse BSP sequencereveals high overall identities
with rat BSP (92%) and human bone sialoprotein (72%).
Conserved regions of a 100% identity, have been iden-
tified at the RNA as well as at the protein level by com-
paring BSP sequences of mouse, rat (OLDBERG et al.,
1988a), porcine (SHAPIRO et al., 1993) and human ( F I ~ R
et al., 1990).
Three of these conserved areas are tyrosine-rich. One
of these tyrosine-rich areas is located at amino-acid posi-
tion 38, two are on both sides of an RGD sequence in the
For personal use only.
carboxy-terminal region of the protein. These tyrosine do-
mains provide potential sites for sulfatation. The high
degree of conservation of this RGD domain supports the
hypothesis that BSP may be involved in cell adhesion pro-
cesses (OLDBERG et al., 1988b).
Mouse BSP contains a large number of acidic amino
acids located in conserved areas. A row of 11 glutamates
starting at amino-acid position 157 is found in one of the
acidic areas. The number of glutamate repeats however,
differs among the species with a minimum of 6 in por-
cine and the largest repeat of 11 found in the mouse BSP.
Serines in this domain can be phosphorylated and together
with the acidic amino acids they create a Caz+binding do-
main. This domain can interact with hydroxyapatite which
might in turn regulate the crystal growth during the
mineralization process (ENDO& GLIMCHER, 1989). The
acidic character of BSP also results in a strong affinity
of the protein for collagen fibrils due to the electrostatic
interaction between these two proteins (FUIISAWA&
KUBOKI, 1992).
This work was partidy supported by the “VlaamsAktieprogram-
ma Biotechnologie” (VLAB-034b) and Innogenetics, Belgium.
References
ENDO,A. & GLIMCHER, M.J. (1989) Connect. Tissue Res. 21,179-1%.
FISHER,L.W., Mc BRIDE,W.O., TERMINE, J.T. & YOUNG,M.F.
(1990) J. Biol. Chem. 265, 2347-2351.
WJISAWA, R. & KUBOKI.Y. (1992) Calcif. Tissue Int. 51,438-442.
A., FRANZBN,
OLDLIERG, A. & HEINEGARD, D. (1988a) J. Biol. Chem.
263, 19430-19432.
OLDBERG, A., FRANZEN, A., HEINEGARD, D., PIERSCHBACHER. M.
& RUOSLAHTI,E. (1988b) J. Biol. Chem. 263, 19433-19436.
SHAPIRO,H.S.,CHEN,J., WRANA,J.L., ZWNG, Q., BLUM,M.&
SODEK, J . (1993) unpublished, EMBL accession
number J04215.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)
B29
Ingrid ZEGERS,A. FATTAHHAIKAL,R. PALMER
&
L. WYNS (Institute of Molecular Biology, Paardemtraat 65,
B-1640 St. Genesius Rode, Belgium and the Department of
Crystallography, Birkbeck College, Malet Street, London,
Great Britain)
Structure of RNase T1 complexed with 3’-GMP
and guanosine
Ribonuclease T1, is the best known member of a fami-
ly of microbial ribonucleases, and a model system for the
study of structure and function. The enzyme cleaves RNA
at the 3’ end of guanosine bases. This cleavage results in
an RNA chain that is terminated by a 2’,3’-cyclic
nucleotide that can, although less efficiently, be hydroly-
sed by the enzyme. ECKSTEIN et al. (1972) used 2’,3’cyclic
guanosine phosphorothioate (cGPS) to determine the
stereochemistry of the catalytic mechanism of RNase T1.
cGPS is a cyclic nucleotide where one of the exocyclicoxy-
gens on the phosphate is replaced by a sulfur. When the
sulfur is in the endo position the cGPS is hydrolysed by
RNase T1. When the sulfur is in the ex0 position the cGPS
is not cleaved during the time scale of a kinetic experi-
ment. By using a modified method for the synthesis and
purification of cGPS, it has been possible to separate the
endo and the ex0 isomers. The ex0 isomer (ex0 cGPS) was
co-crystallised with RNase T1.
The RNase T1 co-crystals were of the traditional or-
thorhombic type, and diffracted to 1.7 A. The refinement
of the structure converged at an R-value of 14.5%, with
good sterochemistry of the model. Inspection of the ini-
tial electron density maps showed that the ex0 cGPS had
hydrolysed during the crystallisation. The final RNase T1
structure contains a 3’-GMP in the active site, and a
guanine in a putative downstream binding site. It can be
interpreted as containing the products of the reaction of
3’,5’-GpG.
In contrast to previous 3’-GMP complexes of RNase
T1 the ribose-phosphate moiety of the inhibitor is in con-
tact with all the active site residues, probably because the
3’-GMP was produced within the crystals and not added
as such to the enzyme. 3’-GMP is a better analogue of
a substrate of the transesterification step than 2’-GMP,
and the protein-inhibitor interactions in the present struc-
ture are different from those found in the RNase T1 2’-
GMP complex (ARNIet al., 1988). The phosphate is now
hydrogen bonded to Asn36, Tyr38, Arg77, and His92. The
ribose 2’-OH is hydrogren bonded to His40 and Glu58.
The orientations and positions of Glu58 and His92 are
consistent with an in-line mechanism for catalysis, where
Glu58 acts as the general base and His92 as the general
acid. The fact that His40 interacts with the 2’-OH could
indicate that this residue helps the general base (Glu58)
by stablizing a negative charge that is produced on the 2’-
oxygen upon deprotonation. The proton on the 2’-OH
would thus be abstracted by the concerted action of Glu58
and Hisru).
We thank the VLAB and the FKFO for financial support.
References
ARNIet al. (1988) J. Biol. Chem. 263, 15358-15368.
ECKSTEIN et a/. (1972) Biochemistry 11, 3507-3512.
 B30 socrCd BELGE DE BIOCHIMIE, UCL, 20 NOVEMBRE 1993

P.L.N. Z Q ~ ~ ~ E R Mand
A NG.G.
N ROUSSEAU
(Hormorreand
Metabolic Research Unit. Universitt! Catholique de Louvain
and International Institute of Cellular and Molecular
Pathology, Avenue Hippocrate 75, B-I200 B m e l l ~ )
Liver-specific DNase-I hypersensitive sites and
DNA methylation pattern in the promoter region
of a 6-phosphofnrcto-2-kinase/fructose-2,6-bis-
phosphatase gene
6-Phosphofructo-2- kinase/fructose-2,6-bisphos-
phatase (PFK-2/FBPase-2) catalyzes the synthesis and
degradation of fructose-2,6-bisphosphate, a potent
stimulator of glycolysis. One gene codes for the liver,
muscle and hepatoma isozymes of PFK-Z/FBPase-2 by
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Toronto on 01/28/15

the alternative use of three promoters (DARVILLE et al.,

1989; DUPRIEZ et al., 1993). We focus on the mecha-
nisms that control the liver promoter. To identify on
the gene in situ potential cis-acting sequences, we have
examined 15 kb of its 5’ region for DNase-I-
hypersensitive sites detectable on chromatin. We also
have evaluated the DNA methylation status of the
3.7 kb encompassing the liver promoter.
Five DNase-I-hypersensitivesites were detected on
liver chromatin, three upstream (M1 at -4500, L2 at
-1000, L1 at -200) and two downstream (I1 at + 3000,
I2 at + 3500) from the liver-type mRNA cap site. Their
presence correlated with transcriptional activity as they
were not observed on chromatin from kidney, a tissue
where the gene is not expressed. Sites L l and M1 cor-
For personal use only.

responded to the liver and to the muscle promoter,

respectively. They had been characterized previously
in vitro by transient transfection (LEMAIGRE et al., 1991;
DARVJLLE et ul., 1992). The presence of M1 on liver
chromatin was consistent with the fact that it is weak-
ly active in this tissue. Site L2 coincided a
glucocorticoid-responsive unite described by LAN= et
al. (1992), but its presence did not depend on glucocor-
ticoids, Thus, sites L2 and L1 could correspond to novel
control elements.
While DNA was methylated around -2000 both in
liver and kidney, downstream from that position it was
fully demethylated in liver but not in kidney. This pat-
tern changed during development of fetal liver. The
data suggest mechanisms for the inactivation of the L
promoter in kidney and for its activation in develop-
ing and adult liver.
This work was supported in part by the FRSM, and the “PGles
d’Attraction Interuniversitaire (PAI). P.L.N.Z. holds an IRSIA
Fellowship.

References
DARVILLE.M.I.. CRSPIN, K.M., HUE, L. & ROUSSEAU,G.G. (1989)
Proc. Natl. Acad. Sci. USA 86. 6543-6547.
DARVILLE, M.I., ANTOINE, I.V. & ROUSSEAW,G.G. (1992) Nucleic
Acidr Res. 20, 3515-3583.
DUPRIEZ,V.J., D A R ~M.,I.,ANTOINE,I.V., GEOONNE,A.,
GWSDAEL.J. 8 ROUSSEAU, G.G. (1 993) Roc. Natl. Acad. Sci.
USA 90, 8224-8228.
LANOE, A.J., ESPINET,G., HAU, R., EL-MAGHRABI,M.R.,
V m w , A . M . , M m s I c s ~R.Y.,GRA”ER,
, D.K.8rPILKIS.S.J.
(1992) J. Biol. Chem. 267, 15673-15680.
LEMAIORE, F.P., DURVIAUX, S.M. & ROUSSEAU,G.G. (1991) Mol.
Cell. Biol. 11, 1099-1106.

Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1994, 102 (1)