Bariloche protein symposium argentine society for biochemistry and molecular biology

148
BIOCELL, 27 (Suppl. I), 2003
LI-P3.
EFFECT OF AN EXTERNAL ELECTRIC FIELD ON AN
ORDERED LIPID LAYER
Wilke, Natalia and Baruzzi, Ana. M.
Instituto de Fisicoquímica de Córdoba, Dpto. de Fisicoquímica,
Facultad de Ciencias Químicas, Universidad Nacional de
Córdoba. E-mail: [e-mail]
Galactocerebroside layers were transferred onto conducting
substrates (electrodes) using the Langmuir-Shaeffer method. Then,
the covered electrodes were immersed in NaNO
3
 20 mM, and
different potential values were applied. Using Electrochemical
Impedance Spectroscopy (EIS), the effect of the applied potential
on the layers structure was analyzed. This technique provides the
electrical capacitance of the interface as well as the electrical
resistance of the film.
The galactocerebroside film has been investigated in our laboratory
previously using epifluorescence microscopy, ellipsometry
(Manuscript in preparation, J. Phys. Chem), infrared reflection
spectroscopy and other electrochemical techniques (Langmuir
2001,17, 3980-3986, J. Electroanal. Chem. 537 (2002) 67-76,
Langmuir 2003, 19 (17), 6876-6880). The experiments indicate
that in the aqueous environment the lipid film leave holes onto
the electrode, allowing water molecules to penetrate. Besides, a
negative potential applied to the film would increase the holes
proportion and/or sizes. In the EIS experiments this is evidenced
by the membrane resistance decrease at negative potential values.
The interfacial capacitance of the covered electrode is lower than
that of the bare interface, due to the low permittivity of the lipid
layer.
LI-P4.
ISOLATION OF A NOVEL START DOMAIN CONTAINING
PROTEIN HIGHLY EXPRESSED IN GESTATIONAL
TROPHOBLASTIC TUMOUR
Durand S, Angeletti S, and Genti-Raimondi S.
Dpto. de Bqca. Clínica, Fac. de Cs. Qcas., U.N.C., Córdoba,
Argentina. E-mail: [e-mail]
To identify genes whose expression is specifically associated to
“gestational trophoblastic diseases” the differential display
technique was performed. This strategy resulted in the isolation
of an EST named UNCDD1 which identified a clone containing a
3315bp insert from HeLa cell line cDNA library. This cDNA
encodes a protein called GTT1/StarD7 of 295 aminoacids that
shows 49% of similarity with the phosphatidylcholine transfer
protein and has a START domain. Northern blot assays performed
with normal, benign and malignant gestational trophoblastic
samples revealed a 3.5kb transcript exclusively expressed in the
JEG-3 choriocarcinoma cell line. However, semiquantitative RT-
PCR analysis carried out with the same samples demonstrated
GTT1/StarD7 expression throughout all of them but the expression
level was more than 3 times higher in JEG-3 cell line.
Semiquantitative RT-PCR assays performed in diverse human
tumour cell lines revealed GTT1/StarD7 expression in all of them
with the highest levels detected in JEG-3, JAR, HT-29 and HepG2
cells. Finally, an increase of 100% in GTT1/StarD7 expression
was observed when dexamethasone was added to JEG-3, HT-29
and HepG2 cell cultures. In conclusion, the high GTT1/StarD7
expression profile in JEG-3 cells, its lipids binding domain and
dexametasone regulation suggest that GTT1/StarD7 may play an
important role in the phospholipid-mediated signaling of
gestational trophoblastic tumours cellular events.
LI-P5.
GANGLIOSIDES, GANGLIOSIDE GLYCOSYL
TRANSFERASES AND  NEWLY  SYNTHESIZED
GANGLIOSIDES IN SPHINGOLIPID/CHOLESTEROL-
ENRICHED DOMAINS OF GOLGI AND PLASMA
MEMBRANES FROM CHO-K1 CELLS
Pilar Crespo, Adolfo Zurita, Claudio Giraudo, Hugo J. Maccioni
and Jose L. Daniotti.
CIQUIBIC, Fac. de Ciencias Químicas, UNC, Córdoba. E-mail:
[e-mail]
Glycosphingolipid-enriched microdomains (GEM) are specialized
detergent-resistant domains of the plasma membrane (PM) in
which some gangliosides concentrate. Although genesis of GEM
is consider to occur in the Golgi complex, where the synthesis of
gangliosides also occurs, the issue concerning the incorporation
of ganglioside species into GEM is poorly understood. Using CHO-
K1 cells with different glycolipid composition, we compared the
behaviour to cold Triton X-100 solubilization of PM ganglioside
species with the same species newly synthesized in Golgi
membranes. We also investigated if three ganglioside
glycosyltransferases are included or excluded from GEM in Golgi
membranes. Results show that a fraction of PM GM3, and most
GD3 and GT3 reside in GEM. Immunocytochemical examination
of GD3 expressing cells showed GD3 distributed as cold detergent
resistant patches in the PM. In Golgi membranes we were unable
to find evidence for GEM localization of either ganglioside
glycosyltransferases or newly synthesized gangliosides. Since the
same ganglioside species appear in PM GEM, it was concluded
that  in vivo nascent GD3, GT3 and GM3 segregate from their
synthesizing transferases and then enter GEM. This event could
have taken place shortly after synthesis in the Golgi cisternae,
along the secretory pathway and/or at the cell surface.
Supported by Foncyt, HHMI, F. Antorchas, SECyT-UNC, ISN.
LI-P2.
EFFECTS OF THALLIUM (I) AND THALLIUM (III) ON
LIPOSOMES MEMBRANE PHYSICAL PROPERTIES
Marcela S. Villaverde and Sandra V. Verstraeten.
Department of Biological Chemistry, IQUIFIB (UBA-CONICET),
School of Pharmacy and Biochemistry, University of Buenos Aires,
Junín 956, C1113AAD, Buenos Aires, Argentina. E-mail:
[e-mail]
The hypothesis that thallium (Tl) interaction with membrane
phospholipids could result in the alteration of membrane physical
properties was investigated.  Working with liposomes composed
of brain phosphatidyl choline and phosphatidyl serine, we found
that Tl
+
, Tl
3+
, and Tl(OH)
3
 (0.5 to 25 
µM): (a) increased membrane
surface potential, (b) decreased the fluidity of the anionic regions
of the membrane, in association with an increased fluidity in the
cationic regions, and (c) promoted the rearrangement of lipids
through lateral phase separation.  The magnitude of these effects
followed the order Tl
3+
, Tl(OH)
3
 > Tl
+
.  In addition, Tl
3+
 also
decreased the hydration of phospholipid polar headgroups, and
induced membrane permeabilization.  The present results show
that Tl-interacts with membranes inducing major alterations in
the rheology of the bilayer, which could be partially responsible
for the neurotoxic effects of this metal.

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