Bariloche protein symposium argentine society for biochemistry and molecular biology

59
BIOCELL, 27 (Suppl. I), 2003
BT-C6.
DEVELOPMENT OF DNA VACCINES AGAINST
HEMOLYTIC UREMIC SYNDROME (HUS)
Bentancor Leticia
1,2
; Pistone Creydt Virginia
3
; Giambartolomei
Guillermo
4
; Meiss Roberto
1
; Ghiringhelli Daniel
2
; Palermo
Marina
1
.
1
Acad Nac Medicina; 
2
LIGBCM, UNQ; 
3
Cat. Fisiología, Fac.
Medicina,UBA; 
4
Lab Inmunogenética, Htal de Clínicas. Buenos
Aires, Argentina. E-mail: [e-mail]
We have reported that murine immunization with pCDNA
expressing the B subunit gene plus the first codons of the A subunit
of Shiga toxin-2 (pStx2
∆A) triggered a Th1-immune response
which induced a partial protection from Stx2 toxicity in the mouse
model of HUS. Here we studied acute and chronic toxicity of this
plasmid and its protein, and the improvement of this vaccine
through a novel pStx2
∆A construction and immunization protocols.
We purified Stx2
∆A protein from JM109 lysates FPLC. Serial
fractions were eluted and analyzed for specificity by dot blot
revealed with anti-Stx2 antibody. Mice were injected with pStx2
∆A
(50
µg), purified Stx2∆A protein, or with a combined protocol of
both. Animals received one injection of 500ng or three injections
of 2-500 ng of the protein for acute or chronic toxicity studies.
Mice were monthly bled and after 2, 4 and 6 months from the first
injection, lungs, livers and kidneys were excised for
hystopatological studies. We found low neutralizing activity in
sera from protein-immunized mice and different levels of renal
damage in mice injected with pStx2
∆A, highest concentrations of
protein, or with combined protocol. Finally, we obtained and
sequenced a new pStx2
∆A construction using Stu I and Ava I
enzymes, carrying the B subunit, and the last 31 aa of the A subunit
of Stx2.
BT-C7.
SELECTION OF CAMELID ANTI-IDIOTYPIC VHHs
BEARING DNA INTERNAL IMAGE BY PHAGE-DISPLAY
LM Zarebski, M Urrutia, G Vila Melo, FA Goldbaum
Lab. de Inmunología Molecular y Estructural, Fund. Instituto
Leloir, Buenos Aires, Argentina. E-mail: [e-mail]
In camelids, a subset of the immunoglobulins consists of heavy
chain homodimers devoid of light chains, and are thus called heavy
chain IgGs (hcIgGs). Their variable region (VHH) is the smallest
antigen-binding fragment possible, and being just one polypeptide
chain it is especially suitable for engineering. From llamas
immunized with an anti-DNA mouse IgG, we sought to obtain anti-
idiotypic VHHs able to back-elicit anti-DNA antibodies by
molecular mimicry. Upon immunization of llamas with ED84, a
monoclonal anti-DNA antibody with sequence specificity to a ds18-
mer (Site35), high titers (>30000) towards ED84 Fab fragment
were obtained. Total IgGs were purified and hcIgGs and
conventional IgGs were isolated. By ELISA, both fractions showed
significant binding to ED84. On the other hand, the VHHs were
amplified from cDNA of mononuclear cells and were subsequently
used to construct a phage display library of 10
8
 
individual clones.
After two rounds of solid phase panning with specific elution (i.e.,
with Site35), a 58% of anti-idiotypic VHHs was obtained. Those
monoclonal VHHs, and the idiotypic fraction of polyclonal hcIgGs
and conventional IgGs were injected in mice. Preliminary results
show that it is possible to obtain anti-DNA antibodies from the
response to anti-idiotypic antibodies, confirming the hypothesis
of molecular mimicry  being attained from an "internal image" of
the antigen.
BT-C8.
PROTEOMIC ANALYSIS OF A MELANOMA CELL LINE:
INSIGHT INTO A MOLECULAR PATHWAY OF
TUMORIGENESIS
Sosa, María Soledad
1
; López, Juan Antonio
2
; Camafeita, Emilio
2
;
Juárez, Silvia
2
; Albar, Juan Pablo
2
; Podhajcer,Osvaldo
1
 and Llera,
Andrea S.
1
1
Fundación Instituto Leloir, Buenos Aires, Argentina, and 
2
Centro
Nacional de Biotecnología, Madrid, España. E-mail:
[e-mail]
SPARC is a matricellular glycoprotein that elicits changes in cell
shape and proliferation. While its normal expression is restricted
to tissues under remodeling, SPARC was found to be overexpressed
in different tumors, in association with tumor progression and
metastasis. Our previous results showed that stable antisense
SPARC expression abolished tumorigenicity in an in vivo
melanoma murine model. Traditional approaches to define a
molecular pathway for SPARC-mediated tumor progression have
failed to render meaningful results. Thus, we started a proteomic
analysis of proteins expressed by melanoma cells with modulated
SPARC expression, in order to identify new SPARC-interacting
proteins. We studied conditioned culture medium from human
MEL-LES clone 1-D (L-1D, showing an 80% decrease of SPARC
mRNA and protein expression), as compared with the control MEL-
LES clone CMV (L-CMV). Proteomic analysis was done by two-
dimensional electrophoresis followed by tryptic digestion of spots
and MALDI-TOF peptide fingerprinting analysis. Up to date we
have identified 94 proteins, among them those related with SPARC
function, as well as extracellular matrix structural proteins and
oncoproteins. We are currently focusing in establishing which
proteins are differentially expressed in the non-tumorigenic clone
L-1D, as opposed to the tumorigenic clone L-CMV, aiming at
identifying proteins that may be involved in SPARC molecular
pathway.
MI-P1.
ARGININE KINASE OVEREXPRESSION IMPROVES
TRYPANOSOMA CRUZI SURVIVAL CAPABILITY
Alonso, Guillermo D.; Pereira, Claudio A.; Ivaldi, M. Soledad;
Silver, Ariel M.; Torres, Héctor N. and Flawiá, Mirtha M.
INGEBI, Buenos Aires, Argentina.
E-mail: [e-mail]
Arginine kinase catalyzes the reversible transphosphorylation
between N-phospho-L-arginine and ADP. Phosphoarginine and
phosphocreatine, generally called phosphagens, play an essential
role as energy reserve because the high-energy phosphate can be
transferred to ADP when ATP is needed. The molecular and
biochemical characterization of arginine kinases in Trypanosoma
cruzi and Trypanosoma brucei, the etiological agents of Chagas´
disease and human sleeping sickness respectively, have been
reported by this laboratory. It was established that a single-copy
gene encodes for a functional arginine kinase in Trypanosoma
cruzi. Here we demonstrate that the homologous overexpression
of the Trypanosoma cruzi arginine kinase improves the transfected
cells’ ability to grow and resist under nutritional and pH stress
conditions. The stable-transfected parasites showed an increased
cell density from the day 10 of culture, when the carbon sources
became scarce, which resulted in a 2.5-fold respect to the control
group on day 28. Additional stress conditions were also tested.
We propose that arginine kinase is involved in the parasites’
adaptation to environmental changes. Moreover, here we report a
successful overexpression model of a T. cruzi endogenous enzyme,
and the characterization of its biological effects.

---