Bariloche protein symposium argentine society for biochemistry and molecular biology

72
BIOCELL, 27 (Suppl. I), 2003
MI-P50.
INTRAVENOUS LIPOSOMAL BENZNIDAZOL AS
TRYPANOCIDAL AGENT:  INCREASING  DRUG
DELIVERY TO LIVER IS NOT ENOUGH
M.J. Morilla
1
, P.E. Benavidez
1
, J.A. Montanari
1
, M.J. Prieto
1
, M.O.
Lopez
1
, P. Petray
2
, E.L. Romero
1,*
.
1
Laboratorio de Diseño de Transportadores de Drogas,
Universidad Nacional de Quilmes, Roque Saenz Peña 180, Bernal
B1876BXD, Bs As, Argentina. 
2
Laboratorio de Virologia, Hospital
de Niños Dr. R. Gutierrez, Gallo 1330, C1425EFD, Bs As,
Argentina.
With the aim of investigating if delivery of benznidazole (BNZ)
to liver could be increased by incorporating the drug in
multilamellar liposomes, single bolus of free BNZ, or liposomal
BNZ formulations (MLV-BNZ) composed of HSPC:DSPG:Chol
2:1:2 mol:mol at 0,7% w/w drug/total lipid ratio, were injected by
intramuscular (i.m.),subcutaneous (s.c.) and intravenous (i.v.)
routes, at 0.2 mgBNZ/kg, in rats. The resulting blood
concentrations were followed along 9 h post injection (p.i.) and
drug accumulation in liver was determined after 4 and 9 hours pi.
Only upon i.v. injection of MLV-BNZ, a threefold higher BNZ
accumulation in liver was obtained, together with blood BNZ
concentrations of 1,1 
µg/ml (30% lower than the BNZ blood
concentration achieved upon iv administration of free drug). The
increased uptake of BNZ by liver raised every 56 h showed to
exert no effect on parasitaemia levels of mice infected with a RA
strain of Trypanosoma cruzi. Our results indicate that relationship
between increased selectivity for an infected tissue and therapeutic
effect is not straightforward, at least for the MLV-BNZ regimen
used in the present study.
MI-P51.
GLUTATHIONE DEFICIENCY INDUCES A PLEIOTROPIC
RESPONSE IN Rhizobium Tropici CIAT899
Muglia, Cecilia; Lodeiro, Aníbal and Aguilar, O. Mario.
IBBM, Fac. Cs. Exactas, UNLP. 47 y 115, La Plata. E-mail:
[e-mail]
Glutathione is involved in many cellular functions including stress
tolerance. In the common bean symbiont R. tropici it is essential
for protection against environmental stresses, such as acidity,
salinity and oxidative stress. We have previously cloned the gshB
R. tropici wild type gene (which encodes glutathione synthetase)
and demonstrated that its transcription is transiently activated when
shifted to acidic conditions. We have now mapped the transcription
initiation of gshB gene by primer extension analysis, and found it
to be located 71 bp upstream from the predicted start codon.
Centered at  position –40 we have found a palindromic sequence
of the general form TTGN
13
CAA, similar to the TTGN
11
CAA
palindromic sequence found in the Rhizobium etli, Sinorhizobium
meliloti and Agrobacterium tumefaciens  recA promotor, known
as the SOS box. recA gene is activated in response to DNA damage,
suggesting that gshB gene should be regulated by a SOS type
response. We also show that catalase activation by oxidative stress
is impaired in glutathione deficient mutant, which we propose
occurs in an OxyR mediated manner. We have also studied the
effects of glutathione deficiency in symbiosis. We have found that,
although  gshB deficient mutant is able to nodulate Phaseolus
vulgaris, the resulting nodules are small and in a higher number
compared to plants inoculated with the wild type. We have also
found that bacterial glutathione is necessary to maintain an efficient
symbiosis, since lack of rhizobial glutathione is associated to
premature nodule senescence.
MI-P52.
CONSTRUCTION OF A SINGLE-CHAIN FV ANTIBODY
FRAGMENT WITH Trypanosoma cruzi  trans-SIALIDASE
NEUTRALIZING  ACTIVITY
Muiá Romina, Pitcovsky Tamara A, and Campetella Oscar.
Instituto de Investigaciones Biotecnológicas, Universidad
Nacional de Gral. San Martín, San Martín Prov. Buenos Aires,
Argentina. E-mail: [e-mail]
Trypanosoma cruzi trans-sialidase (TS) is considered a key factor
in the establishment of the chagasic infection. TS is able to direct
transfer sialyl residues among macromolecules an, activity of
interest in glycoconjugates research. Specific inhibitors are
required to understand both, TS involvement in the chagasic
infection and its biochemical properties. TS-neutralizing antibodies
constitutes the only inhibitors available. Then, defined inhibitory
antibodies-derived peptides provide a useful tool for the further
rational development of TS-targeted drugs. We report here the
construction of a single-chain Fv (scFv) derived from a mouse
monoclonal antibody displaying TS-neutralizing activity. By
employing the Recombinant Phage Antibody System, the variable
regions of the heavy and light chains of the IgG from the 13G9
hybridoma   were cloned, sequenced and assembled into an sc Fv.
This construction was cloned into pCANTAB vector, and expressed
both, as phage displayed in Escherichia coli XL1Blue and in the
soluble form cleaved to the supernatant in E. coli Top10F’ cultures.
Both constructions retained the TS-neutralizing activity.
MI-P53.
INTERACTION BETWEEN NATURAL POLYAMINES AND
SYNTHETIC DEOXYOLIGONUCLEOTIDES: AN NMR STUDY
Niemevz F
1
, Buldain GY
1
, De Candia A
2
, and Fernández CO.
2
1
Departamento de Química Orgánica, Facultad de Farmacia y
Bioquímica (UBA), Buenos Aires, Argentina. 
2
LANAIS RMN 300
(CONICET-UBA), Buenos Aires, Argentina. E-mail:
[e-mail]
Putrescine, spermidine and spermine are natural polyamines
distributed in living organisms. The intracellular concentration of
polyamines has been shown to influence cell growth and
carcinogenesis. The structural rationale for the physiological effects
of polyamines has relied in the understanding of the polyamine-
nucleic acid interactions. Nuclear Magnetic Resonance (NMR)
spectroscopy has provided valuable insights into tRNA-polymine
interaction, giving support and enhancing structure-based drug
design. In this work we extended our studies to explore the
interaction between natural polyamines and synthetic
deoxyoligonucleotides. The 
1
H 2D NMR spectra of
d(CGCGAATTCGCG)
2 
 are typical of a B DNA conformation. The
31
P NMR spectrum of d(CGCGAATTCGCG)
2 
  remains unchanged
upon addition of putrescine and spermidine. However, the
interaction with spermine reveals changes in the chemicals shifts
of a discrete number of 
31
P resonances. 
1
H NMR titration
experiments of d(CGCGAATTCGCG)
2 
 with polyamines exhibits
a similar behavior. The 
15
N relaxation times of 
15
N-labeled
spermine are strongly affected by the presence of the
deoxyoligonucleotide, whereas purtrescine and spermidine show
similar relaxation properties in the absence and presence of
d(CGCGAATTCGCG)
2 
. These results clearly show that spemine
is the natural polyamine exhibiting binding features in the presence
of the d(CGCGAATTCGCG)
2
 fragment. These conclusions are
relevant for understanding the molecular basis of polyamine affinity
and specificity and for enhanced polyamine analogs design.

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