Biomedical subjects

Cristina Ibarra

Publications and source records attributed to Cristina Ibarra.

11 recordsLinked to original sources

PubMed2006

Simple multichannel system for the measurement of the net water flux across biological tissues.

This paper describes the development of a simple system for measurement of net water movement through biological membrane barriers. The system is based on the detection of a water meniscus inside a polyethylene tube, which reflects the water movement inside one hemichamber of a modified Ussing chamber containing a membrane barrier. The detection device consists of a commercial computer-controlled flat bed scanner and specifically developed software. This system allows one to perform a relatively high number of individual experiments per physical unit. It is a flexible and affordable device, which allows comparatively more information per unit to be obtained than previously described methods.

Body Water↗

PubMed2006

[Characterization of the epithelial sodium channel in human pre-eclampsia syncytiotrophoblast].

The syncytiotrophoblast (SCT), a multinucleated epithelium forming the outer layer of chorionic villi, acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated Na+ transport. The present study was conducted to analyze the presence of the epithelial Na channel (ENaC) in placental tissue from normal and pre-eclamptic women and in BeWo cell, a model of a human SCT. Changes in the expression of these proteins during sodium transport across the placenta may be related to the pathogeny of pre-eclampsia. The role that ENaC and Na+ transport deregulation play on human placental tissues still remains unknown although in aldosterone-responsive epithelial cells (kidney, colon), abnormalities upregulating its activity lead to increased Na+ uptake and hypertension (i.e. Liddle's syndrome) whereas a diminished channel activity can result in the pseudohypoaldosteronisn syndrome with salt loss and hypotension. Our results show that ENaC is expressed in the apical membrane of normal syncytiotrophoblast. The amplified fragment of alpha-ENaC was cloned and sequenced having a 100% identity with the sequence of (alpha-ENaC obtained from GenBank (SCNN1A, accession number Z92981). We found that the transcription of the alpha-ENaC mRNA was not detectable in preeclamptic placentas and the protein was not observed with immunohistochemistry staining, probably indicating a low protein expression level. In BeWo cells ENac was found and its expression is regulated by aldosterone, vasopressin, progesterone and estradiol. With patch clamp techniques we studied the currents trough ENaO channels in Bewo cells. We observed currents that were blocked by 10 microM amiloride in cells incubated in 100 nM aldosterone for 12 hs. The amplitude of this current was 20-fold the basal current, a reversal potential of 3 mV and a conductance of 127 +/- 26 pS/pF with pulses between -60 and -140 mV. These characteristics are similar to those reported in ENaC channels in several tissues. Although their roles in placenta are still poorly understood, the differences in the expression of ENaC in pre-eclamptic placentas may have consequences for ion transport and these data could lead to future studies concerning the mechanism involved in the pathophysiology of pre-eclampsia.

Blotting, Western↗

PubMed2005

Cytotoxic effect of Shiga toxin-2 holotoxin and its B subunit on human renal tubular epithelial cells.

Shiga toxin-producing Escherichia coli produces watery diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome (HUS). In Argentina, HUS is the most common cause of acute renal failure in children. The purpose of the present study was to examine the cytotoxicity of Stx type 2 (Stx2 holotoxin) and its B subunit (Stx2 B subunit) on human renal tubular epithelial cells (HRTEC), in the presence and absence of inflammatory factors. Cell morphology, cell viability, protein synthesis and apoptosis were measured. HRTEC are sensitive to both Stx2 holotoxin and Stx2 B subunit in a dose- and time-dependent manner. IL-1, LPS and butyrate but not TNF, IL-6 and IL-8, increased the Stx mediated cytotoxicity. The effects of Stx2 B subunit appear at doses higher than those used for Stx2 holotoxin. Although the physiological importance of these effects is not clear, it is important to be aware of any potentially toxic activity in the B subunit, given that it has been proposed for use in a vaccine.

Adult↗

PubMed2005

Ionic currents in multidrug resistant K562 human leukemic cells.

In this study, the expression and functional characterization of currents through the CFTR (cystic fibrosis transmembrane regulator) and ORCC (outwardly rectifying chloride channels) were determined in wild-type K562 chronic human leukemia cells (K562-WT) and in its resistant counterpart, the vincristine resistant cell line (K562-Vinc). Expression of the CFTR and MDR1 (multidrug resistant) gene products was determined by a semi-quantitative RT-PCR protocol. The amplified products in K562-WT and K562-Vinc showed two bands corresponding to CFTR and MDR1. MDR1 mRNA increased by 20-fold in K562-Vinc whereas no change in CFTR mRNA levels was observed. CFTR and ORCC channel activity were measured with a whole cell configuration of the patch clamp technique. Forskolin (40 microM n activator of adenylate cyclase, added to the extracellular side increased the current in both cell lines. A fraction of the activated whole cell currents was inhibited by 500 microM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and subsequent addition of 500 microM diphenylamine-2-carboxylate (DPC plus DIDS) further inhibited the remaining currents. The levels of forskolin-activated currents and subsequent blockade were similar in both cell lines. The effect of forskolin was prevented in cells previously exposed to 500 microM DPC. The effects of DIDS and DPC on the forskolin-activated whole cell currents support the idea that both CFTR and ORCC are generating a significant fraction of these currents with DIDS inhibiting ORCC currents and DPC inhibiting CFTR currents when the blockers are added one after another to the extracellular side. Finally, we show that exposure of K562 cells to vincristine which results in the over expression of MDR1 is not accompanied by a significant down regulation of CFTR as in other cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

[Cytotoxic effect of Shiga toxin type 2 and its B subunit on human renal tubular epithelial cell cultures].

Shiga toxin (Stx)-producing E. coli causing watery diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome (HUS). In Argentina, HUS is the most common cause of acute renal failure in children. The purpose of the present study was to examine the cytotoxicity of Stx type 2 (Stx2) and its B subunit (Stx2B) on human renal tubular epithelial cells (HRTEC), in the presence and absence of inflammatory factors. Cytotoxic effects were assessed in terms of functionality of the epithelium, histological damage, cell viability, protein synthesis and cellular apoptosis. Results show that Stx2 regulates the passage of water through the HRTEC within an incubation period of 1h. Within longer periods, up to 72 hours, the study of morphology, viability, protein synthesis and apoptosis shows that HRTEC were sensitive to the cytotoxic action of Stx2 and Stx2B in a dose- and time-dependent manner. These effects were potentiated by lipopolysaccharides (LPS), IL-1beta, and butirate.

Adult↗

PubMed2004

Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells.

In this study, the presence of Na(+)-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na(+)-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an approximately 3:1 Na(+)/K(+) permeability-selectivity ratio. The Na(+)-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na(+) channel activity. Spontaneous and vasopressin-induced Na(+) channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the alphaENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although alphaENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on alphaENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na(+)-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na(+) transport mechanisms in the mammalian proximal nephron.

Adenosine Triphosphate↗

PubMed2003

Characterization of urea transport in Bufo arenarum oocytes.

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.

Animals↗

PubMed2003

Development of DNA vaccines against hemolytic-uremic syndrome in a murine model.

Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2 Delta A). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.

Animals↗

PubMed2003

Genetic diversity of Vibrio cholerae O1 in Argentina and emergence of a new variant.

The genetic diversity of Vibrio cholerae O1 strains from Argentina was estimated by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-nine isolates carrying the virulence genes ctxA, zot, ace, and tcpA appeared to represent a single clone by both typing methods; while 11 strains lacking these virulence genes exhibited several heterogeneous RAPD and PFGE patterns. Among the last group, a set of isolates from the province Tucumán showed a single RAPD pattern and four closely related PFGE profiles. These strains, isolated from patients with diarrhea, did not produce the major V. cholerae O1 virulence determinants, yet cell supernatants of these isolates caused a heat-labile cytotoxic effect on Vero and Y-1 cells and elicited significant variations on the water flux and short-circuit current in human small intestine mounted in an Ussing chamber. All these effects were completely abolished by incubation with a specific antiserum against El Tor hemolysin, suggesting that this virulence factor was responsible for the toxic activity on both the epithelial cells and the small intestine specimens and may hence be involved in the development of diarrhea. We propose "Tucumán variant" as the designation for this new cluster of cholera toxin-negative V. cholerae O1 strains.

Argentina↗

PubMed2003

CFTR in K562 human leukemic cells.

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 microM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2002

Membrane currents in the oocyte of the toad Bufo arenarum.

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes.

Action Potentials↗