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C Ibarra

Publications and source records attributed to C Ibarra.

At least 19 recordsLinked to original sources
PubMed2006

Interferon regulatory factor-1 regulates reconstituted extracellular matrix (rECM)-mediated apoptosis in human mammary epithelial cells.

Interactions between extracellular matrix (ECM) and mammary epithelial cells are critical for mammary gland homeostasis and apoptotic signaling. Interferon regulatory factor-1 (IRF-1) is a transcriptional regulator that promotes apoptosis during mammary gland involution and p53-independent apoptosis. We have recently shown that rapid cell surface tamoxifen (Tam) signaling promotes apoptosis in normal human mammary epithelial cells that were acutely damaged by expression of human papillomavirus type-16 E6 protein (*HMEC-E6). Apoptosis was mediated by recruitment of CREB-binding protein (CBP) to the gamma-activating sequence (GAS) element of the IRF-1 promoter, induction of IRF-1 and caspase-1/-3 activation. Here, we show that growth factor-depleted, reconstituted ECM (rECM), similar to Tam, promotes apoptosis in *HMEC-E6 cells through induction of IRF-1. Apoptosis was temporally associated with recruitment of CBP to the GAS element of the IRF-1 promoter, induction of IRF-1 expression and caspase-1/-3 activation. Small interfering RNA-mediated suppression of IRF-1 protein expression in *HMEC-E6 cells blocked (1) induction of IRF-1, (2) caspase-1/-3 activation and (3) apoptosis. These observations demonstrate that IRF-1 promotes rECM-mediated apoptosis and provide evidence that both rECM and rapid Tam signaling transcriptionally activate IRF-1 through recruitment of CBP to the IRF-1 GAS promoter complex.

Antineoplastic Agents, Hormonal↗
PubMed2006

Functional and molecular expression of AQP9 channel and UT-A transporter in normal and preeclamptic human placentas.

Transcellular flux of urea across human placenta may be facilitated by aquaglyceroporins and/or by urea transporters (UT). Previously we have reported the presence of AQP3 and AQP9 in the syncytiotrophoblast of human term placenta (hST). In the present study, we detected a significant uptake of water, urea and mannitol sensitive to mercury and phloretin in explants from normal term placenta, which indicates a functional AQP9. In addition, we observed an increase of AQP9 expression in preeclamptic placenta with a lack of functionality of AQP9 for water and mannitol. Our data showed a molecular and functional expression of UT-A in hST from normal and preeclamptic placentas. In the last case, urea uptake sensitive to phloretin and mercury increased and the UT-A protein seems to be augmented. These results suggest that the increase of urea uptake in preeclamptic pregnancies could be attributed to an increase of expression of UT-A more than AQP9 proteins. In conclusion, our results provide new evidences that suggest the involvement of AQP9 and UT-A in the urea excretion mechanism across hST from mother to fetus in physiological conditions. Much further work is needed to define whether the overexpression of AQP9 plays a direct role either in the pathogenesis or in the adaptative response of preeclampsia.

Amino Acid Sequence↗
PubMed2005

Clostridium perfringens enterotoxin damages the human intestine in vitro.

In vitro, Clostridium perfringens enterotoxin (CPE) binds to human ileal epithelium and induces morphological damage concurrently with reduced short-circuit current, transepithelial resistance, and net water absorption. CPE also binds to the human colon in vitro but causes only slight morphological and transport changes that are not statistically significant.

Colon↗
PubMed2004

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops.

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

Adult↗
PubMed2003

CodY is a nutritional repressor of flagellar gene expression in Bacillus subtilis.

Expression of the sigma(D)-dependent flagellin gene, hag, is repressed by the CodY protein in nutrient-rich environments. Analysis of a codY mutant bearing a hag-lacZ reporter suggests that the availability of amino acids in the environment is the specific signal that triggers this repression. Further, hag-lacZ expression appears to be sensitive to intracellular GTP levels, as demonstrated by increased expression upon addition of decoyinine. This result is consistent with the postulate that the availability of amino acids in the environment effects intracellular GTP levels through the stringent response. However, the levels of hag-lacZ measured upon the addition of subsets of amino acids suggest an additional mechanism(s). CodY is a DNA binding protein that could repress flagellin expression directly by binding to the hag promoter region, or indirectly by binding to the fla/che promoter region that governs expression of the sigma(D) transcriptional activator required for hag gene expression. Using an electrophoretic mobility shift assay, we have demonstrated that purified CodY protein binds specifically to both the hag and fla/che promoter fragments. Additionally, CodY acts as a nutritional repressor of transcription from the fla/che promoter region that contains two functional promoters. CodY binds to both the sigma(D)- and sigma(A)-dependent promoters in this region, as demonstrated by DNase I footprint analyses. Footprint analyses of the hag gene demonstrated that CodY binds downstream of its sigma(D)-dependent promoter. Taken together, these results identify new members of the CodY regulon that encode motility functions in Bacillus subtilis and are controlled by the sigma(D) alternate sigma factor.

Amino Acids, Branched-Chain↗
PubMed2002

Adenylyl cyclase types I and VI but not II and V are selectively inhibited by nitric oxide.

Adenylyl cyclase (AC) isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO) is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP), a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (approximately 98.5%) of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with ionomycin. A high inhibition (approximately 76%) of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM) for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V.

Adenylyl Cyclase Inhibitors↗
PubMed2001

Contribution of aromatic-aromatic interactions to the anomalous pK(a) of tyrosine-9 and the C-terminal dynamics of glutathione S-transferase A1-1.

Most cytosolic glutathione S-transferases (GSTs) exploit a hydrogen bond between an active site Tyr and the bound glutathione (GSH) cofactor to lower the pK(a) of the GSH and generate the nucleophilic thiolate anion, GS(-). In human (hGSTA1-1) and rat (rGSTA1-1) homologues, the active site Tyr-9 has a low pK(a) of 8.1-8.3, for which the functional significance is unknown. Crystal structures of GSTA1-1 suggest that weakly polar interactions between the electropositive ring edge of Phe-10 and the pi-cloud of Tyr-9, in the apoenzyme, could stabilize the tyrosinate anion and also modulate the pK(a) of GSH. Upon binding a product GSH conjugate, Phe-10 moves away from Tyr-9, allowing the highly dynamic C-terminus to "close" over the active site. To explore the role of Phe-10 in modulating the Tyr-9 pK(a) and in ligand binding, rGSTA1-1 mutants F10Y, F10L, and F10A were characterized. The pK(a)s of Tyr-9 in the apoenzymes were 8.2 +/- 0.2, 8.7 +/- 0.2, and 9.3 +/- 0.1, respectively, for F10Y, F10L, and F10A, compared to 8.3 +/- 0.2 for the "wild type". The experimentally determined pK(a)s qualitatively paralleled the energies required to remove a proton predicted by ab initio calculations using model compounds constrained to the coordinates of rGSTA1-1. The pK(a) of GSH in the binary complex was significantly less affected by these substitutions. In contrast, F220I and F220Y C-terminal mutations caused the pK(a) of Tyr-9 to decrease modestly. For the binary complex with S-hexyl-GSH, which induces the "closed" conformation, Tyr-9 retains a low pK(a) and the Phe-10 substitutions have significant effects. Presumably, Phe-10 plays a critical structural role in stabilizing the closed conformation. The mutations F10L and F10A also slowed the rate of GSH conjugate binding by 10-20-fold, as measured by stopped-flow fluorescence. The effects of Phe-10 substitution were large for both steps of the biphasic binding reaction, suggesting the importance of aromatic interactions throughout the reaction coordinate. A unified view of the C-terminal dynamics of GSTA1-1 is discussed, which emphasizes the coupling between Tyr-9 ionization, active site solvation, and C-terminal dynamics.

Animals↗
PubMed2001

Regional and age-dependent expression of the nitric oxide receptor, soluble guanylyl cyclase, in the human brain.

Nitric oxide (NO), synthesized by neuronal NO synthase (NOS-I), plays essential physiological roles in the brain. The major molecular target for NO is soluble guanylyl cyclase (sGC), a heterodimeric hemoprotein composed of a larger alpha and a smaller beta subunit. Both subunits of sGC are needed to generate the second messenger cyclic GMP (cGMP). Here we show using subunit-specific antibodies and Western blot analysis that sGCalpha1 and sGCbeta1 protein subunits are present in all examined human brain regions. The relative distribution of the two subunits was similar and also correlated well with the known distribution of NOS-I. The highest expression levels of sGC were found in cortex, basal ganglia and the limbic system. These regions display the most prominent biochemical and histological changes during ageing. In cortex, a negative correlation between the amounts of sGC and age was found, while sex and post-mortem delay time did not affect sGC levels significantly. Our data suggest that sGCalpha1 and sGCbeta1 subunits are widely distributed in human brain, consistent with a major role in NO signaling. Moreover, the NO/cGMP pathway appears to be affected by ageing in the human brain.

Aged↗
PubMed2001

Formation and structure of a complex of sucrose with cobalt(III)bis(phenanthroline).

Sucrose forms a dicationic complex with cobalt(III)bis(phenanthroline) which can be isolated as the sparingly water-soluble triiodide salt or the water-soluble chloride. The circular dichroism (CD) spectrum demonstrates the Delta configuration at Co(III) and the presence of two phenanthroline and one sucrose residues in the complex. The O-2(g)--O-1(f) distance in crystalline sucrose permits strain-free coordination of these centers with Co(III) and in the complex the H-1,1'(f) singlet of sucrose separates into a pair of doublets. The 1H NMR spectrum in DMSO-d(6) shows that OH-2(g) is deprotonated and the signal of OH-1(f) is shifted strongly downfield by complexation with Co(III). Coordination involving glucose and fructose residues is consistent with neither alpha-methyl glucoside nor fructofuranose forming mixed complexes with phenanthroline. Structure simulation with the semi-empirical PM3(tm) basis set indicates that complexation by O-2(g) and OH-1(f) can give a Delta-complex with little structural distortion, whereas in hypothetical Lambda-complexes there is distortion of the sucrose residue. Observation of an NOE involving the sucrose and phenanthroline residues supports the postulated structure of the Delta-complex.

Circular Dichroism↗
PubMed2001

The C-terminus of glutathione S-transferase A1-1 is required for entropically-driven ligand binding.

Binding of a hydrophobic glutathione product conjugate to rGST A1-1 proceeds via a two-step mechanism, including rapid ligand docking, followed by a slow isomerization to the final [GST.ligand] complex, which involves the localization of the flexible C-terminal helix. These kinetically resolved steps have been observed previously by stopped-flow fluorescence with the wild-type rGST A1-1, which contains a native Trp-21 approximately 20 A from the ligand binding site at the intrasubunit domain-domain interface. To confirm this binding mechanism, as well as elucidate the effects of truncation of the C-terminus, we have further characterized the binding and dissociation of the glutathione-ethacrynic acid product conjugate (GS-EA) to wild-type, F222W:W21F, and Delta209-222 rGST A1-1 and wild-type hGST A1-1. Although modest kinetic differences were observed between the hGST A1-1 and rGST A1-1, stopped-flow binding studies with GS-EA verified that the two-step mechanism of ligand binding is not unique to the GST A1-1 isoform from rat. An F222W:W21F rGST A1-1 double mutant provides a direct fluorescence probe of changes in the environment of the C-terminal residue. The observation of two relaxation times during ligand binding and dissociation to F222W:W21F suggests that the C-terminus has an intermediate conformation following ligand docking, which is distinct from its conformation in the apoenzyme or localized helical state. For the wild-type, Delta209-222, and F222W:W21F proteins, variable-temperature stopped-flow experiments were performed and activation parameters calculated for the individual steps of the binding reaction. Activation parameters for the binding reaction coordinate illustrate that the C-terminus provides a significant entropic contribution to ligand binding, which is completely realized within the initial docking step of the binding mechanism. In contrast, the slow isomerization step is enthalpically driven. The partitioning of entropic and enthalpic components of binding energy was confirmed by isothermal titration calorimetry with wild-type and Delta209-222 rGST A1-1.

Animals↗
PubMed2001

Localization of the C-terminus of rat glutathione S-transferase A1-1: crystal structure of mutants W21F and W21F/F220Y.

Twelve C-terminal residues of human glutathione S-transferase A1-1 form a helix in the presence of glutathione-conjugate, or substrate alone, and partly cover the active site. According to X-ray structures, the helix is disordered in the absence of glutathione, but it is not known if it is helical and delocalized, or in a random-coil conformation. Mutation to a tyrosine of residue 220 within this helix was previously shown to affect the pK(a) of Tyr-9 at the active site, in the apo form of the enzyme, and it was proposed that an on-face hydrogen bond between Tyr-220 and Tyr-9 provided a means for affecting this pK(a). In the current study, X-ray structures of the W21F and of the C-terminal mutation, W21F/F220Y, with glutathione sulfonate bound, show that the C-terminal helix is disordered (or delocalized) in the W21F crystal but is visible and ordered in a novel location, a crystal packing crevice, in one of three monomers in the W21F/F220Y crystal, and the proposed hydrogen bond is not formed. Fluorescence spectroscopy studies using an engineered F222W mutant show that the C-terminus remains delocalized in the absence of glutathione or when only the glutathione binding site is occupied, but is ordered and localized in the presence of substrate or conjugate, consistent with these and previous crystallographic studies. Proteins 2001;42:192-200.

Animals↗
PubMed2001

Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel.

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

Animals↗
PubMed2000

Tissue engineering meniscus: cells and matrix.

Tissue engineering, a relatively new field of research, was born as a result of a search for solutions to the limited alternatives currently used to replace structure and function lost because of failure or loss of tissues throughout the body. The field merges advances in cell biology, growth factor therapy, and material design. It encourages creation of tissue-engineered menisci for repair and replacement therapy and offers a means for developing a better understanding of meniscal cell and matrix biology.

Animals↗
PubMed2000

Meniscal injury and repair: clinical status.

Repair or resection of meniscal injuries is one of the most common operative procedures in orthopedics today. A variety of techniques for reconstruction have been attempted and experts are still unsure which treatment of meniscal lesions is best. This article reviews different techniques of meniscal repair and some novel approaches that may be used for treatment of meniscal lesions in the coming years.

Animals↗
PubMed2000

Tissue-engineered ligament: cells, matrix, and growth factors.

Current treatment modalities for anterior cruciate ligament (ACL) tears rely on the use of grafts for reconstruction. Treatment can be divided into three categories: autografts, allografts, and synthetic graft replacements. The varied success rates and associated advantages and disadvantages of each method have resulted in controversy as to the best treatment for ACL injuries.

Animals↗
PubMed2000

Effect of Shiga toxin 2 on water and ion transport in human colon in vitro.

Shiga toxin-producing Escherichia coli (STEC) colonize the lower segments of the human gastrointestinal tract, causing gastrointestinal and systemic diseases. In this study, the effects of Shiga toxin 2 (Stx2) on fluid absorption and ion transport in the human colon were examined. Net water movement (Jw) and short-circuit current (Isc) were simultaneously measured across the colonic mucosa incubated with crude or purified Stx2. Stx2 significantly inhibited the absorptive J(w) with no effect on the basal I(sc) after 60 min of exposure. These effects may be due to the inhibition of a nonelectrogenic transport system present in the surface colonic villus cells. Morphological studies of the colonic mucosa treated with crude or purified Stx2 demonstrated a selective damage in the absorptive villus epithelial cells. These findings suggest that Stx2 inhibits water absorption across the human colon by acting on a specific cell population: the mature, differentiated absorptive villus epithelium.

Bacterial Toxins↗
PubMed1999

Diarrheagenicity evaluation of attenuated Vibrio cholerae O1 and O139 strains in the human intestine ex vivo.

The recent spread of El Tor cholera in Latin America highlights the need for a safe and economical vaccine. The main approach for developing live recombinant vaccines has been to disarm known pathogenic strains of cholera toxin leaving intact antigens involved in protection. These recombinant vaccine candidates do not cause severe diarrhea, but they are too reactogenic for wide scale usage. We describe here a test capable of determining the diarrheagenic potential of attenuated V. cholerae strains. The functional test consists in the simultaneous recording of net water movement, electrical potential difference and short-circuit current across the human intestine ex vivo. We found that human tissues incubated with supernatants from the attenuated 638, 413 and 251a V. cholerae strains caused no changes in the ion conductances and water absorption in ileal and colon tissues allowing them to be assayed in volunteers.

Animals↗