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M Iriarte

Publications and source records attributed to M Iriarte.

At least 19 recordsLinked to original sources
PubMed2002

Polymer-solvent interaction parameters in polymer solutions at high polymer concentrations.

Molecular mass and temperature dependences of the polymer-solvent interaction parameter have been investigated in the extreme interval of high polymer concentration using inverse gas chromatography (IGC). The observed molecular mass dependence has been compared with the predictions of a theoretical model, which emphasises the role of intramolecular contacts in the polymer chain. The model reproduces reasonably well the molecular mass dependence. However, enthalpic and entropic contributions of the interaction parameter, obtained from IGC measurements at different temperatures, exhibit behaviours difficult to explain in the framework of the current models.

Chromatography, Gas↗
PubMed2000

Effects of diabetic cardiomyopathy on regional electrophysiologic characteristics of rat ventricle.

AIMS/HYPOTHESIS: To identify the possible causes of the lengthening of the action potential duration described in patients affected by diabetes mellitus. METHODS: We studied the effects of streptozotocin-induced diabetes on the current density of the repolarising potassium currents It(o), IK, Iss and IK1 in enzymatically isolated myocytes from three different regions of rat heart: total right ventricle, subepicardium at the apex of the left ventricle and subendocardium at the base of the left ventricle. RESULTS: No changes in IK1 were found due to diabetes, but there was a uniform decrease in It(o) (50%) and Iss (40%) current densities in the three regions. In contrast, IK diminished unevenly, with the greatest decrease in the subendocardium at the base of the left ventricle (48%), followed by the subepicardium at the apex of the left ventricle (32%) and right ventricle (10%). CONCLUSION/INTERPRETATION: These findings suggest the existence of regional differences in ion channel expression associated with diabetes. The decrease of these repolarising currents could account for the lengthening of action potential and the consequent change in the Q-T interval of the ECG observed in diabetic rats.

4-Aminopyridine↗
PubMed2000

Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins.

Y. enterocolitica translocates virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Here we investigated whether Y. enterocolitica could translocate Yops into a range of eukaryotic cells including neurons and insect cells. Y. enterocolitica translocated the hybrid reporter protein YopE-Cya into each of the eukaryotic cell types tested. In addition, Y. enterocolitica was cytotoxic for each of the adherent cell types. Thus we detected no limit to the range of eukaryotic cells into which Y. enterocolitica can translocate Yops. The Yop effectors YopE, YopH and YopT were each cytotoxic for the adherent cell types tested, showing that not only is Y. enterocolitica not selective in its translocation of particular Yop effectors into each cell type, but also that the action of these Yop effectors is not cell type specific. Invasin and/or YadA, two powerful adhesins were required for translocation of Yop into non-phagocytic cells but not for translocation into macrophages. To use the Yersinia translocation system for broad applications, a Y. enterocolitica translocation strain and vector for the delivery of heterologous proteins into eukaryotic cells was constructed. This strain + vector combination lacks the translocated Yop effectors and allows delivery into eukaryotic cells of heterologous proteins fused to the minimal N-terminal secretion/translocation signal of YopE. Using this strategy translocation of a YopE-Diphtheria toxin subunit A hybrid protein into several cell types has been shown.

Adhesins, Bacterial↗
PubMed1998

TyeA, a protein involved in control of Yop release and in translocation of Yersinia Yop effectors.

Extracellular Yersinia spp. disarm the immune system by injecting the effector Yersinia outer proteins (Yops) into the target cell. Yop secretion is triggered by contact with eukaryotic cells or by Ca2+ chelation. Two proteins, YopN and LcrG, are known to be involved in Yop-secretion control. Here we describe TyeA, a third protein involved in the control of Yop release. Like YopN, TyeA is localized at the bacterial surface. A tyeA knock-out mutant secreted Yops in the presence of Ca2+ and in the absence of eukaryotic cells. Unlike a yopN null mutant, the tyeA mutant was defective for translocation of YopE and YopH, but not YopM, YopO and YopP, into eukaryotic cells. This is the first observation suggesting that Yop effectors can be divided into two sets for delivery into eukaryotic cells. TyeA was found to interact with the translocator YopD and with residues 242-293 of YopN. In contrast with a yopN null mutant, a yopNDelta248-272 mutant was also unable to translocate YopE and YopH. Our results suggest that TyeA forms part of the translocation-control apparatus together with YopD and YopN, and that the interaction of these proteins is required for selective translocation of Yops inside eukaryotic cells.

Apoptosis↗
PubMed1998

Differences in regional distribution of K+ current densities in rat ventricle.

The objective of the present work is to study the ionic mechanisms for the regional differences in action potential duration in rat ventricle. This regional diversity has been related to differences in the regional distribution of some potassium currents in several species. Single cells were obtained by enzymatic dispersion of tissue segments from rat ventricular muscle. Whole cell voltage-clamp methods were used to identify the K+ currents involved in action potential repolarisation in the different regions. 4-Aminopiridine, TEA and voltage protocols were used to isolate the following potassium currents: transient outward, Ito, delayed rectifier, Ik, and sustained current, Iss. In the present work, we have studied the distribution of these three repolarising currents, and that of the inward rectifier, Ikl, in the free wall of the right ventricle, the subepicardium of the apex of the left ventricle and in the subendocardium of the base of the left ventricle. Action potential duration was longer in the left than in the right ventricle, and in the former it was longer in the subendocardium of the base than in the subepicardium of the apex. The main difference was in the phase 1, suggesting the implication of Ito. This was confirmed with voltage-clamp experiments. In conclusion, this work shows that Ito current density is higher in the regions with the shorter action potential, whereas there are no differences in the regional distribution of Ik, Iss or Ikl.

4-Aminopyridine↗
PubMed1998

Heparin interferes with translocation of Yop proteins into HeLa cells and binds to LcrG, a regulatory component of the Yersinia Yop apparatus.

Yersiniae are equipped with the Yop virulon, an apparatus that allows extracellular bacteria to deliver toxic Yop proteins inside the host cell cytosol in order to sabotage the communication networks of the host cell or even to cause cell death. LcrG is a component of the Yop virulon involved in the regulation of secretion of the Yops. In this paper, we show that LcrG can bind HeLa cells, and we analyse the role of proteoglycans in this phenomenon. Treatment of the HeLa cells with heparinase I, but not chondroitinase ABC, led to inhibition of binding. Competition assays indicated that heparin and dextran sulphate strongly inhibited binding, but that other glycosaminoglycans did not. This demonstrated that binding of HeLa cells to purified LcrG is caused by heparan sulphate proteoglycans. LcrG could bind directly to heparin-agarose beads and, in agreement with these results, analysis of the protein sequence of Yersinia enterocolitica LcrG revealed heparin-binding motifs. In vitro production and secretion by Y. enterocolitica of the Yops was unaffected by the addition of heparin. However, the addition of exogenous heparin decreased the level of YopE-Cya translocation into HeLa cells. A similar decrease was seen with dextran sulphate, whereas the other glycosaminoglycans tested had no significant effect. Translocation was also decreased by treatment of HeLa cells with heparinitase, but not with chondroitinase. Thus, heparan sulphate proteoglycans have an important role to play in translocation. The interaction between LcrG and heparan sulphate anchored at the surface of HeLa cells could be a signal triggering deployment of the Yop translocation machinery. This is the first report of a eukaryotic receptor interacting with the type III secretion and associated translocation machinery of Yersinia or of other bacteria.

Adenylyl Cyclases↗
PubMed1998

YopT, a new Yersinia Yop effector protein, affects the cytoskeleton of host cells.

Extracellular Yersinia disarm the immune system of their host by injecting effector Yop proteins into the cytosol of target cells. Five effectors have been described: YopE, YopH, YpkA/YopO, YopP and YopM. Delivery of these effectors by Yersinia adhering at the cell surface requires other Yops (translocators) including YopB. Effector and translocator Yops are secreted by the type III Ysc secretion apparatus, and some Yops also need a specific cytosolic chaperone, called Syc. In this paper, we describe a new Yop, which we have called YopT (35.5kDa). Its secretion required an intact Ysc apparatus and SycT (15.0kDa, pl4.4), a new chaperone resembling SycE. Infection of macrophages with a Yersinia, producing a hybrid YopT-adenylate cyclase, led to the accumulation of intracellular cAMP, indicating that YopT is delivered into the cytosol of eukaryotic cells. Infection of HeLa cells with a mutant strain devoid of the five known Yop effectors (deltaHOPEM strain) but producing YopT resulted in the alteration of the cell cytoskeleton and the disruption of the actin filament structure. This cytotoxic effect was caused by YopT and dependent on YopB. YopT is thus a new effector Yop and a new bacterial toxin affecting the cytoskeleton of eukaryotic cells.

Amino Acid Sequence↗
PubMed1998

LcrG is required for efficient translocation of Yersinia Yop effector proteins into eukaryotic cells.

Extracellular Yersinia disables the immune system of its host by injecting effector Yop proteins into host cells. We show that a Yersinia enterocolitica nonpolar lcrG mutant is severely impaired in the translocation of YopE, YopH, YopM, YpkA/YopO, and YopP into eukaryotic cells. LcrG is thus required for efficient internalization of all the known Yop effectors.

Bacterial Outer Membrane Proteins↗
PubMed1998

The virulence plasmid of Yersinia, an antihost genome.

The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds.

Animals↗
PubMed1997

YscM1 and YscM2, two Yersinia enterocolitica proteins causing downregulation of yop transcription.

Synthesis of the Yop proteins by yersiniae is downregulated when secretion is prevented by closure or destruction of the contact (type III) secretion channel. In Yersinia pseudotuberculosis, a mutation in the IcrQ gene, encoding a secreted protein, reduces this feedback inhibition mechanism. Surprisingly, mutation in the yscM gene, the IcrQ homologue in Y. enterocolitica, does not lead to the same deregulated phenotype. In this paper, we addressed the question of this discrepancy. We found a new gene on the Y. enterocolitica pYV plasmid that encodes a protein with 57% identity to YscM (now called YscM1). Overexpression of this gene, called yscM2, like overexpression of IcrQ and yscM1, blocked Yop secretion. A double yscM1, yscM2 mutant had the same phenotype as that of the IcrQ mutant. The discrepancy can thus be explained by the existence of two functionally equivalent copies of yscM in Y. enterocolitica. Overexpression of yscM1 drastically reduced the expression of a yopH-cat reporter gene when tested in a pYV+ background. However, no effect could be observed in the absence of a pYV plasmid, indicating that YscM1 does not act directly as a transcriptional repressor or as an anti-VirF factor. We have also ruled out that YscM acts by obstructing the secretion channel.

Bacterial Outer Membrane Proteins↗
PubMed1997

Virulence and arsenic resistance in Yersiniae.

The genus Yersinia contains three pathogenic species: Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica. Only a few biotypes and serotypes of Y. enterocolitica are pathogenic, and these form two distinct groups: some are of low virulence, and they are encountered worldwide; others, mainly encountered in North America, are markedly more virulent. All pathogenic yersiniae possess a 70-kb virulence plasmid called pYV which encodes secreted antihost proteins called Yops as well as a type III secretion machinery that is required for Yop secretion. Genes encoding Yop synthesis and secretion are tightly clustered in three quadrants of the pYV plasmid. We show here that in the low-virulence strains of Y. enterocolitica, the fourth quadrant of the plasmid contains a new class II transposon, Tn2502. This transposon encodes a defective transposase, but transposition can be complemented in trans by Tn2501, another class II transposon. Tn2502 was not detected in the pYV plasmids of the more virulent American strains of Y. enterocolitica, of Y. pseudotuberculosis, and of Y. pestis. Tn2502 confers arsenite and arsenate resistance. This resistance involves four genes; three are homologous to the arsRBC genes present on the Escherichia coli chromosome, but no homolog of the fourth one, arsH, has been found. The systematic presence of such a resistance operon on a virulence plasmid is unusual and could be related to the recent spread of low-virulence Y. enterocolitica strains. The presence of this ars operon also constitutes the first significant difference between the pYV plasmids from different Yersinia species.

Amino Acid Sequence↗
PubMed1996

Status of YopM and YopN in the Yersinia Yop virulon: YopM of Y.enterocolitica is internalized inside the cytosol of PU5-1.8 macrophages by the YopB, D, N delivery apparatus.

The Yersinia Yop virulon is an anti-host system made up of four elements: (i) a type III secretion system called Ysc; (ii) a system designed to deliver bacterial proteins into eukaryotic target cells (YopB, YopD); (iii) a control element (YopN); and (iv) a set of intracellularly delivered proteins designed to disarm these cells or disrupt their communications (YopE, YopH and possibly others). YopM, another Yop protein, binds thrombin and is thus presumed to act as an extracellular effector. Here, we analyzed YopM from Y.enterocolitica and we wondered whether it could also be delivered inside eukaryotic cells. To answer this question we applied the Yop-Cya reporter strategy. Hybrids made of 141 or 100 N-terminal residues of YopM fused to Cya were delivered inside PU5-1.8 macrophages by recombinant Y.enterocolitica strains. YopB and YopD were required as translocators. Leakage of the reporters into the macrophage culture supernatant during the bacterial infection increased strongly when YopN was missing, showing that YopN is involved in the control of delivery of YopM inside eukaryotic cells. YopN itself was not delivered into the macrophages. In conclusion, YopM is translocated inside the eukaryotic cells and its physiopathological role should be revised or completed.

Adenylate Cyclase Toxin↗
PubMed1996

Molecular determinants of Yersinia pathogenesis.

The genus Yersinia contains three pathogenic species: Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica. Even though the three species use different routes to infect their host and provoke diseases of different intensity, they share a common tropism for the lymphoid tissue and they are able to resist the primary immune response of the host. The main genetic determinants involved in this resistance are encoded by a highly conserved 70-kb virulence plasmid. The genes harbored by the pYV plasmid encode the lipoprotein YlpA, the outer membrane protein YadA, and a group of at least 11 secreted proteins called Yops. The pYV plasmid also encodes the apparatus necessary for the secretion of the Yop proteins, as well as those involved in the regulation of Yop synthesis. The Yop proteins are secreted by a specific secretion system which is considered as the archetype of a new secretion pathway called type III. After their secretion they are immediately internalized into the cytosol of a target eukaryotic cell, which represents a new phenomenon in microbial pathogenesis. The chromosome of Y. enterocolitica completes the virulence panoply of the bacteria by encoding an enterotoxin called Yst, fibrillae named Myf and an invasin called Inv.

Adhesins, Bacterial↗
PubMed1995

Microvascular angina pectoris in hypertensive patients with left ventricular hypertrophy and diagnostic value of exercise thallium-201 scintigraphy.

In a series of 120 hypertensive patients, 60 were found to have echocardiographic left ventricular (LV) hypertrophy (Devereux's method). Of these, 18 (30%) had typical stress-induced angina and underwent coronary angiography, which showed that 11 (61%) had normal coronary arteries, and 7 (39%) (p < 0.05) had coronary stenosis of the epicardial arteries. Stress-rest thallium-201 scintigraphy (Burow's quantitative method) yielded abnormal results in 21 of the 60 patients with LV hypertrophy. Five of 30 (17%) were asymptomatic, 14 of 18 (78%) had angina, and 2 of 12 (17%) had dyspnea on exertion. In 5 normal patients used as a control group, coronary flow reserve after administration of papaverine (10 coronary arteries) was 6.25 +/- 1.4 versus 3.7 +/- 0.8 in 10 thallium-negative, asymptomatic hypertensive patients with LV hypertrophy (p < 0.001). The mean coronary flow reserve of 21 patients with abnormal thallium-201 results was 2.71 +/- 0.96 (p < 0.01 compared with the group with normal thallium-201 findings) and 2.5 +/- 0.6 in the segments with lowest uptake (p < 0.05 compared with normal segments in these same patients). Thus, stress-induced angina pectoris in hypertensive patients with LV hypertrophy was due to small-vessel disease in over half of our patients (62%).

Adult↗
PubMed1995

The rpoS gene from Yersinia enterocolitica and its influence on expression of virulence factors.

The chromosome of Yersinia enterocolitica encodes a heat-stable enterotoxin called Yst and a surface antigen called Myf, which closely resembles enterotoxin-associated fimbriae. Both factors could act in conjunction to produce diarrhea. Production of the enterotoxin is regulated by temperature, osmolarity, and pH and occurs only when bacteria reach the stationary phase. Myf production is regulated by temperature and pH and, as we show in this work, also occurs after the exponential growth phase. In an attempt to understand the late-phase expression of yst and myf, we cloned, sequenced, and mutagenized the gene encoding RpoS, an alternative sigma factor of the RNA polymerase involved in expression of stationary-phase genes in other enterobacteria. An intact rpoS gene was necessary for full expression of yst in the stationary phase but not for the expression of myf and of pYV-encoded virulence determinants.

Amino Acid Sequence↗
PubMed1995

MyfF, an element of the network regulating the synthesis of fibrillae in Yersinia enterocolitica.

The Yersinia enterocolitica surface antigen Myf is a fibrillar structure that resembles CS3 fimbriae. Gene myfA encodes the 21-kDa major subunit of the antigen, while genes myfB and myfC are required for the transport and assembly of pilin subunits at the bacterial cell surface. Here we show that the expression of Myf is regulated at the transcriptional level by temperature and pH. Gene myfA is transcribed at 37 degrees C and in acidic medium. The transcription start is preceded by a putative -10 box for the vegetative RNA polymerase as well as by sequences resembling the consensus sequence recognized by sigma 28. Thus, myfA could be transcribed either from a classical sigma 70 promoter or from a sigma 28 promoter. Transcription of myfA requires at least two genes, myfF and myfE, situated immediately upstream from myfA. The myfF product does not show similarity to any known regulatory protein. It is an 18.5-kDa protein with no typical helix-turn-helix motif and a unique hydrophobic domain in the NH2-terminal part. T7 expression, osmotic shock, fractionation experiments, and TnphoA fusion analyses carried out in Escherichia coli suggest that MyfF is associated with the inner membrane by means of its hydrophobic domain whereas the hydrophilic part protrudes in the periplasm. These features strikingly evoke ToxS, a protein involved in regulation of Tcp pilus production in Vibrio cholerae. MyfE resembles PsaE, a protein involved in regulation of pH6 antigen in Yersinia pestis. Genes myfF and myfE are presumably part of a whole regulatory network. MyfF could be an element of the signal transducing system.

Amino Acid Sequence↗