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Biomedical subjects

E Hidalgo

Publications and source records attributed to E Hidalgo.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cross-clamping of the three hepatic veins in the piggyback technique is a safe and well tolerated procedure.

A common stump of the three hepatic veins has always been used to fashion the upper vena cava anastomosis in 205 liver transplantations with the piggyback (PGB) technique performed in our Unit, to avoid outflow problems. The aim was to study the repercussion of lateral inferior vena cava (IVC) clamping on IVC flow and pressure as well as on systemic hemodynamics. We have studied 42 orthotopic liver transplantations performed with the PGB technique. Intraoperative IVC blood flow measurements by transit time ultrasonic volume flowmetry, IVC pressure, and systemic hemodynamics were taken before and after lateral IVC clamping. Graft outflow complications, stenosis or kinking of the upper vena cava anastomosis have not been found in any of the 205 PGB procedures. A significant decrease of IVC flow (23%) and cardiac output (12%) occurred after IVC clamping, whereas mean arterial and central venous pressures were not altered significantly, probably due to an increase (25%) of systemic vascular resistance. Only in one case was an almost total clamping of IVC needed. Venovenous bypass was not needed in any case. Renal perfusion pressure was adequate in all cases. We conclude that the use of a common stump of the three hepatic veins for upper vena cava anastomosis in the PGB technique is safe because any outflow problem of the graft is avoided and, at the same time, is well tolerated hemodynamically because most of the IVC flow is preserved.

Constriction↗
PubMed1998

Biliary complications secondary to late hepatic artery thrombosis in adult liver transplant patients.

Biliary complications (BC) are the usual presentation of late hepatic artery thrombosis (HAT) of the liver graft. Our aim was to study the clinical features and outcome of BC secondary to HAT compared to BC which occurred in liver transplant (LT) patients with patent vessels. We present a retrospective study of 224 LTs performed in 204 patients between 1988 and 1996. The mean recipient x s age was 51 years. A choledochocholedochostomy without T-tube was used as biliary reconstruction in most cases (67%); in 12%, a choledochojejunostomy was performed. An iliac conduit was necessary in 15% of cases and back-table arterial reconstruction was performed in 10% of cases of anatomic variants in graft arteries. Different donor, recipient and intraoperative variables, as well as treatment and outcome. were studied in the two groups of patients presenting BC with or without HAT. BC occurred in 38 cases (17%) whereas HAT was diagnosed in 11 cases (4.9%). Therefore, 23% of BC encountered after LT were secondary to HAT. Nine cases of late HAT manifested as BC, septicaemia (88%) and hepatic bilomas (8 cases). Percutaneous or surgical drainage of hepatic bilomas was performed in all cases, followed by retransplantation in six cases (66%). BC secondary to HAT appeared later than the rest of BC. Donor age was the only significant predisposing factor found in our study. Graft survival is significantly reduced as most patients needed retransplantation. In conclusion, BC secondary to HAT presented later in livers from older donors in the form of biliary sepsis and hepatic biloma. Retransplantation was ultimately required in most cases and graft survival was significantly diminished.

Bile Duct Diseases↗
PubMed1998

Early use of tacrolimus as rescue therapy for refractory liver allograft rejection.

The aim of this study was to compare two different periods of tacrolimus rescue therapy for intractable rejection. From January 1992 to May 1996, 140 liver transplants (LTx) were performed in our hospital under cyclosporine A-based immunosuppression. Twenty-four (17.1%) patients were switched to tacrolimus because of chronic rejection, steroid-resistant rejection or cholestasic hepatitis C recurrence. Mean follow-up was 21 months (range 12-56 months). In the first period (January 1992-March 1994), conversion to tacrolimus was indicated later, after unsuccessful repeated rejection therapy. In the second period (April 1994-May 1996), conversion to tacrolimus was indicated early, immediately after unsuccessful rejection therapy or directly at the moment of diagnosis with no further treatment. Eleven of 54 LTx were treated with tacrolimus in period 1 (20.3%), and 13 of 86 LTx in period 2 (15.1%). Only 4 of 11 (36.6%) grafts converted were rescued during the first period, while 11 of 13 (84.6%) were rescued in the second (P < 0.03). Patients in the first period received more courses of steroids than those of the second (1.7 +/- 0.7 vs 0.9 +/- 0.7, P < 0.02). Furthermore, six patients received one or two courses of OKT3 in period 1 while only one received one course in period 2 (P < 0.03). Preconversion mean bilirubin levels of patients in the first period were higher than those in the second (15.9 +/- 7.3 mg/dl vs 9.7 +/- 5.8 mg/dl, P < 0.05). Preconversion mean bilirubin levels of 6.8 +/- 5.4 mg/dl and 21.8 +/- 18.5 mg/dl were observed in patients with successful and unsuccessful tacrolimus rescue therapy, respectively, independent of the treatment period (P < 0.05). Mortality rates were higher in the first period than in the second (82% versus 23%; P < 0.02). In conclusion, conversion to tacrolimus as rescue therapy for intractable rejection or cholestasic hepatitis C recurrence is an efficacious alternative, particularly when tacrolimus is initiated early.

Graft Rejection↗
PubMed1998

Risk factors for acute renal failure requiring dialysis after liver transplantation.

Acute renal failure (ARF) is a common and severe complication after liver transplantation (LT). The aim of this study was to ascertain the impact of ARF requiring dialysis in the outcome of LT and to analyze the risk factors leading to this event in the early post-operative period. From October 1988 to December 1994, 172 LT were performed in 158 patients. Postoperative ARF occurred in 88 transplants (51.1%) during the early postoperative period: mild ARF was found in 46 (serum creatinine 1.5-3 mg/dl), moderate ARF in 12 (serum creatinine > 3 mg/dl) and severe ARF in 30 (serum creatinine > 3 mg/dl with dialysis requirement). Preoperative, intraoperative, and postoperative variables were studied, comparing patients presenting severe ARF with the remaining patients. Postoperative mortality in the dialysed group was much higher than in the non-dialysis group (50% vs. 13.4%)(p < 0.001) and 1-yr actuarial graft survival was 73.4% for the non-dialysed group compared with 40.9% for the dialysed group (p < 0.05). Among 38 variables investigated, only two factors had independent prognostic value in multivariate analysis: preoperative serum creatinine > 1.5 mg/dl (OR = 4.4, p = 0.006) and graft dysfunction grades III-IV (OR = 8.9, p = 0.001). In conclusion, ARF is a severe complication post-LT; its appearance could be predicted in patients with pre-transplant renal dysfunction, severe graft dysfunction, or both. However, in many cases renal function may revert to normal if treated aggressively with early dialysis support.

Acute Kidney Injury↗
PubMed1997

Cysteine-to-alanine replacements in the Escherichia coli SoxR protein and the role of the [2Fe-2S] centers in transcriptional activation.

The Escherichia coli soxRS regulon activates oxidative stress and antibiotic resistance genes in two transcriptional stages. SoxR protein becomes activated in cells exposed to excess superoxide or nitric oxide and then stimulates transcription of the soxS gene, whose product in turn activates>/=10 regulon promoters. Purified SoxR protein is a homodimer containing a pair of [2Fe-2S] centers essential for soxS transcription in vitro . The [2Fe-2S] centers are thought to be anchored by a C-terminal cluster of four cysteine residues in SoxR. Here we analyze mutant SoxR derivatives with individual cysteines replaced by alanine residues (Cys-->Ala). The mutant proteins in cell-free extracts bound the soxS promoter with wild-type affinity, but upon purification lacked Fe or detectable transcriptional activity for soxS in vitro . Electron paramagnetic resonance measurements in vivo indicated that the Cys-->Ala proteins lacked the [2Fe-2S] centers seen for wild-type SoxR. The Cys-->Ala mutant proteins failed to activate soxS expression in vivo in response to paraquat, a superoxide- generating agent. However, when expressed to approximately 5% of the cell protein, the Cys-->Ala derivatives increased basal soxS transcription 2-4-fold. Overexpression of the Cys119-->Ala mutant protein strongly interfered with soxS activation by wild-type SoxR in response to paraquat. These studies demonstrate the essential role of the [2Fe-2S] centers for SoxR activation in vivo ; the data may also indicate oxidant-independent mechanisms of transcriptional activation by SoxR.

Alanine↗
PubMed1997

Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor.

SoxR protein of Escherichia coli governs a global response against superoxide-generating agents (such as paraquat) or nitric oxide, and provides broad antibiotic resistance. A redox signal activates SoxR post-translationally to trigger transcription of a second regulatory gene, soxS. Activated and non-activated SoxR bind the soxS promoter with the same high affinity, but only the activated protein stimulates soxS transcription. SoxR acts by an unusual mechanism of positive control: the protein binds the soxS promoter between near-consensus -10 and -35 elements that are separated by an unusually long 19 bp (versus the optimal 17 bp). We have constructed and analyzed site-specific deletions that alter the promoter element spacing. Reducing the spacer length to 16-18 bp dramatically elevated basal expression of soxS in vivo and in vitro, and nearly eliminated additional activation by SoxR in response to paraquat. More strikingly, shortening the spacer converted SoxR from an activator into a repressor regardless of paraquat treatment. Gel mobility-shift assays show that repression by SoxR of the promoters with 17 and 16 bp spacers is due to interference with binding by RNA polymerase. Thus, activated SoxR remodels the unusual configuration of the wild-type soxS promoter into a highly active form, probably by compensating for the suboptimal distance between the -10 and the -35 elements.

Bacterial Proteins↗
PubMed1997

Redox signal transduction: mutations shifting [2Fe-2S] centers of the SoxR sensor-regulator to the oxidized form.

SoxR is a [2Fe-2S] transcription factor triggered by oxidative stress and activated in vitro by one-electron oxidation or assembly of the iron-sulfur centers. To distinguish which mechanism operates in cells, we studied constitutively active SoxR (SoxRc) proteins. Three SoxRc proteins contained [2Fe-2S] centers required for in vitro transcription and, like wild-type SoxR, were inactivated by chemical reduction. However, in vivo spectroscopy showed that even without oxidative stress, the three SoxRc proteins failed to accumulate with reduced [2Fe-2S] (< or = 4% compared to > or = 40% for wild type). One SoxRc protein had a redox potential 65 mV lower than wild type, consistent with its accumulation in the oxidized (activated) form in vivo. These results link in vitro and in vivo approaches showing novel redox regulation that couples an iron-sulfur oxidation state to promoter activation.

Aerobiosis↗
PubMed1997

Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator.

Protein iron-sulfur (FeS) centers have recently been implicated in the regulation of gene expression. In the redox-sensing SoxR protein, the oxidation state of [2Fe-2S] centers controls its activity as a transcription activator independent of DNA-binding ability. Thus, FeS centers allosterically link cellular oxidative stress to the expression of defense genes.

Bacterial Proteins↗
PubMed1996

The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor.

SoxR protein is a redox-responsive transcription factor that governs a regulon of oxidative stress and antibiotic resistance genes in Escherichia coli. Purified SoxR contains oxidized [2Fe-2S] clusters and stimulates in vitro transcription of its target gene soxS up to 100-fold. SoxR transcriptional activity, but not DNA binding, is completely dependent on the [2Fe-2S] clusters; apo-SoxR prepared in vitro binds the soxS promoter with unchanged affinity but does not have transcription activity. Thus, modulation of the SoxR [2Fe-2S] clusters was proposed to control the protein's function in transcription. Here, we provide evidence that SoxR with reduced [2Fe-2S] clusters is inactive. Redox titration of purified SoxR revealed a midpoint potential of -285 +/- 10 mV (pH 7.6). In vitro transcription assays showed that SoxR was inactivated when the [2Fe-2S] cluster was reduced (-380 mV), and full activity was restored upon reoxidation (+100 mV). The results suggest that one-electron oxidation and reduction of the [2Fe-2S] cluster regulate SoxR transcriptional activity.

Anaerobiosis↗
PubMed1996

Activation of SoxR-dependent transcription in vitro by noncatalytic or NifS-mediated assembly of [2Fe-2S] clusters into apo-SoxR.

SoxR is a transcriptional activator that senses superoxide and nitric oxide stress in Escherichia coli. The active protein isolated from E. coli contains a pair of [2Fe-2S] clusters per SoxR dimer. We previously demonstrated that the iron-free protein (apo-SoxR), isolated during purification in thiol-containing buffers, binds soxS promoter DNA with an affinity equal to that of the metalloprotein (Fe-SoxR), but lacks significant ability to activate transcription in vitro. Here we demonstrate the reversibility of this process: the full transcriptional activity of SoxR can be restored by in vitro assembly of iron-sulfur clusters into the apoprotein. Two methods were used to synthesize the metallocenters of SoxR: (i) nonenzymatic, in which apo-SoxR, incubated in the presence of iron, inorganic sulfide, and a reducing agent, regained full transcriptional activity in 5-6 h; (ii) enzymatic, in which NifS protein of Azotobacter vinelandii regenerated active Fe-SoxR in as little as 2 min. Analysis by electron paramagnetic resonance spectroscopy indicated that binuclear [2Fe-2S] clusters were restored by both the enzymatic and nonenzymatic reconstitutions. A mutant SoxR protein missing one of its four cysteine residues failed to undergo either transcriptional activation or the formation of [2Fe-2S] centers, even in the presence of NifS. Thus, only the presence of an iron-sulfur center is required to restore transcriptional activity to apo-SoxR. Moreover, the catalytic generation of [2Fe-2S] centers extends the known specificity of this enzyme beyond that already shown for [4Fe-4S] centers. Catalytic generation of [2Fe-2S]-containing SoxR could allow for rapid activation of this transcription factor in vivo.

Apoproteins↗
PubMed1996

Errors evolution and analysis in antineoplastic drug preparation during one year.

We analyzed the errors occurring in the preparation circuit of cytotoxic mixtures of the Centralized Cytotoxic Preparation Unit during one year. Analysis of their evolution meant the investigation of twenty parameters susceptible to error. Each parameter was considered one error opportunity. Error has been defined either by the lack of data or mistake in the controlled parameter. In 4,734 preparations (94,680 parameters) there were 314 errors. The percentage of error per parameter in the first month of study was 0.74; at sixth was 0.34 and the last month was 0.26. Only in four months the day of maximum number of preparations coincided with the day of maximum number of errors. We conclude that the percentage of errors in the preparation process is low with a tendency to decrease and that the number of daily preparations is not the single factor that influences the production of errors.

Antineoplastic Agents↗
PubMed1996

Anesthetic efficacy of eutectic prilocaine-lidocaine cream in pediatric oncology patients undergoing lumbar puncture.

OBJECTIVE: To evaluate the efficacy of eutectic mixture of local anesthetics 5% (Emla) in reducing pain associated with lumbar punctures in children. DESIGN: Prospective, double-blind, randomized, placebo-controlled trial. SETTING: University pediatric hospital. PATIENTS: Eleven pediatric oncology patients (mean age 6.6 y, range 4-16) who underwent 31 lumbar punctures. MAIN OUTCOME MEASURES: The analgesic effect was measured by using two methods. The first was a 10-point visual analog scale reported by the patient and the second was an 8-point behavioral pain scale assessed by the nurse who applied the cream. RESULTS: Emla cream was associated with significantly lower pain scores than those with placebo as measured by the patient when the puncture was successful on the first attempt (2.0 +/- 1.6 Emla group, 3.8 +/- 1.9 placebo group; p < 0.05). CONCLUSIONS: The use of Emla cream may reduce pain substantially only in patients who undergo a successful lumbar puncture on the first attempt.

Adolescent↗
PubMed1996

A second Escherichia coli gene with similarity to gapA.

An open reading frame has been found downstream of the ald gene at 31 min in the Escherichia coli chromosome and has been designated gapC because of its high similarity with gapA (min 39, encoding glyceraldehyde-3-phosphate dehydrogenase), and with gapB (min 62, a gene with high similarity to gapA, encoding erythrose-4-phosphate dehydrogenase). The gapC gene (min 31) encodes a polypeptide of 204 amino acids, 126 residues shorter than glyceraldehyde-3-phosphate dehydrogenase. In this 204-codon open reading frame several amino acids important for catalysis are conserved. However, the cofactor binding site is lost. The results illustrate a case of a gene, encoding a glycolytic enzyme, for which at least three copies maintaining a certain degree of similarity are apparent in the E. coli genome. It seems likely that the genes encode products with different cellular functions. The origin of these three copies of the gap gene by horizontal transfer or by duplication of an ancestral gene is discussed.

Amino Acid Sequence↗
PubMed1995

Binuclear [2Fe-2S] clusters in the Escherichia coli SoxR protein and role of the metal centers in transcription.

SoxR protein of Escherichia coli is activated by superoxide-generating agents or nitric oxide as a powerful transcription activator of the soxS gene, whose product activates approximately 10 other promoters. SoxR contains non-heme iron essential for abortive initiation of transcription in vitro. Here we show that this metal dependence extends to full-length transcription in vitro. In the presence of E. coli sigma 70 RNA polymerase, iron-containing SoxR mediates open complex formation at the soxS promoter, as determined using footprinting with Cu-5-phenyl-1,10-phenanthroline. We investigated the nature of the SoxR iron center by chemical analyses and electron paramagnetic resonance spectroscopy. Dithionite-reduced Fe-SoxR exhibited an almost axial paramagnetic signature with g values of 2.01 and 1.93 observable up to 100 K. These features, together with quantitation of spin, iron, and S2-, and hydrodynamic evidence that SoxR is a homodimer in solution, indicate that (SoxR)2 contains two [2Fe-2S] clusters. Treatment of Fe-SoxR with high concentrations of dithiothreitol caused subtle changes in the visible absorption spectrum and blocked transcriptional activity without generating reduced [2Fe-2S] centers, but was also associated with the loss of iron from the protein. However, lowering the thiol concentration by dilution allowed spontaneous regeneration of active Fe-SoxR.

Bacterial Proteins↗
PubMed1994

An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein.

The soxRS oxidative stress regulon of Escherichia coli is triggered by superoxide (O2.-) generating agents or by nitric oxide through two consecutive steps of gene activation. SoxR protein has been proposed as the redox sensing gene activator that triggers this cascade of gene expression. We have now characterized two forms of SoxR: Fe-SoxR contained non-heme iron (up to 1.6 atoms per monomer); apo-SoxR was devoid of Fe or other metals. The spectroscopic properties of Fe-SoxR indicated that it contains a redox active iron-sulfur (FeS) cluster that is oxidized upon extraction from E. coli. Fe-SoxR and apo-SoxR bound the in vivo target, the soxS promoter, with equal affinities and protected the same region from DNase I in vitro. However, only Fe-SoxR stimulated transcription initiation at soxS in vitro > 100-fold, similar to the activation of soxS expression in vivo. This stimulation occurred at a step after the binding of RNAP and indicates a conformational effect of oxidized Fe-SoxR on the soxS promoter. The variable redox state of the SoxR FeS cluster may thus be employed in vivo to modulate the transcriptional activity of this protein in response to specific types of oxidative stress.

Bacterial Proteins↗
PubMed1994

High similarity among the tomato yellow leaf curl virus isolates from the west Mediterranean basin: the nucleotide sequence of an infectious clone from Spain.

An isolate of tomato yellow leaf curl geminivirus, from the first epidemic outbreaks that occurred in Murcia, Spain (TYLCV-M) in 1992, was cloned and its nucleotide sequence was determined. The circular single stranded DNA consisted of 2777 nucleotides. The genome organization resembled that of other TYLCV sequenced so far; regulatory signal sequences for bidirectional transcription and for polyadenylation of the transcripts were localized in the sequence. Infectivity of the cloned DNA was demonstrated by subcloning a 1.8 mer of TYLCV-M in pBin19 and agroinoculating it into tomato and Nicotiana benthamiana plants. Symptoms and viral DNA forms in agroinfected plants did not differ from those of field infected ones. Sequence comparisons with other TYLCV isolates show a high homogeneity between isolates from the West Mediterranean Basin, suggesting the presence of a geographical cluster.

Amino Acid Sequence↗