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Biomedical subjects

E Duque

Publications and source records attributed to E Duque.

At least 19 recordsLinked to original sources
PubMed2006

Detection of amastigotes of Trypanosoma cruzi in a kidney graft with acute dysfunction.

We described a case of allograft kidney dysfunction associated with renal parenchymal infection with amastigotes of Trypanosoma cruzi. The patient was diagnosed as being chronically infected prior to transplantation. The infection was probably acquired by blood transfusion. He could not complete antiparasitic treatment due to drug toxicity. He was transplanted from a cadaver who showed a negative test for Chagas' disease. One year after transplantation the serum creatinine progressively increased. Histological examination of renal biopsy revealed intracytoplasmic amastigotes of T cruzi. No evidence of other specific alterations in the graft was detected. It was unknown whether graft dysfunction was only due to parasitic infection. The present case confirmed that T cruzi can infect kidney grafts and that immunosuppression in kidney transplantation is potentially a cause of dissemination of Chagas' disease.

Adult↗
PubMed2006

The perfusion pressure in non-heart-beating donors may expand the glomeruli.

INTRODUCTION: We have observed expanded glomeruli in biopsies of kidneys from non-heart-beating donors (NHBDs). We sought to determine the differences in glomerular size between NHBDs and brain-dead donors and to assess whether glomerular size impacted graft outcome. METHODS: We estimated the glomerular area using the maximal planar area (MPA) method in 198 pretransplant biopsies from 119 donors: 54 (45.4%) NHBDs and 65 (54.6%) brain-dead donors. Donor data and graft outcomes were correlated with MPA and percentage of glomerulosclerosis. The range of follow-up was 1 to 3 years. Uni- and multivariate analyses were performed. RESULTS: MPA was larger among NHBDs. MPA and GS both significantly correlated with donor age. The association between MPA and age was independent of nephron loss (ie; GS). Increased glomerular size was only observed among donors younger than 50 years. Graft survival and function were not independently associated with MPA. Donor age was a better predictor of graft outcome. CONCLUSIONS: The perfusion pressure used in NHBDs may expand the glomeruli, but this maneuver does not have any effect on graft outcome. Among donors without severe changes, glomerular size increment shows a limit around the sixth decade of life. In our study, MPA was not an independent predictor of graft survival or function.

Adolescent↗
PubMed2001

A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks.

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.

Amino Acid Sequence↗
PubMed2001

Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida.

Pseudomonas putida DOT-T1E grows on a water-toluene double liquid phase. Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family. The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene. The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA. The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence. In a TtgR-deficient background, expression from the ttgA promoter increased about 20-fold, suggesting that TtgR represses expression from the ttgA promoter. In this mutant, background expression of the ttgR gene was also much higher than in the wild-type background; however, its level of expression increased in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type. Analysis of a P. putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp-like gene. The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon. The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp-like protein is a global regulatory protein involved in the solvent-tolerant response of this strain.

Bacterial Proteins↗
PubMed2001

Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E.

In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.

Anti-Bacterial Agents↗
PubMed2001

Mutations in genes involved in the flagellar export apparatus of the solvent-tolerant Pseudomonas putida DOT-T1E strain impair motility and lead to hypersensitivity to toluene shocks.

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn5-'phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn5-'phoA-Km was inserted within the fliP gene, which is part of the fliLMNOPQRflhBA cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, the P. putida fliP mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An flhB knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the fliL gene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in P. putida DOT-T1E to the periplasm.

Alkaline Phosphatase↗
PubMed1998

Survival in soil of different toluene-degrading Pseudomonas strains after solvent shock.

We assayed the tolerance to solvents of three toluene-degrading Pseudomonas putida strains and Pseudomonas mendocina KR1 in liquid and soil systems. P. putida DOT-T1 tolerated concentrations of heptane, propylbenzene, octanol, and toluene of at least 10% (vol/vol), while P. putida F1 and EEZ15 grew well in the presence of 1% (vol/vol) propylbenzene or 10% (vol/vol) heptane, but not in the presence of similar concentrations of octanol or toluene. P. mendocina KR1 grew only in the presence of heptane. All three P. putida strains were able to become established in a fluvisol soil from the Granada, Spain, area, whereas P. mendocina KR1 did not survive in this soil. The tolerance to organic solvents of all three P. putida strains was therefore assayed in soil. The addition to soil of 10% (vol/wt) heptane or 10% (vol/wt) propylbenzene did not affect the survival of the three P. putida strains. However, the addition of 10% (vol/wt) toluene led to an immediate decrease of several log units in the number of CFU per gram of soil for all of the strains, although P. putida F1 and DOT-T1 subsequently recovered. This recovery was influenced by the humidity of the soil and the incubation temperature. P. putida DOT-T1 recovered from the shock faster than P. putida F1; this allowed the former strain to become established at higher densities in polluted sites into which both strains had been introduced.

Benzene Derivatives↗
PubMed1998

Efflux pumps involved in toluene tolerance in Pseudomonas putida DOT-T1E.

The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-'phoA mutagenesis of wild-type P. putida DOT-T1E cells. The mutant, called P. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[14C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[14C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of 14C-labeled substrate in whole cells increased in competition assays in which toluene-and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.

Amino Acid Sequence↗
PubMed1997

Mechanisms for solvent tolerance in bacteria.

The development of tolerance in Pseudomonas putida DOT-T1 to toluene and related highly toxic compounds involves short- and long-term responses. The short-term response is based on an increase in the rigidity of the cell membrane by rapid transformation of the fatty acid cis-9,10-methylene hexadecanoic acid (C17:cyclopropane) to unsaturated 9-cis-hexadecenoic acid (C16:1,9 cis) and subsequent transformation to the trans isomer. The long-term response involves in addition to the changes in fatty acids, alterations in the level of the phospholipid polar head groups: cardiolipin increases and phosphatidylethanolamine decreases. The two alterations lead to increased cell membrane rigidity and should be regarded as physical mechanisms that prevent solvent penetrance. Biochemical mechanisms that decrease the concentration of toluene in the cell membrane also take place and involve: (i) a solvent exclusion system and (ii) metabolic removal of toluene via oxidation. Mutants unable to carry out cis --> trans isomerization of unsaturated lipids, that exhibit altered cell envelopes because of the lack of the OprL protein, or that are unable to exclude toluene from cell membranes are hypersensitive to toluene.

Adaptation, Physiological↗
PubMed1997

Removal of high concentrations of nitrate from industrial wastewaters by bacteria.

Klebsiella oxytoca isolate 15 was isolated from the grounds of a nitration factory and was found to be tolerant to nitrate at concentrations up to 0.5 to 1 M. Physicochemical parameters for optimal growth conditions for K. oxytoca isolate 15 were established. Growth took place when the nitrate concentration in the medium was less than 150 mM, and full nitrate consumption required about 14 g of C per g of N. This strain was able to remove nitrate without accumulating nitrite. The system was scaled up to a 40-liter pilot plant and was operated on-site satisfactorily.

Journal Article↗
PubMed1996

Metabolism of nitrate esters by a consortium of two bacteria.

The products of condensation of organic alcohols and nitric acid are nitrate esters with the general structure C-O-NO2. These products are widely employed as vasodilators and explosives, and are true xenobiotic compounds, as they do not occur in nature. We have isolated and characterized a consortium of two microorganisms, Arthrobacter ilicis and Agrobacterium radiobacter, that mineralized recalcitrant ethylene glycol dinitrate. The Arthrobacter strain was the actual degrading microorganism, although the second microbe facilitated mineralization. The biodegradation of ethylene glycol dinitrate by A. ilicis involved the progressive elimination of the nitro groups from the organic molecule to generate ethylene glycol, which was then mineralized. Waters polluted with ethylene glycol dinitrate have been shown amenable to biological treatment in a pilot plant with wastewaters generated during the synthesis of the chemical in a factory.

Arthrobacter↗
PubMed1995

Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons.

Pseudomonas putida DOT-T1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole C source. The strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to organic solvents whose log P(ow) (octanol/water partition coefficient) was higher than 2.3. Solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. Mg2+ ions in the culture medium improved solvent tolerance. Electron micrographs showed that cells growing on high concentrations of toluene exhibited a wider periplasmic space than cells growing in the absence of toluene and preserved the outer membrane integrity. Polarographic studies and the accumulation of pathway intermediates showed that the strain used the toluene-4-monooxygenase pathway to catabolyze toluene. Although the strain also thrived in high concentrations of m- and p-xylene, these hydrocarbons could not be used as the sole C source for growth. The catabolic potential of the isolate was expanded to include m- and p-xylene and related hydrocarbons by transfer of the TOL plasmid pWW0-Km.

Benzene Derivatives↗
PubMed1993

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.

Hybridization, Genetic↗
PubMed1993

Mineralization of p-methyl-14C-benzoate in soils by Pseudomonas putida (pWW0).

Pseudomonas putida bearing the archetypal TOL plasmid pWW0 metabolizes p-methylbenzoate through a meta-cleavage pathway. In complex environments such as soils that are relatively rich in organic matter, we observed metabolism of p-methyl-14C-benzoate, which could be monitored as 14CO2 evolution from the labelled alkylaromatic. Linear 14CO2 evolution in soils took place for at least a month, although efficient mineralization of the alkylaromatic required appropriate mass transfer to allow the bacterial cell and target chemical to remain in close contact.

Benzoates↗
PubMed1992

Behavior in agricultural soils of a recombinant Pseudomonas bacterium that simultaneously degrades alkyl- and haloaromatics.

Pseudomonas sp. FR1 (pFRC20P) is a recombinant bacterium able simultaneously to degrade alkyl- and haloaromatics due to the xylXYZL and xylS genes from the TOL plasmid borne on the bacterial chromosome, and to the gene encoding for a gamma-lactone isomerase from Alcaligenes eutrophus on pFRC20P. The survival of this strain in sterile soils was shown to depend on the physicochemical properties of the soil. The recombinant information was stable in bacteria introduced in soils and conferred selective advantage to the host bacterium when the soils were amended with low amounts of p-methylbenzoate. However, relatively high amounts of this chemical significantly reduced survival of the bacterium. Survival was more seriously affected by chlorobenzoates than by methylbenzoates. Evolution of 14CO2 from p-methyl-14C-benzoate in soils confirmed that Pseudomonas sp. FR1 (pFRC20P) functions in a complex system in accordance with its design. Survival of Pseudomonas sp. FR1 (pFRC20P) was better at 4 degrees C and 25 degrees C than at 37 degrees C. On plates, the pFRC20P plasmid was mobilized by helper plasmids to P. putida at a frequency of about 10(-6) transconjugants per recipient. However, no transfer of recombinant DNA integrated on the chromosome was found, nor was lateral transfer of the recombinant DNA borne by Pseudomonas sp. FR1 (pFRC20P) detected in soils.

Benzoates↗
PubMed1991

Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid.

Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.

Aminobutyrates↗
PubMed1991

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.

Anti-Bacterial Agents↗