Biomedical subjects

E Díaz

Publications and source records attributed to E Díaz.

At least 55 records · Page 3Linked to original sources

PubMed1998

Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: the phenylacetyl-CoA catabolon.

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a beta-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

Acetyl Coenzyme A↗

PubMed1998

TIP47: a cargo selection device for mannose 6-phosphate receptor trafficking.

Mannose 6-phosphate receptors (MPRs) transport newly synthesized lysosomal hydrolases from the Golgi to prelysosomes and then return to the Golgi for another round of transport. We have identified a 47 kDa protein (TIP47) that binds selectively to the cytoplasmic domains of cation-independent and cation-dependent MPRs. TIP47 is present in cytosol and on endosomes and is required for MPR transport from endosomes to the trans-Golgi network in vitro and in vivo. TIP47 recognizes a phenylalanine/tryptophan signal in the tail of the cation-dependent MPR that is essential for its proper sorting within the endosomal pathway. These data suggest that TIP47 binds MPR cytoplasmic domains and facilitates their collection into transport vesicles destined for the Golgi.

Amino Acid Sequence↗

PubMed1998

[Smoking initiation in students: cross-sectional and longitudinal study of predictive factors].

BACKGROUND: This study examines the evolution of tobacco use among schoolchildren in sixth and seventh grades in Barcelona (11-13 years) and identifies smoking risk factors through cross-sectional and follow-up analysis. POBLATION AND METHODS: The cohort consists of 1,003 students attending 13 schools who served as control units for a study assessing the effects of a school-based addictive substances prevention program. Participants answered the same questionnaire in 1990 and 1991. Risk factors for the experimentation and for regular smoking are studied through cross-sectional and prospective studies. RESULT: In a year, smokers increase from 9.7% to 18.9% and regular smokers rise from 1.1% to 5.5%. The initial cross-sectional analysis finds stronger associations than the follow-up one. Some variables loose their significance in the longitudinal analysis. Among non-smokers, the strongest predictors to emerge are intention to smoke, the belief that smoking is fun, peers, siblings and mother consumption, and being male; however girls appear to consolidate the consumption more than boys among the cohort of triers. CONCLUSIONS: Tobacco use prevalences are consistent with other studies developed in our city. Social environment emerges as an important predictor; friends, siblings and mothers tobacco use and gender are the main risk factors.

Adolescent↗

PubMed1998

Programmed cell death in the pathogenesis of rabbit hemorrhagic disease.

Rabbit hemorrhagic disease is a rapidly lethal infection caused by a calicivirus, characterized by acute liver damage and disseminated intravascular coagulation (DIC). Following morphological criteria and using a specific in situ labeling technique, we have found that liver cell death induced upon infection is due to apoptosis, and that programmed cell death is a constant feature in rabbits experimentally infected with RHDV. The process affected mainly hepatocytes, but also macrophages and endothelial cells presented morphologic hallmarks of apoptosis, expressing all these cell types viral antigens as determined by immunohistochemistry. The occurrence of programmed cell death was correlated with the appearance of the RHDV induced pathology in tissues by DNA fragmentation detection in situ. Hepatocyte apoptosis produced extensive parenchymal destruction causing a lethal, acute fulminant hepatitis that is characteristic of RHD. Apoptosis of intravascular monocytes and endothelial cell was observed together with fibrin thrombi in blood vessels. Since apoptotic cells are known sites of enhanced procoagulant activity, apoptosis of these cell populations might constitute a first step in the pathogenesis of DIC and a common pathway to other viral hemorrhagic fevers. In conclusion, apoptosis in RHD may be determinant in the development of the pathogenesis of this disease.

Animals↗

PubMed1998

Characterization of the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid in Escherichia coli K-12.

We have identified, cloned, and sequenced the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (PP) in Escherichia coli K-12. This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a putative operon (hcaA1A2CBD) and two additional genes transcribed in the opposite direction that encode a potential permease (hcaT) and a regulator (hcaR). Sequence comparisons revealed that while hcaA1A2CD genes encode the four subunits of the 3-phenylpropionate dioxygenase, the hcaB gene codes for the corresponding cis-dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in E. coli. The inducible expression of the hca genes requires the presence of the hcaR gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family of regulators. Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP) but also cinnamic acid (CI) to its corresponding 2, 3-dihydroxy derivative. Further catabolism of DHPP requires the mhp-encoded meta fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A. Ferrández, J. L. García, and E. Díaz, J. Bacteriol. 179:2573-2581, 1997). Expression in Salmonella typhimurium of the mhp genes alone or in combination with the hca cluster allowed the growth of the recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Thus, the convergent mhp- and hca-encoded pathways are also functional in S. typhimurium, and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.

Amino Acid Sequence↗

PubMed1998

Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp. strain Y2.

The chromosomal region of Pseudomonas sp. strain Y2 involved in the conversion of styrene to phenylacetate (upper catabolic pathway) has been cloned and sequenced. Four catabolic genes, styABCD, and two regulatory genes, stySR, were identified. This gene cluster when transferred to Escherichia coli W confers to this phenylacetate-degrading host the ability to grow on styrene as the sole carbon and energy source. Genes styABCD are homologous to those encoding the styrene upper catabolic pathway in Pseudomonas fluorescens ST. Northern blot analyses have confirmed that genes styABCD constitute a transcription unit. The transcription start site of the sty operon was mapped 33 nucleotides upstream of the styA translational start codon. The styS and styR genes, which form an independent transcriptional unit, are located upstream of the styABCD operon, and their gene products show high similarity to members of the superfamily of two-component signal transduction systems. The styS gene product is homologous to histidine kinase proteins, whereas the styR gene product exhibits similarity at its N-terminal domain with cluster 1 of receiver modules and at its C terminus with the LuxR/FixJ family 3 of DNA-binding domains. Expression of the catabolic operon decreased significantly in the absence of the stySR genes and was restored when the stySR genes were provided in trans in the presence of styrene, suggesting that the stySR system behaves as a styrene-inducible positive regulator of the styABCD operon. Finally, a gene encoding a phenylacetyl-coenzyme A ligase that catalyzes the first step in the phenylacetate catabolism (styrene lower catabolic pathway) has been identified upstream of the styS gene. This activity was found to be induced in Pseudomonas sp. strain Y2 cells grown on styrene but not present in cells grown on glycerol. These results strongly suggest that the genes responsible for the complete mineralization of styrene are clustered in the chromosome of Pseudomonas sp. strain Y2.

Amino Acid Sequence↗

PubMed1998

The role of Candida sp isolated from bronchoscopic samples in nonneutropenic patients.

STUDY OBJECTIVES: To determine the clinical significance of Candida sp isolated from bronchoscopic samples in patients with suspected pneumonia. DESIGN: A retrospective chart review was performed in all nonneutropenic adult patients with Candida sp isolates from respiratory secretions obtained by bronchoscopy over a 5-year period (1991 to 1995). Potential risk factors, therapeutic decisions, and outcome were recorded. Microbiological findings, chest radiograph reports, and pathologic material were reviewed. Isolates were classified as definite, probable, or indeterminate contamination, or as definite pulmonary candidiasis, on the basis of histologic findings, therapeutic decisions, and outcome. SETTING: A 600-bed teaching hospital with 16 beds in a medical-surgical ICU. PATIENTS: Thirty-seven consecutive patients with positive cultures for Candida sp from respiratory samples obtained by bronchoscopy were evaluated. Thirty-two of these 37 patients (86.5%) received antibiotic therapy prior to sampling, and 23 (62.2%) were intubated. RESULTS: Contamination was classified as definite in 3 patients (8.1%) and probable in 30 others (81.0%). Contamination was indeterminate in two cases (5.4%). Two additional patients (5.4%) received antifungal agents for systemic candidiasis. No cases of pulmonary candidiasis could be demonstrated, although 24 of 28 patients showed protected specimen brush cultures > or = 10(3) cfu/mL. CONCLUSIONS: Nonneutropenic patients with isolation of Candida sp from bronchoscopic samples, even in high concentrations, are unlikely to have invasive candidiasis. Indication for initiation of antifungal therapy should be based on histologic evidence or identification from sterile specimens.

Adult↗

PubMed1998

[Visual evoked potentials (VEP). Current perspectives].

INTRODUCTION: The objective of this study is to describe current tendencies in the recording and interpretation of visual evoked potentials (VEP). In the introduction we describe the history of the three main classical schools and the modern methodology which combines them, so as to study the photopic and scotopic systems together and thus be better able to determine the origin of the underlying pathology. MATERIAL AND METHODS: We describe recording techniques using flash and chequer-board and a strategy for study of the visual pathway. RESULTS: The application of this technique is described for pathologies in which its findings are of particular interest: Silent multiple sclerosis, Parkinson, hepatic encephalopathy, dementia and psychiatric disorders in which certain neurotransmitters play a part; visual acuity; coma and brain death; cortical blindness. A separate section deals with paediatrics, in view of its special characteristics. We call for a return to the use of this technique with great future diagnostic potential.

Brain Death↗

PubMed1997

A novel Rab9 effector required for endosome-to-TGN transport.

Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9-GTP with roughly fourfold preference to Rab9-GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (approximately 30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.

Amino Acid Sequence↗

PubMed1997

Visuospatial discrimination deficit in rats after ibotenate lesions in anteromedial visual cortex.

To assess the role of the rat anteromedial extrastriate cortex (AM) in a visuospatial discrimination task, restricted bilateral ibotenic acid lesions were placed stereotaxically in this region. Gray rats with lesions in AM were trained in a task requiring them to discriminate the location of a light stimulus placed vertically at different elevations. Correct responses required pressing right or left levers to obtain rewards. In contrast to unoperated controls, lesioned rats failed in learning the visuospatial discrimination task. A correlation was found between the bilateral extent of the lesion in area AM and the behavioral deficit. Another group of lesioned rats was trained to discriminate brightness differences of the light stimulus but requiring the same egocentric right/left motor response. The performance of these rats was similar to that of controls. From these results we conclude that extrastriate area AM in the rat is necessary for visuospatial discrimination, but not for correct egocentric motor responses. The visuospatial functions of area AM in the rat are reminiscent of visuospatial functions ascribed to the parietal streams of extrastriate visual areas in the monkey.

Animals↗

PubMed1997

Effect of itraconazole on lytic antibodies levels in chronic Chagas disease.

We report the chemotherapeutic effect of itraconazole in chronic chagasic patients. The efficacy of treatment was analyzed by xenodiagnosis (XD), conventional serology (CS) and antibody dependent-complement mediated lysis test (CoML). Twenty chronic chagasic persons from endemic areas of Chile were grouped according to the results of xenodiagnosis (XD) performed before therapy. The follow-up of patients was performed at an average time of 42 months after treatment. No significative statistical differences were observed in the result of ELISA and Indirect Immunofluorescence test performed on serq before and after therapy with itraconazole. In the group of patients with positive XD before therapy, sera from six of them displayed lytic activity against Trypanosoma cruzi. The lytic activity of only one of these sera became negative after therapy. In relation to XD, five of those patients showed a positive tests at least once during the follow-up period. In the group of ten patients with negative XD before therapy, sera from eight of them displayed lytic activity. At the end of the follow-up period seven of them became negative. Nine of the ten patients maintained their original condition with respect to XD. These results suggests that parasitaemia level before therapy condition could be an important parameter to consider in the chemotherapy of chronic Chagas disease.

Adult↗

PubMed1997

Designing recombinant Pseudomonas strains to enhance biodesulfurization.

The dsz biodesulfurization cluster from Rhodococcus erythropolis IGTS8 has been engineered under the control of heterologous broad-host-range regulatory signals to alleviate the mechanism of sulfur repression, and it was stably inserted into the chromosomes of different Pseudomonas strains. The recombinant bacteria were able to desulfurize dibenzothiophene more efficiently than the native host. Furthermore, these new biocatalysts combine relevant industrial and environmental traits, such as production of biosurfactants, with the enhanced biodesulfurization phenotype.

Biodegradation, Environmental↗

PubMed1997

Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl)propionate catabolic pathway of Escherichia coli K-12.

We report the complete nucleotide sequence of the gene cluster encoding the 3-(3-hydroxyphenyl)propionate (3-HPP) catabolic pathway of Escherichia coli K-12. Sequence analysis revealed the existence of eight genes that map at min 8 of the chromosome, between the lac and hemB regions. Six enzyme-encoding genes account for a flavin-type monooxygenase (mhpA), the extradiol dioxygenase (mhpB), and the meta-cleavage pathway (mhpCDFE). The order of these catabolic genes, with the sole exception of mhpF, parallels that of the enzymatic steps of the pathway. The mhpF gene may encode the terminal acetaldehyde dehydrogenase (acylating) not reported previously in the proposed pathway. Enzymes that catalyze the early reactions of the pathway, MhpA and MhpB, showed the lowest level of sequence similarity to analogous enzymes of other aromatic catabolic pathways. However, the genes mhpCDFE present the same organization and appear to be homologous to the Pseudomonas xyl, dmp, and nah meta-pathway genes, supporting the hypothesis of the modular evolution of catabolic pathways and becoming the first example of this type of catabolic module outside the genus Pseudomonas. Two bacterial interspersed mosaic elements were found downstream of the mhpABCDFE locus and flank a gene, orfT, which encodes a protein related to the superfamily of transmembrane facilitators that might be associated with transport. All of the genes of the 3-HPP cluster are transcribed in the same direction, with the sole exception of mhpR. Inducible expression of the mhp catabolic genes depends upon the presence, in the cis or trans position, of a functional mhpR gene, which suggests that the mhpR gene product is the activator of the 3-HPP biodegradative pathway. The primary structure of MhpR revealed significant similarities to that of members of the IclR subfamily of transcriptional regulators. A 3-HPP catabolic DNA cassette was engineered and shown to be functional not only in enteric bacteria (E. coli and Salmonella typhimurium) but also in Pseudomonas putida and Rhizobium meliloti, thus facilitating its potential application to improve the catabolic abilities of bacterial strains for degradation of aromatic compounds.

Amino Acid Sequence↗

PubMed1996

Restricting the dispersal of recombinant DNA: design of a contained biological catalyst.

To restrict horizontal gene spread and, thus, create organisms whose behavior in the field is more predictable, we have combined a mini-Tn5 cloning system that allows stable insertion of foreign genes into the chromosomes of a variety of Gram-negative bacteria with a gene-containment circuit based on the universal lethal-function colicin E3. Use of the system is exemplified by the construction of a micro-organism designed to degrade polychlorinated biphenyls, important environmental pollutants. The relevant genotype of the microorganism is subject to a stringent gene-containment mechanism that provides neither an advantage nor a disadvantage to the host cell for survival, but decreases frequencies of productive chromosomal transfer frequencies by at least four orders of magnitude.

Catalysis↗

PubMed1996

A stringently controlled expression system for analysing lateral gene transfer between bacteria.

The lateral transfer of genetic information among microorganisms is a major force driving the outstanding adaptability of microbial communities to environmental changes. Until now little information has been obtained on gene transfer in natural ecosystems. We present here a genetic circuit for detecting and quantifying horizontal gene transfer from a defined donor microorganism to recipient organisms in the absence of selection for a recipient-specific phenotype. The system consists of an engineered lacZ (encoding beta-galactosidase) reporter gene whose expression is controlled by a synthetic regulatory element based on a fusion between the Pr promoter-operator from lambda bacteriophage and the 5' non-coding leader region of the inp gene encoding the IS 10 transposase function. Expression of this reporter cassette in the recombinant microorganism is completely shut down by two chromosomally encoded trans-acting repressors working at the level of transcription (the Cl-EK117 protein from the lambda phage), and at the level of translation (the antisense RNA-OUT of the IS 10 element). When the reporter element is transferred to a different host by any mechanism, it escapes repression and becomes expressed. The system was validated with Pseudo-monas putida, and conjugational transfer frequencies of the reporter element as low as 10(-6) were detected. The modular design and broad host range of the genetic circuit, in combination with biomarkers which permit real-time in situ detection, will facilitate the monitor-ing of gene flow in a non-disruptive manner within the environment.

Bacteria↗

PubMed1996

Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster.

We have determined and analyzed the nucleic acid sequence of a 14,855-bp region that contains the complete gene cluster encoding the 4-hydroxyphenylacetic acid (4-HPA) degradative pathway of Escherichia coli W (ATCC 11105). This catabolic pathway is composed by 11 genes, i.e., 8 enzyme-encoding genes distributed in two putative operons, hpaBC (4-HPA hydroxylase operon) and hpaGEDFHI (meta-cleavage operon); 2 regulatory genes, hpaR and hpaA; and the gene, hpaX, that encodes a protein related to the superfamily of transmembrane facilitators and appears to be cotranscribed with hpaA. Although comparisons with other aromatic catabolic pathways revealed interesting similarities, some of the genes did not present any similarity to their corresponding counterparts in other pathways, suggesting different evolutionary origins. The cluster is flanked by two genes homologous to the estA (carbon starvation protein) and tsr (serine chemoreceptor) genes of E. coli K-12. A detailed genetic analysis of this region has provided a singular example of how E. coli becomes adapted to novel nutritional sources by the recruitment of a catabolic cassette. Furthermore, the presence of the pac gene in the proximity of the 4-HPA cluster suggests that the penicillin G acylase was a recent acquisition to improve the ability of E. coli W to metabolize a wider range of substrates, enhancing its catabolic versatility. Five repetitive extragenic palindromic sequences that might be involved in transcriptional regulation were found within the cluster. The complete 4-HPA cluster was cloned in plasmid and transposon cloning vectors that were used to engineer E. coli K-12 strains able to grow on 4-HPA. We report here also the in vitro design of new biodegradative capabilities through the construction of a transposable cassette containing the wide substrate range 4-HPA hydroxylase, in order to expand the ortho-cleavage pathway of Pseudomonas putida KT2442 and allow the new recombinant strain to use phenol as the only carbon source.

Amino Acid Sequence↗

PubMed1996

[Etiologic and pathologic study of respiratory disease in lambs from intensive breeding facilities in southern Spain].

Between 1991 and 1993, it was observed epidemiologically that respiratory disturbances in lambs are associated with high temperatures during the summer. The etiological agent isolated is principally Mycoplasma ovipneumoniae; moreover, Actinobacillus pleuropneumoniae biovar A has been isolated in a high number of samples. Histopathologically, an interstitial bronchopneumonia was the main lesional finding; this lesion is associated with previous mycoplasma infection.

Animal Husbandry↗

PubMed1995

The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria.

The Gram+ bacterium Rhodococcus globerulus P6 (RgP6) catabolizes a range of polychlorinated biphenyl (PCB) congeners, thus being of interest in bioelimination processes for PCB. The first step in the pathway, a dioxygenase attack of one of the biphenyl (BP) rings, is catalyzed by biphenyl dioxygenase (BDO). In this study, the nucleotide (nt) sequences of the four clustered cistrons, bphA1A2A3A4, encoding the subunits of BDO and forming part of the bph operon of RgP6 for BP degradation, were determined. A conserved motif proposed to bind a Rieske-type [2Fe-2S] cluster was identified in the deduced amino acid (aa) sequence of both the a subunit of the terminal oxygenase (BphA1) and ferredoxin (BphA3). The ferredoxin reductase subunit (BphA4) contains conserved sites for FAD and NADH binding. Deduced aa sequences of the BDO subunits shared homologies to multicomponent aromatic ring-hydroxylating dioxygenases from Gram- microorganisms. Stronger identity was found to toluene dioxygenase (TDO) of Pseudomonas putida F1 than to other BDO. Aa sequence comparisons suggest that BP degradation genes of RgP6 may have originated in Gram- microorganisms, probably Pseudomonas, and subsequently transferred to this Gram+ bacterium.

Amino Acid Sequence↗