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Biomedical subjects

M A Prieto

Publications and source records attributed to M A Prieto.

At least 19 recordsLinked to original sources
PubMed2001

Superimposed levels of regulation of the 4-hydroxyphenylacetate catabolic pathway in Escherichia coli.

The regulation of the Pg promoter, which controls the expression of the meta operon of the 4-hydroxyphenylacetic acid (4-HPA) catabolic pathway of Escherichia coli W, has been examined through in vivo and in vitro experiments. By using Pg-lacZ fusions we have demonstrated that Pg is a promoter only inducible in the stationary phase when cells are grown on glucose as the sole carbon and energy source. This strict catabolite repression control is mediated by the cAMP receptor protein (CRP). This event does not require the presence of the specific HpaR repressor or the 4-HPA permease (HpaX), excluding the involvement of a typical inducer exclusion mechanism. However, the acetic acid excreted in the stationary phase by the cells growing in glucose acts as an overflow metabolite, which can provide the energy to produce cAMP and to adapt the cells rapidly to the utilization of a new less preferred carbon source such as the aromatic compounds. Although Pg is not a final sigma(38)-dependent promoter, it is activated by the global regulator integration host factor (IHF) in the stationary phase of growth. Gel retardation assays have demonstrated that both CRP and IHF simultaneously bind to the Pg upstream region. DNase I footprint experiments showed that cAMP-CRP and IHF binding sites are centered at -61.5 and -103, respectively, with respect to the transcription start site +1 of the Pg promoter.

Antigens, Human Platelet↗
PubMed2001

[Professionals and managers in face of training].

OBJECTIVES: To analyse ongoing education within primary care teams from managers' perspective. DESIGN: Cross-sectional descriptive study. SETTING: The Mother-Infant Health Programme of Andalusia in primary care. INTERVENTION: The views of health centre professionals were obtained through 8 groups. The type of health district and the number of years in operation were used as criteria for division. The views of the area managers were collected through a self-filled questionnaire from all the managers in the autonomous community. MEASUREMENTS AND MAIN RESULTS: Professionals believed it necessary to keep up to date and train in new technology. They thought that training activities should focus on questions such as evaluation of programmes, focus of risk, counselling and communication, and certain techniques such as IUD insertion. Both managers and professionals coincided in recognising the effect of training on the development of mother-child services. There was consensus among professionals in affirming that training increases the quality of care delivered. Access to training was the main incentive element used by most area managers and one of those most highly valued by professionals. The professionals affirmed that in recent years there had been obstacles to their education by attending scientific events outside their areas. Many professionals stated that the economic assistance of the pharmaceutical industry for courses caused inequalities between doctors and nurses in access to training. Professionals were very critical of training combined with specialist training. CONCLUSIONS: Professionals think they have sufficient training for mother-child health care. It is generally recognised that family doctors are better trained for working on health programmes than other professionals at the centres. Ongoing training was rated very highly by both professionals and directors, as it was seen as a motivating element and thought to be an activity almost solely carried out at health centres.

Child↗
PubMed2001

Biodegradation of aromatic compounds by Escherichia coli.

Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications.

Amines↗
PubMed2000

[Professionals and managers on internal communication in the Program of Mother-Child Health of Andalucia. Evaluation Team of the PSMI].

OBJECTIVE: To find the views of primary health area directors and of professionals in health centres (HC) in Andalusia on internal communication within the Mother-Child Health Programme introduced in this autonomous community. DESIGN: Descriptive study that uses qualitative and quantitative methodology. SETTING: Primary care (PC) areas and HC in Andalusia, between July 1994 and February 1995. PARTICIPANTS: 51 PC area directors and 91 HC professionals. MEASUREMENTS AND MAIN RESULTS: Self-administered questionnaire (directors) and focus groups (professionals) were used. Analysis by non-parametric tests compared averages and analysed discourse contents. Internal communication was an important weakness in the Mother-Child Health Programme and in PC in general. There were important differences between the perceptions of directors and professionals in relation to the guidelines of the Mother-Child Programme. As such, in all the area administrations it was affirmed that there were programme objectives agreed with the professionals, and transmission channels for these objectives between the area and the centres, although these were transmitted in writing in under 25% of the areas. However, professionals thought that the objectives and guidelines of the programme were unclear and ill-defined. In addition, the view that the annual objectives are not negotiated was quite common. CONCLUSIONS: Internal communication in PC is a considerable weakness in HC management. The professionals perceive considerable conflicts of interest between them and the organisation's directors.

Communication↗
PubMed2000

Bacterial promoters triggering biodegradation of aromatic pollutants.

Unraveling the complex transcriptional regulation of bacterial catabolism of aromatic pollutants is a prerequisite for engineering efficient biological systems for many biotechnological applications. A first level of regulation relies on specific regulator-promoter pairs. There have been new insights into the molecular mechanisms that regulatory proteins use to sense a given signal and to activate transcription initiation from the cognate promoters. A second level of regulation allows adjustment of the expression of the particular catabolic operons in response to the global environmental conditions of the cells, and recent findings provide some clues about the mechanisms underlying such complex regulatory checkpoints.

Biodegradation, Environmental↗
PubMed2000

Functional analysis of the small component of the 4-hydroxyphenylacetate 3-monooxygenase of Escherichia coli W: a prototype of a new Flavin:NAD(P)H reductase subfamily.

Escherichia coli W uses the aromatic compound 4-hydroxyphenylacetate (4-HPA) as a sole source of carbon and energy for growth. The monooxygenase which converts 4-HPA into 3,4-dihydroxyphenylacetate, the first intermediate of the pathway, consists of two components, HpaB (58.7 kDa) and HpaC (18.6 kDa), encoded by the hpaB and hpaC genes, respectively, that form a single transcription unit. Overproduction of the small HpaC component in E. coli K-12 cells has facilitated the purification of the protein, which was revealed to be a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH. Subsequently, the reduced flavins diffuse to the large HpaB component or to other electron acceptors such as cytochrome c and ferric ion. Amino acid sequence comparisons revealed that the HpaC reductase could be considered the prototype of a new subfamily of flavin:NAD(P)H reductases. The construction of a fusion protein between the large HpaB oxygenase component and the choline-binding domain of the major autolysin of Streptococcus pneumoniae allowed us to develop a rapid method to efficiently purify this highly unstable enzyme as a chimeric CH-HpaB protein, which exhibited a 4-HPA hydroxylating activity only when it was supplemented with the HpaC reductase. These results suggest the 4-HPA 3-monooxygenase of E. coli W as a representative member of a novel two-component flavin-diffusible monooxygenase (TC-FDM) family. Relevant features on the evolution and structure-function relationships of these TC-FDM proteins are discussed.

Amino Acid Sequence↗
PubMed2000

Influence of triflusal on platelet activation after coronary artery bypass graft.

The aim of the study was to investigate the effects of the antiplatelet agent triflusal on the changes in platelet function in patients who underwent a cardiopulmonary bypass for coronary arteries (CABG). In 20 surgical patients, blood was sampled before and at the conclusion of surgery, 48 h later (in the intensive care unit), and after 10 days of treatment with 600 mg/day triflusal (triflusal was administered from the first day after surgery). Adenosine diphosphate (ADP) and collagen-induced platelet aggregation in whole blood, granular release of beta-thromboglobulin and platelet release of thromboxane B2 were measured. Basal values were compared with results in a group of ten healthy volunteers. All platelet determinations of activation were higher in coronary patients than in healthy volunteers. Immediately after CABG, the platelet reactivity to ADP and collagen were significantly lower, and release of beta-thromboglobulin and thromboxane B2 were higher, than in the pre-CABG samples. During the patient's stay in the intensive care unit, all values tend to return to pre-CABG values. Triflusal inhibits both platelet beta-thromboglobulin (63% with respect to the post-CABG value) and thromboxane B2 (91% with respect to the post-CABG value) release. Platelet aggregation after 10 days of triflusal treatment tended to return to the pre-CABG values. In conclusion, Triflusal reduces platelet activation caused by the coronary artery bypass graft surgery.

Adenosine Diphosphate↗
PubMed1999

Novel biodegradable aromatic plastics from a bacterial source. Genetic and biochemical studies on a route of the phenylacetyl-coa catabolon.

Novel biodegradable bacterial plastics, made up of units of 3-hydroxy-n-phenylalkanoic acids, are accumulated intracellularly by Pseudomonas putida U due to the existence in this bacterium of (i) an acyl-CoA synthetase (encoded by the fadD gene) that activates the aryl-precursors; (ii) a beta-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the pha locus) integrated by two polymerases (PhaC1 and PhaC2) and a depolymerase (PhaZ). The complete assimilation of these compounds requires two additional routes that specifically catabolize the phenylacetyl-CoA or the benzoyl-CoA generated from these polyesters through beta-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the pha locus (phaC1, phaC2, and phaZ) as well as those related to the biosynthesis of precursors (fadD) or to the catabolism of their derivatives (acuA, fadA, and paa genes). Additional experiments showed that the blockade of either fadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade of phaZ hampered the mobilization of the polymer and decreased its production. Mutations in the paa genes, encoding the phenylacetic acid catabolic enzymes, did not affect the synthesis or catabolism of polymers containing either 3-hydroxyaliphatic acids or 3-hydroxy-n-phenylalkanoic acids with an odd number of carbon atoms as monomers, whereas the production of polyesters containing units of 3-hydroxy-n-phenylalkanoic acids with an even number of carbon atoms was greatly reduced in these bacteria. Yield-improving studies revealed that mutants defective in the glyoxylic acid cycle (isocitrate lyase(-)) or in the beta-oxidation pathway (fadA), stored a higher amount of plastic polymers (1.4- and 2-fold, respectively), suggesting that genetic manipulation of these pathways could be useful for isolating overproducer strains. The analysis of the organization and function of the pha locus and its relationship with the core of the phenylacetyl-CoA catabolon is reported and discussed.

Acetyl Coenzyme A↗
PubMed1999

Engineering of stable recombinant bacteria for production of chiral medium-chain-length poly-3-hydroxyalkanoates.

In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for phaC1, which encodes one of the mcl-PHA polymerases of Pseudomonas oleovorans. We used the mini-Tn5 system as a tool to construct Escherichia coli 193MC1 and P. oleovorans POMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection. The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level. E. coli 193MC1 produced considerably shorter polyesters than P. oleovorans produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7. Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution. In addition, we found that E. coli 193MC1 incorporated exclusively the R configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.

Acyltransferases↗
PubMed1999

PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes.

The phaC1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of Pseudomonas oleovorans GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant P. oleovorans strain carrying a phaC1::lacZ reporter system, that the phaC1 gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon source. Moreover, a P. oleovorans GPo1 mutant (strain GPG-Tc6) expressing higher levels of the reporter gene than the wild-type strain in the presence of glucose or citrate has been generated by mini-Tn5 insertional mutagenesis. Characterization of this mutant allowed us to conclude that phaF, a gene located downstream of the pha gene cluster, was knocked out in this strain. P. oleovorans GPG-Tc6 regained the ability to control phaC1 gene expression when complemented with the phaF wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 and phaI and phaF genes. The amino acid sequences of the phaI gene product and the N-terminal half of the PhaF protein showed a significant degree of similarity. Furthermore, the primary structure of the PhaF C terminus identifies this protein as a member of the histone H1-like group of proteins. Northern blot analysis showed two transcription units containing phaF, i.e., phaF and phaIF transcripts. Expression of the phaIF operon is more efficient in the presence of octanoic acid and is enhanced by the lack of the PhaF protein. In addition, it has also been demonstrated that both PhaF and PhaI proteins are bound to PHA granules produced by P. oleovorans. A model for the role of PhaF in regulating PHA synthesis is presented.

Acyltransferases↗
PubMed1998

[Motivation and incentives: different perceptions of administrators and professionals. Evaluation Team of the Materno-Infantile Health Program].

OBJECTIVE: To find the views of Primary Care District managers and Health Centre (HC) professionals in Andalusia on systems of motivation and incentive in use in this autonomous community. DESIGN: A descriptive study which uses qualitative and quantitative methodology. SETTING: Primary Care districts and Health Centres in Andalusia. PARTICIPANTS: Primary Care district managers (51) and HC professionals (91). MATERIAL AND METHODS: Information-gathering: self-administered questionnaire (managers) and focus groups (professionals). ANALYTICAL TECHNIQUES: Non-parametric tests to compare means and analysis of contents of discourse. Field-work. July 1994 and February 1995. RESULTS: 55% of the district managers thought that the incentive systems in use were inadequate and insufficient. Enabling professionals to be trained is the main motivating and incentive mechanism in place. Variable productivity was the next most common mechanism. However, variable productivity, in the view of the professionals, was the main demotivating factor of recent years. Professionals thought that the economic rewards of the incentives system were slight, the target variables were unsuitable, there were no objective evaluation mechanisms and the economic differences between the incentives earned by managers and professionals were too great. CONCLUSIONS: The District managers indicated that improvements in the incentives systems used are needed. The HC professionals affirmed that the Andalusian incentives system has demotivated Primary Care teams and has had little effect on orienting professional practice.

Administrative Personnel↗
PubMed1998

[Hemolytic uremic syndrome in children of Mendoza, Argentina: association with Shiga toxin-producing Escherichia coli infection].

Shiga toxin-producing Escherichia coli (STEC) has been associated with pathogenesis of hemolytic uremic syndrome (HUS) worldwide. The aim of the present study was to characterize the HUS cases reported in Mendoza and to determine their association with STEC infection. From July 1994 through June 1996 thirty-six patients with HUS were admitted to Hospital Pediátrico "Dr. HJ Notti" (Mean age 22.8 +/- 14.9 months, 44% females). The children developed HUS following an acute diarrheal illness in 94.4% of the cases. Bloody diarrhea was observed in 83.3% of them. Antimicrobial therapy had been administered to 69.4% of the patients. Most of the patients were well-nourished (88.9%), belong to middle-low socioeconomical condition (91.7%), from urban areas (72.2%) and they were mostly assisted during summer and the beginning of autumn. The acute stage of the disease occurred with presentation of pallor (100%), edema (25%), anuria (38.9%), oliguria (41.7%), hemolytic anemia (97.2%), thrombocytopenia (86.1%) and neurological involvement (41.7%). Twenty-five of them presented the full clinical syndrome. Peritoneal dialysis were performed in 50% and packed blood cell transfusion in 88.9%. The mean days of hospitalization was 15.1 +/- 9.2 [range 1-32]. A 91.7% of the patients recovered renal function, two developed chronic renal failure and one died. Cumulative evidence of STEC infection was found in 19 (86.4%) of 22 HUS patients. STEC O157:H7, biotype C was found in 8 (36.4%). The prevalent Stx type was Stx2 in STEC, free fecal Stx (STMF) and Stx-neutralizing antibodies (a-Stx). In Mendoza, as in the rest of Argentina E. coli O157:H7, biotype C, Stx2 producer is the most frequently detected pathogen in HUS cases.

Argentina↗
PubMed1997

Identification of the 4-hydroxyphenylacetate transport gene of Escherichia coli W: construction of a highly sensitive cellular biosensor.

The mechanism of uptake of 4-hydroxyphenylacetate (4-HPA) by Escherichia coli W was investigated. The 4-HPA uptake was induced by 4-HPA, 3-hydroxyphenylacetate (3-HPA) or phenylacetate (PA) and showed saturation kinetics with apparent Kt and Vmax values of 25 microM and 3 nmol/min per 10(9) cells, respectively. Transport of 4-HPA was resistant to N,N'-dimethylcarbodiimide (DCCD), but was completely inhibited by cyanide and 4-nitrophenol, and, to a lower extent, by arsenate and azide, suggesting that energy is required for the uptake process. Competition studies showed that 4-HPA uptake was inhibited by 3-HPA or 3,4-dihydroxyphenylacetate (3,4-DHPA) but not by 2-hydroxyphenylacetate (2-HPA), L-tyrosine or other structural analogues, indicating a narrow specificity of the transport system. We have demonstrated, using two experimental approaches, that the hpaX gene of the 4-HPA catabolic cluster, which encodes a protein of the superfamily of transmembrane facilitators, is responsible for 4-HPA transport. Aside from the aromatic amino acid transport systems, hpaX is the first transport gene for an aromatic compound of enteric bacteria that has been characterized. A highly sensitive cellular biosensor has been constructed by coupling the 4-HPA transport system to a regulatory circuit that controls the production of beta-galactosidase. This biosensor has allowed us to demonstrate that the transport system performs efficiently at very low external concentrations of 4-HPA, similar to levels that would be expected to occur in natural environments.

Binding, Competitive↗
PubMed1997

Molecular characterization of PadA, a phenylacetaldehyde dehydrogenase from Escherichia coli.

The padA gene encoding the phenylacetaldehyde dehydrogenase involved in the catabolism of 2-phenylethylamine in Escherichia coli has been cloned, sequenced, and located at 31.0 min on the chromosome. The deduced PadA polypeptide contains 499 amino acid residues with a predicted molecular mass of 53.7 kDa, and its primary structure reveals significant similarity with that of members of the aldehyde dehydrogenase superfamily. By engineering optimal transcription and translation elements, a high expression of the padA gene has been achieved. The active PadA enzyme is a homodimer that prefers NAD+ over NADP+ as coenzyme. The enzyme efficiently oxidizes only phenylacetaldehyde-like aromatic aldehydes, and has a weak esterase activity with p-nitrophenol. The padA gene constitutes a new catabolic tool for designing DNA cassettes to expand the abilities of microorganisms to degrade toxic aromatic compounds.

Aldehyde Oxidoreductases↗
PubMed1997

Identification of a novel positive regulator of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli.

The hpa cluster of Escherichia coli W ATCC 11105 encodes the enzymes involved in the catabolism of 4-hydroxyphenylacetate (4-HPA). The catabolic genes are organized in two operons, the hpaBC operon, which produces the hydroxylase activity, and the meta operon, which encodes the enzymes that cleave the aromatic ring and allows its further metabolism. Using a monocopy or multicopy lacZ reporter system, we have demonstrated that the hydroxylase genes are transcribed from the PBC promoter which is positively regulated by the hpaA gene product. HpaA is activated by 4-HPA, 3-HPA, or phenylacetate and represents a novel member of the AraC/XylS family of regulators that recognizes aromatic effectors. The -35 box of the PBC promoter clearly deviates from the consensus sigma 70 promoters of E. coli, but upstream of this box we observed two direct repeats, a common characteristic of promoters regulated by the AraC family of proteins. The hpaA gene, which appears to form a transcriptional unit with the putative hpaX transport gene, is also expressed from an alternative promoter that is located within the hpaX structural sequence. On the basis of these findings, we propose a working model for the regulation of the unique aromatic catabolic pathway thus far characterized at a molecular level in E. coli. This regulatory circuit opens a new scenario for the development of novel microbiological tools for environmental purposes.

Amino Acid Sequence↗