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M A Prieto

Publications and source records attributed to M A Prieto.

25 records · Page 2Linked to original sources
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↗
PubMed1995

Cloning and sequencing of the pac gene encoding the penicillin G acylase of Bacillus megaterium ATCC 14945.

The pac gene encoding the penicillin G acylase (PGA) of Bacillus megaterium ATCC 14945 has been cloned in Escherichia coli HB101 (proA, leuB) using a selective minimal medium containing phenylacetyl-L-leucine instead of L-leucine. The nucleotide sequence of this gene has been determined and contains an open reading frame of 2406 nucleotides. The deduced amino acid sequence shows significant similarity with other beta-lactam acylases. Although the PGA of B. megaterium is extracellular, the enzyme produced in E. coli appears to have a cytoplasmic localization.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of 4-hydroxyphenylacetate 3-hydroxylase of Escherichia coli. A two-protein component enzyme.

The nucleotide sequences of the hpaB and hpaC genes encoding the 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli W ATCC 11105 have been determined. These genes appear to be part of an operon and encode two proteins of 58,781 and 18,679 Da, respectively, that are required for hydroxylase activity. This aromatic hydroxylase is NADH-dependent and uses FAD as the redox chromophore. The largest component (HpaB) has been purified by affinity chromatography in Cibacron blue. E. coli cells that express exclusively hpaB showed only a very low hydroxylase activity that was enhanced in the presence of extracts containing the smallest protein HpaC. This behavior resembles that of the coupling protein of the 4-hydroxyphenylacetate 3-hydroxylase from Pseudomonas putida, and it might prevent the wasteful oxidation of NADH in the absence of substrate. Using a promoter-probe plasmid we have demonstrated that the hpaBC operon is expressed by a promoter inducible by 4-hydroxyphenylacetic acid. A gene, named hpaA, encoding a protein homologous to the XylS/AraC family of regulators, was identified upstream of the hydroxylase operon. The role played by HpaA in the regulation of the hpaBC operon remains to be elucidated. Since HpaB is not homologous to other aromatic hydroxylases, we suggest that the E. coli 4-hydroxyphenylacetate 3-hydroxylase is the first member of a new family of two-component aromatic hydroxylases sequenced so far.

Amino Acid Sequence↗
PubMed1993

Characterization of an Escherichia coli aromatic hydroxylase with a broad substrate range.

The hpaB gene encoding an aromatic hydroxylase of Escherichia coli ATCC 11105, a penicillin G acylase-producing strain, has been cloned and expressed in E. coli K-12. This gene was located near the pacA gene coding for penicillin G acylase. The hydroxylase has a molecular mass of 59,000 Da, uses NADH as a cosubstrate, and was tentatively classified as a 4-hydroxyphenylacetic acid hydroxylase, albeit it exhibited a rather broad substrate specificity acting on different monohydric and dihydric phenols. E. coli W, C, and B as well as Klebsiella pneumoniae M5a1 and Kluyvera citrophila ATCC 21285 (a penicillin G acylase-producing strain) but not E. coli K-12 contained sequences homologous to hpaB. Our results support the hypothesis that hpaB is a component of the 4-hydroxyphenylacetic acid degradative pathway of E. coli W.

Blotting, Southern↗
PubMedDate not supplied

[Problems and proposals for improvement in the health care of economic immigrants].

OBJECTIVES: To analyse existing problems in health care to economic immigrants from the point of view of health and social professionals, health authorities and economic immigrants and to know which will be their proposals and priorities to improve economic immigrants's health assistance. METHODS: Qualitative approach study made in Majorca island. (Spain). Three focus groups and three nominal groups with health and social workers from primary health care and hospital services were carried out as well as two partially structured interviews to health authorities, one Autonomic Authority and one Governmental Authority, and twelve partially structured interviews to economic immigrants identified trough key informants. RESULTS: Health and social workers perceive the increase in the number of economic immigrants as a massive arrival. The main problems identified are: access restrictions to public health services for economic immigrants, the follow-up of these patients and diagnostic difficulties because lack of training in tropical medicine. Health and social workers's first proposal for improving this situation is their own training in tropical medicine. Autonomic Authority priorities are to guarantee access to public health services for all immigrants and to look actively for health problems in these population. Governmental Authority, instead, holds the legalization for all. In fact, economic immigrants are not concerned on health care because legalization and housing problems. CONCLUSIONS: The main barriers identified by health and social workers in health care to economic immigrants are: access restrictions, follow-up and lack of training in tropical medicine. Health and social first proposal to improve this situation is their own training in tropical medicine while Autonomic and Central Authorities's priorities are not the same. For economic immigrants, health assistance is not their biggest concern.

Delivery of Health Care↗
PubMedDate not supplied

[Dietary behavior disorders: opinions and expectancies on prevention and treatment strategies from the perspective of the several social actors].

AIMS: The prevalence increase of Eating Disorders in the last decades emphasizes the necessity to develop strategies from the public healthcare system which include the perspective of the principal implicated social actors. The present study aims to know their opinions and expectations regarding the prevention and treatment of Eating Disorders. METHODS: Realization of 5 focus groups (with relatives, adolescents, professionals from the health and educational sector, representatives from institutions and support groups) and 14 semi-structured interviews (with Eating Disorder patients and relatives) about strengths and weaknesses of actual prevention and treatment programs, expectations of change and suggestions for improvement. Analysis of contents and triangulation of results. RESULTS: The absence of resources and knowledge about Eating Disorders, the scarcity of transversal programs and insufficient coordination were named as the main weaknesses whereas interdisciplinary pilot programs were positively valued. As principal demands, major endowment of human and material resources, increased coverage of specialized services, promotion of specific training and improvement of coordination was asked for. Regarding prevention, preference toward an unspecific approach to the subject in order to hinder imitation between young people was highlighted. The care in Day Hospitals was considered one of the most appropriate treatment modalities for the characteristics of this disease being valued the potentially therapeutic effect of contact between Eating Disorder patients. The support network, the education field and the family were named as other important pillars in a multidisciplinary approach to the disease. There was also a demand for the development of combined programs. CONCLUSIONS: The discussion of different psychotherapeutic approaches, the role of support groups in the caring network as well as the post-hospital follow-up were identified as issues open to future research.

Adolescent↗