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P Barbosa

Publications and source records attributed to P Barbosa.

29 records · Page 2Linked to original sources
PubMed1995

Exploring the role of histidines in the catalytic activity of duck delta-crystallins using site-directed mutagenesis.

The duck delta 2-crystallin gene encodes an enzymatically-active argininosuccinate lyase while the delta 1-crystallin gene product, although 94% identical, is enzymatically inactive. Four histidine residues in the duck delta 2-crystallin. His91, His110, His162 and His178, were converted to asparagine residues in an effort to define the role of histidines in the catalytic process of this enzyme-crystallin and to explain the lack of enzyme activity in the delta 1-crystallin protein. The recombinant mutant proteins were expressed in E. coli and purified to homogeneity for analysis. These four residues were chosen because they fall within highly conserved regions of argininosuccinate lyases from several species. This analysis revealed that change of His91 or His162 for asparagine resulted in complete loss of activity. The His110 enzyme had a reduced Vmax and the His178 enzyme was near normal in its kinetic properties. These data confirm the roles of histidine in the catalytic process of this enzyme-crystallin and suggest that the change of His91 to Gln91 observed in the duck and chicken delta 1-crystallin molecules may be sufficient to account for the lack of enzymatic activity of those proteins.

Amino Acid Sequence↗
PubMed1994

Kinetic analysis of HIV-1 early replicative steps in a coculture system.

During an acute human immunodeficiency virus (HIV) infection in vitro, different forms of unintegrated viral DNA accumulate in target cells. They include linear full-length HIV DNA molecules, which are the precursors of the integrated provirus, and two types of circular molecules (with one or two LTRs), whose role and mode of formation are not fully understood. To evaluate the intracellular fate of HIV unintegrated DNA, and to follow the formation of the two types of circular DNA molecules, the nuclear transport of viral DNA, and its integration in host cell DNA, we have designed a "DNA chase" assay. This assay is based on cocultivation of persistently HIV-1-infected H9 cells with uninfected MT4, allowing rapid accumulation of viral DNA, which is then blocked by addition of AZT. In this highly efficient, synchronous, one-step cycle infection system, HIV linear DNA can be detected on Southern blots as early as 4 hr after the start of the coculture. Subsequently, viral DNA that had been synthesized before the addition of AZT could be "chased," establishing that almost all linear DNA molecules are rapidly transported to the nucleus, where they are either processed into the two types of circles or integrated. We could estimate that from the number of viral DNA molecules synthesized in 6 hr in this system, at least a third will become integrated and another third will circularize within 24 hr.

Biological Transport↗
PubMed1993

[Mitral valvuloplasty with Inoue balloon catheter. Analysis of 112 cases].

PURPOSE: To present immediate results of percutaneous mitral valvuloplasty (PVM) with Inoue balloon for the treatment of mitral stenosis. METHODS: One hundred and twelve consecutive patients mean age 31.26 +/- 9.5 years, 82% females, the majority in functional class III/IV (69%) underwent percutaneous mitral valvuloplasty (PVM) with Inoue balloon. The procedure was undertaken by the transseptal technique. RESULTS: PMV achieved significant reduction in mean left atrial pressure pre 24.57 +/- 5.37 post 12.36 +/- 4.82, mitral gradient pre 15.31 +/- 4.28 post 3.48 +/- 2.09 and increased mitral valve area pre 0.88 +/- 0.12 post 2.0 +/- 0.28 (P < 0.001). An increase of at least 25% of the mitral valve area with a final result greater than 1.5 cm2 was obtained in 96% of the cases. Cardiac tamponade occurred in five cases (4.4%). In 4 a successful treatment was accomplished in the catheterization laboratory. Significant mitral regurgitation was seen in one case (0.9%) and there was one death (0.9%). CONCLUSION: The results of this series attest to the effectiveness of the PMV with Inoue balloon in selected cases of mitral stenosis.

Adolescent↗
PubMed1991

Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase.

The major soluble protein in the lenses of most birds and reptiles is delta-crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2-crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity.

Animals↗
PubMed1991

Analysis of naturally occurring and site-directed mutations in the argininosuccinate lyase gene.

Argininosuccinic aciduria is an inborn error of metabolism due to the genetic deficiency of argininosuccinate lyase. In order to determine the molecular basis for the disease, RNA isolated from cultured skin fibroblasts derived from four unrelated patients was reverse-transcribed and amplified using the polymerase chain reaction and the products were cloned and sequenced. Three single base missense mutations were identified: Arg111----Trp, Gln286----Arg, and Arg193----Gln. One single base amber mutation was identified at Gln454. One mutation involved a 13-base pair deletion within exon 13, and it was noted that the majority of the mature RNA derived from this allele was deleted for the entire exon rather than containing the exon with the 13 bases deleted. A final mutation was observed in which exon 2 was deleted from the mature RNA. The molecular basis for this deletion was not determined. Of the eight potential mutations present in the four cell lines studied, six mutations were identified and further data indicate that the remaining two unidentified mutations were different from those identified. Two site-directed mutations were created in the cDNA, Lys51----Asn and His89----Gln, and these were expressed in yeast. The Lys51 mutation caused an approximate 2-fold reduction in activity and the His89 mutation resulted in an approximate 10-fold reduction in activity. The combination of determination of naturally occurring mutations and the study of the effect of site-directed mutations on the activity of argininosuccinate lyase provide insight into the amino acid residues critical to the function of the enzyme.

Amino Acid Sequence↗
PubMed1991

Characterization of the human argininosuccinate lyase gene and analysis of exon skipping.

A cosmid clone containing the entire human argininosuccinate lyase gene was isolated and characterized. The gene is approximately 35 kb in length and is divided into 16 exons. The sequence surrounding all splice sites was determined, and one unusual 5' splice site sequence in which a GC dinucleotide occurred instead of the consensus GT dinucleotide was observed. The exon structure of the gene is identical to that of the rat and similar to that of the delta-crystallin genes in the chicken. The transcription initiation site was identified, and 252 bases 5' of that site were sequenced. Within that region, the GC content is 75%, no TATA element was observed, and multiple potential Spl and CACCC binding sites were noted. Also, a putative consensus sequence that may play a role in the regulation of urea cycle genes was identified within this region. Analysis of the structure of the mature mRNA derived from this gene revealed that the sequences encoded by exon seven were deleted in approximately 5-10% of the mature mRNA in all tissue sources examined.

Argininosuccinate Lyase↗
PubMedDate not supplied

[The family physician and the human immunodeficiency virus seropositive patient].

OBJECTIVES: To characterize a subject who is HIV positive and closely observed in the consultation of clinical immunology (Santo António General Hospital), from a demographic and socio-economic point of view; to determine the percentage of these subjects who have a family doctor; how often they go to a family doctor; the reasons for a more frequent visit to the doctor; if there has been any alteration in the reasons for consulting the family doctor after establishing the diagnosis of HIV infection; who made the diagnosis of HIV infection; to evaluate, from these patient's point of view, if there has been any alteration in the family doctor's attitude or vice versa after the diagnosis. CHARACTERISATION OF THE STUDY: A descriptive, transversal study was carried out from 30/01/97 to 13/03/1997. METHOD: A questionnaire with twenty-two questions was used in a personal interview of a random sample of 100 patients observed in the Consultation of Clinical Immunology at Santo António General Hospital and represented 40% of the population studied. RESULTS: One hundred patients answered the questionnaire, 73 were male and 27 female, with an mean age of 34.73 years. The majority were single, representing 44% of the random sample, 33% had completed primary education (or equivalent), and 28% had attended secondary school (complete or incomplete), 35% of the subjects were employed and 34% unemployed. In what concerns area of residence, the majority live in the metropolitan area of Oporto. The majority of the patients (85%) had a family doctor. Nevertheless, 1/3 had never paid a visit to their doctor and, as for the others, the majority rarely did. After the diagnosis of HIV infection, the reasons that led patients to see their doctors were merely administrative (prescriptions--24.56%, sick leave--21.05%). On the other hand, before the diagnosis, health problems were their major concern and priority (27.54%). The diagnosis was made in equal proportion (28%) by the hospital services and by institutions providing health care for drug addicts. In most cases, from the patient's point of view, there had not been any change in the family doctor's attitude after the diagnosis of HIV infection and vice versa. COMMENTS: Although the majority of the subjects have a family doctor, their demand is very low, therefore, there is a waste of opportunities in terms of primary, secondary and tertiary medical care.

Adult↗