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A Prado

Publications and source records attributed to A Prado.

At least 19 recordsLinked to original sources
PubMed2001

Structural and functional properties of Escherichia coli-derived nucleoplasmin. A comparative study of recombinant and natural proteins.

Fourier transform infrared spectroscopy, circular dichroism and prediction techniques have been used to investigate the conformational properties of nucleoplasmin isolated from oocytes and eggs of Xenopus. laevis and overexpressed in Escherichia coli. A simple and fast method allows purification of recombinant nucleoplasmin free of truncated and/or aggregated forms, and therefore provides a suitable sample to carry out the structural and functional comparison between these proteins. The secondary structure of the three proteins estimated from both spectroscopic techniques was very similar, and was found to be 31--33% loops, 27--34% beta structure, 22--26% turns and 9-14% alpha helix. Prediction studies, in good agreement with experimental data, also suggest that beta structure is the major regular conformation, and that loops and turns are the most abundant conformational features within the secondary structure of nucleoplasmin. Furthermore, the spectroscopic characterization of a truncated version of the protein, lacking 80 residues at the C-terminus, and the prediction data indicate that the secondary structure elements of the protein are segregated into two regions. The N-terminal fragment (comprising residues 1--120) which holds all the putative beta strands, and the solvent-exposed C-terminal region, that is suggested to be enriched in turn and loop structures. The phosphate/protein monomer molar ratios, obtained from chemical analysis and mass spectrometry, are 0, 3 and 7--10 for recombinant, oocyte and egg nucleoplasmin, respectively. Phosphorylation does not significantly affect the secondary structure of the protein, but clearly modulates its ability to decondense sperm nuclei and to remove basic proteins from DNA.

Amino Acid Sequence↗
PubMed2000

Mushroom body defect, a gene involved in the control of neuroblast proliferation in Drosophila, encodes a coiled-coil protein.

Neurogenesis relies on the establishment of the proper number and precisely controlled proliferation of neuroblasts, the neuronal precursor cells. A role for the mushroom body defect (mud) gene in both of these aspects of neuroblast behavior, as well as possible roles in other aspects of fruit fly biology, is implied by phenotypes associated with mud mutations. We have localized mud by determining the sequence change in one point mutant, identifying a predicted ORF affected by the mutation, and showing that an appropriate segment of the genome rescues mud mutant phenotypes. An analysis of mud cDNAs and a survey of mud transcripts by Northern blotting indicate that the gene is subject to differential splicing and is expressed primarily during embryogenesis but also, at lower levels, during subsequent developmental stages in a sexually dimorphic manner. The gene is predicted to encode a polypeptide without obvious homologs but with two prominent structural features, a long coiled coil that constitutes the central core of the protein and a carboxyl-terminal transmembrane domain.

Alternative Splicing↗
PubMed2000

Evaluation of an immunocapture-agglutination test (Brucellacapt) for serodiagnosis of human brucellosis.

We evaluated the validity and the usefulness of a new test for the diagnosis of human brucellosis based on an immunocapture-agglutination technique. A total of 315 sera from 82 patients with a diagnosis of brucellosis, 157 sera from patients in whom brucellosis was suspected but not confirmed, and 412 sera from people living in rural areas with endemic brucellosis were studied. The seroagglutination test (SAT), Coombs anti-Brucella test, and Brucellacapt test were evaluated. All the initial sera from the 82 patients proved to be positive in Brucellacapt and Coombs tests, while only 75 (91.4%) were positive in the SAT. If a >/=1/160 diagnostic threshold titer was defined for the Brucellacapt test, Coombs test, and SAT, the sensitivities were 95.1, 91.5, and 65.8%, respectively. Taking the same diagnostic threshold titer for the 157 sera from the unconfirmed but suspected patients, the specificities of the Brucellacapt, Coombs, and SAT were 81.5, 96.2, and 100%, respectively; for the 412 control sera, the specificities were 99.0, 99.8, and 100%. The diagnostic efficiency (area below the receiver operating characteristic curve) of Brucellacapt was 0.987852 (95% confidence interval [CI], 0.95109 to 0.99286), very similar to the diagnostic efficiency of the Coombs test (0.97611; 95% CI, 0.94781 to 0.99146) and higher than that of SAT (0.91013; 95% CI, 0.86649 to 0.94317). The results of the Brucellacapt test were compared with those of the Coombs test (correlation coefficient, 0.956; P = 0.000) and SAT (correlation coefficient, 0.866; P = 0.000). The study shows very good correlation between the Brucellacapt and Coombs tests, with a high concordance between titers obtained in the two tests. Nevertheless, lower correlation and concordance were found between the Brucellacapt and Coombs tests when the results for titers of >/=1/160 were compared (0.692; P = 0.000). In acute brucellosis, the Brucellacapt and Coombs tests render positive titers of >/=1/160. When the titers are lower, they increase significantly in the following 30 days, despite the evolution of SAT titers. In contrast, Brucellacapt and Coombs titers are always high (>/=1/640) in brucellosis with long evolution, whether SAT titers are higher or lower than 1/160.

Adolescent↗
PubMed1999

Evaluation of six serological tests in diagnosis and postoperative control of pulmonary hydatid disease patients.

Latex agglutination (LA), passive hemagglutination (PHA), immunoelectrophoresis (IEP) and specific IgE, IgM, IgG enzyme-linked immunosorbent assay (ELISA) tests for diagnosis and postoperative follow-up of 79 patients with surgically confirmed pulmonary hydatidosis were evaluated. Specific IgG ELISA was the most sensitive test (83.5%) and the least sensitive tests were specific IgE ELISA (44.3%) and IEP (50.6%). The specificity obtained for all the serologic test was above 97% in all cases. The greatest number of false positives in all tests (except IEP) occurred in patients with Taenia saginata and Taenia solium cysticerci infestations and in patients with lymphoma and leukemia. Specific IgG ELISA demonstrated the highest negative predictive value (93.8%). No statistically significant differences (p > 0.050) were found in the sensitivity of the tests when patients with only one cyst and patients with various cysts were compared. Considering only the patients without relapse, the percentage of seropositive patients increased in all tests at 1 and 3 months after surgery. After that time the percentage of seropositive patients decreased. At 48 months after surgery all patients without relapse became negative in IEP, specific IgE ELISA, and specific IgM ELISA. The antibody titers in all seropositive patients increased during the 3 months after surgery. From these 3 months onward, antibody levels decreased in all serologic tests studied in the group of patients without relapse. The patients who had relapses during the first year after surgery presented persistently elevated antibody titers in all postoperative sera. The antibody titers of the patients who relapsed between the third and fourth years after surgery decreased progressively the third month after surgery, and increased in the serum obtained at the moment of relapse diagnosis. Our results show that persistence of elevated antibody titers in patients with pulmonary hydatidosis in the year after surgery or titer increase after a progressive decrease are indicative of relapse or reinfection.

Antibodies, Helminth↗
PubMed1999

The haplolethal region at the 16F gene cluster of Drosophila melanogaster: structure and function.

Extensive aneuploid analyses had shown the existence of a few haplolethal (HL) regions and one triplolethal region in the genome of Drosophila melanogaster. Since then, only two haplolethals, 22F1-2 and 16F, have been directly linked to identified genes, dpp and wupA, respectively. However, with the possible exception of dpp, the actual bases for this dosage sensitivity remain unknown. We have generated and characterized dominant-lethal mutations and chromosomal rearrangements in 16F and studied them in relation to the genes in the region. This region extends along 100 kb and includes at least 14 genes. The normal HL function depends on the integrity of a critical 4-kb window of mostly noncoding sequences within the wupA transcription unit that encodes the muscle protein troponin I (TNI). All dominant lethals are breakpoints within that window, which prevent the functional expression of TNI and other adjacent genes in the proximal direction. However, independent mutations in these genes result in recessive lethal phenotypes only. We propose that the HL at 16F represents a long-range cis regulatory region that acts upon a number of functionally related genes whose combined haploidy would yield the dominant-lethal effect.

Animals↗
PubMed1998

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.

Animals↗
PubMed1997

[Occult gallbladder disease or microlithiasis in patients with acute pancreatitis: a frequent clinical event].

BACKGROUND: Patients with acute pancreatitis (AP) and a normal gallbladder by standard echographic evaluation may have "occult" gallbladder disease or microlithiasis with recurrent episodes of AP. AIM: To conduct a prospective evaluation of patients with the diagnosis of non-biliary AP in order to detect "occult" gallbladder disease and to compare its clinical presentation with that of biliary AP. PATIENTS AND METHODS: Patients admitted with the diagnosis of AP to a clinical hospital were included in the study. According to an abdominal ultrasound study, patients were classified as having or not cholelithiasis. A duodenal biliary drainage was performed in 15 patients with AP and without gallbladder stones. RESULTS: Patients without cholelithiasis had recurrent AP more often than patients with biliary AP (53 and 3.3% respectively). Excessive alcohol ingestion did not rule out the possibility of biliary etiology. In 6 patients, the analysis of duodenal bile showed cholesterol crystals, and cholecystectomy confirmed the existence of gallbladder disease in 5. All of them remained asymptomatic during a follow-up period of four years. One patient refused surgery, with subsequent development of gallstones and recurrent episodes of AP. In other 4 patients, gallbladder disease was confirmed by percutaneous gallbladder puncture or during cholecystectomy. No recurrence of AP were observed during the follow-up CONCLUSIONS: Microlithiasis or "occult" gallbladder disease accounts for at least 67% of the original "non-biliary" AP. Duodenal bile analysis is a useful and necessary technique for the evaluation of patients with "non-biliary" acute pancreatitis. Careful clinical and echographic follow-up of this subgroup of patients with AP is mandatory.

Acute Disease↗
PubMed1996

The Turbidity of Cell Nuclei in Suspension: A Complex Case of Light Scattering.

The turbidity of a suspension of cell nuclei isolated from animal tissue homogenates is a complex case of non-Rayleigh scattering. As a first approximation to this system, we have characterized a number of factors that may contribute to the observed turbidity: cation-dependent chromatin condensation, thermal denaturation of chromatin, nuclear shrinking, and changes in the optical properties of the membrane bilayer. Small differences in cation concentration, particularly in the case of divalent cations, lead to large changes in chromatin supramolecular organization, thus to large turbidity effects; thermally-induced changes in turbidity have a similar origin, although they are less pronounced. Under certain circumstances, either salts or heat may induce condensation of chromatin, the latter being connected to the inner side of the nuclear envelope, nuclear shrinking ensues, and this in turn modifies the suspension turbidity. Finally, changes in the physical properties of the lipid bilayers or of the phospholipids in the nuclear envelopes may also have significant effects, though smaller than chromatin changes, in the overall turbidity of the nuclear suspension.

Journal Article↗
PubMed1995

Functional recovery of troponin I in a Drosophila heldup mutant after a second site mutation.

To identify proteins that interact in vivo with muscle components we have used a genetic approach based on the isolation of suppressors of mutant alleles of known muscle components. We have applied this system to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. We show that the structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle.

Alleles↗
PubMed1994

An assessment of the biochemical applications of the non-ionic surfactant Hecameg.

A number of properties and effects of the novel non-ionic detergent Hecameg (6-O-(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside) have been examined in view of its possible biochemical applications. In particular, its critical micellar concentration has been measured, and its effects on pure lipid membranes, soluble and membrane-bound enzymes have been recorded. Hecameg has some advantageous and some less advantageous properties; its relatively high critical micellar concentration (16.5 mM), almost insensitive to pH or ionic strength changes, makes it suitable for reconstitution procedures in which detergent must be removed by dialysis. It is also an effective lipid-solubilizing agent, producing leakage of vesicle contents at detergent concentrations well below the solubilizing range. Among the drawbacks, the presence of an amide group in the molecule may interfere with the protein amide group in spectroscopic measurements. It also appears to be less gentle than other nonionic surfactants towards certain enzyme activities.

Adenosine Triphosphatases↗
PubMed1993

Abnormal muscle development in the heldup3 mutant of Drosophila melanogaster is caused by a splicing defect affecting selected troponin I isoforms.

The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6a1, 6a2, 6b1, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6b1, resulting in the failure to produce all exon 6b1-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor.

Amino Acid Sequence↗
PubMed1992

[Characteristics of severe esophagitis in patients with collagen diseases].

Connective tissue disease may alter esophageal function generating symptoms due to gastroesophageal reflux and motor disturbances. Fifteen patients with connective tissue diseases and severe esophagitis defined by the presence of esophageal stenosis or ulcerations were studied. Diagnosis was made with radiologic, endoscopic and manometric studies. Dysphagia was present in 11 and gastroesophageal reflux in all. All patients has an hypotensive and shorter lower esophageal sphincter. Better therapeutic results were obtained with surgical treatment.

Adult↗
PubMed1991

Electrical activity of the lateral rectus muscle and abducens nerve during unconditioned eye retraction induced by corneal stimulation in the cat.

Horizontal eye movements, the electromyogram of the lateral rectus muscle and the evoked electroneurogram of the VIth nerve were recorded in the alert cat during air puff stimulation of the cornea. A burst of activity was observed in the lateral rectus muscle following air puff stimulation of the ipsilateral or contralateral eye. This activity produced rotational eye movement. The VIth nerve produced two volleys of activity that were interpreted as the R1 and R2 parts of the reflex. It can be concluded that the lateral rectus muscle participates in the ocular retraction following trigeminal stimulation. It is further suggested that motoneurons in the main abducens nucleus participate in the corneal reflex, permitting several final pathways for conditioned and unconditioned nictitating membrane extension.

Abducens Nerve↗
PubMed1990

Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate. A Fourier-transform infrared spectroscopic study.

In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2(+)-ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.

Animals↗
PubMed1990

[Peritonitis in continuous ambulatory peritoneal dialysis. Microbiological and clinical evaluation].

In 1976, Popovich et al. described a technique of peritoneal dialysis using bottled dialysate. Later Oreopoulos et al. modified the technique by using plastic bags. But peritonitis still is a major and potentially serious complication of peritoneal dialysis. We have evaluated a) microbiologic diagnostic methods for infectious peritonitis, b) incidence of etiologic agents, and c) the evolution during antimicrobial treatment. Eighteen patients with chronic renal failure of diverse causes were followed from initiation of the CAPD program since January 1981 until June 1988. There were 80 episodes of infectious peritonitis during 17 patient-years of dialysis with an overall incidence of peritonitis of 4.7 episodes/patient-year. The total volume centrifuged technique and culture of sediment showed a sensibility of 85% in 73 episodes where cultures were obtained. The 59.1% of episodes of peritonitis were caused by gram negative bacilli; 11.6% were due to Acinetobacter calcoaceticus and Gram positive cocci accounted for 37.3%. These results are different from those found in other countries because most of our patients had received antimicrobial agents which probably changed their body flora, some did not have manual ability, others were of bad hygienic habits and finally, all of them had frequent contact with hospital environment. The species most frequently isolated were coagulase negative staphylococci (12.8%), probably from patients' skin flora. (ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗