Biomedical subjects

A S Pereira

Publications and source records attributed to A S Pereira.

At least 19 recordsLinked to original sources

PubMed1999

Biochemical and spectroscopic characterization of overexpressed fuscoredoxin from Escherichia coli.

Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfido-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mössbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data.

Amino Acid Sequence↗

PubMed1999

Reactions of nitric oxide with the reduced non-heme diiron center of the soluble methane monooxygenase hydroxylase.

The soluble methane monooxygenase system from Methylococcus capsulatus (Bath) catalyzes the oxidation of methane to methanol and water utilizing dioxygen at a non-heme, carboxylate-bridged diiron center housed in the hydroxylase (H) component. To probe the nature of the reductive activation of dioxygen in this system, reactions of an analogous molecule, nitric oxide, with the diiron(II) form of the enzyme (Hred) were investigated by both continuous and discontinuous kinetics methodologies using optical, EPR, and Mössbauer spectroscopy. Reaction of NO with Hred affords a dinitrosyl species, designated Hdinitrosyl, with optical spectra (lambdamax = 450 and 620 nm) and Mössbauer parameters (delta = 0.72 mm/s, DeltaEQ = 1.55 mm/s) similar to those of synthetic dinitrosyl analogues and of the dinitrosyl adduct of the reduced ribonucleotide reductase R2 (RNR-R2) protein. The Hdinitrosyl species models features of the Hperoxo intermediate formed in the analogous dioxygen reaction. In the presence of protein B, Hdinitrosyl builds up with approximately the same rate constant as Hperoxo ( approximately 26 s-1) at 4 degrees C. In the absence of protein B, the kinetics of Hdinitrosyl formation were best fit with a biphasic A --> B --> C model, indicating the presence of an intermediate species between Hred and Hdinitrosyl. This result contrasts with the reaction of Hred with dioxygen, in which the Hperoxo intermediate forms in measurable quantities only in the presence of protein B. These findings suggest that protein B may alter the positioning but not the availability of coordination sites on iron for exogenous ligand binding and reactivity.

Bacterial Proteins↗

PubMed1998

Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization.

Rapid freeze-quench (RFQ) Mössbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mössbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of approximately 80 s-1 and the remainder at a much slower rate of approximately 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mössbauer parameters (DeltaEQ = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mössbauer method match those of a transient blue species (lambdamax = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

Animals↗

PubMed1998

Spectroscopic characterization of a novel tetranuclear Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans.

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2. Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.

Bacterial Proteins↗

PubMed1997

Rapid and parallel formation of Fe3+ multimers, including a trimer, during H-type subunit ferritin mineralization.

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms. At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified. These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the "young core." The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+-tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem. J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

Animals↗

PubMed1996

Characterization of representative enzymes from a sulfate reducing bacterium implicated in the corrosion of steel.

This communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed.

Corrosion↗

PubMed1995

Variations in the anatomical organization of the human aortic arch. A study in a Portuguese population.

We have studied the anatomical features of human aortic arches in a sample of the Portuguese population using postmortem measurements of the arches and their branches in 33 cadavers. We found that there is a significant minority of individuals (18% of the total) who did not present the usual 3-branched aortic arch; interestingly, all 2-branched arches were seen in women and all 4-branched arches were observed in men. The arterial branches could spring either from the ascending segment of the arch (61% of total) or from its horizontal segment (39% of total). Our measurements showed that the springing of the branches was associated with the narrowing of the aortic arch, so that the difference in the diameter of the aortic arch between the origin of the first and last branches could reach up to 11 millimeters. We found that the relative caliber of the arterial branches of the aortic arch depended on the position of its origin at the arch: in aortic arches with branches that were originated at the horizontal segment of the arch the first arterial branch was always the widest of the three, whereas in aortic arches with branches coming out at the ascending segment of the arch the widest arterial branch was the third one. Our data support the view that there is a wide spectrum of variations in the anatomical arrangement of the aortic arch in humans, and offer information on the prevalence of aortic arch variations in a Portuguese population.

Adult↗

PubMed1994

Mammalian ferrochelatase, a new addition to the metalloenzyme family.

A [2Fe-2S] cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway. Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy. In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of [2Fe-2S]+ cluster. Mössbauer spectra of the recombinant ferrochelatase, purified from a strain of E. coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in 57Fe-enriched medium, demonstrated unambiguously that the cluster is a [2Fe-2S] cluster. No change in the cluster oxidation state was observed during catalysis. The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the [2Fe-2S] center in regulation of mammalian ferrochelatases.

Amino Acid Sequence↗

PubMed1994

Anatomical basis for the separation of four cardiac zones in the walls of human heart ventricles.

The coronary vessels of 70 human hearts were visualized postmortem by injection of the coronary arteries with a X-ray opaque substance (for angiographic studies) or with a low viscosity resin (to obtain vascular casts). Analysis of the data suggests a new anatomical systematization of the vascularization of the myocardial tissue of human heart ventricles: it can be divided into four zones each having a different origin of the arterial vessels. These four components of the heart ventricles are the antero-septal (AS), postero-septal (PS), left-lateral (LL), and right-lateral (RL) zones. They correspond to the territories of the anterior interventricular branch of the left coronary artery (AS zone), of the posterior interventricular branch of the right coronary artery (PS zone), of the left circumflex artery (LL zone), and of the right coronary artery (RL zone) up to the origin of the posterior interventricular artery. This systematization of the arterial heart ventricles in zones offers a balanced division of the myocardial tissue, since each of the four zones occupied about one fourth of the total volume of the ventricles. In our samples, the most common distribution of segments in the wall of heart ventricles was the following: 16 segments in the AS zone, 11 segments in the PS zone, 5 segments in the LL zone, and 4 segments in the RL zone. The separation of four zones in the walls of heart ventricles, each of them made up of different segments, may be helpful in the understanding of the pathophysiology of myocardial ischemia, and also in the choice of surgical strategies to treat aneurisms of the heart ventricle wall.

Adolescent↗

PubMed1993

Scanning electron microscopy of the deep lymphatic network of the murine lung as viewed in corrosion casts.

The lymphatic microvessels of the deep lung tissue were studied in corrosion casts, which were observed by scanning electron microscopy (SEM) after injection of a methacrylate resin (Mercox) through the trachea of CD-1 mice. We found that the deep lymphatics of the murine lung were composed of two interconnecting networks: a poorly developed capillary system located at the interacinar region, and a rich plexiform complex surrounding the bronchus. Lymphatic capillaries did not penetrate the alveolar area, thus leaving most of the lung parenchyma devoid of direct access to lymphatic drainage. Lung lymphatic vessels showed a small luminal surface and a low density of endothelial nuclei. Pulmonary lymphatic capillaries often formed star-like anastomoses. The structural features of pulmonary lymphatics, including their three-dimensional organization, were distinctly separate from those of the blood microvasculature of the lung.

Animals↗

PubMed1992

Blood vasculature of the lymph node in the dog: anatomical evidence for participation of extrahilar arterial vessels in the blood supply of the cortex.

The organization of the arterial vessels of dog lymph nodes (LN) was studied using methods of visualization of the vasculature by systemic injection of different tracers (colloidal carbon, Micropaque resin and methylmethacrylate) followed by observation of the samples by light microscopy (after clearing of the thick sections of LN) or scanning electron microscopy (corrosion casts). LN from all of the three groups of nodes studied (tracheobronchial, paratracheal and popliteal) showed an extensive network of arterial vessels encircling the capsule of the organ. We found that branches of these capsular arteries penetrated deeply into the cortical domain of LN. The capsule-originating vessels appeared to have a significant participation in the blood supply of the LN parenchyma at the cortical domains of the organs. Our findings are in contrast with current views on the angiology of the LN that consider that virtually all of the arterial capillaries of the LN parenchyma come from hilar arteries. We propose, therefore, that important segments of the LN cortex receive their blood supply from capsular arteries rather than from hilar vessels.

Animals↗

PubMed1992

Particle clearance from the canine pleural space into thoracic lymph nodes: an experimental study.

We instilled tungsten powder (CaWO4) into the pleural space of the dog and studied the kinetics and distribution of particle translocation from the pleural space to the thoracic lymph nodes over 1-7 days. We found that the transport of tungsten particles to regional lymph nodes was present at day 1, and reached its peak at day 3. In situ detection of tungsten by elemental particle analysis of lymph node sections by scanning electron microscopy complemented by light microscopy and X-ray analysis allowed precise mapping of the marker in the thoracic nodes. The first lymph nodes to become tungsten-laden was the parasternal group (day 1-3). From day 3 to 7 tungsten inclusions decreased in these parasternal nodes while moderately increasing in the remaining intrathoracic lymph nodes. Retrocardiac pleural folds containing numerous "milky spots" also accumulated prominent amounts of tungsten early after intrapleural injection of CaWO4. These data indicate that 1) particle translocation from the pleural space to regional lymph nodes is a rapid process and is first directed to the parasternal lymph nodal subgroup; 2) particle dissemination to virtually all other lymph nodes within the thorax follows thereafter; 3) retrocardiac pleural folds contribute to the clearance of particles from the pleural space.

Animals↗

PubMed1992

Microanatomy of the blood vasculature of lymph node follicles in the dog.

We studied the anatomical features of the arterial vasculature of the lymphoid follicles (LF) of tracheobronchial lymph nodes in the dog. The microvascular organization of the lymph node was visualized after systemic perfusion of the canine arterial system with gelatinated colloidal carbon. We found that LF contained a substantial number of blood capillaries forming a vascular network with a density comparable to that of neighboring domains of the node. The density of the vascular supply was greater in the secondary than in the primary LF. The geometric arrangement of LF capillaries was also different in the two types of LF. These findings are in contrast with the current view that the LF is a vascular-poor domain of the lymph node. We also document the plasticity of the LF blood microvasculature that appears to proliferate in conjunction with lymphoblast turnover and immune reactivity characteristic of secondary LF.

Animals↗

PubMed1992

[Image processing in medicine].

In this paper some basic concepts of Digital Image processing are presented, such as image enhancement and segmentation, and some of the activities in Medical Imaging of the Image Processing group of the University of Aveiro/INESC are described.

Diagnostic Imaging↗

PubMed1992

Blood vessel architecture in lymph nodes of the dog viewed by scanning electron microscopy.

Scanning electron microscopy was used to investigate the submicroscopic organization of the blood vessels of dog lymph nodes (LN). The LN vessels were casted by systemic perfusion of the animals vasculature with two resins of distinct viscosity. This resulted in the retrieval of two types of LN vascular replicas that depicted either the arterial blood system alone (methacrylate casting) or both the arterial and venous blood systems (Mercox casting). We found that the dog LN showed a significantly higher density of arterial vessels in the cortex than in the medulla. In the cortical domain, a subcapsular layer stood out because of its rich content in arterial capillaries. The use of the high-resolution resin (mercox) resulted in excellent structural detail of the luminal surface of the LN vessels that allowed a clear distinction between arterioles and venules based on the geometrical pattern of the imprints left in the replicas by the nuclei of endothelial cells. At the cortex-medulla frontier, most arterioles showed narrowings of their lumen that suggested the existence of sphincters at this level. Our findings document that the microanatomical arrangement of blood vessels in the LN of the dog is different from that of LN from other mammals studied so far, in particular from rodents where vascular-poor microdomains have been reported in the LN cortex. The arteriolar sphincters that we detected at the innermost zone of the cortex may represent the structural counterpart of the physiological modulation of the blood supply of the cortex exerted by arterial branches coming from the hilus.

Animals↗

PubMed1982

Determination of lipids, including medium-chain fatty acids, in human feces.

In this method for determining fecal lipids, feces are saponified with ethanolic potassium hydroxide, the ethanol is evaporated, and the acidified residue extracted with hexane. An aliquot of the hexane solution is then titrated with ethanolic sodium hydroxide, the solvents are evaporated, and the residue (soaps and unsaponifiable matter) is weighed. The unsaponifiable matter is extracted with acetone and determined gravimetrically. The method thus permits the determination of milli-equivalents of fatty acids, total lipids, and fatty acids in one sample and is applicable to feces containing medium-chain fatty acids and their triglycerides.

Fatty Acids↗

PubMed1976

Total fatty acid composition of duck fatty tissues.

Total lipids extracted from duck fatty tissues were fractionated on thin layer plates into polar lipids and neutral lipids. Neutral lipids were similarly fractionated into their components. Fatty acid methyl esters from total lipids were fractionated by gas-liquid-chromatography. Results indicated that duck fatty tissues are mostly formed by neutral lipids and that triglycerides comprise the vast majority of neutral lipids. Results also indicated that the major fatty acids in duck lipids are: oleic greater than linoleic greater than stearic greater than palmitoleic. About 73% of all fatty acids present belong to the C-18 series. The unsaturation level for duck lipids is about 73%.

Adipose Tissue↗

PubMed1975

Jejunal biopsies in protein-calorie malnutrition and intestinal parasitic infestation in Brazil.

Jejunal biopsies were performed in 71 subjects, who were classified into three groups as follows: Forty-one patients, on a poor diet, without clinical signs of malnutrition, with and without parasitic infestation; nineteen patients with severe protein-calorie malnutrition, with and without parasitic infestation; control group, ten medical students and one of the authors on a normal diet and without parasitic infestation. The dissecting microscope appearance showed mild alteration of the intestinal villi of Group A patients with parasitic infestation and severe alteration on Group B patients, especially those with parasitism. On histological examination, only the total mucosal thickness showed statistically significant differences between the Group B (severe protein malnutrition) and the other groups.

Adolescent↗