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Biomedical subjects

J Salgado

Publications and source records attributed to J Salgado.

At least 37 records · Page 2Linked to original sources
PubMed1997

Possible nosocomial transmission of psittacosis.

OBJECTIVES: To investigate a cluster of seven pneumonia cases among persons exposed to a pet-shop worker hospitalized with psittacosis. DESIGN: Epidemiological study of the outbreak, sero-diagnostic evaluation of exposed persons with pneumonia; and retrospective review of the pneumonia cases. SETTING: 450-bed teaching community hospital. PARTICIPANTS: Employees of the nursing unit where the patient with psittacosis was hospitalized. RESULTS: The index case had laboratory and clinical evidence of psittacosis. Four of the seven exposed persons who developed pneumonia had serologic results that met criteria for a diagnosis of psittacosis, and three met criteria for Chlamydia pneumoniae infection, possibly reflecting cross-reactivity between the microimmunofluorescence assays for the two diseases. The epidemiological graph suggested a relationship between the index case and the cluster of pneumonias in exposed individuals. CONCLUSIONS: Psittacosis may have been transmitted person to person in a hospital setting.

Chlamydophila psittaci↗
PubMed1996

Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A.

Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.

Amino Acid Sequence↗
PubMed1996

Analysis of the paramagnetic copper(II) site of amicyanin by 1H NMR spectroscopy.

Application of the tailored pulse sequences like super-WEFT allows the direct observation of the hyperfine-shifted signals of the paramagnetic Cu(II) forms of blue copper proteins in solution. The signals can be assigned by applying 2D NMR techniques, like EXSY, to solutions containing a mixture of reduced and oxidized species. The Fermi contact shift is separated from the pseudocontact shift on the basis of the known g-tensor anisotropy of the Cu(II) state, allowing the determination of a number of hyperfine-splitting constants between protons on the Cu ligands and the unpaired electron. These results are used to quantify the spin density distribution over the Cu ligands. In amicyanin about 50%-60% of the unpaired electron density is found on the ligands. It appears possible to quantify the Cu-S(Met) interaction on the basis of the NMR results. Application of the technique to the wild type forms of amicyanin and azurin and to two active site mutants of amicyanin (His96Asp and a plastocyanin-amicyanin loop exchange mutant) shows that the Cu-S(Met) interaction parallels the rhombicity and axial distortion of the Cu site.

Amino Acid Sequence↗
PubMed1996

Paramagnetic cobalt and nickel derivatives of Alcaligenes denitrificans azurin and its M121Q mutant. A 1H NMR study.

Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic 1H NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin, Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His46, and His117 metal ligands suggest a movement of the metal ion out of the equatorial plane, indicating that the metal site is tetrahedral. These effects are less pronounced in the Ni(II) M121Q azurin than in the Co(II) metalloderivative. The similarity between the NMR spectra of the Co(II) derivatives of stellacyanin and the M121Q azurin is in agreement with a very similar metal site in both proteins and supports the existence of a coordinated Gln in stellacyanin.

Alcaligenes↗
PubMed1996

Characterization of allergoids from ovalbumin in vitro and in vivo.

Several in vivo and in vitro methods for monitoring immunological properties of two allergoids obtained by formaldehyde treatment of ovalbumin (OA) were developed. The calculated molecular weight of allergoids was 80 kD (OA-F1) and 165 kD (OA-F2), respectively. The allergenic activity in vitro of allergoids in mast-cell histamine release assay was 1000 times lower than of OA. Both allergoids showed reduced ability to induce passive cutaneous anaphylaxis in the Sprague-Dawley rats or systemic anaphylaxis in Dunkin-Harley guinea-pigs. The ability of OA and allergoids to bind to the OA-specific IgE antibodies was measured in vivo by the inhibition of passive cutaneous anaphylaxis (PCA-inhibition). Allergoid binding to IgE was 51-66% lower than the native allergen. Moreover, the avidity of OA-specific IgG antibodies, measured by ELISA-inhibition, for allergoids and allergen was of the same order. Allergoids induced a different pattern of humoral immune response from that, induced by the native allergen. Thus, after immunization of BALB/c mouse, both allergoids induced a higher production of IgG and a lower production of IgE than OA, only OA-F2 induced a lower production of IgG1. The differences in the IgA response to the immunogens was not significant. Delayed hypersensitivity studies in the BALB/c mouse showed that allergoids were 5- to 12-times less effective in inducing a cell-mediated immune response than OA. The present study provides a battery of immunological methods for preclinical testing of modified allergens.

Allergens↗
PubMed1995

Metastability of dimiristoylphosphatidylethanolamine as studied by FT-IR and the effect of alpha-tocopherol.

The metastability of dimiristoylphosphatidylethanolamine (DMPE) has been studied by means of Fourier transform infrared spectroscopy (FT-IR), both in the absence and in the presence of alpha-tocopherol. Two different methods of hydration were used to prepare the samples, poorly hydrated and well hydrated, and the results have been compared with anhydrous DMPE. Poorly hydrated DMPE gave place to a high-melting phase formed upon melting from gel to L alpha at approx. 49 degrees C, with a new transition to L alpha at approx. 55 degrees C. However, well hydrated DMPE incubated at 4 degrees C for 49 days gave place to a subgel phase which was transformed by heating into a L beta phase at about 40 degrees C and this into a L alpha phase after further heating at 52 degrees C. The subgel phase was more hydrated and less rigid than the high-melting phase. On the other hand, alpha-tocopherol, when included in poorly hydrated DMPE, stabilized a high-melting phase, which was transformed by heating, directly into a L alpha. However, when a sample of DMPE containing alpha-tocopherol was incubated for 49 days at 4 degrees C a dehydrated solid phase different from the subgel and the high-melting phases was formed.

Antioxidants↗
PubMed1995

1H-NMR study of a cobalt-substituted blue copper protein: Pseudomonas aeruginosa Co(II)-azurin.

Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 1H-NMR spectrum of Pseudomonas aeruginosa Co(II)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact-shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(II)-azurin, i.e. His46, His117, Cys112, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(II) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N2S plane towards the Gly45 O axial position and weakly interacting with the Met121 sulfur.

Azurin↗
PubMed1995

The crystal structure of nickel(II)-azurin.

The nickel(II)-azurin metalloderivative has been crystallized and its structure solved at 0.205-nm resolution by X-ray diffraction. The overall structure is not modified by the metal exchange and the only differences with regard to the native copper(II)-azurin occur in the metal site region. These variations affect principally the axial ligands. Nickel co-ordinates more strongly to the carbonyl oxygen of Gly45 while its distance to the Met121 S4 enlarges up to 0.330 nm. The resulting metal center structure is intermediate between those of the Cu(II) and Zn(II) azurins, and can be described as distorted tetrahedral. However, the existence of contact interaction between Met121 and the nickel ion is still possible as has been shown by paramagnetic 1H-NMR studies in solution.

Azurin↗
PubMed1995

Effects of cyclosporine and dexamethasone on IgE antibody response in mice, and on passive cutaneous anaphylaxis in the rat.

The suppressive effects of cyclosporine A (CsA) and dexamethasone (Dxt) on antigen-specific IgE responses to ovalbumin (OA) were studied in BALB/c mice. The effects upon other isotypes were also analyzed. The antiovalbumin IgE response did not change when low doses of CsA [8 mg/kg intraperitoneally (i.p.)] were administered; IgA also remained unchanged, while IgG and IgG1 decreased significantly. At higher CsA doses (16 mg/kg i.p. or orally), a decrease was noted for all the ispotypes assayed. Dxt administered orally at 0.3 mg/kg selectively inhibited IgE and IgA but did not influence IgG or IgG1 levels. Delayed hypersensitivity reactions to OA were not modified by CsA, but were depressed by Dxt. Although CsA had not effect on passive cutaneous anaphylaxis in the rat, Dxt significantly reduced this reaction when it was administered 6 h before challenge. These results suggest that Dxt has more specific antiallergic activity than CsA.

Animals↗
PubMed1994

Interaction of cobalt ions with carboxypeptidase A.

The interaction of cobalt(II) with native and cobalt(II)-substituted carboxypeptidase has been investigated, at pH 7.5, by electronic absorption and 1H NMR spectroscopies. The reaction of the cobalt(II) uptake by the metalloenzyme [MCPA] (M = Zn or Co) occurs very slowly and a bimetallic complex, [MCPA(Co)], is formed. On the basis of the 1H NOE experiments, the isotropically shifted proton resonances were assigned as belonging to a coordinated histidine residue. 1H NMR titrations of [ZnCPA(Co)] with zinc(II) show that the zinc ion does not compete with cobalt for binding to the noncatalytic site. The temperature dependence of the isotropic shifts, molar absorbance, and longitudinal relaxation time values are indicative of a five-coordinated geometry for the cobalt ion. The identification of the noncatalytic cobalt binding site is also discussed.

Animals↗
PubMed1993

Alpha-tocopherol interacts with natural micelle-forming single-chain phospholipids stabilizing the bilayer phase.

The effect of the association of alpha-tocopherol (vitamin E) with single chain or very asymmetric phospholipids has been studied by 31P nuclear magnetic resonance, Fourier transform infrared spectroscopy, and light microscopy. Our results demonstrate that alpha-tocopherol stabilizes and forms bilayer structures in systems composed of either 1-palmitoyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-sn-glycero-3-phosphocholine, or 1,2-dipalmitoyl-sn-glycero-3- phosphocholine/1-palmitoyl-sn-glycero-3-phosphocholine, whereas in the absence of alpha-tocopherol all these systems produce micellar structures. The free hydroxyl group of alpha-tocopherol is shown to be important in stabilizing the bilayer structure by comparing the effects produced by an analog of alpha-tocopherol, alpha-tocopheryl acetate, which has the hydroxyl group blocked by an acetyl group. It is suggested that the complementary shapes of alpha-tocopherol and the asymmetric phospholipids may be the reason for the stabilization of the bilayer structure.

Lipid Bilayers↗
PubMed1993

Two-dimensional 1H NMR spectra of ferricytochrome c551 from Pseudomonas aeruginosa.

The full assignment of 1H NMR signals of heme proton resonances of ferricytochrome c551 from Pseudomonas aeruginosa has been performed by means of 2D NMR experiments. This technique allows the complete and unequivocal assignment of all heme resonances, including methylene resonances of the propionic groups, directly implicated in the pH dependence of the redox properties of cytochrome c551.

Bacterial Proteins↗
PubMed1993

Effects of platelet-activating factor and related lipids on dielaidoylphosphatidylethanolamine by DSC, FTIR and NMR.

The effect of platelet-activating factor (1-O-hexadecyl-2-acetyl-sn- glycero-3-phosphocholine, PAF) and two related molecules, 1-O-hexadecyl-sn-glycero-3-phosphocholine (LPAF) and 1-palmitoyl-sn-glycero-3- phosphocholine (LPC) on dielaidoylphosphatidylethanolamine (DEPE) lipid structure and polymorphism has been studied by differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) and 31P nuclear magnetic resonance (31P-NMR) spectroscopies. From the interaction of these molecules with DEPE it is concluded that all of them stabilize the lamellar phase with respect to the hexagonal HII phase and this effect is clear even at concentrations of these compounds as low as 1 mol%. It is also shown that, although they perturb the gel to liquid-crystalline phase transition of DEPE up to a similar extent, fluidizing the membrane, PAF but not LPAF or LPC, induces the presence of more than one peak in the calorimetric profile. Moreover, FTIR data indicate that lateral phase separations formed by PAF-rich phases are taking place. Remarkably, delta H of the main transition decreases at concentrations lower than 2 mol% but remains nearly constant up to 30 mol%. 31P-NMR measurements showed that all these molecules were capable of inducing isotropic signals in the spectra produced by molecules associated to membranes before micellization of the vesicles.

Calorimetry, Differential Scanning↗