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Biomedical subjects

A Pacheco

Publications and source records attributed to A Pacheco.

At least 19 recordsLinked to original sources
PubMed2000

An MCD spectroscopic study of the molybdenum active site in sulfite oxidase: insight into the role of coordinated cysteine.

Temperature-dependent magnetic circular dichroism (MCD) spectroscopy has been used for the first time to probe the electronic structure of the Mo active site in sulfite oxidase (SO). The enzyme was poised in the catalytically relevant [Mo(V):Fe(II)] state by anaerobic reduction of the enzyme with the natural substrate, sulfite, in the absence of the physiological oxidant cytochrome c. The [Mo(V):Fe(II)] state is of particular importance, as it is proposed to be a catalytic intermediate in the oxidative half reaction, where SO is reoxidized to the resting [Mo(VI):Fe(III)] state by two sequential one-electron transfers to cytochrome c. The MCD spectrum of the enzyme shows no charge transfer transitions below approximately 17000 cm(-1). This has been interpreted to result from (1) a severe reduction in ene-1,2-dithiolate sulfur in-plane and out-of-plane p orbital mixing, (2) a decrease in the dithiolate sulfur out-of-plane p-Mo d(xy) orbital overlap, and (3) an orthogonal orientation between the vertical cysteine sulfur p (perpendicular to the Mo-Scys sigma-bond) and Mo d(xy) orbitals. The spectroscopically determined cysteine sulfur p-Mo d(xy) bonding scheme in the [Mo(V):Fe(II)] state is consistent with the crystallographically determined O-Mo-Scys-C dihedral angle of approximately 90 degrees and precludes a covalent interaction between the vertical cysteine sulfur p orbital and Mo d(xy), effectively decoupling the cysteine from an effective through-bond electron transfer pathway. We have tentatively assigned a 22250 cm(-1) positive C-term feature in the MCD as the cysteine S(sigma)-->Mo d(xy) charge transfer that becomes allowed by a combination of configuration interaction and low-symmetry; however, the orbital overlap is anticipated to be quite small due to the near orthogonality of these orbitals. Therefore, we propose that the primary role of the coordinated cysteine is to decrease the effective nuclear charge on Mo by charge donation to the metal, statically poising the active site at more negative reduction potentials during electron transfer (ET) regeneration. Finally, the results of this study are consistent with the pyranopterin ene-1,2-dithiolate acting to couple the Mo site into efficient superexchange pathways for ET regeneration following oxygen atom transfer to the substrate.

Animals↗
PubMed2000

Primary cutaneous CD30-positive large cell lymphoma.

Primary cutaneous CD30-positive large cell lymphoma is a recently described entity, recognized by immunohistochemical criteria. Spontaneous regression may occur and although relapses are frequent, most patients have a favourable prognosis. We describe a 83-year-old woman with a large ulcerated tumour on her right forearm, with 2 months evolution. Histological and immunohistochemical examination revealed a CD30-positive large cell lymphoma. Systemic involvement was not detected. The tumour regressed spontaneously. Cutaneous relapses occurred one year later, all with spontaneous regression. After a 2-year follow-up period, the patient is well, without concurrent extracutaneous disease. This case is in keeping with other reports of spontaneous regression and favourable outcome of this type of cutaneous lymphoma.

Aged↗
PubMed2000

Irritant-induced asthma: clinical and functional aspects.

We report on three patients who experienced persistent asthma symptoms after repetitive irritant exposure which took place over a period from several days to months. Airway inflammation was assessed by induction of sputum and functional follow-up information was obtained from serial lung function tests. All patients had bronchial hyperresponsiveness to methacholine at the time of diagnosis. However, induced sputum samples did not show increased differential count of eosinophils. Treatment with inhaled corticosteroids was started in all of the patients and two of them were removed from work. In the two patients who left the workplace, methacholine inhalation test became negative when symptoms disappeared, whereas the patient who continued working had persistent asthma symptoms and a deterioration of bronchial hyperresponsiveness.

Adult↗
PubMed1999

The pH dependence of intramolecular electron transfer rates in sulfite oxidase at high and low anion concentrations.

The individual rate constants for intramolecular electron transfer (IET) between the Mo(VI)Fe(II) and Mo(V)Fe(III) forms of chicken liver sulfite oxidase (SO) have been determined at a variety of pH values, and at high and low anion concentrations. Large anions such as EDTA do not inhibit IET as dramatically as do small anions such as SO4(2-) and Cl-, which suggests that specific anion binding at the sterically constrained Mo active site is necessary for IET inhibition to occur.IET may require that SO adopt a conformation in which the Mo and Fe centers are held in close proximity by electrostatic interactions between the predominantly positively charged Mo active site, and the negatively charged heme edge. Thus, small anions which can fit into the Mo active site will weaken this electrostatic attraction and disfavor IET. The rate constant for IET from Fe(II) to Mo(VI) decreases with increasing pH, both in the presence and absence of 50 mM SO4(2-) . However, the rate constant for the reverse process exhibits no significant pH dependence in the absence of SO4(2-), and increases with pH in the presence of 50 mM S04(2-). This behavior is consistent with a mechanism in which IET from Mo(V) to Fe(III) is coupled to proton transfer from Mo(V)-OH to OH-, and the reverse IET process is coupled to proton transfer from H2O to Mo(VI) = O. At high concentrations of small anions, direct access of H2O or OH- to the Mo-OH will be blocked, which provides a second possible mechanism for inhibition of IET by such anions. Inhibition by anions is not strictly competitive, however, and Tyr322 may play an important intermediary role in transferring the proton when an anion blocks direct access of H2O or OH- to the Mo-OH. Competing H-bonding interactions of the Mo-OH moiety with Tyr322 and with the anion occupying the active site may also be responsible for the well-known equilibrium between two EPR-distinct forms of SO that is observed for the two-electron reduced enzyme.

Anions↗
PubMed1999

Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in </=3 ps, and I(0)(double dagger), which is formed in 220 ps, as well as stimulated emission from the PYP excited state. In the present study, kinetic measurements at two excitation wavelengths (395 nm and 460 nm) on either side of the PYP absorption maximum (446 nm) were undertaken using 100-fs pump and probe pulses. Global analysis over a range of probe wavelengths yielded time constants of 1.9 ps for the photochemical formation of the I(0) intermediate via the PYP excited state, and 3.4 ps for the repopulation of the ground state from the excited state. In addition to these pathways, 395 nm excitation also initiated an alternative route for PYP excitation and photochemistry, presumably involving a different excited electronic state of the chromophore. No photochemical intermediates formed before I(0) were observed. Based on these data, a quantum yield of 0.5-0.6 for I(0) formation was determined. The structural and mechanistic aspects of these results are discussed.

Bacteria↗
PubMed1999

Fluoxetine versus amitriptyline in the treatment of major depression with associated anxiety (anxious depression): a double-blind comparison.

Although common in clinical settings, major depressive disorder with associated anxious symptoms ('anxious depression') has not been well studied in antidepressant clinical trials. The aim of this study was to compare the effects of fluoxetine versus amitriptyline in this group of patients. After a single-blind placebo run-in period of 2 weeks, patients were treated on a double-blind basis with fluoxetine or amitriptyline for 8 weeks. Assessment instruments included: 21-item Hamilton Rating Scale for Depression, Hamilton Rating Scale for Anxiety, Clinical Global Impressions, Raskin Depression Scale and Covi Anxiety Scale. A total of 157 patients were randomized to either fluoxetine or amitriptyline. Fluoxetine was given at a fixed dose of 20 mg/day and amitriptyline was given in a range of 50-250 mg/day (mean of 138.1 mg/day). Fluoxetine was comparable to amitriptyline in all efficacy measures except the HAMD sleep factor. Unwanted effects were more frequent and more severe in the amitriptyline-treated patients. Fluoxetine was comparably efficacious to amitriptyline in the treatment of major depression with associated anxiety. Since fluoxetine was far better tolerated, it is a promising alternative for this frequent and disabling condition.

Adult↗
PubMed1998

Eosinophilic lung disease associated with Candida albicans allergy.

A significant number of cases of chronic eosinophilic pneumonia remain idiopathic in spite of a comprehensive search of associated causes. This study reports a patient with a classical clinical presentation of chronic eosinophilic pneumonia and peripheral blood eosinophilia in whom selective sensitization to Candida albicans was demonstrated. This yeast was present in the bronchoalveolar lavage culture and specific serum immunoglobulin (Ig)E and IgG against C. albicans were found in the patient's serum. Levels of these specific immunoglobulins diminished with corticosteroid treatment and increased coinciding with a new outbreak of symptoms after lowering the dosage of corticosteroids. To the author's knowledge, this is the first case described of chronic eosinophilic pneumonia associated with sensitization to C. albicans. Evaluation of allergy to C. albicans should be performed in chronic eosinophilic pneumonia before labelling cases as idiopathic.

Adult↗
PubMed1997

Molecular basis of sulfite oxidase deficiency from the structure of sulfite oxidase.

The molybdenum-containing enzyme sulfite oxidase catalyzes the conversion of sulfite to sulfate, the terminal step in the oxidative degradation of cysteine and methionine. Deficiency of this enzyme in humans usually leads to major neurological abnormalities and early death. The crystal structure of chicken liver sulfite oxidase at 1.9 A resolution reveals that each monomer of the dimeric enzyme consists of three domains. At the active site, the Mo is penta-coordinated by three sulfur ligands, one oxo group, and one water/hydroxo. A sulfate molecule adjacent to the Mo identifies the substrate binding pocket. Four variants associated with sulfite oxidase deficiency have been identified: two mutations are near the sulfate binding site, while the other mutations occur within the domain mediating dimerization.

Amino Acid Sequence↗
PubMed1996

Herpes virus saimiri transformation of T cells in CD3 gamma immunodeficiency: phenotypic and functional characterization.

The characterization of T cell immunodeficiencies could in part be supported by using stable cell lines in which biochemical and molecular studies of the defect could be carried out thereby omitting frequent bleeding of patients. First attempts to obtain such cell lines included HTLV-I transformation and exogenous IL-2 administration, but both models have important disadvantages. Recently, a virus isolated from the squirrel monkey, Herpes virus saimiri (HVS), has been reported to have the ability to transform T cells. A stable IL-2-dependent HVS-transformed T cell line from a CD3 gamma deficient patient has been obtained; and this cell line displays both the phenotypic and the functional characteristics of the patient's lymphocytes. Moreover, the line down-modulates TCR/CD3 surface expression upon CD3 engagement, as do the patient's lymphocytes, showing that CD3 gamma and its phosphorylation are not necessary for TCR/CD3 internalization. In addition, the abnormal staining pattern of different anti-TCR/CD3 monoclonal antibodies is preserved in the HVS-patient line. Since HVS is capable of transforming CD3 gamma- T cells, the CD3 gamma chain does not seem to be involved in the HVS receptor process. The fact that it is not possible to obtain a CD8+ HVS line from the CD3 gamma- patient supports the existence of a functional anomaly in his scanty CD8+ peripheral lymphocytes. Thus, HVS transformation is a suitable model for T cell immunodeficiency studies and characterization. It may also be used in the future in cellular models for in vitro gene therapy trials.

CD3 Complex↗
PubMed1994

Primary T lymphocyte immunodeficiency associated with a selective impairment of CD2, CD3, CD43 (but not CD28)-mediated signal transduction.

A 2-year-old female with important signs of immune response failure against virus, bacteria, fungi and protozoa and no obvious humoral or lymphocyte phenotypical defect was studied. Both peripheral blood mononuclear cells and IL-2-dependent T cell lines derived from the patient showed a severe selective T cell activation impairment via CD2, CD3 and CD43; however, this defect was reversible with the addition of either IL-2, or phorbol myristate acetate (PMA) or anti-CD28 antibodies. Concordantly, the induction of IL-2 (and, in part, IL-3 and IL-4) messenger RNA was severely reduced in stimulated T cells, but that of other cytokines was either normal (IL-5) or only slightly diminished (interferon-gamma (IFN-gamma)). It is concluded that an activation T cell defect exists previous to protein kinase C (PKC) and between membrane receptors and the activation pathway of certain response genes encoding for interleukins involved in proliferation (i.e. IL-2, IL-3 and IL-4), but not of others (i.e. IL-5). The use of T cell lines from human T lymphocyte activation deficiencies allows dissection of T cell pathology and the corresponding physiological pathways. In the present description, there is an evident independence of the CD28 T cell activation pathway from those induced through CD2 or CD3, and the differential gene regulation of the different interleukins.

Antigens, CD↗
PubMed1994

Is sodium acetate dextran superior to sodium chloride dextran for small volume resuscitation from traumatic hemorrhagic shock?

Small volumes (4 mL/kg body weight (bw)) of hypertonic sodium chloride dextran effectively restore cardiac output and nutritional blood flow and increase arterial pressure in severe hemorrhagic shock. It has been suggested that the chloride anion be replaced with acetate to provide a solution that avoids the risk of hyperchloremia and has the advantage of supplying a buffering base to optimize hypertonic resuscitation. This study compares the effects of hypertonic sodium chloride dextran solution (7.2% NaCl/10% dextran 60 [NaCl-Dx]; n = 7) with sodium acetate dextran (10.4% Na-Ac/10% dextran 60 [NaAc-Dx]; n = 6) on hemodynamic, oxygen transport, and metabolic variables. Both solutions had the identical osmolality (2400 mOsmol/kg). Dogs (16.9 +/- 1.9 kg) were anesthetized and mechanically ventilated. Shock was induced by exteriorization of intestine and blood withdrawal (50% of blood volume) to maintain mean arterial blood pressure (MAP) at 40 mm Hg for 75 min. Thereafter, resuscitation was performed either with NaCl-Dx (4 mL/kg over 2 min) or NaAc-Dx (4 mL/kg over 4 min). During hypertonic resuscitation, there was a short-lasting decrease in MAP, which was more pronounced in the NaAc-Dx group (delta MAP -7.3 +/- 2.5 mm Hg). Cardiac index and oxygen consumption were normalized within 5 min after resuscitation with both solutions. In NaAc-Dx-treated animals, MAP remained at lower values as compared to NaCl-Dx-treated dogs at 5 and 30 min after resuscitation (52 +/- 3 vs 74 +/- 6, and 61 +/- 7 vs 79 +/- 12 mm Hg; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1993

A decrease in the estimated frequency of the extended HLA haplotype B18 CF130 DR3 DQw2 is common to non-insulin-dependent diabetes, juvenile rheumatoid arthritis, and Berger's disease.

Extended HLA haplotypes frequencies were estimated from the HLA, C2, Bf and C4 phenotypes of 74 patients with non-insulin-dependent diabetes (NIDD), 92 with juvenile rheumatoid arthritis (JRA), 44 with Berger's disease (BD), 83 with insulin-dependent diabetes (IDD), and 140 healthy controls. The extended HLA haplotype B18 CF130 DR3 DQw2, which is common (around 10% phenotype frequency) in healthy Spaniards and in other populations of paleo-North African origin, was found to be significantly less frequent in NIDD, JRA and BD, whereas its frequency was normal in IDD (although DR3 DQw2 haplotypes were increased in the latter disease). These data support the existence of a common HLA-linked pathogeneic mechanism in NIDD, JRA and BD, and point to a genetic difference between IDD and NIDD at the HLA level. This effect is readily detectable in our population because the uncommon BfF1 allele marks that haplotype instead of the more common BfS, which marks B8 CS01 DR3 DQw2 in other Caucasians. Our results support the hypothesis of strong selective pressures operating at the HLA level to preserve extended HLA haplotypes with advantageous gene sets from dilution by crossing-over. Imbalanced incomplete haplotypes may give rise to inappropriate T-cell repertoire selection in the thymus and/or antigen handling in the periphery, and be partly responsible for the pathogenesis of certain HLA-linked diseases (i.e. NIDD, JRA, and BD).

Alleles↗
PubMed1993

Selective disbalances of peripheral blood T lymphocyte subsets in human CD3 gamma deficiency.

The selection of T lymphocytes in the thymus and their activation upon the encounter with foreign antigens in the periphery require the aggregation and signals of the T cell receptor (TcR)/CD3 complex and several surface molecules termed coreceptors (notably CD4 or CD8 and CD45). The spatial arrangement and interactions of the different molecules in the resulting multimolecular recognition structure are mostly unknown. Here we report, from studies on a healthy human CD3 gamma deficiency, that the lack of the CD3 gamma component of the TcR/CD3 complex is associated with a long-term severe defect of peripheral blood CD4+ CD45RA+ and CD8+ lymphocytes, whereas CD4+CD45RO+, B and natural killer lymphocytes are unaffected. These results suggest that the CD3 gamma site of the TcR/CD3 complex is required for the peripheral representation of certain T cell types.

CD3 Complex↗