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Biomedical subjects

P A Fernandes

Publications and source records attributed to P A Fernandes.

At least 19 recordsLinked to original sources
PubMed2006

Detailed microscopic study of the full zipA:FtsZ interface.

Protein-protein interaction networks are very important for a wide range of biological processes. Crystallographic structures and mutational studies have generated a large number of information that allowed the discovery of energetically important determinants of specificity at intermolecular protein interfaces and the understanding of the structural and energetic characteristics of the binding hot spots. In this study we have used the improved MMPB/SA (molecular mechanics/Poisson-Boltzmann surface area) approach that combining molecular mechanics and continuum solvent permits to calculate the free energy differences upon alanine mutation. For a better understanding of the binding determinants of the complex formed between the FtsZ fragment and ZipA we extended the alanine scanning mutagenesis study to all interfacial residues of this complex. As a result, we present new mutations that allowed the discovery of residues for which the binding free energy differences upon alanine mutation are higher than 2.0 kcal/mol. We also observed the formation of a hydrophobic pocket with a high warm spot spatial complementarity between FtsZ and ZipA. Small molecules could be designed to bind to these amino acid residues hindering the binding of FtsZ to ZipA. Hence, these mutational data can be used to design new drugs to control more efficiently bacterial infections.

Alanine↗
PubMed2006

Determination of the DeltapKa between the active site cysteines of thioredoxin and DsbA.

Thioredoxin superfamily members share a considerable degree of structural similarity, with a conserved CX(i)X(j)C motif at the active site, where C stand for two cysteines that alternate between a reduced thiol and oxidized disulfide states, and X(i)and X(j) are two amino acids different in each family member. Despite these similarities, they display very different redox potentials and pKas for the active site dithiol, and fulfill different physiological roles. Thioredoxin, for example, promotes the reduction of disulfide bonds, while DsbA promotes their oxidation in prokaryotic cells. The factors that promote these differences are still not fully understood. However, it is generally accepted that the different stabilities of the redox active disulfide bond depends on the degree of stabilization, in the reduced state, of the thiolate of one of the active site cysteines (nucleophilic cysteine). In this work we have used QM/MM methods to compare and characterize the active site dithiols of both enzymes, and to shed some light on the structural features responsible for the large differences in pKa and redox potential between two homologous enzymes, thioredoxin and DsbA. We have also analyzed the main factors pointed out in the literature as responsible for their different properties. We obtained the value of 4.5 for pKa difference (DeltapKa) between the nucleophilic cysteines of both enzymes, which is in excellent agreement with most of the experimental values. Additionally, we found that the principal differentiating factor responsible for this observed DeltapKa are the alpha2-alpha helices, which greatly contribute to the mentioned value, by stabilizing the DsbA thiolate in a much greater extend than the thioredoxin thiolate. A double mutation of the conserved residues Asp26 and Lys57, in thioredoxin, and Glu24 Lys58, in DsbA, by alanines did not change the DeltapKa value; this supports the hypothesis that these residues are not involved in the differentiation of the properties of the active centre dithiol. However, we found out that these residues are important for the stabilization of the nucleophilic thiolate. The X(i) and X(j) residues also do not seem to promote the stabilization of the thiolates. In fact, the corresponding double alanine mutants are more stable than the wild-type enzymes. However, these residues are involved in the differentiation between thioredoxin and DsbA, stabilizing the DsbA thiolate by a larger extent than the thioredoxin thiolate.

Amino Acid Substitution↗
PubMed2006

Molecular dynamics model of unliganded HIV-1 reverse transcriptase.

HIV-1 RT is one of the most important antiviral targets in the treatment of acquired immunodeficiency syndrome (AIDS). Several crystallographic structures are available for this enzyme, mostly with bound inhibitors. Despite their importance for structure based drug design towards new anti-HIV retrovirals, the X-ray structures of the unliganded enzyme could only be obtained incomplete, with a low resolution and until recently even the conformation of the p66 thumb was controversial. In this work we have aligned different X-ray RT structures, and built up a computational model of RT using homology modeling, which was afterwards refined and validated through MD simulations with explicit solvent. The model enzyme was structurally stable through the whole MD simulation, showing a RMSD of 2 Angstrom from the starting geometry. The Ramanchandram plot has improved along the simulation. Both intra-domain and interdomain movements were observed. The thumb kept its closed conformation through the whole simulation. A contact map, hydration sites study and a detailed analysis of the solvation of the nucleotide binding site are also presented.

Binding Sites↗
PubMed2003

New designs for MRI contrast agents.

New designs for Magnetic Resonance Imaging contrast agents are presented. Essentially, they all are host-guest inclusion complexes between y-cyclodextrins and polyazamacrocycles of gadolinium (III) ion. Substitutions have been made to the host to optimise the host-guest association. Molecular mechanics calculations have been performed, using the UFF force field for metals, to decide on the suitability of the substitutions, and to evaluate the host-guest energies of association. Interesting general conclusions have been obtained, concerning the improvement of Magnetic Resonance Imaging contrast agents; namely, a set of rational methodologies have been deduced to improve the association between the gadolinium (III) chelates and the cyclodextrins, and their efficiency is demonstrated with a large set of substituted complexes, opening new doors to increase the diagnostic capabilities of Magnetic Resonance Imaging.

Contrast Media↗
PubMed2000

Flocculation of Saccharomyces cerevisiae is induced by transformation with the GAP1 gene from Kluyveromyces marxianus.

A non-flocculent strain of Saccharomyces cerevisiae was transformed with the GAP1 gene which encodes p37, a GAPDH-like protein present in the cell wall of Kluyveromyces marxianus flocculent cells. The transformed cells were characterized with respect to flocculation behaviour, morphology, growth, cell wall integrity and GAPDH activity. A flocculent phenotype was acquired by the transformed cells, showing a behaviour in respect to flocculation/deflocculation very similar to that of K. marxianus. The presence of p37 in the cell wall was assessed by immunoprecipitation of biotinylated cell wall proteins and an accumulation of p37 was evident in the cell wall of transformed cells. This result was confirmed by studies using a chimeric protein resulting from fusing the p37 with a yeast-enhanced green fluorescent protein, yEGFP. The recombinant protein was localized mainly in the cell wall of the transformed strain, although the presence of p37 in the cytosol was indicated by an increase in GAPDH activity. Calcofluor white sensitivity tests indicated that the cell wall structure is affected by the accumulation of p37. These results provided further evidence of p37 function regarding flocculation and that although lacking a N-terminal signal peptide p37 is targeted to the cell wall.

Biomass↗
PubMed1995

Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene family from Kluyveromyces marxianus--polymerase chain reaction-single-strand conformation polymorphism as a tool for the study of multigenic families.

Three glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Kluyveromyces marxianus were identified and characterized. The coding region of two of them (GAP2 and GAP3) is very similar (99.6% homology). The other gene (GAP1) is only 86% homologous to GAP2 or GAP3 and is responsible for the expression of Gap1p. This protein is extremely homologous to the K. marxianus cell wall protein p37, presumably involved in flocculation. However, no leader sequence could be detected in this gene. The identification of the three genes was possible with the use of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), as it permits us to overcome the difficulties caused by the high homology amongst the genes. Expression of the GAPDH genes under different carbon sources (glucose or ethanol) was assessed either by Northern blot or reverse transcription-PCR-SSCP analysis, revealing that genes GAP1 and GAP2, but not GAP3, are transcribed. The results also indicate that the transcription of the gene encoding the cell wall protein p37 (Gap1p) is not dependent on the carbon source, in contrast with the expression of the gene GAP2, which is affected in cells growing in a glucose-depleted medium.

Amino Acid Sequence↗
PubMed1993

Flocculation of Kluyveromyces marxianus is induced by a temperature upshift.

An upshift of the growth temperature from 26 to 40 degrees C in the presence of calcium leads to the aggregation of Kluyveromyces marxianus cells and to the formation of flocs. Analysis of cell wall proteins, either by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of extractable mannoproteins or by immunolocalization, revealed an accumulation of a protein with Mr 37 kDa(p37), upon flocculation. Immunological studies confirmed the homology of this protein with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When mRNA isolated from cells growing at 40 degrees C was translated in vitro, a 35 kDa newly labelled protein was synthesized and immunoprecipitation assays showed that this protein is recognized by p37-antiserum, suggesting that the 35 kDa polypeptide might be an unglycosylated precursor for of p37. The results indicated that the presence of this cell wall mannoprotein closely related to GAPDH is dependent on the growth temperature, suggesting its role as adhesin.

Calcium↗
PubMed1992

A protein homologous to glyceraldehyde-3-phosphate dehydrogenase is induced in the cell wall of a flocculent Kluyveromyces marxianus.

A protein with an apparent molecular weight of 37,000 (p37) is present in very large amounts in the cell wall of Kluyveromyces marxianus, after the induction of flocculation of the yeast. This protein was isolated by preparative gel electrophoresis and its purity checked by SDS-PAGE and reverse-phase HPLC. SDS-PAGE, endoglycosidase-H treatment and peptide sequencing indicated that p37 is a glycoprotein with a high identity to cytosolic glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae. Polyclonal antibodies were used for Western blot analysis and immunocytochemistry, which showed that p37 is present in the cell wall of non-flocculent K. marxianus and, therefore, is a constitutive protein of the cell wall.

Amino Acid Sequence↗
PubMed1990

Quantitative analysis of the effect of freezing on donor sperm motion kinetics.

One hundred ninety-two semen specimens from 14 donors were analyzed on a CellSoft Semen Analyzer (CRYO Resources, New York, NY) before and after freezing. Mean post-thaw motility decreased by 52%. Correlation of the percent decrease in post-thaw versus prefreeze motility was significant but of poor predictive value. Comparisons of the percent change in post-thaw motility and sperm motion kinetics between four discrete ranges of prefreeze motility (32% to 66%, 67% to 76%, 77% to 84%, 85% to 94%) revealed that the effect of freezing on sperm cell survival was equivalent between all ranges. However, significant differences occurred between these ranges for curvilinear velocity, straight line velocity, and mean amplitude of lateral head displacement but not for linearity nor beat cross frequency. All correlations between prefreeze and post-thaw motion variables were significant but closest for curvilinear velocity and straight line velocity. Furthermore, correlations of prefreeze versus post-thaw velocity measurements were strongest for those cells within the 65% to 85% range of prefreeze motility. We suggest that sperm survival is independent of prefreeze motility. However, velocity kinetics appear stable after freezing for those specimens that had an initial motility of 65% to 85%.

Analysis of Variance↗
PubMed1989

Concentration of receptors for estradiol and progesterone in canine endometrium during estrus and diestrus.

Receptors for estrogen and progesterone were measured in cytosols prepared from specimens of canine endometrium obtained at late proestrus, day 4 of estrus, day 2 of diestrus, and at 10 day intervals from days 10 through 80 of diestrus. Twenty nine adult bitches were used, with 2 to 4 dogs used at each time point. Concentrations of estradiol receptors measured in endometrial cytosols from late proestrus through day 10 of diestrus were similar (mean +/- SEM: 9.9 +/- 2.2, 10.5 +/- 1.2, 16.3 +/- 1.6, and 16.2 +/- 2.9 pmol/g of tissue at proestrus, day 4 of estrus, days 2 and 10 of diestrus, respectively). As serum concentrations of progesterone increased during early diestrus, the concentration of estradiol receptors decreased and were significantly (P less than 0.05) lower on days 30 (4.9 +/- 1.3 pmol/g of tissue) and 40 (3.7 +/- 0.6 pmol/g of tissue) of diestrus. After day 40 of diestrus, when serum concentrations of progesterone were approaching basal concentrations, the concentration of estradiol receptors increased and remained significantly (P less than 0.05) higher from days 60 to 80 of diestrus (day 60, 13.4 +/- 2.9; day 70, 15.7 +/- 1.7; day 80, 19.8 +/- 2.4 pmol/g of tissue). As observed for estrogen receptors, the concentration of endometrial receptors for progesterone also gradually increased from late proestrus (4.9 +/- 1.3 pmol/g of tissue) to day 2 of diestrus (6.4 +/- 0.3 pmol/g of tissue).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Luteal function in the bitch: changes during diestrus in pituitary concentration of and the number of luteal receptors for luteinizing hormone and prolactin.

The concentration of unoccupied luteal receptors for luteinizing hormone (LH) and prolactin, and the concentration of these two hormones in the pituitary was determined in 11 groups of bitches (n = 3 or 4/group) representing stages from proestrus through Day 80 of diestrus. Despite dramatic changes in serum concentrations of progesterone, the concentration of luteal receptors for LH and prolactin was quite constant throughout the entire luteal phase. In association with the ovulatory surge of LH, pituitary concentration of LH decreased abruptly from proestrus to Day 2 of diestrus, and was then gradually replenished during the remainder of diestrus. The concentration of prolactin in the pituitary did not vary significantly from proestrus through late diestrus.

Animals↗
PubMedDate not supplied

Phasic maternal prolactin secretion during spontaneous labor is associated with cervical dilatation and second-stage uterine activity.

OBJECTIVE: In this study, we attempted to determine the physiologic roles of prolactin (PRL) and hCG during labor. METHODS: Serial blood samples were collected from 19 women (15 multiparous and four nulliparous) before, during, and after labor; assayed for PRL and hCG; and compared with cervical dilatation and uterine contraction frequency. RESULTS: Prolactin levels decreased in association with progressive cervical dilatation during the first stage of labor and reached a nadir at full dilatation. Thereafter, PRL increased rapidly during the second stage of labor and peaked within 1 hour postpartum. Uterine contraction frequency also increased during the second stage and correlated significantly (r2 = 0.60; P = .0006) with levels of PRL. Levels of hCG increased during labor and peaked at about the time of delivery. CONCLUSION: These data suggest that phasic PRL secretion is associated with cervical dilatation and that after transition to the second stage of labor, uterine contraction frequency may be associated with the increase in PRL levels.

Cervix Uteri↗
PubMedDate not supplied

The contribution of mild thiamine deficiency and ethanol consumption to central cholinergic parameter dysfunction and rats' open-field performance impairment.

We studied at the biochemical, morphological, and behavioral levels the effect of chronic ethanol consumption, associated or not with a mild thiamine deficiency episode. We found that (i) thiamine deficiency induced a significant decrease of the acetylcholinesterase (AChE) activity both in cortex and hippocampus; (ii) chronic ethanol treatment has no effect on cortical AChE activity, but induced a significant decrease of hippocampal enzyme activity; (iii) the reduction in cortical and hippocampal AChE activity induced by chronic ethanol treatment associated with a 1-week thiamine deficiency was also significant and was greater than that induced by ethanol alone. Furthermore, either chronic ethanol or thiamine deficiency induced a significant decrease in the release of acetylcholine (ACh) in the stimulated condition using high potassium concentration; and when both treatments were associated the decrease was even greater. In the unstimulated condition, the reduction in the release of ACh was greater for ethanol treatment than for thiamine deficiency. Open-field tests showed that only in the "sniffing" category were there significant differences among the experimental groups. No morphological change was detected by optical microscopy, suggesting that the injury process was in its initial stages in which only functional and behavioral changes are displayed. In addition, our biochemical results indicate that cortical cholinergic susceptibilities to ethanol and thiamine deficiency are significantly different.

Acetylcholine↗
PubMedDate not supplied

Effect of parity on pituitary prolactin response to metoclopramide and domperidone: implications for the enhancement of lactation.

OBJECTIVE: The gastrointestinal motility agents metoclopramide and domperidone are known to increase pituitary prolactin (PRL) secretion and breast milk production. This study compared the effect of single doses of two strengths of metoclopramide and a single dose of domperidone on PRL secretion. METHODS: Ten nonpregnant women had baseline evaluation of serum PRL concentrations. The PRL concentrations were then determined after random oral administration of metoclopramide 10 mg, metoclopramide 5 mg, and domperidone 10 mg. Blood samples were drawn in the first 7 days of the menstrual cycle, at 13 time points over a 6-hour period (0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300, and 360 minutes), with the zero time point beginning at 0800 hours. Variables such as weight, height, age, gravidity, parity, and oral contraceptive use were recorded. RESULTS: Baseline PRL concentrations showed the natural circadian rhythm. Metoclopramide and domperidone both caused a significant increase in PRL. However, PRL secretion was most influenced by parity. Nulliparous women had the quickest and highest PRL secretion with metoclopramide 10 mg, compared with the PRL response with metoclopramide 5 mg and domperidone 10 mg. Conversely, multiparous women had PRL secretion patterns that were equivalent between the medications. CONCLUSIONS: The PRL response to the medications was most influenced by parity. Therefore, we suggest that the medication therapy of choice for enhancing lactation may not be the same in all women, but may instead be determined by parity.

Cross-Over Studies↗
PubMedDate not supplied

The acute release of maternal prolactin by instrumental cervical dilatation simulates the second stage of labor.

OBJECTIVE: We hypothesize that artificial stimulation of the cervix causes an acute secretion of maternal prolactin (PRL) that mimics PRL secretion during the second stage of human labor. METHODS: Eighteen women scheduled for first-trimester therapeutic abortion had a blood sample drawn at the following times: before and after laminaria tent (LT) insertion for cervical ripening, 18-24 hours later upon entry in the procedure room, after LT removal, during instrumental cervical dilatation and uterine evacuation by suction curretage, soon after evacuation, and 1/2 hour and 1 hour after evacuation. Local cervical anesthesia, intravenous analgesics, and sedation were administered before the procedure. Blood serum was assayed for PRL, human chorionic gonadotropin (hCG), and estradiol. RESULTS: The initial PRL levels were 34 ng/mL +/- 4.2 standard error of the mean (SEM) and did not change after 18-24 hours of LT placement nor after LT removal. However, PRL increased significantly (t = 5.8; P = .00001) over basal levels by 204% +/- 15 at the time of instrumental dilatation and rose to 378% +/- 33 at 1/2 hour postevacuation. After 1 hour, PRL had declined to 173% +/- 10%. Serum levels of estradiol and hCG changed only after evacuation. CONCLUSION: Artificial stimulation of the cervix causes an immediate surge in PRL levels. We suggest that the acute increase in pituitary PRL secretion that occurs during the second stage of labor results from a neural signal from the cervix and not from endocrine changes associated with labor.

Abortion, Therapeutic↗