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F Leal

Publications and source records attributed to F Leal.

At least 37 records · Page 2Linked to original sources
PubMed1995

An immunodominant 90-kilodalton Aspergillus fumigatus antigen is the subunit of a catalase.

We have identified, purified, and characterized structurally and functionally a 90-kDa immunodominant antigen associated with the water-soluble fraction of Aspergillus fumigatus. This antigen is recognized by 90.3% of serum samples from patients with aspergilloma and should be considered either by itself or better in combination with other purified antigens as a candidate for developing a standardized immunoassay for the detection of aspergilloma. p90 is a glycoprotein containing at least two two N-linked sugar chains of 2 and 5 kDa, respectively, which are not necessary for its reactivity with aspergilloma serum samples. Using specific anti-p90 rabbit serum, we have demonstrated that under native conditions, p90 exists in oligomeric form and has associated catalase activity. This activity is resistant to extreme temperatures (> 60 degrees C), reducing agents (40 mM dithiothreitol), high concentrations of denaturing agents such as 8 M urea and 8% sodium dodecyl sulfate, and treatments with ethanol-chloroform-water (5:3:10 [vol/vol]) mixtures.

Animals↗
PubMed1995

[The effect of the woman's age, the rate of cleavage and embryo quality on obtaining a pregnancy by in-vitro fertilization].

Multiple factors influence the probability of obtaining a pregnancy through in vitro fertilization (IVF) and embryo transfer (ET). This retrospective study was designed to assess their importance in order to improve prognostic ability and treatment success. 341 consecutive embryo transfer cycles using the same ovarian stimulation protocol were considered and divided in two main groups: 92 cycles in which a clinical pregnancy was achieved and 249 cycles without success. All the embryo transfers were performed in patients from the in vitro fertilization program of the Human Reproductive Unit, Santa Maria Hospital, Lisbon, between January 1991 and December 1993. No significant differences were found between the two groups studied concerning the IVF indications, ovarian response to the stimulation, sperm quality, oocyte maturation and mean number of oocytes retrieved per patient. The women's age was higher in the group which did not achieve a pregnancy, when compared with the pregnant group (p < 0.001), showing a decline of success after the age of 35. Overall oocyte fertilization rate was 88.2% in cycles with pregnancy and 83.5% in cycles without pregnancy (p < 0.02). In the pregnant patients, there was a significantly higher rate of embryo transfers in which all the embryos received had reached at least the four-cell stage at 42-43 hr postinsemination, compared with the non pregnant patients (82% versus 63%, p < 0.001). All the 92 pregnancies originated from transfers of at least one embryo that had undergone two or more mitotic divisions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Variability of Aspergillus nidulans antigens with media and time and temperature of growth.

The influence of culture medium and time and temperature of growth on the appearance of Aspergillus nidulans antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining or Western blot (immunoblot), of the proteins present in total cellular extracts or culture supernatants. Samples in the exponential, deceleration, and stationary growth phases were selected by biochemical, morphological, and ultrastructural criteria. Protein and antigen patterns (detected with rabbit antibodies) from total extracts were very similar in all cases, and the major differences observed seemed to depend on the age of the cultures. Culture supernatant patterns were highly dependent on the type of medium (complex or defined) and the age of the culture. Temperature did not significantly influence these results. The reproducible reactivity of selected human sera from aspergilloma-affected individuals was strictly associated with the use of defined media, especially Czapek Dox-AOAC, in both total extracts and culture supernatants. Extended growth times were necessary in the case of metabolic antigens (those obtained from culture supernatants). Screening of a battery of 10 selected human serum samples from patients with aspergilloma or invasive aspergillosis demonstrated that two of the antigens (96 to 98 and 45 kDa) from stationary-phase culture supernatants in Czapek Dox-AOAC medium were consistently reactive. When considered together as one unit, both antigens reacted with more than 50% of the sera, and at least one or the other of the antigens reacted with more than 90% of the sera. Less consistent results were obtained for two somatic antigens (from total cell extracts) of 45 to 50 and 20 to 22 kDa.

Animals↗
PubMed1993

Fractured zona oocytes in in-vitro fertilization cycles stimulated with gonadotrophin-releasing hormone analogue and human menopausal gonadotrophin.

In order to assess the possible influence of gonadotrophin-releasing hormone analogue and human menopausal gonadotrophin on the occurrence of fractured zona oocytes (FZOs) in in-vitro fertilization (IVF) treatment cycles, we analysed 267 consecutive cycles in 199 patients. In 87 cycles, at least one fractured zona oocyte was recovered, and in 180 cycles only intact zona oocytes (IZOs) were recovered. FZOs represented 5.8% of all oocytes retrieved and 14.8% when only cycles with FZOs were considered. Serum oestradiol concentrations were significantly higher at day -3 and day -2 (P < 0.02) in cycles yielding at least one fractured zona oocyte compared to IZO cycles (day 0 = retrieval day), and there was a higher incidence of G terminal pattern of oestradiol curve (P < 0.01) in cycles with FZOs. The mean numbers of all oocytes retrieved and of mature oocytes were significantly higher in FZO than in IZO cycles (P < 0.001). The fertilization rate of mature oocytes was significantly reduced (P < 0.05) in cycles with one or more oocytes with fractured zonae. There was no significant difference in the number of embryos transferred, pregnancy and abortion rates in both groups. We conclude that although the occurrence of fractured zona oocytes is a frequent event, it does not affect the overall results of our IVF programme. Zona pellucida fragility may be the result of over-maturation of some oocytes.

Adult↗
PubMed1991

Analysis of Aspergillus nidulans conidial antigens and their prevalence in other Aspergillus species.

Aspergillus nidulans is an ascomycetous fungus that reproduces asexually by forming multicellular conidiophores and uninucleate spores called conidia. These elements constitute the main vehicle for the transmission of this and other pathogenic Aspergillus species and are the starting point of the different forms of aspergillosis. In order to use A. nidulans as a potential source of useful antigens for the immunodiagnosis of these diseases, we have examined the total protein composition of conidial extracts of this fungus by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in gels of different percent T. Injection of SDS-extracted conidial proteins into rabbits allowed us to raise a battery of polyclonal antibodies which have defined some important immunogenic polypeptides. Several of these immunogens were both present in mycelial extracts and recognized by antimycelium antibodies. Four of them, designated cdA, cdB, cdC, and cdE, were also found in conidial extracts of other pathogenic Aspergillus species. Only cdE was undetectable in cell extracts of the nonrelated species Fusarium culmorum and Phycomyces blakesleeanus.

Antigens, Fungal↗
PubMed1990

Fluid-phase endocytosis in yeasts other than Saccharomyces cerevisiae.

A FITC-dextran internalization assay with Saccharomyces cerevisiae as positive control was used to determine whether fluid-phase endocytosis is a general characteristic of yeasts. Schizosaccharomyces pombe, Pichia polymorpha, Kluyveromyces phaseolosporus, Yarrowia lipolytica and Candida albicans were clearly positive, whereas results obtained with Debaryomyces marama were inconclusive. In all cases internalized FITC-dextran was found to be localized in the vacuoles and the process was always time- and temperature-dependent. Lower eucaryotes, particularly yeasts, appear to have the ability to incorporate substances from the extracellular medium through fluid-phase endocytosis.

Dextrans↗
PubMed1988

Group G Streptococci: susceptibility patterns and the effect of the inoculum size and growth phase on the bactericidal activity of penicillin.

Twenty-seven independent group G streptococcal isolates were studied by in vitro susceptibility testing against 22 different antimicrobial agents. Penicillin with a MIC90 of 0.03 micrograms/ml and ampicillin with a MIC90 of less than or equal to 0.015 micrograms/ml remain the agents of first choice for treatment of group G streptococcal infections. Tolerance was not demonstrated using the macrobroth dilution method in four media, Todd-Hewitt, Mueller-Hinton, Mueller-Hinton (cation-supplemented), and Tryptose Phosphate broths. Multiple regression analyses of time-kill curves of group G streptococci showed that the rate of cell death with penicillin at 0.04 micrograms/ml (five times greater than each organism's MIC) for both logarithmic- and stationary-phase cells with low-inocula were the same, but were five to six times greater in rate of death compared to the high-inocula cultures. Increasing the concentration to 1 microgram/ml of penicillin (125 times greater than each organism's MIC) did not significantly affect the rate of cell death for low-inocula cultures of either phase. Therefore, the size of the inoculum was found to be more significant than the phase of bacterial growth. These findings may explain the therapeutic discrepancy of relapses or prolonged group G streptococcal infections despite the organism being susceptible to the given antibiotic.

Aminoglycosides↗
PubMed1987

Detection of PDGF-2 homodimers in human tumor cells.

The v-sis oncogene encodes a protein structurally and functionally related to human platelet-derived growth factor (PDGF). In the present studies, we show that the primary translational product of the human sis proto-oncogene is a 26-kd protein, p26c-sis. This product is processed to yield a disulfide-linked homodimer, p56c-sis, which is further processed to a 35,000-dalton dimer, p35c-sis. Like the v-sis gene product, the PDGF-2 precursor undergoes N-linked glycosylation, implying its processing through the endoplasmic reticulum. The PDGF-2 product was shown to possess functional properties of PDGF. Whereas lysates of control COS-1 cells lacked mitogenic activity, lysates of COS-1 cells transfected with a c-sis/PDGF-2 expression vector specifically stimulated DNA synthesis of quiescent fibroblasts. Moreover, this activity was completely inhibitable by PDGF antibody. Identical forms of the sis/PDGF-2 product were identified in human tumor cells that expressed c-sis/PDGF-2 transcripts. These proteins were shown to be specifically associated with the membrane component of the tumor cells and were not detectably secreted into the culture medium. These findings support the concept that expression of the sis/PDGF-2 product in human cells responsive to its proliferative actions can be an important step in the processes leading to malignancy.

Cell Division↗
PubMed1985

Evidence that the v-sis gene product transforms by interaction with the receptor for platelet-derived growth factor.

A scheme for partial purification of biologically active v-sis-coded protein from cells transformed with simian sarcoma virus (SSV) has made possible a functional comparison of the transforming protein with platelet-derived growth factor (PDGF). The SSV-transforming gene product is capable of specifically binding PDGF receptors, stimulating tyrosine phosphorylation of PDGF receptors, and inducing DNA synthesis in quiescent fibroblasts. Each of these activities was specifically inhibited by antibodies to different regions of the v-sis gene product. Moreover, viral infection of a variety of cell types revealed a strict correlation between those cells possessing PDGF receptors and those susceptible to transformation by SSV. These findings provide evidence that SSV-transforming activity is mediated by the interaction of a virus-coded mitogen with PDGF receptors.

Animals↗
PubMed1985

Endogenous factors that modulate yeast glucan synthetase in cell-free extracts.

Yeast 1,3-beta-D-glucan synthetase (EC 2.4.1.34) activity is modulated by endogenous factors obtained by the extraction of different subcellular fractions with hot water. Cell wall fractions were enriched in activators while supernatant fractions also contained appreciable amounts of inhibitors. The action of these compounds requires the presence of EDTA. Maximal activation by the stimulatory material was reached when assayed in sonicated enzymatic preparations that had been obtained by mechanical breakage of cells in water. The activating material derived from cell wall fractions contained a mixture of low molecular weight compounds. They were found to be different from GTP as deduced from their resistance to alkaline phosphatase and different elution profile in gel filtration. The supernatant material was also heterogeneous with regard to both activators and inhibitors. The combined effect of GTP and activating material derived from cell wall fractions was supraadditive. The polymers synthesized in the absence and in the presence of the endogenous activator were characterized as beta-1,3-glucans on the base of their resistance to periodate and susceptibility to beta-glucanases. However, the length of the radioactive chains was greater when synthesized in the presence of the activator. This was mediated by an increase in the Vmax of the synthetase.

Cell Membrane↗
PubMed1985

The v-sis/PDGF-2 transforming gene product localizes to cell membranes but is not a secretory protein.

The v-sis transforming gene encodes the woolly monkey homologue of human platelet-derived growth factor (PDGF) polypeptide 2. After its synthesis on membrane bound polyribosomes, the glycosylated precursor dimerizes in the endoplasmic reticulum and travels through the Golgi apparatus. At the cell periphery, the precursor is processed to yield a dimer structurally analogous to biologically active PDGF. Small amounts of two incompletely processed forms are detectable in tissue culture fluids of simian sarcoma virus (SSV) transformants. However, the vast majority remains cell associated. Thus, this growth factor-related transforming gene product is not a classical secretory protein. These findings define possible cellular locations where the transforming activity of the sis-PDGF-2 protein may be exerted.

Animals↗
PubMed1984

An examination of factors affecting the instability of Saccharomyces cerevisiae glucan synthetase in cell free extracts.

Yeast beta(1--3) glucan synthetase is stimulated and stabilized by EDTA. Sucrose protects the enzyme from self-inactivation. Preincubation of cell free extracts at low sucrose concentrations indicates a slow transition of the enzyme towards dissociation. Transition kinetics at 30 degrees C and 0 degrees C in the presence and in the absence of sucrose are interpreted assuming that a subunit is thermolabile in the free state and that sucrose increases its stability. Magnesium is deleterous for glucan synthetase in cell-free extracts. Chaotropic agents inactivate glucan synthetase according to their capacity to solubilize and depolymerize biological compounds. Fluoride plays a special role in the activation of glucan synthetase. Its action appears to be dependent on the presence of GTP (or other nucleotides). The role of all these agents on the activity and stability of the enzyme is interpreted in a unified scheme.

Edetic Acid↗
PubMed1982

The plasma membrane of Saccharomyces cerevisiae. Molecular structure and asymmetry.

The molecular structure of the plasma membrane of the haploid strain Saccharomyces cerevisiae X-2180 1 A has been studied by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein and glycoprotein components have been identified and their apparent Mr determined. A glycoprotein showing an apparent Mr of 27500 has been shown to be the main structural component. Treatment of the cells with cycloheximide prior to plasma membrane isolation resulted in a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff positive bands. It is postulated that treatment with this drug rids the plasma membrane of glycoprotein secretory components which are in the process of being secreted to the periplasmic space, thus allowing the study of the basic structural components of the organelle. The electrophoretic pattern of the internal membranes revealed close similarities with that of the plasma membrane and though two-dimensional electrophoresis might disclose greater differences, these similarities suggest a common origin for most of the components of both membranous systems. Finally, radioiodination techniques, have been used in studying the asymmetric disposition of some of the components of the plasma membrane. At least five polypeptides were identified as located to the outer layer of the plasma membrane and two more glycopeptides were shown to span across the bilayer.

Cell Membrane↗
PubMedDate not supplied

Immunogenic potential of Aspergillus nidulans subcellular fractions and their polypeptide components.

Cell-free extracts of the ascomycetous fungus Aspergillus nidulans were separated into three subcellular fractions: cell walls, total membranes and cytosol, and two different immunization protocols were used to raise antibodies against them in 12 New Zealand rabbits. The immune response was followed over time by dot and Western blot analyses to determine the immunogenic potential of each individual fraction and their polypeptide components. The IgG fractions, purified from pools of the best sera, were used to analyze in detail the antigenic composition of A. nidulans mycelium. The fast immunization protocol provided a much earlier response and higher sera titres. Cytosols and membranes were more immunogenic than cell walls and, in most cases, a positive correlation was shown between the titre of each serum and the number of detected antigens. The polypeptides of A. nidulans included six major immunodominant antigens of the molecular weights ranging between 13 and 200 kDa.

Animals↗