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Biomedical subjects

L Franco

Publications and source records attributed to L Franco.

At least 19 recordsLinked to original sources
PubMed1992

Presence and turnover of adenosine diphosphate ribose in human erythrocytes.

ADP-ribose was detected in human red blood cells (RBC) at 0.45 +/- 0.1 microM concentrations. These levels could be estimated after purification of ADP-ribose by means of three sequential HPLC fractionations of RBC extracts. Extraction was performed by sonication of RBC either in trichloroacetic acid, followed by centrifugation, or in carbonate-bicarbonate buffer, pH 10.0, followed by rapid ultrafiltration. Neither procedure of extraction caused artefactual formation of ADP-ribose. Prolonged incubation of intact RBC in isotonic buffer containing labeled orthophosphate resulted in the slow incorporation of radioactivity into ADP-ribose. Identification of the labeled ADP-ribose was confirmed upon incubation of the purified metabolite with nucleotide pyrophosphatase, yielding radioactive 5'-AMP and ribose 5-phosphate, while its exposure to a nonspecific deaminase resulted in the quantitative formation of labeled inosine diphosphate ribose.

Adenosine Diphosphate↗
PubMed1992

Chromatin structure of the yeast SUC2 promoter in regulatory mutants.

We have previously suggested that two positioned nucleosomes are removed from the promoter of the Saccharomyces cerevisiae SUC2 gene upon depression by glucose starvation. To gain further insight into the changes accompanying derepression at the chromatin level we have studied the chromatin structure of the SUC2 promoter in several mutants affecting SUC2 expression. The non-derepressible mutants snf1, snf2 and snf5 present a chromatin structure characteristic of the repressed state, irrespective of the presence or absence of glucose. The non-repressible mutants, mig1 and ssn6, as well as the double mutant snfs sn6 exhibit an opened chromatin structure even in the presence of glucose. These results suggest that the DNA-binding protein encoded by MIG1 is necessary to produce the characteristic pattern of repressed chromatin and that the SNF1 protein kinase is sufficient to produce the derepressed chromatin pattern. A model is presented for the transitions that result in opening up of the chromatin structure.

Base Sequence↗
PubMed1992

Study of the safety and immunogenicity of the synthetic malaria SPf66 vaccine in children aged 1-14 years.

Safety and immunogenicity tests of the SPf66 malaria vaccine have been carried out on a population of children, aged 1 to 14 years, in the town of Tumaco, Colombia. Adverse reactions measured after each vaccination were local and minimal, and observed in only a small percentage of the vaccinated children. One year later, no delayed reaction was evident. The majority of the child population developed high antibody titres against SPf66 and the degree of response did not vary with age. These induced antibodies recognize the native parasite proteins, in particular the molecules from which the amino acid sequence of this vaccine was deduced. These studies demonstrate that the SPf66 vaccine is safe and highly immunogenic for use in children greater than 1 year old.

Adolescent↗
PubMed1992

A Pan-American 5-year study of fluconazole therapy for deep mycoses in the immunocompetent host. Pan-American Study Group.

Eighty-eight immunocompetent patients with deep mycoses from eight countries were evaluated with the same protocol for efficacy of fluconazole monotherapy. Entry doses were raised from 100 to 400 mg as safety was shown in initial cohorts, and dosages up to 2,400 mg daily and durations up to 44 months were studied. Results were very similar in different countries. Twenty-seven of 28 evaluable patients with paracoccidioidomycosis, 13 of 19 with sporotrichosis, 14 of 16 with coccidioidomycosis, and eight of eight with histoplasmosis demonstrated objective responses to therapy, as did one patient each with zygomycosis and alternariosis. For these patients, relapses have been unusual thus far. In contrast, one patient with chromoblastomycosis responded but relapsed, and six did not respond; one patient with mycetoma responded but relapsed, and two did not respond. The drug was well tolerated by patients, including six who received intravenous therapy. In vitro susceptibility tests suggested that clinical response was correlated with susceptibility but that resistance did not preclude clinical response. Fluconazole therapy appears efficacious for several deep mycoses; dosages of greater than 200 mg daily may be needed for some diseases. The further evaluation of fluconazole for these entities is warranted.

Adolescent↗
PubMed1992

The POT1 gene for yeast peroxisomal thiolase is subject to three different mechanisms of regulation.

The Saccharomyces cerevisiae POT1 gene is, as are other yeast peroxisomal protein genes, inducible by fatty acids and repressible by glucose. We have now found that it is also induced during the stationary phase of the culture. To investigate these three regulatory circuits, we have studied the mRNA levels of regulatory mutants as well as the changes in chromatin structure upon gene activation. We conclude that the regulation of transcriptional activity in glucose repression, oleate induction, and stationary phase induction follow different molecular mechanisms. We suggest that this multiplicity of regulatory mechanisms may represent a general rule for the yeast peroxisomal protein genes.

Acetyl-CoA C-Acetyltransferase↗
PubMed1992

Determination of the immunization schedule for field trials with the synthetic malaria vaccine SPf 66.

The synthetic malaria vaccine SPf 66 has been shown to be safe, immunogenic and effective in trials performed with controlled groups naturally and experimentally exposed to the disease. In order to continue the trials in open populations, it was necessary to standardize the vaccination characteristics. We have performed four field trials with soldier volunteers with the aim, among others, of defining the number of doses required, the intervals between applications, the protein concentration, and the adjuvant to be used. In these trials, the vaccinated individuals' immune responses were evaluated by assaying anti-SPf 66 antibody titres, in vitro growth inhibition of the P. falciparum parasite, and the vaccinees' capacity to recognize P. falciparum native proteins. From these results we conclude that the best vaccination schedule, for adults, is three doses administered subcutaneously on days 0, 30 and 180, each containing 2 mg of the synthetic polymerized petide SPf 66 adsorbed to alum hydroxide.

Adjuvants, Immunologic↗
PubMed1992

Saperconazole in the treatment of systemic and subcutaneous mycoses.

In a 2-year period, 30 patients with culture-proven mycoses (chromoblastomycosis, sporotrichosis, and paracoccidioidomycosis) were treated with the new orally administered triazole, saperconazole (SPZ) (R66905). The daily dose varied from 100 to 200 mg. All patients responded to treatment; the mean time required to heal the lesions and convert the cultures to negative was 3.5 months for sporotrichosis, 4.6 for paracoccidioidomycosis, and 9.0 for chromoblastomycosis. Evaluation by a scoring system indicated that 36.6% of the patients achieved complete resolution of the pretherapy abnormalities, while the remaining (63.3%) experienced major improvement. No collateral effects were reported; there were no bone-marrow or liver toxicities. SPZ is an effective drug for the treatment of the above-mentioned mycoses and appears to be suitable for the control of chromoblastomycosis.

Administration, Oral↗
PubMed1991

Subcellular localization and nucleosome specificity of yeast histone acetyltransferases.

We have previously reported [López-Rodas et al. (1989) J. Biol. Chem. 264, 19028-19033] that the yeast Saccharomyces cerevisiae contains four histone acetyltransferases, which can be resolved by ion-exchange chromatography, and their specificity toward yeast free histones was studied. In the present contribution we show that three of the enzymes are nuclear, type A histone acetyltransferases and they are able to acetylate nucleosome-bound histones. They differ in their histone specificity. Enzyme A1 acetylates H2A in chicken nucleosomes, although it is specific for yeast free H2B; histone acetyltransferase A2 is highly specific for H3, and histone acetyltransferase A3 preparations acetylate both H3 and H4 in nucleosomes. The fourth enzyme, which is located in the cytoplasm, does not accept nucleosomes as substrate, and it represents a canonical type B, H4-specific histone acetyltransferase. Finally, histone deacetylase activity is preferentially found in the nucleus.

Acetyltransferases↗
PubMed1991

The nucleotide sequence of a third cyclophilin-homologous gene from Saccharomyces cerevisiae.

The nucleotide sequence of a 1558 bp DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae contains an open reading frame of 954 nucleotides with coding potential for a protein with high similarity to the ubiquitous cyclophilins which are both peptidyl-prolyl cis-trans isomerases and cyclosporin A-binding proteins. It should, therefore, represent the third gene (SCC3) of this kind from S. cerevisiae. SCC3 is present in a single copy in the genome of S. cerevisiae and results in a constitutively expressed 1.2 kb transcript during cell growth. Its putative protein product (Scc3) contains two hydrophobic cores, one at the amino terminal, 20 amino acids long, which could serve as a signal peptide, and the other one at the carboxyl end with a structure similar to a transmembrane helix. These findings suggest that Scc3 could be a secretory or, more likely, a transmembrane protein. The only cyclophilin with similar structure to that of Scc3 is ninaA from Drosophila melanogaster, a transmembrane protein which seems to be implicated in the correct folding and/or intercalation of rhodopsin in the endoplasmic reticulum of the fly photoreceptors (Stamnes, M.A. et al., Cell 65, 219-227, 1991). In addition, the amino and the carboxy regions of Scc3 and ninaA share a significant level of homology, which suggests that they have a similar function, albeit for different target proteins.

Amino Acid Isomerases↗
PubMed1991

Heterogeneity of guanine nucleotide binding proteins in human red blood cell membranes.

Membranes from human erythrocytes bind radioactive GTP and GTP analogs according to apparently homogeneous patterns. In spite of this uniform type of association, multiple guanine nucleotide binding proteins have been identified both by SDS-PAGE analysis of native and of variously ADP-ribosylated membrane preparations and by FPLC chromatography of solubilised erythrocyte membranes preliminarily incubated with [alpha-32P] GTP in the presence of 5 mM MgCl2. From eight to nine peak fractions of pronase-digestible GTP-binding activity were separated on a MA7Q anion exchange column, this pattern being highly reproducible with different membrane preparations. Prior incubation of membranes with [alpha-32P] GTP in the presence of excess unlabeled GDP resulted in displacement of bound labeled nucleotide from all FPLC fractions. The patterns of GTP binding were also markedly modified by preliminary treatment of membranes with N-ethylmaleimide. Detectable GTPase activity was present in each of the FPLC peak fractions. This wide heterogeneity of guanine nucleotide binding proteins raises so far unanswered questions as to their physiological significance in the mature erythrocyte.

Blood Protein Electrophoresis↗
PubMed1990

Theophylline inhibition of BCG-induced pulmonary inflammatory responses.

The present study was performed to evaluate the effect(s) of theophylline on BCG-induced pulmonary inflammatory responses in an experimental rat model system. Five groups of six animals each received the following: group 1: theophylline-treated, BCG-challenged, and sacrificed three days later; group 2: saline-treated, BCG-challenged, and sacrificed three days later; group 3: theophylline-treated, BCG-challenged, and sacrificed five days later; group 4: saline-treated, BCG-challenged, and sacrificed five days later; and group 5: no treatment and no BCG-challenge ("absolute controls"). Quantitative recovery of bronchoalveolar lavage (BAL) cells was performed and compared with histopathologic changes in lung specimens from each animal in all five groups. No significant differences in quantitative BAL cellular recovery were observed in theophylline-treated animals from saline-treated controls. There was a significant reduction in BAL cell number in saline-treated animals sacrificed five days after BCG-challenge (group 4) from that seen in saline-treated animals sacrificed three days after BCG-challenge (group 2). The most striking finding, however, was a marked diminution in granuloma formation induced by theophylline five days after BCG challenge (group 3). These findings suggest an antiinflammatory effect of theophylline.

Animals↗
PubMed1989

Yeast contains multiple forms of histone acetyltransferase.

We have assayed several methods to quantitatively recover yeast histone acetyltransferases in an attempt to study the multiplicity of enzymatic activities. Two methods, namely (NH4)2SO4 precipitation and salt dissociation of chromatin in 0.5 M NaCl, yielded convenient preparations of total histone acetyltransferases. DEAE-Sepharose chromatography of the crude extracts resulted in the separation of three peaks of activity when total yeast histones were used as substrate. However, the scanning of the enzymatic activity toward individual histones along the chromatography, achieved by determining the specific activity of the individual histones after incubating whole histones and [14C]acetyl-CoA with the chromatographic fractions, yielded four peaks. The first two peaks showed specificity toward H2B and H3, respectively. Although they partially overlapped, rechromatography on cation exchangers allowed us to resolve the two activities, and several criteria were used to prove that they correspond to different enzyme molecules. The last two peaks were H4-specific, but the present data suggest that one of the activities is chromatin-bound, whereas the other surely corresponds to the cytoplasmic B-form of the enzyme. The enzyme specific for yeast H2B acetylates chicken erythrocyte H2A, rather than H2B. The detected multiplicity of yeast histone acetyltransferases may correspond to the multiplicity of roles proposed for histone acetylation.

Acetylation↗
PubMed1989

Chromatin structure of transposon Tn903 cloned into a yeast plasmid.

Transposon Tn903 contains the APH gene for kanamycin resistance, which is active in yeast [A. Jiménez and J. Davies (1980) Nature (London) 287, 869-871] and is flanked by two inverted repeats (IR) 1057 bp long. When plasmid pAJ50, carrying Tn903 and the 2-microns circle origin of replication, is cloned into Saccharomyces cerevisiae, nucleosomes are assembled in vivo on the prokaryotic DNA of the transposon. Indirect end labeling revealed that three nucleosomes are preferentially positioned on symmetrical sequences from both IRs. DNase I digestion also confirmed that the chromatin structure is symmetrical in both IRs. This suggests that sequence determinants are decisive for chromatin structure in these regions. We have calculated the rotational and translational fits [H. R. Drew and C. R. Calladine (1987) J. Mol. Biol. 195, 143-173] for the Tn903 sequence and the results indicate that the nucleosome positioning on the IRs is sequence-directed. Nucleosome deposition on the APH gene also occurs, but no clear positioning exists. Some sequence preference for positioning nucleosomes on the promoter can be predicted, especially from the translational fit. Experimental data indicate, however, that nucleosomes are absent from the promoter. Therefore, chromatin can be organized on prokaryotic DNA in a manner that resembles the typical eukaryotic chromatin structure.

Chromatin↗
PubMed1989

In vivo assembly of chromatin on pBR322 sequences cloned into yeast plasmids.

In order to study the in vivo assembly of chromatin on prokaryotic DNA templates, we have transformed yeast cells with plasmids pAJ50 and pRB58, which contain pBR322 sequences. In both cases nucleosomes are assembled in vivo on pBR322 DNA, although the nucleosomes are not homogeneous in size. To explore whether there is any preference for nucleosome assembly along pBR322 sequences, we have used an indirect end labeling method. The results indicate that most nucleosomes are placed at random on pBR322, although the probability for histone octamers to interact with some short regions is somewhat reduced. These regions coincide with sequences in which the frequency distribution of nucleosomes reconstituted in vitro (E. Caffarelli et al. (1988) Eur. J. Biochem. 171, 497-501) is low. Sequence determinants that direct chromatin assembly in vitro seem thereby to act to some extent in vivo.

Blotting, Southern↗
PubMed1988

Xanthine oxidase inhibitory activity of Paraguayan Myrtaceae.

Nineteen Myrtaceae collections belonging to 15 species, 12 of which are used in Paraguayan folk medicine, were assayed for inhibitory activity towards the enzyme xanthine oxidase. Most leaf and stem extracts were active showing IC50 values ranging from 3.0 to 50 micrograms/ml. The reported activities support existing ethnobotanical data concerning their use for the treatment of gout.

Gout↗