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Biomedical subjects

C Monteiro

Publications and source records attributed to C Monteiro.

At least 19 recordsLinked to original sources
PubMed2000

Differentiation of O-acetyl and O-carbamoyl esters of N-acetyl-glucosamine by decomposition of their oxonium ions. Application to the structure of the nonreducing terminal residue of Nod factors.

Nod factors are substituted N-acyl chito-oligomers secreted by plant symbiotic bacteria of the Rhizobium family. Substitutions on the oligosaccharide core specify their recognition by host plants. A method using tandem mass spectrometry is proposed to locate the O-acetyl and O-carbamoyl substituents on the nonreducing terminal residue of the chito-oligomers. As model compounds, all the positional isomers of monoacetyl and monocarbamoyl esters of 1-O-methyl-N-acetyl-alpha-D-glucosamine were synthesized. Oxonium ions (MH - CH3OH)+ were generated by liquid secondary ion mass spectrometry (LSIMS) and their decomposition was recorded on a tandem magnetic instrument. Large differences were observed in the relative abundances of ions resulting from elimination of water and of the O-ester substituent from metastable oxonium ions. Deuterium exchange reactions indicated parallel elimination pathways involving either exchangeable or carbon-linked hydrogens. The intensity ratios of some of the ions generated by collisions with helium atoms allowed the isomers to be distinguished. The main dissociation routes were identified. Metastable and collision-induced decomposition of the B1 ions from Nod factors of Sinorhizobium meliloti and Azorhizobium caulinodans resembled that of the 6-O-substituted N-acetylglucosamine models. Decomposition of the B1 ion from Mesorhizobium loti and Rhizobium etli Nod factors, was similar to that of 3-O-carbamoyl N-acetyl-glucosamine and different to that of the 4-O isomer. 6-O- and 3-O-carbamoylation specified by the nodU and nolO genes, respectively, of Rhizobium. sp. NGR234 were confirmed.

Acetylglucosamine↗
PubMed2000

Beta globin gene inhibition by antisense RNA transcripts.

Sickle cell disease is caused by a mutation in the beta globin gene leading to hemoglobin S (Hb S) production. Several approaches have been explored to prevent Hb S polymerization in red blood cells and the symptoms associated with this disorder. To this end we tested a mammalian expression vector carrying a human beta globin antisense cDNA (pZeobetaAS) fragment in a mouse erythroleukemia cell line expressing the human gamma and beta globin genes. We observed a relative reduction in beta globin mRNA levels compared with gamma mRNA levels in the presence of pZeobetaAS. Moreover, analysis at the protein level showed an average 76% decrease in beta chains and a 517% increase in gamma chain biosynthesis. The inhibitory effect of the antisense vector on globin expression was maintained long term in culture. The expression vector pZeobetaAS was also transfected into primary erythroid progenitors to test its effects on globin genes undergoing normal developmental switching during differentiation. We observed a relative reduction of beta globin mRNA levels compared with gamma mRNA levels. These results support a novel role for antisense cDNA expression vectors as an alternative gene therapy strategy to inhibit betas gene expression in sickle cell disease. Gene Therapy (2000) 7, 438-444.

Anemia, Sickle Cell↗
PubMed1999

Association of p53 genomic instability with the glutathione S-transferase null genotype in gastric cancer in the Portuguese population.

AIMS: p53 gene mutations are the most common genetic changes known to occur in human cancer. In previous studies, the presence of alterations to the p53 gene has been linked to the null phenotype of the glutathione S-transferase mu gene (GSTM1). GSTM1 appears to be part of a protective mechanism against the development of cancers in which environmental chemical carcinogens are involved. To screen for such an association in stomach cancer, p53 allelic loss and genomic instability and GSTM1 genotypes were investigated in gastric tumour DNA samples from 113 patients. METHODS: The polymerase chain (PCR) reaction was used to amplify a (CA) repeat array in the p53 locus; electrophoresis, genotyping, and allele quantification were performed using an automated DNA sequencer and Genescan software. The presence of the GSTM1 gene was determined by means of a differential PCR in which multiple genes were co-amplified in the same reaction tube. RESULTS: Loss of heterozygosity (LOH) of the p53 gene was found in 36 of 87 informative cases and genomic instability was present in eight of 113 cases. Further analysis into histological subtypes and sites of tumours did not show any positive association with p53 loss. An association between the presence of LOH and the GSTM1 null genotype was not seen; however, all the samples with genomic instability of the p53 gene (eight of 113) also showed a GSTM1 null genotype. CONCLUSION: This study does not support the hypothesis of an association between LOH in the p53 gene and the GSTM1 null genotype, but suggests that the GSTM1 null genotype might influence p53 genomic instability.

Alleles↗
PubMed1998

Overproduction of SM28GST in a baculovirus expression vector and its use to evaluate the in vivo immune responses of mice vaccinated against Schistosoma mansoni with naked DNA encoding the SM28GST gene.

To evaluate the immune responses of mice vaccinated intramuscularly with naked DNA encoding a single parasite-derived gene, sufficient quantities of protein are necessary for use in the immunological assays. A plasmid carrying the cDNA encoding the entire sequence for the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST) was used as a source of naked DNA to vaccinate mice. Using polymerase chain reaction employing custom primers to add Eco RI and Hind III restriction sites at the 5' and 3' ends, respectively, a 651-bp fragment was amplified from the vaccine plasmid. This product was isolated, ligated into the pFastBac HTb donor plasmid containing a 6X histidine (6X-his) tag, and transposed into the baculovirus expression vector system. Following blue white selection screening, high molecular weight DNA was isolated and transfected in Sf21 insect ovary cells using a liposomal preparation. Culture medium containing infective virus particles was used to infect a series of Sf21 cultures and the cells were lysed after 3-5 days. The lysates were subjected to immobilized metal (Ni-NTA) affinity chromatography from which the 6X-his-tagged recombinant Sm28GST was eluted in 250 mM imidazole. The eluted protein was probed with a polyclonal rabbit antibody specific for the Sm28GST and subsequently recognized using a monoclonal antibody specific for the 6X-his tag following concentration of the pooled fractions. Mice were vaccinated intramuscularly with purified plasmid DNA encoding either the Sm28GST or firefly luciferase. Skin tests performed using recombinant Sm28GST were positive in only those mice vaccinated with naked DNA encoding the Sm28GST gene. In a different group of experimental mice, only sera from mice vaccinated with naked DNA encoding Sm28GST contained IgG-specific anti-Sm28GST antibodies at 14 days postvaccination, and at 42 days the levels were suggestive of an anamnestic response. These results suggest that naked DNA vaccination of mice is capable of inducing both antigen-specific cell-mediated and humoral immune responses against Sm28GST and further strengthen the case for this antigen being a vaccine candidate.

Animals↗
PubMed1997

Globin gene silencing in primary erythroid cultures. An inhibitory role for interleukin-6.

There are numerous similarities between the erythroid and megakaryocytic lineages which suggest that commitment to either lineage occurs relatively late in hematopoiesis. Commitment toward megakaryocyte development requires obligatory silencing of erythroid-specific genes. Therefore, we investigated the effects of interleukin-6, a known inducer of thrombocyte production, on globin gene expression during erythroid differentiation. Studies in K562 cells demonstrated inhibition of gamma globin gene mRNA production and chain biosynthesis in the presence of exogenous interleukin-6 which was abrogated by anti-interleukin-6 monoclonal antibody. Similar studies in primary erythroid progenitors showed inhibition of burst-forming unit-erythroid colony formation when interleukin-6 was added late in cultures with decreased gamma and beta globin gene mRNA production. Protein binding studies demonstrated an increase in activator protein-1 binding to its consensus sequence by 24 h of interleukin-6 treatment. Inhibition of activator protein-1 gene activity had no effect on gamma gene silencing by interleukin-6. A potential interleukin-6 response element was identified in the gamma globin gene. Interleukin-6 treatment led to a rapid increase in protein binding to the target DNA sequence. These results suggest that interleukin-6 may play an important role in globin gene silencing during megakaryocytic lineage commitment.

Butyrates↗
PubMed1997

Mechanisms underlying degeneration of cryopreserved vascular homografts.

OBJECTIVE: To analyze the mechanism(s) underlying homograft degeneration, we designed an experimental model in which the behavior of cryopreserved autografts and homografts, as well as fresh autografts, implanted in the same animal was compared. METHODS: A cryopreserved homograft was implanted in the aorta of 14 sheep. The excised aortic autologous segment was then subjected to cryopreservation, and 1 to 8 weeks later it was implanted 1 to 2 cm below the cryopreserved homograft. The intermediate segment of the native aorta, the fresh autograft, was dissected at this point. Animals were put to death at different times and the implanted segments were harvested together with a portion of native aorta. Histologic and immunohistochemical analyses, as well as cell viability assessments, were then performed on the explanted segments. Similar studies were also conducted on fragments of cryopreserved autografts and homografts before implantation. RESULTS: With the exception of a partial loss of the endothelium, cryopreserved specimens retained cell viability and morphologic integrity before implantation. Explanted cryopreserved homografts showed profound changes affecting all strata, as well as a decline in cell viability. Lymphocyte infiltrates were found up to 12 months after implantation. Endothelium was always absent in cryopreserved homografts. However, a reendothelialization of the cryopreserved autografts was observed. After an initial period of neuronal degeneration, reenervation of the cryopreserved autograft segment occurred 6 to 12 months after the operation. Findings regarding the fresh autografts were similar to those of the cryopreserved autografts. CONCLUSION: Our results suggest that the immunologic reaction rather than the cryopreservation process is responsible for the degenerative process occurring in cryopreserved homografts.

Animals↗
PubMed1997

Genetic and environmental factors regulating blood pressure in childhood: prospective study from 0 to 3 years.

OBJECTIVES: Blood pressure (BP) regulation depends on the interaction between multiple environmental and genetic factors. Of these, BP sensitivity to dietary sodium intake has been one that has been investigated in adults but not in children. The aim of the present study was to investigate, prospectively, the BP profile in relation to different genetic and hormonal factors, in the first 3 years of life. POPULATION AND METHODS: Thirty-nine children born at term following normal pregnancies, with uncomplicated neonatal periods, were randomly selected to take part in the study. BP, weight and length were evaluated every 3 months from birth to 3 years. At the age of 12 months, haptoglobin phenotypes and plasma active renin concentration were determined as well as random urine evaluation of aldosterone, cAMP, dopamine and digoxin-like immunoreactive substances (DLIS). Family history of cardio-vascular diseases was also recorded. RESULTS: Systolic BP (SBP) demonstrated a gradual increase until the age of 6 months with little variation up to 36 months. Tracking of SBP values was also observed from the first year as infants with high values (above the 75 percentile) maintained this tendency up to, at least, the age of 36 months. The comparison between SBP and diastolic BP (DBP) according haptoglobin phenotypes demonstrated that SBP was systematically higher in allele 1, with apparently an increasing tendency with age, although the differences did not have statistical significance. The comparative study between haptoglobin phenotypes, with correction for the covariates fractional excretion of sodium and potassium, showed that allele 1 carriers had significantly lower plasma renin and urine aldosterone and cAMP concentrations than allele 2, but dopamine excretion was found to be higher in allele 1 than in allele 2. There were no differences among variables relating to family history of cardiovascular disease. CONCLUSIONS: There was an early tracking process of BP values from the first 6 months of life which persists through, at least, to the age of 36 months. Differences in sodium handling between haptoglobin 1 and 2 phenotypes were already present in early childhood, although no significant repercussion in BP values could be demonstrated in the 3-year duration of this study.

Age Factors↗
PubMed1996

Glutathione S-transferase mu polymorphism and susceptibility to lung cancer in the Portuguese population.

Epidemiological studies have led to the suggestion that a genetic basis may exist in the individual variation in predisposition to cancer. Interindividual differences in human toxicological response to carcinogenic exposure have been attributed to heritable polymorphisms in metabolism, namely glutathione S-transferases (GSTs) coding for enzymes that are known to be detoxifiers of carcinogens. Within the human GST mu class, there is a specific isozyme that is frequently lacking. To check whether or not this association exists in the Portuguese population with lung cancer, we used polymerase chain reaction (PCR)-based genotyping to examine GSTM1 polymorphism (nulled and non-nulled) in 84 individuals as a control healthy population and a group of 98 lung cancer patients. In this study we were able to find a frequency of the GSTM1 phenotype among our healthy control subjects consistent with earlier genotyping studies in other Caucasoid populations. For the group of individuals with lung cancer as a whole, or in subsets of histological subtypes, our data for the Portuguese population did not show a positive correlation between the null allele and this neoplasm. In contrast, we found a slight increase in the frequency of the wild-type allele in our lung cancer group.

Carcinoma, Small Cell↗
PubMed1996

APC intragenic haplotypes in familial adenomatous polyposis.

Genetic epidemiological studies are useful for the knowledge of the association of markers and genes involved in diseases. In the present work, we studied the frequency of four adenomatous polyposis coli intragenic RFLP markers often used in risk evaluation in a population of 10 familial adenomatous polyposis patients from 10 unrelated Portuguese familial adenomatous polyposis families not sharing the same mutation, and in a population of 55 unrelated healthy Portuguese volunteers. We compared the frequency obtained to normal and affected populations and to results already reported in other populations. We observed allelic frequencies for the Portuguese population that agree with the published ones. The intragenic polymorphisms show strong gametic disequilibrium suggesting little recombination between them. We observed haplotype frequencies significantly different in patients and controls. The gametic disequilibrium may be due to a common founder for a proportion of apparently unrelated probands.

Adenomatous Polyposis Coli↗
PubMed1996

Damage to UV-sensitive cells by short UV in photographic flashes.

Light emitted by electronic photographic flash units is shown to damage bacteria and human skin fibroblasts deficient in repair systems, with survival curves very similar to those produced by 254 nm short UV. The lesions induced by these flashes are as photorepairable by the photolyase enzyme as those induced by 254 nm UV and result in equivalent survival rates. Biological dosimetry performed with microorganisms highly sensitive to UV (Escherichia coli K12 AB2480, deficient in excision and recombinational-dependent repair systems and Bacillus subtilis UVSSP spores, deficient in excision and in a specific spore repair process) revealed that each 1 ms flash of light from the photographic unit used in this work contained the equivalent of 0.25 J m-2 of 254 nm UV, when measured at a distance of 7.0 cm. This dose of UV was found to be lethal to both repair-deficient E. coli bacteria and repair-deficient human skin fibroblasts obtained from xeroderma pigmentosum donors, as well as mutagenic in B/r wild-type and HCR-mutant bacteria.

Bacillus subtilis↗
PubMed1996

Presymptomatic diagnosis in Portuguese FAP families using intragenic RFLPs and (CA)n flanking markers by fluorescence based semiautomated DNA analysis.

Owing to the large size of the APC gene, responsible for familial adenomatous polyposis, direct screening for individual mutations is not a practical approach. In the present study we establish the methodology of fluorescence based semi-automated DNA analysis to perform presymptomatic diagnosis of members at risk from 11 Portuguese FAP families with three (CA)n markers flanking the APC gene, MBC, CB26, and YN5.64, and four intragenic RFLPs. Haplotypes were constructed on the basis of individual genotypes and their segregation through generations were followed. The study was informative for 12% of subjects using only intragenic RFLPs and increased to 90% when we used the three (CA)n flanking markers. We report two of the 11 families under study in our laboratory and show recombinant events leading to a precise localisation of the CB26 marker between D5S82 and the APC gene. In one family there was a loss of (CA) units of one allele of the CB26 marker from an unaffected mother to her son.

Adenomatous Polyposis Coli↗
PubMed1995

Histologic and genetic assessment of explanted allograft valves.

A possible way of analyzing the immune response triggered by the allograft and the cellular viability is to compare immunocompetent and immunosuppressed patients, such as those having valve replacement and heart transplantation, respectively. These groups differ in immunosuppression therapy, preparation methods, valve hemodynamics. In the present study, we investigated polymerase chain reaction-amplified DNA flanking hypervariable (CA)n regions obtained from valve leaflets taken from patients having valve replacement or heart transplantation and performed a histologic analyses of the cells. In addition, we assessed an autograft valve to compare the hemodynamic effects on the cellular composition of the valve leaflet. We conclude that leaflet cellularity of the heart transplantation and autograft patients is superior to that of the valve replacement patients. These differences were consistent with the occurrence of an immune response in the valve replacement group, which was prevented or abrogated by immunosuppressive therapy administered to the heart transplantation group. However, it cannot be excluded that preparation procedures have a long-term effect on the extracellular matrix, leading to deterioration of cell adhesion and homing conditions.

Adult↗
PubMed1995

The allograft valve in heart transplantation and valve replacement. Genetic assessment of the origin of the cells by means of deoxyribonucleic acid profiles.

Assessment of the cellular origin of allograft valves is essential in comprehending their biologic behavior and in improving preparation methods. In this study we retrospectively analyzed 10 allografts obtained from patients who underwent valve replacement or heart transplantation. Histologic evaluation and deoxyribonucleic acid amplification by polymerase chain reaction technology with fluorescence labeled primers was performed on different parts of the valve leaflets. Automated analyses of the obtained amplimers showed in the heart transplantation group the presence of receptor cells interspersed with native donor cells in three cases. Preliminary results for the valve replacement group are inconclusive as yet.

Adult↗
PubMed1994

On the mechanisms of genotoxicity and metabolism of quercetin.

Quercetin has been the subject of numerous studies on its genetic toxicity and carcinogenicity. Despite its well-proven genetic damaging activity for various genetic end-points (reverse mutations, induction of SOS functions, induction of sister chromatid exchanges, chromosomal aberrations and micronuclei), the mechanisms of genetic damage by quercetin remain, by and large, unknown. The present study aims to further extend the observations on the possible active oxygen species mediated DNA-damaging activity of quercetin and the role of cytochrome P450-dependent metabolism on the genotoxicity of quercetin. The results reported in this work show that quercetin can produce the OH. radical, as assessed by deoxyribose degradation in the presence of Fe3+/EDTA (ethylenediaminetetraacetic acid), and that it induces strand breakage in isolated plasmidic DNA (pUC18). The data support the hypothesis that the production of OH. is mediated by H2O2. The results with genetically engineered V79 cells expressing rat cytochromes 1A1, 1A2 and 2B1 failed to demonstrate metabolism of quercetin, as indicated by the fact that neither an enhancement nor a decrease in the genotoxicity of quercetin was observed. Results obtained on the pH dependence of the induction of chromosomal aberrations by quercetin in V79 cells show that, as the pH value of the medium is increased to 8.0, there is a significant increase in the number of aberrant cells, as expected if oxygen radicals are responsible for the formation of chromosomal aberrations.

Animals↗