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Biomedical subjects

M C López

Publications and source records attributed to M C López.

At least 19 recordsLinked to original sources
PubMed2006

L1Tc non-LTR retrotransposons from Trypanosoma cruzi contain a functional viral-like self-cleaving 2A sequence in frame with the active proteins they encode.

A comparative analysis of 40 Trypanosoma cruzi L1Tc elements showed that the 2A self-cleaving sequence described in viruses is present in them. Of these elements, 72% maintain the canonical 2A motif (DxExNPGP). A high percentage has a conserved point mutation within the motif that has not been previously described. In vitro and in vivo expression of reporter polyproteins showed that the L1Tc2A sequence is functional. Mutations within certain L1Tc2A sequences affect the efficiency of the cleavage. The data indicate that the L1Tc2A sequence may be influencing the L1Tc enzymatic machinery determining the composition and level of the translated products. The residues located immediately upstream of the 2A consensus sequence increase the cleaving efficiency and appear to stabilize the relative amount of translated products.

Amino Acid Sequence↗
PubMed2006

Bactericidal activity of human milk: stability during storage.

Human milk provides infants with defensive factors against many illnesses. This study aims to analyse global bactericidal activity in fresh human milk and evaluate its stability in relation to milk manipulation and its possible alteration following refrigeration. Nineteen milk samples (mature milk) from 19 healthy women are analysed. Viability testing involving a strain of Escherichia coli NCTC 9111, serovar O111:K58(B4):H- was used to determine the bactericidal effect of human milk. Degree of bacteriolysis is calculated as the difference between E. coli counts in controls and in milk samples, expressed as a percentage of the control sample counts. An evaluation of the effect of refrigeration at 4-6 degrees C after 24, 48 and 72 hours, and at -20 degrees C for seven days on bactericidal capacity is made. Bactericidal activity was detected in all milk samples analysed (77.33 +/- 15.14%). This activity persisted after refrigeration for 48 hours and after freezing for 10 days, but showed a significant decrease after refrigeration for 72 hours. In conclusion, maternal milk has bactericidal capacity, providing defence and protection against infection for newborn infants. This property can be altered during the storage of milk. Consequently, if storage in excess of 48 hours is required, freezing is preferable to refrigeration.

Anti-Bacterial Agents↗
PubMed2006

Evaluation of IFN-gamma production by CD8 T lymphocytes in response to the K1 peptide from KMP-11 protein in patients infected with Trypanosoma cruzi.

The cellular response mediated by MHC class I restricted CD8+ T cells has been shown to be crucial in the control of Chagas disease. The K1 peptide derived from T. cruzi KMP-11 protein has a high binding affinity to the HLA-A*0201 molecule. Nevertheless, it is not known whether this peptide is processed and displayed as an MHC class I epitope during natural infection by T. cruzi. The aim of this study was to evaluate, by ELISPOT assay, the ability of K1 peptide to activate CD8+ T lymphocytes to produce IFN-gamma. Therefore, CD8+ T lymphocytes from 22 HLA-A*0201+ individuals, 12 chronic chagasic patients and 10 uninfected controls, were analysed. The results revealed that two of the chagasic patients had IFN-gamma-secreting CD8+ T cells that were able to respond to K1 peptide with a relative frequency of 110 and 230 per million CD8+ T cells. In contrast, none of HLA-A*0201+ uninfected controls responded to K1 peptide. Responses to HLA-A*0201 restricted peptide from the influenza matrix protein were found in six chagasic patients and four uninfected controls with an average frequency of 175 and 111 cells per million CD8+ T cells, respectively. Moreover, a flow cytometric assay for degranulation showed that chagasic responders had K1-specific cytotoxic CD8+ T cells. It is shown here for the first time that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of Chagas disease.

Adult↗
PubMed2005

Molecular characterization of the kinetoplastid membrane protein-11 genes from the parasite Trypanosoma rangeli.

Trypanosomatids are early divergent parasites which include several species of medical interest. Trypanosoma rangeli is not pathogenic for humans but shows a high immunological cross-reactivity with Trypanosoma cruzi, the causative agent of Chagas' disease that affects more than 17 million people throughout the world. Recent studies have suggested that T. cruzi KMP-11 antigen could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection. In the present paper the genes coding for the T. rangeli kinetoplastid membrane protein-11 have been characterized. The results show that the locus encoding this protein is formed by 4 gene units measuring 550 nucleotides in length, organized in tandem, and located in different chromosomes in KP1(+) and KP1(-) strains. The gene units are transcribed as a single mRNA of 530 nucleotides in length. Alignment of the T. rangeli KMP-11 deduced amino acid sequence with the homologous KMP-11 protein from T. cruzi revealed an identity of 97%. Interestingly, the T and B cell epitopes of the T. cruzi KMP-11 protein are conserved in the T. rangeli KMP-11 amino acid sequence.

Amino Acid Sequence↗
PubMed2004

Genotoxic differences by sex in nasal epithelium and blood leukocytes in subjects residing in a highly polluted area.

We describe differences by sex in genotoxic damage found in a population of medical students exposed to a highly oxidative atmosphere, compared with a control group, measured by the single-cell gel electrophoresis assay and histological changes in nasal epithelium smears. Cells were obtained from the nasal epithelium and blood leukocytes. Higher DNA damage in nasal cells and leukocytes was found in males compared to females and control subjects. The percentage of squamous metaplastic changes in the nasal epithelium was also higher in males compared with females and controls. The co-mutation of normal nasal epithelium by squamous cells might modify its protective function in the nose, increasing the risk of damage to the lower respiratory tract. Although, as medical students, males and females were exposed to the same environment and activity patterns, male genotoxicity damage was higher in control and exposed subjects. More research should be done in order to identify direct or indirect sexual hormone intervention.

Adult↗
PubMed2004

Molecular and immunological characterization of L14 ribosomal protein from Leishmania braziliensis.

The isolation and molecular characterization of the gene coding for L14 ribosomal protein from L. braziliensis is described. There are 2 copies of the gene per haploid genome, repeated in a head-to-tail tandem orientation and located in a single chromosome of approximately 950 kb. Northern blot analyses indicate the presence of a single transcript of 0.95 kb which is up-regulated when parasites reach the stationary growth phase. L. braziliensis L14 gene codes for a 175 amino acid long polypeptide showing 75-83% sequence identity with L14 proteins from trypanosomatids and approximately 25% with its counterparts from higher eukaryotic organisms. L14 ribosomal proteins from trypanosomatids and higher eukaryotes share along their molecules a similar distribution pattern of theoretically functional domains. L. braziliensis L14 recombinant protein is not recognized by sera from cutaneous leishmaniasis patients. Immunization of mice with one dose of L14 recombinant protein and a second dose of L14 protein covalently linked to the HSP70 from Trypanosoma cruzi induces a high antibody level against this L14 protein, which is mostly of the IgG2a subtype, as well as a strong increase in splenocyte proliferation index.

Amino Acid Sequence↗
PubMed2003

The endonuclease NL1Tc encoded by the LINE L1Tc from Trypanosoma cruzi protects parasites from daunorubicin DNA damage.

In the present paper we show that the overexpression of the NL1Tc protein, encoded by the L1Tc non-LTR retrotransposon from Trypanosoma cruzi, led to a reduction of about 60% of DNA damage caused by daunorubicin treatment. This repair effect is not observed in transfected parasites overexpressing the NL1Tc mutated in the aspartic acid located in the active site of the enzyme. In addition, NL1Tc overexpression protects the parasite from the negative effect that daunorubicin has on parasite's growth rate. Thus, parasites overexpressing NL1Tc show, after treatment with 4 microM of daunorubicin, growth rate two to three times higher than the growth rate observed in treated control parasites transformed with the empty vector or overexpressing the mutated NL1Tc. Likewise, parasites overexpressing the NL1Tc protein and irradiated with a single dose of gamma-radiation (6 or 9 Gy) show higher growth rates than the parasites overexpressing the mutated NL1Tc or the control transfected parasites.

Animals↗
PubMed2003

Mutations in GCR1 affect SUC2 gene expression in Saccharomyces cerevisiae.

Transcription of SUC2, the gene that encodes the cytoplasmic and secreted forms of the enzyme invertase, is controlled by glucose repression and derepression mechanisms in Saccharomyces cerevisiae. Several regulatory factors such as the Mig1p-Tup1p-Ssn6p repressor complex and the Snf1p kinase complex have been identified previously as regulators of SUC2 expression. We show that, in addition to these factors, expression of SUC2 is affected by mutations in the gene GCR1 that encodes the glycolysis regulatory protein Gcr1p. Expression of Suc2-LacZ was not repressed by glucose in gcr1 mutant yeast cells exposed to glucose. Furthermore, secreted invertase activity was constitutively expressed under glucose-repressed and derepressed conditions in gcr1 mutants. DNA gel mobility shift assays and in-vitro DNase I protection experiments mapped a DNA binding site for Gcr1p in the transcriptional control region of the SUC2 gene, next to a previously mapped Mig1p binding site. However, the mechanism by which gcr1 mutations relieve glucose repression remains obscure.

Base Sequence↗
PubMed2003

Characterization of reverse transcriptase activity of the L1Tc retroelement from Trypanosoma cruzi.

The recombinant protein RTL1Tc, encoded by the non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi, has been shown to have reverse transcriptase (RT) activity using poly(rA)/oligo(dT) and poly(rC)/oligo(dG) homopolymers as template/primers. The optimal RT activity was detected at a concentration of 5 mM Mg2+, pH 8 and between 28 and 37% degrees C. Site-directed mutagenesis in the RT catalytic site proved that substitution of aspartic acid 313 for isoleucine (RT D313IL1Tc) practically abolishes the RT activity of the RTL1Tc protein. RT-polymerase chain reaction assays revealed that the RTL1Tc protein has the ability to use both homologous and heterologous RNA templates. Also, it is shown that the RTL1Tc protein is capable of synthesizing complementary DNA molecules by consecutive switching of the oligo molecule, which the protein uses as a template. This template switching may be involved in the retroelement integration process.

Animals↗
PubMed2003

Differential CD86 and CD40 co-stimulatory molecules and cytokine expression pattern induced by Trypanosoma cruzi in APCs from resistant or susceptible mice.

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.

Animals↗
PubMed2003

Breaking the non-responsiveness of C57BL/6 mice to the malarial antigen EB200--the role of carrier and adjuvant molecules.

Poor immunogenicity and major histocompatibility complex (MHC) restriction of immune responses to certain recombinant proteins or synthetic peptides impose problems in developing effective vaccines. EB200 is one of the vaccine candidate antigens from Plasmodium falciparum, which induces MHC-restricted immune responses in mice of different haplotypes. A way of overcoming this problem is to conjugate the antigen to an immunogenic protein carrier and to use optimal adjuvant substances. We have investigated the carrier effect of glutathione-S-transferase (GST) in CBA and C57BL/6 mice which are high and low responder to EB200, respectively. Our results reveal that the MHC restriction in C57BL/6 mice was broken by the use of GST as a carrier. Studies on the B-cell repertoires in both strains of mice immunized with GST-EB200 by preparing hybridoma cell lines indicate that the B-cell repertoires were similar in both CBA and C57BL/6 mice. However, the antibody affinity and the magnitude of the response were still lower in the low-responder C57BL/6 mice compared with that in CBA even when cholera toxin (CT) was used as adjuvant. To improve the response, the efficacy of various adjuvant substances like alum and Hsp 70 from Trypanosoma cruzi and the combination of various adjuvants was analysed. CT and Hsp 70 together act synergistically and markedly improve the immunogenicity of EB200 by increasing antibody affinity and the magnitude of the responses in C57BL/6 mice, which may be explained by the complementary effect of adjuvants. These results are of importance in the design of efficient vaccines.

Adjuvants, Immunologic↗
PubMed2003

Determination of total arsenic levels by hydride generation atomic absorption spectrometry in foods from south-east Spain: estimation of daily dietary intake.

The total concentration of arsenic in different foods from south-east Spain was determined by hydride generation atomic absorption spectrometry. Mineralization was carried out with an HNO(3)-HClO(4) mixture in a thermostated sand bath. Arsenic determination was performed by the standard addition method. Analyses of NIST and CBR-CEC reference materials demonstrated the reliability and accuracy of the technique. The highest arsenic levels were found in seafood, cereals, meat and meat by-products. In meat and meat by-products, the total arsenic measured in meat was significantly higher than that in sausages (p<0.05). In cereals, arsenic concentrations in corn and white rice samples were significantly higher (p<0.01) than those measured in wheat by-products. Mean arsenic concentrations in cheese were statistically lower than those in other dairy products (p<0.01). New data have been provided on the total arsenic content of a variety of foods in Spain, which is important for making exposure estimates. The estimated daily intake of total arsenic in the Spanish diet was 221 microg As day(-1).

Arsenic↗
PubMed2003

Oral tolerance to dextrin mediated by specific suppressor T-cells induced in the intestinal intraepithelium and their systemic migration.

Antigens presented to the immune system through the oral route induce antigen specific secretory IgA and systemic unresponsiveness, termed oral tolerance (OT). We studied the induction of OT towards a diet antigen: dextrin (DEX) in rats that underwent protein deprivation and were further re-fed. Peyer's patches (PP), mesenteric lymph nodes (MLN) and spleen (Sp) cells from protein re-fed (R) rats mediated hyporesponsiveness after transfer into naïve recipient rats. Low numbers of MLN T cells transferred hyporesponsiveness while higher numbers transferred an enhancement of the delayed type hypersensitivity (DTH) reaction. MLN T cells were further separated based on their ability to bind Vicia villosa (VV). MLN VV- T cells, mainly CD8+, mediated hyporesponsiveness and MLN VV+ T cells (CD45RC+ CD4- CD8- cells) abrogated the hyporesponsiveness. Moreover, Sp DEX adherent T cells were mainly CD8+. Intestinal intraepithelial lymphocytes (iIELs) mainly CD8alpha+ gamma(delta)-TCR+ cells also inhibited the DTH response to DEX after transfer. The positive DTH response to another carbohydrate (levan) indicates the specificity of the suppression to dextrin. Therefore, our data indicate that after oral administration of DEX, two different populations of T cells were generated: one found only in the MLN that mediated DTH responses and the other one capable of migrating from the intestinal intraepithelium through PP and MLN to the Sp, mediating systemic tolerance.

Administration, Oral↗
PubMed2002

Trypanosoma cruzi heat-shock protein-70 kDa,alone or fused to the parasite KMP11 antigen, induces functional maturation of murine dendritic cells.

We analyse the effect of Trypanosoma cruzi heat-shock protein-70 (HSP70) on the maturation of murine dendritic cells (DC)generated from bone marrow precursor cells. The results obtained show that HSP70, both alone and fused to the KMP11 antigen, as well as a HSP70 fragment, is capable of maturing murine DC. Mature DC have enhanced expression of IL12, TNF-alpha cytokines, costimulation molecules and activation markers, showing a clear increase in the allostimulatory capacity. These findings suggest that T. cruzi HSP70 may be a very useful vehicle for developing DC-based immunoprophylaxis and therapy against infections.

Animals↗
PubMed2002

Ascorbic acid in diet supplements: loss in the manufacturing process and storage.

The ascorbic acid content was determined in 25 different diet supplements commercially available to the consumer in two pharmaceutical forms (pills and ampoules). These products are widely consumed by several population groups (elderly people, sportsmen, adolescents, children, etc.). High-performance liquid chromatography was used as the analytical technique. The proposed method has been validated with good linearity, reproducibility, recovery and accuracy, and can be used in routine analyses and in quality control. The ascorbic acid content in pills ranged from 15.62 to 50.16 mg/g, and in that ampoules from 2.12 to 8.83 mg/ml. Depending on the dosage rates, these levels would represent approximately 20-50% of the daily dietary intake recommended by the National Research Council. Possible losses in the manufacturing process and stability during storage for 30 days at 40 +/- 2 degrees C without light, were tested. In relation to the ascorbic acid concentrations stated on the labels, a loss of 12.0-21.9% in pills and of 11.7-18.0% in ampoules was detected. In relation to the stability conditions, the losses are of 1.8-24.8% in pills and of 10.4-19.3% in ampoules. The pasteurisation and sterilisation processes produced a mean loss of ascorbic acid in ampoules of 2.1 and 1.4%, respectively. A statistically significant direct correlation was observed between ascorbic acid loss and content in proteins, humidity, ash, and fructose. The influence of the pharmaceutical form was also tested. Data revealed that the control of losses during the manufacturing and commercialisation process of these products is necessary to ensure the intake of vitamin C from these products by the consumer.

Ascorbic Acid↗
PubMed2001

Chromium in basic foods of the Spanish diet: seafood, cereals, vegetables, olive oils and dairy products.

In the present study, graphite furnace atomic absorption spectrometry (GFAAS) was used to determine the content of total chromium in some basic foods of the Spanish diet. The seafood samples, vegetables and olive oils were mineralized previously with HNO3 and V2O5. A procedure of slurries with Triton X-100 was applied to dairy products. The temperature-time program was optimized for every type of sample. An assessment of the analytical characteristics of the method verifies their reliability. The content of Cr ranged between 0.004 and 0.079 microg/g in seafood (fresh wt.), from 0.007 to 0.456 microg/g in cereals and vegetables (fresh wt.), between not detectable and 0.625 microg/g in dairy products and between not detectable and 0.040 microg/g in olive oils. The high consumption of these products conditions the fact that they should be significant sources of Cr in the diet.

Animals↗
PubMed2001

DNA breakage due to metronidazole treatment.

The mutagenicity of metronidazole [1-(hidroxyethyl)-2-methyl-5-nitroimidazole] (MTZ) has been shown in different prokaryotic systems. However, data on human cells are still contradictory. In this study DNA damage was determined by the single cell gel electrophoresis (SCGE) assay, in lymphocytes from 10 healthy subjects treated with therapeutic doses of this drug. Samples were obtained before treatment, as well as 1 and 15 days after ending treatment. Results showed a significant increase of DNA strand breaks 1 day after ending treatment, although, an inverse correlation between the amount of DNA damage and plasma concentrations of MTZ was obtained. Thus, the observed damage may be induced by some MTZ metabolite rather than by the parent drug. Interestingly, the amount of DNA damage returned to basal levels 15 days after ending treatment, except in two individuals. This persistent damage should be further investigated.

Adolescent↗