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Biomedical subjects

C Puerta

Publications and source records attributed to C Puerta.

18 recordsLinked to original sources
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↗
PubMed2003

NFkappaB and AP-1 DNA binding activity in patients with multiple sclerosis.

Current evidence suggests that multiple sclerosis (MS) results from an autoimmune response mediated by T lymphocytes, which would be activated in the peripheral blood and migrate into the central nervous system. NFkappaB and AP-1 are two main transcription factors involved in T-cell activation. To investigate possible alterations in the activity of these factors in MS individuals, we have assayed NFkappaB and AP-1 DNA binding activity in peripheral blood mononuclear cells (PBMC). Binding activity was analyzed by gel mobility shift assay in MS patients compared with controls. No significant differences were found between the two groups, indicating no evidence of abnormalities associated with MS in NFkappaB or AP-1 binding activities in PBMC, both basally and after PMA+anti-CD3 antibody induction.

Adult↗
PubMed2001

Changes in cannabinoid CB(1) receptors in striatal and cortical regions of rats with experimental allergic encephalomyelitis, an animal model of multiple sclerosis.

Data, initially anecdotal, but recently supported on more solid experimental evidence, suggest that cannabinoids might be beneficial in the treatment of some of the symptoms of multiple sclerosis (MS). Despite this evidence, there are no data on the possible changes in cannabinoid CB(1) or CB(2) receptors, the main molecular targets for the action of cannabinoids, either in the postmortem brain of patients with MS or in animal models of this disease. The present study addressed this question using the model of experimental allergic encephalomyelitis (EAE) in Lewis rats generated by inoculation of guinea pig myelin basic protein in Freund's adjuvant. After inoculation, animals were examined daily to detect the appearance of neurological signs. The first signs appeared around day 10 after inoculation, reaching the highest degree by day 13, when animals were sacrificed and their brains removed and used for analysis of CB(1) receptor binding, mRNA levels, and activation of GTP-binding proteins. CB(1) receptor binding and mRNA levels were not affected in EAE rats in brain areas such as the hippocampus, limbic structures, and cerebellum. However, there was a marked decrease in both parameters in the caudate-putamen, both in the lateral and medial parts, although this decrease did not correspond with decreases in binding in the nuclei recipient of striatal output neurons, which suggests that changes in CB(1) receptors are exclusively located in the cell bodies of striatal neurons. In addition, CB(1) receptor binding, but not mRNA levels, also decreased in the cerebral cortex, both in the deep and the superficial layers. The analysis of [(35)S]GTPgammaS binding after activation of CB(1) receptors with WIN55,212-2, a synthetic agonist, revealed that, despite the decrease in the number of CB(1) receptors in EAE rats, these were more efficiently coupled to GTP-binding protein-mediated signaling mechanisms in both the caudate-putamen and the cerebral cortex of these animals. In summary, these data suggest that the generation of EAE in Lewis rats would be associated with changes in CB(1) receptors in striatal and cortical neurons, which might be related to the alleviation of some motor signs observed after the treatment with cannabinoid receptor agonists in similar models of MS in rodents.

Animals↗
PubMed2000

Aminoguanidine reduces apoptosis of circulating V Beta 8.2 T lymphocytes in Lewis rats with actively induced experimental autoimmune encephalomyelitis. Association with persistent inflammation of the central nervous system and lack of recovery.

Aminoguanidine therapy delayed the onset of actively induced EAE in Lewis rats, but recovery was impaired in most animals. In the central nervous system this was correlated with persistent inflammation and production of proinflammatory cytokines. In the periphery of aminoguanidine-treated animals, T lymphocytes showed increased proliferation against myelin basic protein, and the percentage of Vbeta 8.2(+) T lymphocytes undergoing early apoptosis was markedly decreased, although it was unchanged in Vbeta 8.2(+) T cells isolated from the spinal cord. These results suggest that the prolonged survival of circulating encephalitogenic cells achieved by aminoguanidine would favor a longer lasting entry of these cells into the nervous system resulting in persistent inflammation and lack of recovery.

Animals↗
PubMed2000

Plasticity of the histone H2A genes in a Brazilian and six Colombian strains of Trypanosoma cruzi.

The analysis of three recombinant clones containing the histone H2A locus isolated from a genomic library of Trypanosoma cruzi DNA shows that the H2A gene loci are formed by 1.2 and 0.76 kb long intercalated units organized in a head-to-tail tandem array. The difference in length between the two gene units is due to the presence of a short interspersed nucleotide element (SINE)-like DNA sequence inserted at the 3' end of some of these units. Southern, northern and chromosomal blot analysis of a Brazilian Y strain and six Colombian strains demonstrated the existence of polymorphisms regarding the relative copy number of the H2A gene units, the relative abundance of the H2A transcripts and their chromosomal location. These results show the existence of a dynamic organization in the H2A loci among T. cruzi strains in which a SINE-like sequence may be involved and support the fact that T. cruzi has a high degree of plasticity in its genome.

Animals↗
PubMed2000

The stability and maturation of the H2A histone mRNAs from Trypanosoma cruzi are implicated in their post-transcriptional regulation.

We have recently described that the Trypanosoma cruzi histone H2A genes are actively transcribed as two sized classes of polyadenylated transcripts and that they differ in the 3'-UTRs due to the insertion of a partial SINE sequence in the 3'-end of some of H2A gene units. The expression of the H2A genes in the non-replicative trypomastigote forms is very low, whereas in the replicative forms, there is significant and constitutive transcription of the H2A genes probably regulated in a posttranscriptional way and associated to DNA replication. The data presented in this paper reveal that in epimastigotes, the steady-state levels of the H2A mRNAs are determined by controlling the stability of the messengers in the cytoplasm, most likely mediated by a nuclease attack. The data also indicate that there must be an additional control, associated to the parasite growth phase, which may act at the maturation step of the transcripts. The data suggest, moreover; that the cytoplasmic level of the H2A protein might be involved in the regulation of its own synthesis by controlling translation of existing messengers.

Animals↗
PubMed2000

Molecular characterization of the histone H2A gene from the parasite Trypanosoma rangeli.

The sequence, genomic organization, and transcription of the gene encoding the H2A histone protein of the protozoan parasite Trypanosoma rangeli is described in this paper. The locus encoding the T. rangeli H2A protein is formed by at least 11 gene units measuring 790 nucleotides in length, organized in tandem, and located in a single chromosome of approximately 1.9 Mb. The gene units actively transcribe only one size class of mRNA measuring 0.7 kb in length. The T. rangeli H2A protein contains in the amino-terminal the AGLXFPV motif, which is conserved in a broad range of H2A proteins, and the RSAK motif, which is implicated in repression of the histone's basal transcription in yeast. The carboxyl-terminal of the protein contains a two-lysine residue described as the ubiquitin binding site and the histidine residue implicated in DNA binding.

Amino Acid Sequence↗
PubMed1999

Type IV phosphodiesterase inhibition in experimental allergic encephalomyelitis of Lewis rats: sequential gene expression analysis of cytokines, adhesion molecules and the inducible nitric oxide synthase.

Type IV phosphodiesterase inhibitors are able to suppress EAE. To investigate the effects of this therapy in the central nervous system, we serially analyzed from days 7 to 17 postinoculation the gene expression pattern of tumor necrosis factor (TNF), lymphotoxin, interferon-gamma, interleukin-1beta, the inducible nitric oxide synthase (iNOs), interleukin-10, the vascular cell adhesion molecule-1 (VCAM-1) and the intercellular adhesion molecule-1 (ICAM-1) in the spinal cord of Lewis rats with actively induced EAE, treated with Rolipram. Treated rats had a delayed and milder disease, and reduced numbers of infiltrates in the nervous tissue. The gene expression profile was similar to that of untreated rats, although delayed, with no evidence of IL-10 upregulation during the observation period. The delayed inflammation was not associated with changes in the expression of VCAM-1 and ICAM-1. In peripheral blood mononuclear cells, TNF mRNA levels were decreased and interleukin-10 was unchanged. This therapy did not alter the proliferative ability of T lymphocytes against myelin basic protein. The encephalitogenic potential of splenocytes from treated animals was also unaffected. The high levels of both iNOs mRNA and nitric oxide (NO) found before the appearance of clinical signs, suggests that NO generation might be a contributing factor to the therapeutic benefit achieved by Rolipram in the rat.

Animals↗
PubMed1998

Transcriptional inhibitory role of the tail domains of histone (H3 x H4)2 tetramers.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for this polymerase, (H3 x H4)2 tetramers deprived of their tail domains, and H2A x H2B dimers. Histone (H3 x H4)2 tetramers lacking their terminal domains were obtained from trypsin-digested nucleosomal cores. The oligonucleosomal templates containing (H3 x H4)2 tetramers lacking their tail domains, like the control templates with intact core histone octamers, protect approximately 146 base pairs of DNA against micrococcal nuclease digestion. The transcriptional inhibition caused by the association of DNA with core histone octamers is significantly reduced upon elimination of the tail domains of the (H3 x H4)2 tetramers. Apparently, the terminal domains of (H3 x H4)2 must be present to block transcription efficiently. These results show the important inhibitory role played by the tail domains of the histone (H3 x H4)2 tetramers, suggesting the involvement of these regions in transcriptional regulation.

Animals↗
PubMed1998

Control mechanisms of the H2A genes expression in Trypanosoma cruzi.

In a previous report we have described that the T. cruzi histone H2A gene is encoded in two independent gene clusters located in a single chromosome. In the present paper we show that both gene cluster are actively transcribed as two sized classes of polyadenylated mRNAs demonstrating, moreover, the existence of alternative splicing sites and microheterogeneities at the polyadenylation site. We also describe that while the expression of the H2A genes in the non replicative trypomastigote forms is only residual, in the replicative forms there is constitutive transcription of these genes and that the transcription is not associated to DNA replication. The data show, moreover, that in the replicative forms the steady state levels of the H2A mRNAs are controlled at a post-transcriptional level which is associated to DNA replication.

Amino Acid Sequence↗
PubMed1995

Transcriptional properties of oligonucleosomal templates containing acetylated (H3-H4)2 tetramers.

Direct chemical acetylation of an oligonucleosomal template for bacteriophage T7 RNA polymerase is accompanied by a substantial increase in its capability to support RNA synthesis. The template was assembled from a plasmid, containing a promoter and a terminator for T7 RNA polymerase, plus one (H3-H4)2 tetramer and two H2A.H2B dimers for each 200 base pairs of DNA. Under the employed conditions, acetylation modifies in a preferential way the lysine residues located in the amino-terminal domains of core histones. When the template is assembled with acetylated tetramers and untreated dimers, its efficiency in promoting RNA synthesis is also largely increased. Since a previous work reported transcriptional stimulation upon acetylation of H2A.H2B dimers [Puerta et al. (1995) Biochem. Biophys. Res. Commun. 210, 409], the transcriptional repression brought about by core histone octamers seems to require that the amino-terminal domains of both (H3.H4)2 tetramers and H2A.H2B dimers are not acetylated.

Acetylation↗
PubMed1995

Acetylation of histone H2A.H2B dimers facilitates transcription.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for T7 RNA polymerase, intact (H3.H4)2 tetramers, and either untreated or chemically acetylated H2A.H2B dimers. The nucleosomal particles containing acetylated H2A.H2B dimers protect 145 base pairs of DNA against micrococcal nuclease digestion and prevent the reaction with psoralen of 80 to 145 DNA base pairs. The inhibition of transcriptional initiation caused by the association of DNA with intact core histone octamers decreases significantly when the histone octamers contain acetylated H2A.H2B dimers. These results suggest a role for H2A.H2B dimers in the control of transcription, which might be mediated through acetylation and deacetylation of their lysine residues.

Acetylation↗
PubMed1994

Isolation and characterization of the gene encoding histone H2A from Trypanosoma cruzi.

In the present paper we report the isolation and characterization of the sequence of two genomic DNA fragments coding for the histone H2A of Trypanosoma cruzi. An analysis of the predicted amino acid sequence shows the presence of the amino-terminal motif characteristic of the H2A histones proteins and the Lys-Lys motif reported to be the site for the ubiquitin attachment. Southern blots of total parasite DNA probed with the H2A sequence suggested that the T. cruzi histone H2A gene is encoded in two independent gene clusters. The molecular karyotyping of the parasite indicated that these two clusters locate in a single chromosome of about 700 kb in length. The T. cruzi H2A mRNA is polyadenylated as are the basal histone mRNAs of higher eukaryotes and the histone mRNAs of yeast. By polymerase chain reaction amplification and sequencing and by S1 mapping we determined respectively the 5' and 3' end of the gene showing that the miniexon is added to the mRNA 71 nucleotides upstream of the ATG initiation codon and that the polyadenylation site locates in nucleotide position 773-775 close to invert repeats.

Amino Acid Sequence↗
PubMed1993

Efficient transcription of a DNA template associated with histone (H3.H4)2 tetramers.

A histone-DNA transcription template has been assembled, by dialysis against decreasing salt concentrations, from pGEMEX-1 (4 kilobases), a plasmid containing a promoter for bacteriophage T7 RNA polymerase, and from isolated histone (H3.H4)2 tetramers. Electron microscopy after psoralen cross-linking shows that each histone tetramer protects approximately 80 base pairs of DNA from psoralen action and that, under the employed conditions, an average of 15 tetramer particles are assembled per DNA molecule. This (H3.H4)2-DNA template is efficiently transcribed in vitro by T7 RNA polymerase as compared to naked DNA. The presence of (H3.H4)2 tetramers does not affect initiation, in contrast with the complete histone octamer, (H2A.H2B.H3.H4)2, assembled with the complementary addition of H2A.H2B dimers, which causes transcriptional inhibition mainly by blocking initiation.

DNA↗
PubMed1993

Transcription of mononucleosomal particles acetylated in the presence of n-butyrate.

Although a correlation between chemical acetylation of the amino-terminal tails of core histones and stimulation of RNA synthesis has been reported for nucleosomal core particles (Piñeiro et al. (1991) Biochem. Biophys. Res. Commun. 177:370), no differences in transcription are detected between acetylated and nonacetylated mononucleosomal particles obtained from HeLa cells in the presence and absence of n-butyrate. Apparently, the lysine residues modified in the presence of n-butyrate are not the same responsible for the observed acetylation-induced transcription. The acetylation obtained with n-butyrate might be significantly different from that present in transcriptionally active chromatin.

Acetylation↗
PubMed1991

Yeast nucleosomal particles: structural and transcriptional properties.

Yeast nucleosomal core particles have been characterized by thermal denaturation, circular dichroism, and digestion with DNase I and with trypsin. Practically all nucleosomal DNA melts in one transition centered at 70 degrees C, and the circular dichroism spectrum is displaced to lower wavelengths as compared to that corresponding to chicken nucleosomal cores. The susceptibility of yeast nucleosomal particles to dissociation by salt is significantly higher than that of chicken nucleosomal cores, a substantial dissociation being observed at 0.5 M NaCl. Treatment of yeast nucleosomal particles with the amino group reagent dimethylmaleic anhydride is accompanied by selective release of histones H2A and H2B. The results indicate not large but significant structural differences between yeast and chicken nucleosomal cores. However, the in vitro transcription properties of complete and H2A.H2B-deficient nucleosomal cores are similar in the two kinds of particles: the histone octamer blocks RNA synthesis, this block being eliminated in part by the partial loss of histones H2A and H2B.

Animals↗
PubMedDate not supplied

[Combined therapy in multiple sclerosis].

INTRODUCTION: The availability of new drugs has been a significant advance in the therapy of multiple sclerosis over the last years. However, the control of the disease is far from being complete, and some patients respond poorly or not at all to the new drugs. DEVELOPMENT: The present paper reviews the approved therapies for multiple sclerosis and examines the advantages of the association of drugs, both with known and possible efficacy, as well as the theoretical basis for such associations and their possible clinical indications. Different designs for clinical trials in combination therapy are analyzed with a consideration of the current difficulties for the use of placebo in multiple sclerosis forms associated with relapses. CONCLUSIONS: The achievement of a higher efficacy than the present one is a major challenge in multiple sclerosis therapy. Combination therapy appears to be a reasonable option for that purpose.

Clinical Trials as Topic↗