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J M Requena

Publications and source records attributed to J M Requena.

At least 55 records · Page 3Linked to original sources
PubMed1996

Presence of antibodies in the aqueous humour and cerebrospinal fluid during Leishmania infections in dogs. Pathological features at the central nervous system.

In the present paper we show that in dogs, naturally infected with Leishmania infantum, the aqueous humour and the cerebrospinal fluid contain anti-Leishmania IgGs and that the specificity of antigen recognition of these fluids is similar to that of the sera. We also show that in the encephalon and cerebellum of these dogs there is a pathological sponge-like reaction accompanied by neuronal degeneration, mobilization of glial cells together with accumulation of amyloid deposits. The interstitial and intravascular deposition of IgGs and Leishmania antigens in choroid plexus suggest that in these animals there is a failure of the blood-cerebrospinal and ciliary bodies filtration barriers which may allow the transfer of anti-Leishmania IgGs from the blood stream to these fluids. We suggest that the failure of the blood-cerebrospinal barrier and the in situ concentration of anti-Leishmania IgGs and antigens in brain tissues may predispose to the pathological features detected in this compartment.

Animals↗
PubMed1996

Specific serodiagnosis of human leishmaniasis with recombinant Leishmania P2 acidic ribosomal proteins.

The Leishmania P2 proteins have been analyzed as potential tools for the immunodiagnosis of human mucocutaneous and visceral leishmaniasis. Two recombinant Leishmania infantum proteins, rLIP2a and rLip2b, were used. The analysis indicated that the rLiP2a and rLiP2b proteins are recognized by 76% (16 of 21) and 42% (9 of 21), respectively, of sera from patients with mucocutaneous leishmaniasis and by 50% (5 of 10) and 40% (4 of 10), respectively, of sera from patients with visceral leishmaniasis. The Leishmania P2 proteins were engineered to have deletions of particular amino acids from the carboxyl-terminal region in order to avoid cross-reactivity with sera from patients with systemic lupus erythematosus and Chagas' disease, since it is known that this region is the main target of the autoantibodies present in sera from these patients. The results show that while the modified recombinant proteins rLiP2a-Q and rLiP2b-Q, in which the five carboxyl-terminal amino acids had been deleted, maintain the leishmaniasis-specific epitopes, they do not react with sera from patients with autoimmune disease and Chagas' disease. For this reason, and also because the sera from patients with tuberculosis and leprosy, diseases that have to be considered in a differential clinical diagnosis of infectious diseases, do not react with the rLiP2a-Q or rLiP2b-Q protein, we think that the engineered proteins may be considered specific tools for the immunodiagnosis of mucocutaneous and visceral leishmaniasis.

Adolescent↗
PubMed1995

Assignment of genes to Leishmania infantum chromosomes: karyotype and ploidy.

The use of various pulsed-field electrophoresis methodologies under different conditions allowed us to determine the Leishmania infantum karyotype. A total of 25 chromosomal bands ranging in size from 375 to 3300 kb were resolved amounting to a minimum genomic DNA mass of about 2.6 x 10(7) pb. By molecular hybridization and on the basis of the karyotype, specific gene sequences could be assigned to particular chromosomes. A bias in the chromosomal distribution of different markers was found since 9 out of the 12 analysed gene markers hybridize with chromosomal bands XIXa and XIXb. We infer that chromosomal bands XIXa and XIXb, differing in about 30 kb, could be representing a pair of homologous chromosomes and that another pair of homologs may be also defined by chromosomal bands XVII and XVIII.

Animals↗
PubMed1995

Identification of the Leishmania infantum P0 ribosomal protein epitope in canine visceral leishmaniasis.

In the present work we show that a high percentage of the sera from dogs naturally affected with viscero-cutaneous leishmaniasis contain antibodies reacting with the Leishmania infantum P0 ribosomal protein. In order to map the antigenic determinants of the LiP0 protein during Leishmania-infection, the complete amino acid sequence of the protein was synthesized as overlapping 20-mer peptides. We have identified the sequence AAKEEPEESDEDDFGMG, located adjacent to the C-terminal end of the protein, as the major antigenic determinant. The anti-LiP0 antibodies present in the sera of the infected dogs do not cross-react with a relatively similar antigenic determinant of the LiP2 acidic proteins as an indication that the Leishmania P0 protein is an independent immunogenically functional antigen in the canine form of the infection.

Amino Acid Sequence↗
PubMed1995

Mapping of the linear antigenic determinants from the Leishmania infantum histone H2A recognized by sera from dogs with leishmaniasis.

Antibodies reacting against the H2A histone protein were frequently observed in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum. Using synthetic peptides covering the complete sequence of the protein we have identified the amino terminal region, comprising from amino acids 1 to 20, and the carboxyl terminal region, comprising from amino acids 106 to 132, as conforming the antigenic determinants of the protein. Those regions, exposed in the nucleosome surface, are highly divergent in sequence relative to the mammalian H2A histones. The anti-H2A histone antibodies present in the sera of these dogs specifically recognize the L. infantum H2A histone and they do not react with mammalian histones. The present data indicate that, in spite of the evolutionary conservation of the H2A histone protein among eukaryotic organisms, the humoral response against this protein during natural infection is specifically triggered by the parasite protein antigenic determinants.

Amino Acid Sequence↗
PubMed1995

During active viscerocutaneous leishmaniasis the anti-P2 humoral response is specifically triggered by the parasite P proteins.

In this work we show that in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum there are antibodies that react specifically against the parasite acidic ribosomal proteins LiP2a and LiP2b, and that each one of the Leishmania P proteins elicits a specific humoral immune response. Using synthetic peptides, the antigenic epitope of these proteins has been mapped in a single region located adjacent to the C-terminal domain highly conserved among the eukaryotic P proteins. The anti-P antibodies elicited during the Leishmania infection do not recognize the conserved C-terminal domain of the parasite P proteins, in contrast with the findings reported in Chagas' disease or systemic lupus erythematosus. The antigenic epitopes of the LiP2a and LiP2b are almost identical in amino acid sequence. No reactivity against Trypanosoma cruzi and human P proteins was found in sera from L. infantum-infected dogs.

Amino Acid Sequence↗
PubMed1994

The Leishmania infantum histone H3 possesses an extremely divergent N-terminal domain.

The isolation of a Leishmania cDNA clone coding for an antigen identified as the histone H3 is described. The nucleotide sequence of the cDNA predicts that the Leishmania histone H3 contains 129 residues and that it has a molecular mass of 14,620 Da. Comparison of the amino acid sequence with the consensus sequence of the eukaryotic histone H3 shows that the Leishmania protein has a highly conserved globular region and an extremely divergent amino-terminal portion.

Amino Acid Sequence↗
PubMed1994

Characterization of a short interspersed reiterated DNA sequence of Trypanosoma cruzi located at the 3'-end of a poly(A)+ transcript.

We have carried out the molecular characterization of a highly repeated DNA element, called E12, from Trypanosoma cruzi, which has been found to be interspersed along its genome. The E12 element, repeated about 5.6 x 10(3) times, is found in most of the chromosomal bands of the parasite. Three subregions may be defined within the element on the basis of sequence similarities with other trypanosome genomic sequences. Northern blot analysis demonstrated that sequences of the E12 element are present in several polyadenylated RNA species of T. cruzi. The isolation and characterization of a cDNA clone, pSPFM55, which showed hybridization with the E12 probe, indicated that only one of the E12 subregions, E12A, is found in the cDNA and that it is located at the 3'-end providing the site of polyadenylation addition. The location and high degree of nucleotide conservation of E12A suggest a possible functional role of this sequence in gene expression.

Amino Acid Sequence↗
PubMed1993

The anti Z-DNA reactivity of Z-DNA forming sequences is affected by platinum antitumor drugs.

The effect of binding of platinum antitumor drugs, cis-diamminedichloroplatinum (II) (cis-DDP) and Pt-pentamidine, on the Z-DNA reactivity of potential Z-DNA forming sequences has been studied by enzyme-linked immunosorbent assay. The results indicate that cis-DDP and Pt-pentamidine increase the Z-DNA reactivity of plasmids containing a (dG-dC)16 insert (pUCZ8) and a native Z-DNA forming sequence of Drosophila hydei (pF18). The molar ratio of platinum bound to nucleotides (rb) to produce 50% of Z-DNA reactivity was 0.10 for cis-DDP:pF18, 0.15 for Pt-pentamidine:pF18, and 0.10 for cis-DDP:pUCZ8 and Pt-pentamidine:pUCZ8. The efficacy of Pt-pentamidine to provoke Z-DNA reactivity is 2.5-fold higher than that of cis-DDP. While Pt-pentamidine was capable of inducing Z-DNA reactivity in a GC-rich DNA sequence of the hsp 70 protein of Trypanosoma cruzi (p2M4EO3) and in sequences of pUC8, cis-DDP suppresses Z-DNA reactivity. CD spectra of poly(dG-me5dC).poly(dG-me5dC) modified by the drugs suggest that the increase in Z-DNA reactivity observed in plasmids upon drug binding may be due to shifting towards Z-DNA or a Z-DNA like conformation.

Antineoplastic Agents↗
PubMed1993

Cytoplasmic-nuclear translocation of the Hsp70 protein during environmental stress in Trypanosoma cruzi.

The present study provides immunological evidence of the constitutive presence of the Hsp70 protein in the cytoplasm of logarithmically growing T. cruzi parasites cultured at the normal temperature of 28 degrees C and of the translocation of the protein to the nucleus upon a heat shock treatment (2 hours at 37 degrees C). The nuclear translocation of the protein must depend on other factors beside the temperature per se since at 28 degrees C, in stationary phase growing parasites, the Hsp70 protein was present in both the cytoplasm and the nucleus. During recovery at 28 degrees C the protein leaves the nuclei but the nuclear-cytoplasmic translocation of the protein is a much more gradual process than its initial transport to the nucleus. Since the isoform of the nuclear Hsp70 is different from that found in the cytoplasm it is likely that before translocation to the nucleus the cytoplasmic Hsp70 nuclear precursor must undergo a specific modification.

Animals↗
PubMed1993

Genomic organization and expression of two independent gene arrays coding for two antigenic acidic ribosomal proteins of Leishmania.

In the present paper we describe the isolation and characterization of four novel genes of the parasitic protozoan Leishmania infantum. These genes are organized as two independent gene clusters, and they are related by nucleotide sequence to eukaryotic genes encoding acidic ribosomal proteins. Each gene cluster contains two tandemly linked genes coding for identical proteins. Each of the proteins coded by the gene clusters (called LiP and LiP') are highly divergent in sequence, showing the characteristic features of eukaryotic P-proteins from the P2 group. In spite of the sequence conservation of the coding regions of each of the genes in the cluster, the 5'- and 3'-untranslated regions are heterogeneous in sequence. The analysis of the expression of these genes indicates that logarithmic phase promastigotes show increased levels of LiP- and LiP'-specific transcripts compared with stationary phase promastigotes. The steady state RNA levels of the LiP and LiP' genes show a similar dependence of the growth phase of the parasite. Using specific probes for the divergent 3'-untranslated regions of each of the genes, it was found that the abundance of the mature transcripts is different even when the transcripts are derived from the same gene cluster. These findings probably indicate that the 3'-untranslated regions may influence the stability or turnover of the transcripts derived from both LiP and LiP' gene clusters.

Amino Acid Sequence↗
PubMed1993

Binding of Pt-pentamidine to nucleosomal DNA. Studies of the antiproliferative activity of the drug against human cancer cells.

In the present paper we present data showing that the effect of the binding of the antitumour drug Pt-pentamidine to nucleosomal DNA is the opposite to that of the cis-DDP compound since it causes strong stabilization of the double helix to heat denaturation and because in nucleosome:Pt-pentamidine complexes the nucleosomal denatured DNA is able to reassociate at 71 degrees C. Upon binding, the pentamidine ligand, by itself, also produces stabilization of the nucleosomal DNA but the effect is lower than that induced by Pt-pentamidine. It seems that in Pt-pentamidine:nucleosome complexes about 50% of the adducts are formed during the first hour of incubation of the nucleosomes with the drug since the increase in Tm of the DNA of these complexes is 53% of the total increase in Tm of the DNA of the Pt-pentamidine:nucleosome complexes formed in 48 h. The 'in vitro' screening of the antiproliferative activity of Pt-pentamidine against 60 tumour cell lines indicated that this antitumour compound shows higher antiproliferative activity against small cell lung, non-small cell lung and melanoma cancer lines than against the rest of the cell lines.

Antineoplastic Agents↗
PubMed1993

Mapping of antigenic determinants of the T. cruzi hsp70 in chagasic and healthy individuals.

In the present paper we describe the analysis of the immunological recognition by sera of healthy individuals and chagasic patients of the Trypanosoma cruzi heat shock 70 kDa protein. By a Falcon Assay Screening Test, using as antigen an ATP-agarose purified T. cruzi hsp70, it has been found that the sera of infected patients as well as of that of healthy individuals show reactivity against the hsp70 protein but that the reactivity of the sera of patients is in general significantly higher than that of healthy individuals. The analysis of the reactivity of the chagasic sera against a collection of peptides covering 92% of the protein has shown that more than 50% of the peptides gave a positive response but only against a few peptides did we observe high reactivity in a wide spectrum of sera. Only four peptides (numbers 9, 12, 14 and 47) were recognized by all sera tested with high reactivity values. The sera of healthy individuals also showed reactivity against a large percentage of peptides but with lower values. It was observed that particular peptides showing high reactivity against the sera of healthy donors also show high reactivity against patients' sera. However, the general pattern of reactivity against the peptides is different in chagasic and healthy sera. The immunodominant peptides map in the highly conserved as well as in the less conserved part of the hsp70 molecule. The 1/3 C-terminal, being the least conserved part of the molecule, seems to be the least immunogenic. Mapping of the epitopes led to the identification of particular immunogenic motifs within individual peptides.

Amino Acid Sequence↗
PubMed1993

Isolation, characterization and analysis of the expression of the Leishmania ribosomal PO protein genes.

Two tandemly linked genes are present in the Leishmania infantum genome that code for the acidic ribosomal PO protein. The genes are identical in the coding region, although a striking lack of nucleotide sequence conservation is observed when the boundaries of the coding regions between both genes are compared. The 3' untranslated regions of the two genes are, moreover, different in size. The deduced amino acid sequence of the L. infantum PO protein (LiPO) shows a high degree of sequence conservation, including the highly charged conserved C-terminal domain, with the ribosomal PO proteins of other eukaryotic organisms. Northern blot experiments showed that two different size class transcripts are expressed in the gene cluster and that the steady state level of each of the transcripts in logarithmic phase promastigotes is markedly different. The abundance of both transcripts is down-regulated in parasite cultures on reaching stationary phase. Since it seems that the two Leishmania ribosomal PO genes are expressed in a single polycistronic transcript, it is likely that the different levels of PO mRNAs observed in cultured cells is due to a postranscriptional regulatory mechanism.

Amino Acid Sequence↗
PubMed1993

Isolation of Trypanosoma cruzi specific nuclear repeated DNA sequences.

We described the isolation of two genomic fragments, called E12 and E22, containing highly repeated DNA sequences from Trypanosoma cruzi. The E12 and E22 fragments have molecular sizes of about 1.1 and 0.8 Kb, respectively. Copy number determinations indicate that each one of these sequences are repeated about 5 x 10(3) times in the parasite genome. E12 and E22 elements do not cross-hybridize indicating that they are not related in nucleotide sequence. The Southern blot analysis of total DNA digested with a variety of restriction enzymes shows the existence of a complex pattern of hybridization bands with the repeated elements indicating that they are interspersed along the T. cruzi genome. In fact, pulsed field gel electrophoresis together with hybridization evidenced that both sequences are present in all or nearly all the chromosomal bands. Clones bearing these repetitive elements could be useful for diagnostic of the Chagas' disease and strain classification purposes.

Animals↗
PubMed1992

Molecular characterization of a Leishmania donovani infantum antigen identified as histone H2A.

A Leishmania donovani infantum promastigote cDNA expression library was screened with a serum obtained from a dog naturally infected with this parasite. One of the positive clones obtained revealed nucleotide sequence similarities with the histone H2A genes from various organisms. Northern blot analyses and sequence data of three independently isolated cDNA clones indicated that the Leishmania H2A mRNAs are polyadenylated, as are the basal histone mRNAs of higher eukaryotes and the histone mRNAs of yeast. The analysis of the genomic distribution of the DNA coding for histone H2A suggested that, in L. d. infantum, there are at least four genes coding for the H2A protein. It is likely that there is a simultaneous expression of at least two of the H2A genes since differences in nucleotide sequence between two of the sequenced cDNAs were observed. Affinity-purified antibodies against the beta-galactosidase-fused H2A protein recognize specifically a Leishmania protein band with a molecular mass of 14 kDa.

Amino Acid Sequence↗
PubMed1992

Regulation of hsp70 expression in Trypanosoma cruzi by temperature and growth phase.

The steady-state level of the hsp70 mRNAs of Trypanosoma cruzi cultured at different temperatures and growth conditions has been analyzed by Northern blotting. We show that only one size class of hsp70 mRNA, of about 2.2 kb, is transcribed from the hsp70 cluster and that its transcription is constitutive at 28 degrees C. However, after a heat shock treatment at 37 degrees C for 2 h of logarithmically growing parasites, the abundance of the hsp70 mRNA increased about 4-fold. A similar increase was observed at 28 degrees C when the parasite culture reached the stationary phase of growth. On the other hand, a heat shock at 42 degrees C did not change the steady state level of the 2.2-kb size class of hsp70 mRNA. However, accumulation of transcripts of high molecular weight was detected when stationary growing parasites were cultured at 42 degrees C for 2 h. Also at 37 degrees C the steady state level of the alpha- and beta-tubulin mRNAs of logarithmically growing parasites exhibited a slight increase but only after a period of 24 h. Analysis by one-dimensional immunoblots of the Hsp70 levels showed that at 37 degrees C the abundance of the protein was 4-fold higher than at 28 degrees C. Immunoblots of high-resolution two-dimensional gel electrophoresis showed, moreover, that various isoforms of this protein are constitutively expressed at 28 degrees C and that some of them have a specific pattern of induction at 37 degrees C. We observed, moreover, that the heat shock induces the expression of a series of proteins while it causes repression of others.

Animals↗